FMRP and MOV10 regulate Dicer1 expression and dendrite development

Monica C Lannom; Joshua Nielsen; Aatiqa Nawaz; Temirlan Shilikbay; Stephanie Ceman

doi:10.1371/journal.pone.0260005

. 2021 Nov 30;16(11):e0260005. doi: 10.1371/journal.pone.0260005

FMRP and MOV10 regulate Dicer1 expression and dendrite development

Monica C Lannom ¹, Joshua Nielsen ^2,^¤,^#, Aatiqa Nawaz ^1,^#, Temirlan Shilikbay ^1,^#, Stephanie Ceman ^1,^3,^*

Editor: Barbara Bardoni⁴

PMCID: PMC8631628 PMID: 34847178

Abstract

Fragile X syndrome results from the loss of expression of the Fragile X Mental Retardation Protein (FMRP). FMRP and RNA helicase Moloney Leukemia virus 10 (MOV10) are important Argonaute (AGO) cofactors for miRNA-mediated translation regulation. We previously showed that MOV10 functionally associates with FMRP. Here we quantify the effect of reduced MOV10 and FMRP expression on dendritic morphology. Murine neurons with reduced MOV10 and FMRP phenocopied Dicer1 KO neurons which exhibit impaired dendritic maturation Hong J (2013), leading us to hypothesize that MOV10 and FMRP regulate DICER expression. In cells and tissues expressing reduced MOV10 or no FMRP, DICER expression was significantly reduced. Moreover, the Dicer1 mRNA is a Cross-Linking Immunoprecipitation (CLIP) target of FMRP Darnell JC (2011), MOV10 Skariah G (2017) and AGO2 Kenny PJ (2020). MOV10 and FMRP modulate expression of DICER1 mRNA through its 3’untranslated region (UTR) and introduction of a DICER1 transgene restores normal neurite outgrowth in the Mov10 KO neuroblastoma Neuro2A cell line and branching in MOV10 heterozygote neurons. Moreover, we observe a global reduction in AGO2-associated microRNAs isolated from Fmr1 KO brain. We conclude that the MOV10-FMRP-AGO2 complex regulates DICER expression, revealing a novel mechanism for regulation of miRNA production required for normal neuronal morphology.

Introduction

Neuronal architecture is affected in many neurodevelopmental disorders. Fragile X syndrome (FXS) is caused by loss of the RNA binding protein (RBP) FMRP [1]. Extensive characterization of FMRP loss in Drosophila, mice and humans has led to robust observations revealing the role of FMRP in the development of abnormal dendritic spines [2]. FMRP has also been shown to play an important role in neuronal maturation. FXS patient-derived neurons from induced pluripotent stem cells (iPSCs) and hippocampal neurons from neonatal Fmr1 knockout (KO) mice and adult Fmr1 knockdown (KD) exhibit defects in neurite extension and dendritic maturation [3–7].

FMRP binds to both the coding region of mRNAs and the 3’ UTR [1, 2–9]. It is still poorly understood how loss of a single protein can lead to cognitive impairment although it is known that RBPs seldom act alone, existing in complexes with other RBPs and with the intermediary carrier of information, the mRNA, can enact widespread translational changes depending on their target [2, 10, 11].

FMRP functionally associates with the RNA helicase MOV10 [12], a component of the microRNA (miRNA) pathway and cofactor of Argonaute family members AGO1 and AGO2 [13, 14]. Through Cross-Linking ImmunoPrecipitation (CLIP) experiments, it has been established that FMRP and MOV10 share a common RNA interactome. Unlike FMRP, the Mov10 knockout (KO) is embryonic lethal [15, 16]; however the Mov10 heterozygous (Het) mouse has increased anxiety and hyperactivity, which are features shared with Fragile X syndrome [17] and suggest impaired neuronal function [15]. We were thus interested in investigating the consequences of Mov10 and Fmr1 reduction on dendritic development. Here we show that loss of FMRP and MOV10 leads to impaired dendrite maturation.

MOV10 and FMRP work dynamically to regulate expression of the Dicer1 mRNA. DICER, a type III endonuclease, generates the final functional miRNA from pre-miRNAs, and is highly regulated at every stage of transcription and translation from primary transcript processing to enzyme activity [18]. DICER associates with AGO2 to facilitate the transfer of the mature miRNA. It is unknown what other RBPs participate in this process. Many groups using different organisms have shown that both FMRP and MOV10 associate with DICER and AGO2 [2, 8, 19, 20]). In addition, a recent paper provides compelling evidence that FMRP binds some miRNAs in regions outside of the seed sequence [20]. This would be an intriguing mechanism for FMRP to recruit specific AGO-miRNA complexes to its bound mRNAs. Here, we provide evidence for local miRNA production through regulation of DICER1 expression by FMRP and MOV10.

Materials and methods

Animals

Experiments were performed on C57BL6/J WT, Mov10 Het and Fmr1 KO mice from both sexes (The Jackson Laboratory, Bar Harbor, ME). Animals were kept on a 12/12 hour light/dark cycle with food and water ad libitum. All experiments were performed during the light phase (7 AM-7 PM). Animals were treated in accordance and with compliance with Institutional Animal Care and Use Committee (IACUC) guidelines, IACUC protocol 19112.

Hippocampal neuron culture

Mov10 heterozygotes were genotyped at postnatal day 0 (P0) using tail samples and DNA was extracted with the KAPA Fast Extract Kit (#KK7103, KAPA Biosystems, Wilmington, MA). After genotyping, mouse hippocampi were dissected and cultured as described [21]. Coverslips were coated overnight with 10 μg/mL poly-l-lysine (#P4704, Sigma, St. Louis, MO) and 10⁵ cells/well were plated for immunofluorescence (IF) in minimum essential medium (MEM) supplemented with 10% fetal bovine serum (FBS). After 24 h, the medium was switched to Neurobasal (NB) medium (#21103049, Gibco, Dublin, Ireland) supplemented with B-27 (#17504–044, Gibco). Half of the media was removed and replaced with fresh NB medium every three days. Neuron culture was performed on at least 3 litters from each genotype.

Immunofluorescence and microscopy of cultured neurons

Neurons grown on coverslips were fixed in 4% paraformaldehyde for 10 minutes at room temperature. Samples were blocked in 10% normal donkey serum (#017-000-121, Jackson ImmunoResearch, West Grove, PA) for 30 min at room temperature. MAP2 antibody (1:1000 dilution, # AB5622, RRID: AB_91939, Millipore, Burlington, MA) was incubated overnight at 4°C. Secondary antibody (Alexa 594 goat anti-rabbit [1:4000, RRID:AB_2307325, Jackson ImmunoResearch, 111-585-144,]) was added for 2 h at room temperature. Coverslips were inverted unto glass slides containing mounting media with 1 μg/mL 4′,6-diamidino-2-phenylindole (DAPI). Fluorescence images of DIV14 neurons were obtained with a Zeiss LSM 700 inverted confocal microscope (Zeiss, Oberkochen, Germany) using a 40× and 63× EC Plan-Neufluar 1.30 oil objective respectively. Images were captured with a cooled charge-coupled device (CCD) camera running Zen 2012 software (Zeiss). A total of 10–15 0.2-μM-thick sections were acquired as z-stacks for each neuron imaged.

Neuronal transfection

P0 brains from Fmr1 knockout mice Mov10 heterozygotes mice were harvested for hippocampal neuron culture as above and on DIV2 transfected with pDESTmycDICER, (Addgene plasmid # 19873; RRID:Addgene19873) and empty MYC- or EGFP- expression vector plasmid DNA (Stratagene and Clontech, respectively) using Lipofectamine 2000 (Thermofisher #11668019) for 15 minutes. Half of the transfection media was removed and replaced with Neurobasal (NB) medium (#21103049, Gibco, Dublin, Ireland) supplemented with B-27 (#17504–044, Gibco). After four hours, media was fully replaced, and half of the media was removed and replaced with fresh NB medium every three days. At DIV 7, neurons were prepared for immunofluorescence. Transfection efficiency was measured as percentage of MYC-expressing cells per number of cells plated on coverslip (~50,000) cells and for both conditions, was ~ 0.05%.

Sholl analysis

Anonymized Sholl analysis of all orders of branches (Total Sholl) was performed using confocal z-stacks of WT, Mov10 Het, and Fmr1 KO DIV14 neurons immunostained for MAP2 and imported into ImageJ (Fiji, RRID:SCR_002285). A dendritic complexity analysis, including Sholl analysis, was performed according to the protocol described [22]. Neurite lengths from the soma and soma size area were traced and measured using Image J software and SNT plugin and the data were compiled and analyzed using the Excel program (RRID:SCR_016137, Microsoft, Redmond, WA) and GraphPad Prism (RRID:SCR_002798, San Diego, CA).

Neurite outgrowth

WT and Mov10 KO N2A cells were plated in triplicate (density of 1.5 x 10⁴cells/well) and incubated at 37°C in Dulbecco’s modified Eagle’s medium (DMEM, 10% fetal calf serum). Cells were allowed to differentiate for 48 hours in DMEM (2% FCS) and 20uM Retinoic Acid (Sigma-Aldrich) and imaged under transmitted light using an EVOS cell-imaging microscope (Thermofisher). The images were anonymized and analyzed by an experimenter blinded to the conditions using the Axiovision Image analysis software (Zeiss). 800–1000 differentiated neurons were counted from 10 images per condition.

Golgi staining

Brain tissue was processed separately in three sets of experiments performed at different times from P14 WT (n = 5), Fmr1 KO (n = 5) and Mov10 Het (n = 4). Animals were deeply anesthetized with sodium pentobarbital injection (60 mg/kg, i.p.) and transcardially perfused with 0.9% saline, pH 7.4. The brains were immediately processed for Golgi–Cox analysis using a standard protocol [23], embedded in celloidin [24] and sectioned in a coronal plane at 175 μm. Spine analysis was conducted as outlined by [24] from somatosensory cortex. Briefly, a 10 μM region was selected from each branch (apical and baslar) and all spines in that region were counted and categorized based on morphology. The same region was subsequently utilized for the dendrite width measurements.

Western blot

Samples from at least three biological replicates were prepared for immunoblotting after quantification by Bradford assay and suspension in 1× sample buffer, resolved by SDS-PAGE and analyzed by western/immunoblotting. Briefly, membranes were blocked with 10% non-fat dry milk in phosphate-buffered saline (PBS) containing 1% TWEEN-20 for 1 h at room temperature. Primary antibody was applied for 1 h at room temperature or overnight at 4°C followed by a brief wash in 1% non-fat milk PBS containing 1% TWEEN-20 wash buffer. Horseradish peroxidase (HRP)-conjugated secondary antibody was applied at 1:5000 dilution for 1 h at room temperature and washed 4 × 15 min using wash buffer. The HRP signal was detected using an enhanced chemiluminescent (ECL) substrate and exposed using iBright digital imaging platform. The antibodies used were anti-Dicer (1:100, #sc-393328, RRID:AB_2802128, Santa Cruz Biotechnology, Santa Cruz, CA). anti-eIF5 (RRID:AB_631427, Santa Cruz) at 1:5,000, anti-KIF1A (1ug/ml), (#ab91029, RRID:AB_10862338, Abcam, Cambridge, United Kingdom), 1:1000 anti-MAP1b (# 21633-1-AP, RRID:AB_10793666, Proteintech Group, Rosemont, IL), and HRP-conjugated anti-rabbit and anti-mouse antibodies (RRID:AB_772191, GE Healthcare, Chicago, IL) and Jackson Immunoresearch, (RRID:AB_2338512) respectively. The level of significance and tests performed are described in the Fig legends for each experiment.

Luciferase reporter assays

Luciferase assay constructs were obtained from Addgene (RRID:SCR_002037, Cambridge, MA). N2A or HEK293T cells were seeded at 5x10⁴ cells into a 24 well plate for 24 hours and transfected with irrelevant or Fmr1-specific siRNAs (M-019631-00-0020; D-001810-0X, Dharmacon, Lafayette, CO) using Lipofectamine 2000 (Thermofisher #11668019) for N2A and PEI (Thermofisher #BMS1003-A) for HEK293T for 4 hours. Addition of siRNAs was repeated daily for 72 hours, followed by transfection on Day 4 with luciferase constructs. The procedure was identical for transfection of miRNA mimics (C-310389-05-0002; C-310532-05-0002; C-310427-07-0002, Dharmacon) and pIS1 DICER1 long-mut-miR103/107, (Addgene plasmid # 21652; RRID:Addgene_21652) into N2A cells. For N2A cells, seeding was identical, and transfection of control and luciferase reporter constructs was 24 hours after initial seeding. 1 μg of luciferase (renilla only) containing reporter was transfected along with 10–50 ng of pluc vector (firefly) post knockdown and 24 hours after initial seeding in N2A cells. Luciferase activity was measured in quadruplicate using a dual luciferase reporter assay kit (#E1910, Promega, Madison, WI) on a SynergyTM HT Multi-detection plate reader (Biotek, Winooski, VT) 24 hours post-secondary transfection. Renilla expression from the Dicer “long” 3’UTR construct was normalized to the firefly expression as a control for transfection efficiency. For miRNA mimic and miR deletion and OE experiments, the Dicer "short" 3’UTR was subtracted to account for the effect of MOV10 and FMRP on the luciferase coding sequence itself. Finally, single variate ANOVA was performed to determine if there were any statistically significant differences among the treatment groups versus control followed by a Student’s t-test. All measured data are expressed as means +/- SEM.

eCLIP of P0 WT and Fmr1 KO brain

P0 Brains from Jax WT C57BL/6 and Fmr1ko mice were sent to Eclipse BioInnovations (San Diego, CA). eCLIP was performed per [25], using anti-AGO2 antibody (EAG009, Eclipse BioInnovations). Briefly, single-end (75nt) sequencing was performed on the HiSeq 4000 platform (Illumina, San Diego, CA). The first 10 nt of each read contains a unique molecular identifier (UMI) which was extracted from each read with UMI tools (version 5.2) and appended to the end of the read name. Next, sequencing adapters were trimmed from the 3’ end of each read. Reads were then mapped to a database of mouse repeats using STAR (version 2.6.0c) Reads that mapped to the repeats were removed. The remaining reads were mapped to the mouse genome (mm10) using STAR (version 2.6.0c). PCR duplication removal was performed using UMI tools (version 5.2). CLIP per (version 1.4) was then used to identify clusters within the IP samples, and read density within clusters was compared against the size matched input sample using a custom perl script to identify peaks enriched in the CLIP sample versus the input sample. The significance threshold was -log10(P-value) ≥ 3 and a log2 fold change ≥ 3.

miRNAseq of P0 WT and Fmr1 KO brain

University of Illinois Urbana Champaign sequencing center prepared the libraries from three P0 brains from each genotype and sequenced using NovaSeq 6000 and performed FASTQC (version 0.11.8) on individual samples (N = 3 of each genotype). Average per-base read quality scores are over 30 until ~90 bp and no adapter sequences were found indicating those reads are high in quality. The Sequence Length Distribution plot shows a large spike at 22 bp that are the mature miRNA and a smaller spike at 66 which are tRNAs and hairpin/precursor miRNAs. Weighted counts to mature miRNA, hairpin miRNA and tRNAs were generated. Percentages of total reads that mapped to any of these three ranged from 58.6 to 64.8% (S4 Fig). The mapping percentage did not differ between WT and Fmr1 KO samples. The unmapped reads were discarded while the number of remaining reads (range: 31.5–37.9 million per sample) were kept for further analysis which were then mapped onto each small RNA type.

A total of 3,429 smRNAs (1,978 mature, 1,234 hairpin and 217 tRNA) were detected. The detection threshold was set at 0.25 cpm (counts per million) in at least 3 samples, which resulted in 2,413 genes being filtered out, leaving 1,016 smRNAs (795 mature, 99 hairpin and 122 tRNA) to be analyzed for differential expression that contain 99.99% of the reads. After filtering, Trimmed Mean of M values (TMM) normalization was performed again (S4 Fig) and normalized log2-based count per million values (logCPM) were calculated using edgeR’s cpm() function with prior.count = 2 to help stabilize fold-changes of extremely low expression genes. Differential gene expression (DE) analysis was performed using the edgeR-quasi method (edgeR version 3.30.3). Multiple testing correction was done using the False Discovery Rate method.

Results

Both Mov10 Heterozygote (Het) and Fmr1 Knockout (KO) cultured hippocampal neurons show abnormal morphology

We cultured hippocampal neurons from Mov10 Het mice and showed that they have significantly reduced dendritic arborization compared to wild type (WT) [(p < .0001, Fig 1A and 1C) and [15]. Because MOV10 and FMRP bind a common set of mRNAs [15] and colocalize in dendrites [26], it was logical that FMRP would also be required for normal dendritic arborization of hippocampal neurons, as reported by others [3–7, 27].

Fig 1 — (A) MAP2/DAPI immunostaining of hippocampal neurons from DIV14 WT, Mov10 het, and Fmr1 KO neurons. Neurons were prepared from 3 independent litters of each genotype and thus, 3 independent cultures. The total number, N, was compiled from the three biological experiments. (B) Dendritic morphology analysis of average dendritic length, dendritic nodes and primary branches. Confocal z-stacks of MAP2-stained WT, Mov10 het and Fmr1 KO DIV14 neurons were analyzed. (C) Dendritic morphology analysis. Confocal z-stacks of MAP2-stained WT, Mov10 het and Fmr1 KO DIV14 neurons were analyzed using Sholl. Statistics were calculated using two-way ANOVA followed by Bonferroni multiple comparisons test. Error bars indicate SEM and *p < 0.05; ****p < 0.0001 (n = 56 neurons for WT, n = 94 neurons for Mov10 Het, n = 58 for Fmr1 KO). Scale bar = 10 μm.

We characterized the neurons by measuring average dendrite length, average dendritic branch points, called nodes, and the number of primary branches. Neurons from the Mov10 Het and the Fmr1 KO had significantly shorter dendrites than those of WT; however, dendrite length was not significantly different between the Mov10 Het and the Fmr1 KO neurons (p < .001, Fig 1B). In contrast, the average number of dendritic nodes and the number of primary branches in the Mov10 Het neurons were significantly reduced compared to WT (p < 0.05); furthermore, these same features in Fmr1 KO neurons were significantly reduced compared to both WT and Mov10 Het (p < .0001, Fig 1B), suggesting that the reduction in both the number of nodes and the number of primary branches may underlie the results illustrated in Fig 1A. Concordantly, we observed significantly reduced dendritic branching in the Fmr1 KO neurons compared to WT within 190 micrometers from the cell body. The amount of branching of the Fmr1 KO neurons was also significantly reduced compared to the Mov10 Het neurons within 50 micrometers of the cell body, suggesting that complete loss of FMRP was more detrimental to normal dendritic arborization than a 50% reduction in MOV10 (p < .0001, Fig 1C).

Our results agree with independent studies of neurons in Fmr1 KO mice, which had significant reductions in dendritic complexity, total dendritic length, number of branching points and number of dendritic ends compared to WT neurons in the dentate gyrus [4, 7, 27]. A similar result was observed with FXS human neurons which also exhibited impaired dendritic maturation [7].

It was previously shown that loss of MOV10 and FMRP results in shorter neurites in a murine neuroblastoma cell line (Neuro2A) compared to WT [15]. When we measured the length and width of neurites in Mov10 KO Neuro2A, we confirmed that in the absence of MOV10, neurites are shorter than in the WT cells independent of the amount of retinoic acid (RA) used to differentiate the Neuro2A cells (p < 0.001, S1A–S1C Fig). Furthermore, we found that the neurites have a larger width in the absence of MOV10 (p < 0.001, S1D Fig).

Reduced expression of Mov10 leads to smaller soma size

When we further analyzed our neurons in culture, one individual characteristic stood out: a reduction in soma size between WT and Mov10 Het (Fig 2). A reduction in cell body size is a feature observed in various X-linked disorders, including human FXS neurons [5] and Rett syndrome [28]. It has also been observed in schizophrenia [28–31]. Accordingly, we quantified the cell soma of the Mov10 Het neurons compared to WT neurons and discovered a significant reduction in total soma area in Mov10 Het neurons (p < .05, Fig 2A and 2B). We also measured the soma size of the Fmr1 ko cultured hippocampal neurons and although it, too, was smaller, it was not significantly different than WT (NS, p < .05, S2A and S2B Fig).

Mov10 Het mice have reduced density of immature dendritic spines compared to WT

Because we observed a dendritic phenotype in the hippocampal neurons cultured for 14 days from the three genotypes, we examined dendritic spines in brain sections from animals aged postnatal day 14 (P14) (Fig 3A; [33]). In WT brains, we observed a significantly increased density of the immature spines compared to mature spines (p < .0001, Fig 3B and 3C), which was expected at this particular time point, when rapid pruning is taking place [33, 34]. In contrast, in the Mov10 Het, the mature spine density was the same as the immature spine density, suggesting an increase in the rate of immature spine elimination when MOV10 is reduced (NS, Fig 3B and 3C). Thus, MOV10 appears to stabilize immature spines (Fig 3C). This is a novel role for MOV10 because in Fmr1 KO animals, the ratio of immature to mature spines is skewed towards thinner, immature spines in four-week-old and adult mice [24, 33, 35, 36–39]. When we examined the spine density of mature and immature spines at P14 in Fmr1 ko mice, we observed no difference between WT and Fmr1 ko mice. We conclude that FMRP and MOV10 participate in dendritic arborization but have distinct roles in spine maturation.

Fig 3 — (A) Spine morphology analysis was classified into one of ten different groups, which were further subdivided into immature (yellow) or mature spines (white) based on [34]. (B) Representative images of cortical neurons from WT and *Mov10* Het mice using Golgi staining, scale bar = 2 μm. (C-D) Spine density and average dendrite width were measured per 10 μm dendritic segments of n = 5 (WT animals (8 neurons total) and n = 4 *Mov10* Het animals (8 neurons total) and n = 5 *Fmr1* ko animals. All measured data are expressed as means ± SEM. ****p < 0.0001; NS = not significant; p > 0.05 (Student’s t-test with Welch’s correction).

We observed no difference in the width of the apical dendritic branches between WT and Mov10 Het at P14. However, the widths of the apical oblique and basilar dendritic branches were significantly decreased when MOV10 was reduced (NS, p < .05, p < .0001, Fig 2D). Recent work shows that dendritic widths may be shaped by intracellular transport and forces from the cytoskeleton and the area proportionality accords with a requirement for microtubules to transport materials and nutrients for dendrite tip growth [40]. Thus, reduced MOV10 levels likely perturb dendrite formation because MOV10 binds cytoskeletal mRNAs [15].

The impaired neurite phenotype, reduced soma size, and spine maturation have previously all been found to be associated with impaired miRNA biogenesis [41–44]. miRNAs are 22–26 nucleotides long [45] and are produced upon processing from a longer precursor RNA by the endonuclease DICER [46–51]. Once processed, miRNAs complex with AGO2, forming what is referred to as the RNA induced Silencing Complex (RISC). Since both MOV10 and FMRP are known interactors of AGO2 and are involved in miRNA-associated regulation [12, 26, 52–57], we asked whether miRNA biogenesis could possibly be affected by the loss of MOV10 and FMRP.

Global reduction of AGO2-associated miRNAs in the absence of FMRP

We were interested in the FMRP dependence of AGO2-association with RNAs, thus, queried miRNA association with AGO2 in WT and Fmr1 KO P0 brain using enhanced CLIP (eCLIP) [25]. The advantage of eCLIP over traditional cross-linking immunoprecipitation (IP) experiments is that it includes the amount of input RNA in the calculation of the RNA enrichment in the IP [25]. Using this method, we found that the significantly enriched peaks fell within 261 miRNAs (p < .001, Fig 4A, S1 Table), which is a large subset of the 454 miRNAs identified in an earlier study of P13 brain in association with AGO [58]. The miRNAs are highly correlated between Fmr1 KO and WT but show an average two-fold depletion in the AGO2 IP from Fmr1 KO compared to WT. Thus, FMRP is required for normal AGO2-miRNA complex formation. Previous work [59] showed that the level of AGO2 protein is the same in WT and Fmr1 KO mouse brain thus, different AGO2 levels do not explain our result.

Fig 4 — **(A).** Two-fold less miRNAs associate with AGO2 in the absence of FMRP. Reads per million of WT (X-axis) and Fmr1 KO (Y-axis) AGO2-IPs at each cluster that maps to miRNAs. The IPs had a log2 fold change ≥3 over input and p-value ≤ 0.001. Solid black line = best fit of data. Dashed blue line = actual fit of data. **(B).** Venn diagram showing the overlap between brain-derived iCLIP targets of FMRP [2], MOV10 [15], and AGO2. All three proteins in the brain commonly bound 29 mRNAs (Dicer1 included). **(C).** GO analysis of the shared mRNAs from postnatal brain. *Y axis*: GO terms for Cellular Compartment; *X axis*: negative log (base 10) of the 15 lowest p values showing FMRP binds mRNAs encoding proteins involved in neuron projection.

Because there is a global reduction in AGO2-associated miRNAs in the absence of FMRP, we hypothesized that one of the miRNA processing proteins could be compromised—either decreased or potentially mislocalized in the absence of FMRP. Examination of the original FMRP brain iCLIP list published by Darnell and colleagues revealed Dicer1 as a target among the 842 genes identified [60]. Dicer1 is a MOV10 iCLIP target in brain [15] and Dicer1 is also a target of AGO2 in mouse brain eCLIP [59]. Thus, Dicer1 is one of the 29 genes in the intersection of these gene sets (Fig 4B). DICER1 expression was significantly reduced in MOV10-knockdown HEK293 cells [12], suggesting that MOV10 protects both murine Dicer1 and human DICER1 mRNAs from AGO2-mediated degradation.

Finally, because of the impaired dendrite phenotype in Fig 1, we used the DAVID Gene Functional Classification tool on the significantly changed AGO2-associated mRNAs between WT and Fmr1 KO brain from an eCLIP experiment [59] and found the mRNAs encode proteins involved in neuron projection (Fig 4C), as previously shown [15].

Reduced DICER expression in the absence of FMRP or MOV10

When MOV10 and FMRP bind in the 3’UTR of their mRNA targets, depending on where they bind, the fate of the mRNA changes. When FMRP and MOV10 bind in proximity to each other on the mRNA, it appears that FMRP binds first and recruits MOV10 to unwind miRNA Recognition Elements (MREs) to facilitate AGO2 association [59]. In contrast, when a G-quadruplex is present in the 3’UTR, FMRP binds it first and binding of FMRP to MOV10 through its KH1 domain stabilizes association of FMRP to the G-quadruplex through its RGG box [59]. If an MRE is present in proximity to the G-quadruplex, association with AGO2 is blocked by the FMRP-MOV10 complex and is temporarily protected from degradation [12, 59]. We hypothesized that the FMRP/MOV10/AGO2 complex regulates Dicer1 expression by “protecting” the Dicer1 mRNA, such that in the absence of FMRP and/or MOV10, DICER protein levels would be reduced. When we examined DICER expression in WT and Mov10 heterozygote brains, we saw no significant difference (NS, p > .05, S3 Fig). Because MOV10 is a helicase, we hypothesized that we would need a complete knockout to see an effect. Thus, we examined DICER expression in cells in which Mov10 and Fmr1, respectively, were knocked out. We observed reduced DICER expression in the Mov10 Neuro2A KO and in brain extracts from the Fmr1 KO mouse compared to WT (p < .001, Fig 5A and 5B). It is important to note that the levels of Dicer1 mRNA do not change in N2A cells in the absence of Mov10 [15] (nor in the absence of Fmrp [60, 61]), but Dicer1 mRNA is highly expressed in brain, similarly to Mov10 and Fmr1 mRNA (S3 Fig). Thus, loss of MOV10 or FMRP leads to reduced DICER expression, presumably by allowing access of AGO2 to block translation.

Fig 5 — (A, B) Three representative images of WT and *Mov10* KO N2A cells (50 μg) and brain extract (50 μg) from P2 C57BL/6 WT and *Fmr1* KO mice immunoblotted for DICER and eIF5 as a loading control. Bar graphs of four and six biological replicates, respectively, are shown below. All measured data are expressed as means ± SEM. ***p < 0.001 (Student’s t-test with Welch’s correction).

Changes in the levels of DICER in the absence of MOV10 and FMRP could potentially lead to a defect in miRNA biogenesis, thus a significantly decreased pool of mature miRNAs. In fact, other investigators have examined global miRNA levels in Fmr1 KO brains and found differences from WT [62, 63]. To test this hypothesis, we performed miRNA-seq in P0 WT and Fmr1 KO brains. Weighted counts to mature miRNA, hairpin miRNA and tRNAs ranged from 58.6 to 64.8% (S4A Fig). The mapped reads overwhelmingly came from mature miRNA ranging from 93.5 to 95.8%, with no obvious difference between WT and Fmr1 KO brains (S4B Fig). The tRNA reads made up between 4.2 to 6.5% and did not differ between groups (S4B Fig). The precursor/hairpin only accounted for a tiny of fraction of reads, ranging from 0.03 to 0.05%. We had potentially expected to see an increase in hairpin RNA and a decrease in mature in the Fmr1 KO samples since FMRP regulates Dicer1 and DICER processes hairpin to mature miRNAs. However, we did not observe any consistent percentage changes between WT and Fmr1 KO with the exception of two miRNAs, (mmu-miR-144-5p and mmu-miR-3473c). Our results differ from others likely because the methodologies for miRNA measurement differs as well as the ages of the mice [62, 63]. Therefore, the regulation of DICER expression by FMRP and MOV10 does not lead to a global reduction in miRNA levels, despite observing significantly reduced AGO2-miRNA complexes isolated from the Fmr1 ko brain.

Dicer1 3’UTR regulation by MOV10 and FMRP

From the CLIP-seq data we know that MOV10 and FMRP bind murine Dicer1 mRNA and human DICER1 mRNA in the 3’UTR, respectively. To further dissect the role of MOV10 and FMRP on the 3’UTR of the DICER1 mRNA, we obtained two human DICER1 3’UTR luciferase constructs [64]. One contains the entire 3’UTR (referred to as “long”) and the second has the 3’UTR truncated (referred to as “short”), essentially removing any possibility for miRNA-mediated regulation (Fig 6A). In the absence of MOV10, luciferase expression of the full length DICER1 3’UTR is significantly decreased compared to WT (p < .001 Fig 6C). We also knocked down FMRP in HEK293T cells and observed a similar result (p < .001, Fig 6B and 6D), suggesting that MOV10 and FMRP modulate expression of DICER1 via the 3’UTR.

Fig 6 — MOV10 and FMRP modulate expression of *Dicer1* mRNA via the 3’UTR (A). Schematic illustration of *Dicer1* mRNA with alternative isoforms. Grey boxes show luciferase coding region; black line represents untranslated regions and AAAAAA represents the poly(A) tail. (B). Blot (25ug) and graph showing Hek293T cells were treated with Irr small interfering RNA (*siRNA*) or Fmr1 siRNA (n = 3). (C,D). Effect of Mov10 loss (in N2A cells) and FMRP KD (in Hek293T) on luciferase expression of full-length 3’ UTR of *Dicer1* and the shortest *Dicer1* isoform, which was subtracted, as it represents activity independent of miRNA recognition elements. (E) Effect of miR-103-3p overexpression on *Dicer* long reporter and deletion of the miR-103-3p sites from the *Dicer* long construct in the absence of Mov10. Assays were performed in quadruplicate from three independent experiments. All measured data are expressed as means ± SEM. ***p < 0.001 (single variate ANOVA).

Next we wanted to identify the specific sites in the 3’UTR of DICER1 through which MOV10 and FMRP exert their effect. To determine this, we re-aligned the binding sites from previously published CLIP-seq experiments [12, 65] to map all sequence sets to the same updated transcriptome. Using these data, we used TargetScan software [66] to determine which miRNA recognition elements (MRE) were the closest to the CLIP sites of MOV10 and FMRP. We then tested several different MREs that according to TargetScan, were highly conserved as potential miRNA binding sites of human DICER1 mRNA. miRNA mimics for miR-103-3p, miR-195-3p and miR-206 were transfected into WT and Mov10 KO Neuro2A cells followed by the DICER1 long 3’UTR luciferase reporter. We found that for the miRNAs tested, MOV10 had the strongest effect on the miR-103-3p site (p < .001, Figs 6E and S5A). Addition of miR-103-3p further suppressed the DICER1 long luciferase construct in the absence of MOV10, suggesting that MOV10 blocks AGO2+miR-103-3p (Fig 6E, middle columns, green). Moreover, when we introduced a luciferase construct with the miR-103-3p sites deleted, suppression was lifted and expression of the construct in N2A cells was restored (Fig 6E, right columns, red). The fact that loss of the miR 103–3 sites leads to even more expression than WT in the MOV10 knockout suggests that MOV10 facilitates AGO2 association at other now accessible MREs in WT.

Overexpression of MYC-Dicer1 rescues impaired neuronal phenotype

To definitively show that it is the loss of DICER expression that is the primary cause of the shortened neurites, we expressed MYC-tagged Human DICER1 in the Mov10 KO Neuro2A, Mov10 HET and Fmr1 KO neurons and observed restored neurite length to WT levels (p < .001, Fig 7A) and improved dendritic arborization within 100 μM of the soma (p < .05, Fig 7B), respectively. Although trending, we did not observe restoration of the dendritic arbor in Fmr1 KO neurons when Dicer1 was OE (Fig 7C). Given that Fmr1 KO neurons had a more severe neuronal phenotype, and that MOV10 and FMRP both regulate many different mRNAs, this result was unsurprising. The data together suggest a mechanism for regulating local DICER expression when MOV10 and FMRP are present.

Fig 7 — (A) Brightfield and immunofluorescence images of WT or *Mov10* ko N2A, untransfected or transfected with the empty MYC-vector or the MYC-tagged Human *Dicer1*, as indicated across the top, and stained with the anti-MYC antibody. The average neurite length was measured and shown on the right. Scale bar = 200 μm. Neurite length in micrometers was measured as described in the Methods. All measured data are expressed as means ± SEM. ***p < 0.001 (Student’s t-test with Welch’s correction). (B,C). Empty vectors, either MYC or eGFP, and MYC-tagged Human Dicer1 was transfected (over-expressed [OE]) in Mov10 HET hippocampal neurons (B) and *Fmr1 KO* hippocampal neurons (C) at DIV 2 followed by immunofluorescence for MYC at DIV 7. Sholl statistics were calculated using two-way ANOVA followed by Bonferroni multiple comparisons test. Error bars indicate SEM and *p < 0.05 (n = 3 neurons for Mov10 Het empty MYC-vector control, n = 5 neurons for Mov10 Het MYC Dicer overexpression, n = 5 for Fmr1 KO MYC Dicer overexpression, n = 5 for eGFP vector control. Bubbles were removed from Mov10 Het OE images for easier viewing. Scale bar = 100 μm.

Discussion

Our work reveals a new understanding of how FMRP and MOV10 regulate cobound mRNAs and neuronal development. Fmr1 KO and Mov10 Het neurons share the features of reduced dendritic arborization, including reduced dendritic length, number of nodes, number of primary branches and reduced soma size. Reducing expression of either protein in Neuro2A cells leads to reduced neurite length and simultaneously reducing both proteins does not lead to a shorter phenotype [15] suggesting that FMRP and MOV10 operate in the same neurite outgrowth pathway and dendritic arborization. Although FMRP and MOV10 bind some of the same mRNAs, which includes Dicer1, there is also a large number of RNAs that are unique to FMRP and MOV10 and it is likely that misregulation of these mRNAs cause the unique spine features.

Both proteins participate in the miRNA pathway, which plays an important role in normal dendritic arborization. DICER is the primary producer of miRNAs and its mRNA is directly bound by FMRP, MOV10 and AGO2 [15, 59, 60]. We observed significantly reduced levels of AGO2-associated miRNAs in the Fmr1 knockout brain compared to wild type, initially suggesting a global defect in miRNA production in the absence of FMRP; however, that was not the case based on miRNA-seq of both WT and Fmr1 ko brains, showing that the global miRNA levels were unchanged. To explain our AGO2-eCLIP results, we propose that FMRP participates in loading AGO2 with miRNAs (Fig 8A and 8B). In fact, it was recently shown that FMRP is able to bind miRNAs in regions outside of the seed sequence [20] and miRNAs are present in the FMRP CLIP lists [8].

Fig 8 — (A). Part I of model showing the recruitment of AGO to a miRNA following interaction with FMRP (B). Part II of model showing fate of Dicer1 mRNA bound by MOV10 and FMRP. Binding of both FMRP and MOV10 in proximity of MRE blocks association with AGO2 [12]. In the absence of MOV10 and FMRP, AGO2 is free to bind the MRE and translational regulation takes place. Pink line indicates MRE.

We observed reduced DICER expression in the absence of MOV10 and FMRP in cell lines and brain, respectively, although since DICER is an enzyme, there must be enough present to produce normal levels of miRNAs in brain. Thus, we propose that the role of FMRP and MOV10 on Dicer translation affects local expression of DICER in dendrites. In fact, local activation of DICER activity in neurons has been demonstrated before [67]. Dynamically altering local production of miRNAs by changing expression levels of DICER is one way in which neurons could respond to a wide range of temporal and environmental signals [68–70]. The DICER protein itself, along with FMRP and MOV10, is found in neuronal granules and thus can be rapidly dendritically and synaptically localized [71–74]. In fact, neuronal granules typically consist of one mRNA and a large composition of RNA binding proteins [71], suggesting a mechanism in place for rapid control of miRNA synthesis upon signal stimulation.

We hypothesize that FMRP, like most RNA binding proteins, has many different functions in the cell, based on its phosphorylation state, its binding partners and its location in the cell. We might envision that in the cell body, DICER translation is facilitated by the association of FMRP and MOV10 with its mRNA. When the DICER-AGO-MOV10-FMRP complex is transported in the dendrite to the synapse, it awaits stimulation which activates DICER to produce miRNAs locally, which associate with AGO2 through its association with FMRP. Presumably translation is occurring of FMRP-MOV10 bound mRNAs because this complex blocks AGO association. However, translation must eventually stop. Perhaps methylation of FMRP’s RGG box releases the FMRP-MOV10 complex from the 3’UTR of synaptically localized mRNAs and AGO now associates with the mRNAs to block translation.

In addition, there may be many roles for MOV10 in the regulation of miRNA production. A recent study showed that shRNA-mediated knockdown of MOV10 in spermatogonial progenitor cells resulted in a significant decrease in most miRNAs. The authors suggest that MOV10 regulates miRNA biogenesis through nuclear RNA metabolism and splicing control, as levels of the miRNA processing proteins were unchanged [75].

In summary, we describe a novel mechanism that has many potential implications. FMRP and MOV10—by regulating the Dicer1 mRNA, an indispensable element of the canonical miRNA processing machinery—could alter downstream expression of many genes. It has been long known that there is a global increase in protein translation in the absence of FMRP [76]. Much research has focused on finding a single target of FMRP to develop novel therapeutics for patients with FXS [77]. The work presented here might explain why this granular approach has so far not yielded a single gene whose deficiencies explain all of the features of FXS. Our work elucidates how it is possible that so many different genes can be affected by the loss of Fmr1 and increases our understanding of the role of miRNA biogenesis elements in maintaining proper neuronal cell homeostasis.

Conclusions

Our work provides a new understanding of how the microRNA processing pathway is regulated and a novel mechanism for how FMRP loss leads to a global increase in translation. DICER is the primary producer of miRNAs and its mRNA is directly bound by FMRP, MOV10 and AGO2. We observed significantly reduced AGO2-associated microRNAs in the Fmr1 knockout brain compared to WT. DICER protein is also significantly reduced in both the Fmr1 knockout brain and in the Mov10 knockout Neuro2A cells, suggesting that FMRP and MOV10 act to block AGO2-mediated suppression of Dicer1. This work describes how the FMRP/MOV10/AGO2 complex regulates Dicer1 expression and suggests that FMRP facilitates loading AGO2 with miRNAs.

Supporting information

S1 Fig. Mov10 KO Neuro2A cells have shorter and wider neurites compared to WT.

(A, B). Brightfield images of N2A WT and Mov10 KO cells. Scale bar = 200 μm. (C). Quantification of neurite length of WT and Mov10 KO in the presence of different concentrations of retinoic acid (RA). Between 800–1000 proliferating and differentiated cells were counted from triplicate experiments, and a total of 10 images were counted per condition. (D). Average neurite width in differentiated WT and Mov10 KO N2A cells (1 mM RA) were measured n = 100–250 (WT and Mov10 KO). All measured data are expressed as means ± SEM. ***p < 0.001 (Student’s t-test with Welch’s correction).

(TIF)

Click here for additional data file.^{(655.9KB, tif)}

S2 Fig. No change in total soma area in vitro between WT and Fmr1 KO neurons.

(A) Immunofluorescence microscopy of control (WT) and Fmr1 KO primary hippocampal neural cultures at 14 days in vitro (DIV14) showing MAP2 (red) and DAPI (Jentarra et al., 2010). The dashed box indicates the region shown at higher magnification. (B) Measurements of soma size area in DIV14 primary hippocampal neurons in Fmr1 KO (n = 58) compared to WT (n = 56). Scale bar: 25 μm. Data are presented as mean ± SEM; p values in relation to control (NS = p > 0.05), (Student’s t-test with Welch’s correction).

(TIF)

Click here for additional data file.^{(2.8MB, tif)}

S3 Fig. DICER protein levels do not change in brain when Mov10 is reduced.

A) Whole P2 WT and Mov10 Het (25 μg) were immunoblotted against DICER with Ponceau S as a loading control in three independent experiments. Error bars represent SD, and p values were obtained by Student’s t test with Welch’s correction (NS > 0.05).

(TIF)

Click here for additional data file.^{(456.7KB, tif)}

S4 Fig. miRNAseq reveals no difference in the level of mature vs. immature microRNAs in the absence of FMRP.

A). Multidimensional scaling on the top 500 most variable genes in brain samples. B). Percentages of total reads in the sequencing report that mapped to any of these three: mature, hairpin and tRNA ranged from 58.6 to 64.8%. The mapping percentage does not differ between WT and FMRKO samples. C). Percentage of mapped reads coming from mature miRNA, hairpin miRNA, and tRNA.

(TIF)

Click here for additional data file.^{(626.7KB, tif)}

S5 Fig. Overexpression of miR-195-3p and miR-206 does not significantly decrease Dicer 3’UTR expression in the absence of Mov10.

(A). Effect of Mov10 KO on luciferase Dicer1 3′ UTR and miR-195-3p and miR-206 site overexpression. (B) Screenshot from Integrated Genome Browser (IGB) of Dicer1 3’UTR (running from right to left) with human AGO2 CLIP sites (pink), human MOV10 CLIP sites (green) and human FMRP CLIP sites showing relative locations to the MRES containing sites for miR-195-3p and miR-206 binding. (C). TargetScan screenshot of Dicer1 3’UTR (running from right to left) with red circles showing location of MRE sites whose miRNAs were overexpressed in A. Assays were performed in quadruplicate from three independent experiments. All measured data are expressed as means ± SEM. ***p < 0.001 (Student’s t-test with Welch’s correction).

(TIF)

Click here for additional data file.^{(6.6MB, tif)}

S1 Table. AGO2 eCLIP miRNAs.

(XLSX)

Click here for additional data file.^{(34.7KB, xlsx)}

S2 Table. Soma size area in WT, Mov10 Het and Fmr1 KO neurons.

(XLSX)

Click here for additional data file.^{(62.9KB, xlsx)}

S1 Raw image

(TIF)

Click here for additional data file.^{(600.5KB, tif)}

Acknowledgments

We thank Dr. Roberto Galvez and Dr. Amogh Belagodu for advice on Golgi staining, spine measurement and interpretation of the data. We thank Dr. Hee Jung Chung and Dr. Sung-Soo Jang for instruction on preparing hippocampal neuron cultures. We thank Adriana Tienda for measuring the spines, Malaak Yeyha and Megan Ringling for neurite measurements, and Dr. Lisa Stubbs for critical reading of this manuscript.

Data Availability

eCLIP data files are available from the NCBI Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo/) under the accession numbers GSE129885 (AGOeCLIP-SEQ).

Funding Statement

This study was supported by NIH grant # R01 MH093661 and NSF grant 1855474 to SC. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

References

1.Ashley CT, Sutcliffe JS, Kunst CB, Leiner HA, Eichler EE, Nelson DL, et al. (1993) Human and murine FMR-1: alternative splicing and translational initiation downstream of the CGG-repeat. Nat Genet 4:244–251. doi: 10.1038/ng0793-244 [DOI] [PubMed] [Google Scholar]
2.Irwin SA, Christmon CA, Grossman AW, Galvez R, Kim SH, DeGrush BJ, et al. (2005) Fragile X mental retardation protein levels increase following complex environment exposure in rat brain regions undergoing active synaptogenesis. Neurobiol Learn Mem 83:180–187. doi: 10.1016/j.nlm.2004.11.004 [DOI] [PubMed] [Google Scholar]
3.Doers ME, Musser MT, Nichol R, Berndt ER, Baker M, Gomez TM, et al. (2014) iPSC-derived forebrain neurons from FXS individuals show defects in initial neurite outgrowth. Stem Cells Dev 23:1777–1787. doi: 10.1089/scd.2014.0030 [DOI] [PMC free article] [PubMed] [Google Scholar]
4.Guo W, Polich ED, Su J, Gao Y, Christopher DM, Allan AM, et al. (2015) Fragile X Proteins FMRP and FXR2P Control Synaptic GluA1 Expression and Neuronal Maturation via Distinct Mechanisms. Cell reports 11:1651–1666. [DOI] [PMC free article] [PubMed] [Google Scholar]
5.Telias M, Kuznitsov-Yanovsky L, Segal M, Ben-Yosef D (2015) Functional Deficiencies in Fragile X Neurons Derived from Human Embryonic Stem Cells. The Journal of neuroscience 35:15295–15306. doi: 10.1523/JNEUROSCI.0317-15.2015 [DOI] [PMC free article] [PubMed] [Google Scholar]
6.Telias M, Segal M, Ben-Yosef D (2016) Immature Responses to GABA in Fragile X Neurons Derived from Human Embryonic Stem Cells. Frontiers in cellular neuroscience 10:121. doi: 10.3389/fncel.2016.00121 [DOI] [PMC free article] [PubMed] [Google Scholar]
7.Shen M, Wang F, Li M, Sah N, Stockton ME, Tidei JJ, et al. (2019) Reduced mitochondrial fusion and Huntingtin levels contribute to impaired dendritic maturation and behavioral deficits in Fmr1-mutant mice. Nat Neurosci 22:386–400. doi: 10.1038/s41593-019-0338-y [DOI] [PMC free article] [PubMed] [Google Scholar]
8.Ascano M, Mukherjee N, Bandaru P, Miller JB, Nusbaum JD, Corcoran DL, et al. (2012) FMRP targets distinct mRNA sequence elements to regulate protein expression. Nature 492:382–386. doi: 10.1038/nature11737 [DOI] [PMC free article] [PubMed] [Google Scholar]
9.Tran SS, Jun HI, Bahn JH, Azghadi A, Ramaswami G, Van Nostrand EL, et al. (2019) Widespread RNA editing dysregulation in brains from autistic individuals. Nat Neurosci 22:25–36. doi: 10.1038/s41593-018-0287-x [DOI] [PMC free article] [PubMed] [Google Scholar]
10.Skariah G, Seimetz J, Norsworthy M, Lannom MC, Kenny PJ, Elrakhawy M, Forsthoefel C, Drnevich J, Kalsotra A, Ceman S (2017) Mov10 suppresses retroelements and regulates neuronal development and function in the developing brain. BMC Biology 15:54. doi: 10.1186/s12915-017-0387-1 [DOI] [PMC free article] [PubMed] [Google Scholar]
11.Brown V, Jin P, Ceman S, Darnell JC, O’Donnell WT, Tenenbaum SA, et al. (2001) Microarray identification of FMRP-associated brain mRNAs and altered mRNA translational profiles in fragile X syndrome. Cell 107:477–487. doi: 10.1016/s0092-8674(01)00568-2 [DOI] [PubMed] [Google Scholar]
12.Miyashiro KY, Beckel-Mitchener A, Purk TP, Becker KG, Barret T, Liu L, et al. (2003) RNA cargoes associating with FMRP reveal deficits in cellular functioning in Fmr1 null mice. Neuron 37:417–431. doi: 10.1016/s0896-6273(03)00034-5 [DOI] [PubMed] [Google Scholar]
13.Kenny PJ, Zhou H, Kim M, Skariah G, Khetani RS, Drnevich J, et al. (2014) MOV10 and FMRP regulate AGO2 association with microRNA recognition elements. Cell reports 9:1729–1741. doi: 10.1016/j.celrep.2014.10.054 [DOI] [PMC free article] [PubMed] [Google Scholar]
14.Meister G, Landthaler M, Peters L, Chen PY, Urlaub H, Luhrmann R, et al. (2005) Identification of novel argonaute-associated proteins. Curr Biol 15:2149–2155. doi: 10.1016/j.cub.2005.10.048 [DOI] [PubMed] [Google Scholar]
15.Banerjee S, Neveu P, Kosik KS (2009) A Coordinated Local Translational Control Point at the Synapse Involving Relief from Silencing and MOV10 Degradation. Neuron 64:871. doi: 10.1016/j.neuron.2009.11.023 [DOI] [PubMed] [Google Scholar]
16.Skariah G, Perry KJ, Drnevich J, Henry JJ, Ceman S (2018) RNA helicase Mov10 is essential for gastrulation and central nervous system development. Dev Dyn 247:660–671. doi: 10.1002/dvdy.24615 [DOI] [PMC free article] [PubMed] [Google Scholar]
17.Zafarullah M, Tassone F (2019) Molecular Biomarkers in Fragile X Syndrome. Brain Sci 9. doi: 10.3390/brainsci9050096 [DOI] [PMC free article] [PubMed] [Google Scholar]
18.Kurzynska-Kokorniak A, Koralewska N, Pokornowska M, Urbanowicz A, Tworak A, Mickiewicz A, et al. (2015) The many faces of Dicer: the complexity of the mechanisms regulating Dicer gene expression and enzyme activities. Nucleic acids research 43:4365–4380. doi: 10.1093/nar/gkv328 [DOI] [PMC free article] [PubMed] [Google Scholar]
19.Chendrimada TP, Finn KJ, Ji X, Baillat D, Gregory RI, Liebhaber SA, et al. (2007) MicroRNA silencing through RISC recruitment of eIF6. Nature 447:823–8. doi: 10.1038/nature05841 [DOI] [PubMed] [Google Scholar]
20.DeMarco B, Stefanovic S, Williams A, Moss KR, Anderson BR, Bassell GJ, et al. (2019) FMRP-G-quadruplex mRNA–miR-125a interactions: Implications for miR-125a mediated translational regulation of PSD-95 mRNA. Plos One 14: e0217275. doi: 10.1371/journal.pone.0217275 [DOI] [PMC free article] [PubMed] [Google Scholar]
21.Beaudoin GM 3rd, Lee SH, Singh D, Yuan Y, Ng YG, Reichardt, et al. (2012) Culturing pyramidal neurons from the early postnatal mouse hippocampus and cortex. Nat Protoc 7:1741–1754. doi: 10.1038/nprot.2012.099 [DOI] [PubMed] [Google Scholar]
22.Ferreira TA, Iacono LL, Gross CT (2010) Serotonin receptor 1A modulates actin dynamics and restricts dendritic growth in hippocampal neurons. Eur J Neurosci 32:18–26. doi: 10.1111/j.1460-9568.2010.07283.x [DOI] [PubMed] [Google Scholar]
23.Glaser EM, Van der Loos H (1981) Analysis of thick brain sections by obverse- reverse computer microscopy: application of a new, high clarity Golgi-Nissl stain. J Neurosci Methods 4:117–125. doi: 10.1016/0165-0270(81)90045-5 [DOI] [PubMed] [Google Scholar]
24.Irwin SA, Idupulapati M, Gilbert ME, Harris JB, Chakravarti AB, Rogers EJ, et al. (2002) Dendritic spine and dendritic field characteristics of layer V pyramidal neurons in the visual cortex of fragile-X knockout mice. Am J Med Genet 111:140–146. doi: 10.1002/ajmg.10500 [DOI] [PubMed] [Google Scholar]
25.Van Nostrand EL, Pratt GA, Shishkin AA, Gelboin-Burkhart C, Fang MY, Sundararaman B, et al. (2016) Robust transcriptome-wide discovery of RNA-binding protein binding sites with enhanced CLIP (eCLIP). Nat Methods 13:508–514. doi: 10.1038/nmeth.3810 [DOI] [PMC free article] [PubMed] [Google Scholar]
26.Wulczyn FG, Smirnova L, Rybak A, Brandt C, Kwidzinski E, Ninnemann O, et al. (2007) Post-transcriptional regulation of the let-7 microRNA during neural cell specification. The FASEB Journal 21:415–426. doi: 10.1096/fj.06-6130com [DOI] [PubMed] [Google Scholar]
27.Guo W, Allan AM, Zong R, Zhang L, Johnson EB, Schaller EG, et al. (2011) Ablation of Fmrp in adult neural stem cells disrupts hippocampus-dependent learning. Nat Med 17:559–565. doi: 10.1038/nm.2336 [DOI] [PMC free article] [PubMed] [Google Scholar]
28.Rangasamy S OS, Gerald B, Hilbert A, Svejda S, Narayanan V. (2016) Reduced neuronal size and mTOR pathway activity in the Mecp2 A140V Rett syndrome mouse model. F1000 Res 5:2269. doi: 10.12688/f1000research.8156.1 [DOI] [PMC free article] [PubMed] [Google Scholar]
29.Rajkowska G SL, Goldman-Rakic PS. (1998) Neuronal and Glial Somal Size in the Prefrontal Cortex: A Postmortem Morphometric Study of Schizophrenia and Huntington Disease. Arch Gen Psychiatry 55:215–224. doi: 10.1001/archpsyc.55.3.215 [DOI] [PubMed] [Google Scholar]
30.Marchetto MC CC, Acab A, Yu D, Yeo GW, Mu Y, Chen G, et al. (2010) A model for neural development and treatment of Rett syndrome using human induced pluripotent stem cells. Cell 143:527–539. doi: 10.1016/j.cell.2010.10.016 [DOI] [PMC free article] [PubMed] [Google Scholar]
31.Chailangkarn T AA, Muotri AR. (2012) Modeling neurodevelopmental disorders using human neurons. Current Opinion Neurobiol 22:785–790. doi: 10.1016/j.conb.2012.04.004 [DOI] [PMC free article] [PubMed] [Google Scholar]
32.Jentarra GM, Olfers SL, Rice SG, Srivastava N, Homanics GE, Blue M, et al. (2010) Abnormalities of cell packing density and dendritic complexity in the MeCP2 A140V mouse model of Rett syndrome/X-linked mental retardation. BMC Neurosci 11:19. doi: 10.1186/1471-2202-11-19 [DOI] [PMC free article] [PubMed] [Google Scholar]
33.Galvez R, Greenough WT (2005) Sequence of abnormal dendritic spine development in primary somatosensory cortex of a mouse model of the fragile X mental retardation syndrome. Am J Med Genet A 135:155–160. doi: 10.1002/ajmg.a.30709 [DOI] [PubMed] [Google Scholar]
34.Greenough WT, Klintsova AY, Irwin SA, Galvez R, Bates KE, Weiler IJ (2001) Synaptic regulation of protein synthesis and the fragile X protein. Proc Natl Acad Sci U S A 98:7101–7106. doi: 10.1073/pnas.141145998 [DOI] [PMC free article] [PubMed] [Google Scholar]
35.Comery TA, Harris JB, Willems PJ, Oostra BA, Irwin SA, Weiler IJ, et al. (1997) Abnormal dendritic spines in fragile X knockout mice: maturation and pruning deficits. Proc Natl Acad Sci U S A 94:5401–5404. doi: 10.1073/pnas.94.10.5401 [DOI] [PMC free article] [PubMed] [Google Scholar]
36.Nimchinsky EA, Oberlander AM, Svoboda K (2001) Abnormal development of dendritic spines in FMR1 knock-out mice. The Journal of neuroscience 21:5139–5146. doi: 10.1523/JNEUROSCI.21-14-05139.2001 [DOI] [PMC free article] [PubMed] [Google Scholar]
37.Hong J, Zhang H, Kawase-Koga Y, Sun T (2013) MicroRNA function is required for neurite outgrowth of mature neurons in the mouse postnatal cerebral cortex. Frontiers in cellular neuroscience 7:151. doi: 10.3389/fncel.2013.00151 [DOI] [PMC free article] [PubMed] [Google Scholar]
38.McKinney BC, Grossman AW, Elisseou NM, Greenough WT (2005) Dendritic spine abnormalities in the occipital cortex of C57BL/6 Fmr1 knockout mice. Am J Med Genet B Neuropsychiatr Genet 136B:98–102. doi: 10.1002/ajmg.b.30183 [DOI] [PubMed] [Google Scholar]
39.Grossman AW, Elisseou NM, McKinney BC, Greenough WT (2006) Hippocampal pyramidal cells in adult Fmr1 knockout mice exhibit an immature-appearing profile of dendritic spines. Brain Res 1084:158–164. doi: 10.1016/j.brainres.2006.02.044 [DOI] [PubMed] [Google Scholar]
40.Levenga J, de Vrij FM, Buijsen RA, Li T, Nieuwenhuizen IM, Pop A, et al. (2011) Subregion-specific dendritic spine abnormalities in the hippocampus of Fmr1 KO mice. Neurobiol Learn Mem 95:467–472. doi: 10.1016/j.nlm.2011.02.009 [DOI] [PubMed] [Google Scholar]
41.Liao M, Liang X, Howard J (2021) The narrowing of dendrite branches across nodes follows a well-defined scaling law. PNAS 118: e2022395118. doi: 10.1073/pnas.2022395118 [DOI] [PMC free article] [PubMed] [Google Scholar]
42.Thomas Treiber NT, Uwe Plessmann, Simone Harlander, Julia-Lisa Daiß, Norbert Eichner, Gerhard Lehmann, et al. (2017) A Compendium of RNA-Binding Proteins that Regulate MicroRNA Biogenesis. Molecular cell 66:270–284.e213. doi: 10.1016/j.molcel.2017.03.014 [DOI] [PubMed] [Google Scholar]
43.Michlewski G CJ (2019) Post-transcriptional control of miRNA biogenesis. Rna 25:1–16. doi: 10.1261/rna.068692.118 [DOI] [PMC free article] [PubMed] [Google Scholar]
44.Ramirez-Moya J, Wert-Lamas L, Riesco-Eizaguirre G, Santisteban P (2019) Impaired microRNA processing by DICER1 downregulation endows thyroid cancer with increased aggressiveness. Oncogene 38:5486–5499. doi: 10.1038/s41388-019-0804-8 [DOI] [PMC free article] [PubMed] [Google Scholar]
45.Lee RC, Feinbaum RL, Ambros V (1993) The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14. Cell 75:843. doi: 10.1016/0092-8674(93)90529-y [DOI] [PubMed] [Google Scholar]
46.Lee Y, Jeon K, Lee JT, Kim S, Kim VN (2002) MicroRNA maturation: stepwise processing and subcellular localization. EMBO J 21:4663–4670. doi: 10.1093/emboj/cdf476 [DOI] [PMC free article] [PubMed] [Google Scholar]
47.Lee Y, Ahn C, Han J, Choi H, Kim J, Yim J, et al. (2003. b) The nuclear RNase III Drosha initiates microRNA processing. Nature 425:415–419. doi: 10.1038/nature01957 [DOI] [PubMed] [Google Scholar]
48.Denli A, Tops B, Plasterk R, Ketting R, Hannon G (2004) Processing of primary microRNAs by the microprocessor complex. Nature 432:231–235. doi: 10.1038/nature03049 [DOI] [PubMed] [Google Scholar]
49.Gregory R, Yan K, Amuthan G, Chendrimada T, Doratoaj B, Cooch N, et al. (2004) The microprocessor complex mediates the genesis of microRNAs. Nature 432:235–240. doi: 10.1038/nature03120 [DOI] [PubMed] [Google Scholar]
50.Han J, Lee Y, Yeom KH, Nam JW, Heo I, Rhee JK, et al. (2006) Molecular basis for the recognition of primary microRNAs by the Drosha- DGCR8 complex. Cell 125:887–901. doi: 10.1016/j.cell.2006.03.043 [DOI] [PubMed] [Google Scholar]
51.Adams-Cioaba MA, Guo Y, Bian C, Amaya MF, Lam R, Wasney GA, et al. (2010) Structural Studies of the Tandem Tudor Domains of Fragile X Mental Retardation Related Proteins FXR1 and FXR2. PloS one 5:e13559. doi: 10.1371/journal.pone.0013559 [DOI] [PMC free article] [PubMed] [Google Scholar]
52.Caudy AA, Myers M, Hannon GJ, Hammond SM (2002) Fragile X-related protein and VIG associate with the RNA interference machinery. Genes & development 16:2491–2496. doi: 10.1101/gad.1025202 [DOI] [PMC free article] [PubMed] [Google Scholar]
53.Ishizuka A, Siomi MC, Siomi H (2002) A Drosophila fragile X protein interacts with components of RNAi and ribosomal proteins. Genes & development 16:2497–2508. doi: 10.1101/gad.1022002 [DOI] [PMC free article] [PubMed] [Google Scholar]
54.Jin P, Alisch RS, Warren ST (2004) RNA and microRNAs in fragile X mental retardation. Nat Cell Biol 6:1048–1053. doi: 10.1038/ncb1104-1048 [DOI] [PubMed] [Google Scholar]
55.Edbauer D, Neilson JR, Foster KA, Wang C-F, Seeburg DP, Batterton MN, et al. (2010) Regulation of Synaptic Structure and Function by FMRP-Associated MicroRNAs miR-125b and miR-132. Neuron 65:373. doi: 10.1016/j.neuron.2010.01.005 [DOI] [PMC free article] [PubMed] [Google Scholar]
56.Kenny PJ, Kim M, Skariah G, Nielsen J, Lannom MC, Ceman S (2020) The FMRP- MOV10 complex: a translational regulatory switch modulated by G-Quadruplexes. Nucleic acids research 48:862–878. doi: 10.1093/nar/gkz1092 [DOI] [PMC free article] [PubMed] [Google Scholar]
57.Lee EK, Kim HH, Kuwano Y, Abdelmohsen K, Srikantan S, Subaran SS, et al. (2010) hnRNP C promotes APP translation by competing with FMRP for APP mRNA recruitment to P bodies. Nature structural & molecular biology 17:732–739. [DOI] [PMC free article] [PubMed] [Google Scholar]
58.Darnell JC, Van Driesche SJ, Zhang C, Hung KYS, Mele A, Fraser CE, Stone EF, Chen C, Fak JJ, Chi SW, Licatalosi DD, Richter JD, Darnell RB (2011) FMRP Stalls Ribosomal Translocation on mRNAs Linked to Synaptic Function and Autism. Cell 146:247–261. doi: 10.1016/j.cell.2011.06.013 [DOI] [PMC free article] [PubMed] [Google Scholar]
59.Kenny P, Ceman S (2016) RNA Secondary Structure Modulates FMRP’s Bi- Functional Role in the MicroRNA Pathway. International Journal of Molecular Sciences 17:985. doi: 10.3390/ijms17060985 [DOI] [PMC free article] [PubMed] [Google Scholar]
60.Chi SW, Zang JB, Mele A, Darnell RB (2009) Argonaute HITS-CLIP decodes microRNA-mRNA interaction maps. Nature 460:479. doi: 10.1038/nature08170 [DOI] [PMC free article] [PubMed] [Google Scholar]
61.Korb E, Herre M, Zucker-Scharff I, Gresack J, Allis CD, Darnell RB (2017) Excess translation of epigenetic regulators contributes to Fragile X syndrome and is alleviated by BRD4 inhibition. Cell 170:1209–1223. doi: 10.1016/j.cell.2017.07.033 [DOI] [PMC free article] [PubMed] [Google Scholar]
62.Liu T., Wan RP., Tang LJ. (2015) A MicroRNA Profile in Fmr1 Knockout Mice Reveals MicroRNA Expression Alterations with Possible Roles in Fragile X Syndrome. Mol Neurobiol 51:1053–1063. doi: 10.1007/s12035-014-8770-1 [DOI] [PubMed] [Google Scholar]
63.Zhang M, Li X, Xiao D, Lu T, Qin B, Zheng Z, et al. (2020) Identification of differentially expressed microRNAsand their target genes in the hippocampal tissues of Fmr1 knockout mice. Am J Transl Res 12:813–824. [PMC free article] [PubMed] [Google Scholar]
64.Mayr C, Bartel DP (2009) Widespread shortening of 3’UTRS by alternative cleavage and polyadenylation activates oncogenes in cancer cells. Cell 138: 673–684. doi: 10.1016/j.cell.2009.06.016 [DOI] [PMC free article] [PubMed] [Google Scholar]
65.Gregersen LH, Schueler M, Munschauer M, Mastrobuoni G, Chen W, Kempa S, et al. (2014) MOV10 Is a 5’ to 3’ RNA helicase contributing to UPF1 mRNA target degradation by translocation along 3’ UTRs. Molecular cell 54:573–585. doi: 10.1016/j.molcel.2014.03.017 [DOI] [PubMed] [Google Scholar]
66.Agarwal V, Bell GW, Nam JW, Bartel DP (2015) Predicting effective microRNA target sites in mammalian mRNAs. Elife 4. doi: 10.7554/eLife.05005 [DOI] [PMC free article] [PubMed] [Google Scholar]
67.Sambandan S, Akbalik G, Kochen L, Rinne J, Kahlstatt J, Glock C, et al. (2017) Activity-dependent spatially localized miRNA maturation in neuronal dendrites. Science 355:634–637. doi: 10.1126/science.aaf8995 [DOI] [PubMed] [Google Scholar]
68.Pilotte J, Dupont-Versteegden EE, Vanderklish PW (2011) Widespread regulation of miRNA biogenesis at the Dicer step by the cold-inducible RNA-binding protein, RBM3. PloS one 6:e28446. doi: 10.1371/journal.pone.0028446 [DOI] [PMC free article] [PubMed] [Google Scholar]
69.Daugaard I, Hansen TB (2017) Biogenesis and Function of Ago-Associated RNAs. Trends Genet 33:208–219. doi: 10.1016/j.tig.2017.01.003 [DOI] [PubMed] [Google Scholar]
70.O’Brien J, Hayder H, Zayed Y, Peng C (2018) Overview of MicroRNA Biogenesis, Mechanisms of Actions, and Circulation. Front Endocrinol 9:402. [DOI] [PMC free article] [PubMed] [Google Scholar]
71.Kiebler MA, Bassell GJ (2006) Neuronal RNA granules: movers and makers. Neuron 51:685–690. doi: 10.1016/j.neuron.2006.08.021 [DOI] [PubMed] [Google Scholar]
72.Mikl M, Vendra G, Doyle M, Kiebler MA (2010) RNA localization in neurite morphogenesis and synaptic regulation: current evidence and novel approaches. J Comp Physiol A Neuroethol Sens Neural Behav Physiol 196:321–334. doi: 10.1007/s00359-010-0520-x [DOI] [PMC free article] [PubMed] [Google Scholar]
73.Bicker S, Khudayberdiev S, Weiss K, Zocher K, Baumeister S, Schratt G (2013) The DEAH-box helicase DHX36 mediates dendritic localization of the neuronal precursor- microRNA-134. Genes & development 27:991–996. doi: 10.1101/gad.211243.112 [DOI] [PMC free article] [PubMed] [Google Scholar]
74.Fritzsche R et al. (2013) Interactome of two diverse RNA granules links mRNA localization to translational repression in neurons. Cell reports 5:1749–1762. doi: 10.1016/j.celrep.2013.11.023 [DOI] [PubMed] [Google Scholar]
75.Fu K, Tian S, Tan H, Wang C, Wang H, Wang M, et al. (2019) Biological and RNA regulatory function of MOV10 in mammalian germ cells. BMC Biol 17:39. doi: 10.1186/s12915-019-0659-z [DOI] [PMC free article] [PubMed] [Google Scholar]
76.Darnell JC, Klann E (2013) The translation of translational control by FMRP: therapeutic targets for FXS. Nat Neurosci 16:1530–1536. doi: 10.1038/nn.3379 [DOI] [PMC free article] [PubMed] [Google Scholar]
77.Bagni C, Tassone F, Neri G, Hagerman R (2012) Fragile X syndrome: causes, diagnosis, mechanisms, and therapeutics. The Journal of clinical investigation 122:4314–4322. doi: 10.1172/JCI63141 [DOI] [PMC free article] [PubMed] [Google Scholar]

PLoS One. doi: 10.1371/journal.pone.0260005.r001

Decision Letter 0

Barbara Bardoni

17 Jun 2021

PONE-D-21-14401

FMRP and MOV10 regulate Dicer1 expression and dendrite development

PLOS ONE

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Reviewer #1: The study by Lannom et al. revealed the importance of MOV10 and/or FMRP in regulating Dicer expression, Ago2 interaction with miRNAs and dendrite development in neuron. They further demonstrate that both proteins act on the 3’-UTR of the Dicer mRNA, and over-expression of Dicer can reverse and neurite deficits in MOV10 KO neurons. Overall the findings appear solid and potentially interesting, but there are a number of concerns that should be addressed.

My biggest concern is the lack of consistency across different experiments that make it hard to draw some of the conclusions. For example, the authors suggest that Dicer expression is only reduced in FMR1 KO brain but not MOV10 Het KO, but the brains they used were P2 while the spine defects of the MOV10 KO mice in Fig. 2 were observed at P14 (which match the 14 DIV neurons in Fig. 1). Is it possible that MOV10 deficiency in the Het KO reduces Dicer expression at P14, which can potentially explain the spine and dendrite phenotypes? Likewise, Fig. 1 examines dendrite defects in cultured hippocampal neurons while Fig. 2 examines spine defects in cortical neuron in vivo using Golgi stain. It would be much better if they correlate dendrite and spine defects in the same group of neurons (either hippocampal or cortical, in vitro or in vivo). Furthermore, the authors claim that the spine defects in MOV10 KO are different from FMRP KO, but the spines from MOV10 KO are at P14 while the spine defects in FMR1 KO mice are implicated from studies by other labs done at different ages (4-wk old and adult, p.14). Without side-by-side comparison in the same experiments, same age and same group of neurons it Is hard to draw this conclusion. In Fig. 6C the MOV10 KO are N2A cells and FMR1 KD is done in 293T cells. They should not be put together in the same graph for comparison.

Other comments:

(1) Fig. 1: how many independent experiments have been performed?

(2) Fig. 2C: is there statistical significance between genotypes for mature spines and immature spines?

(3) Fig. 4B: does MOV10 KO brain show changes in the amount of Ago2-associated miRNAs compared to WT? And what do the two lines in the plot refer to?

(4) Fig. 6D upper panel: Does the green signal represent MYC-Dicer OE cells? It should be stated clearly in the figure. The authors should also show the images for all three experimental conditions that match the quantification in the lower panel.

(5) Fig. 6D: Top left image of the upper panel indicates WT + Dicer OE but there is no such condition in the histogram of the lower panel. Does the blue bar represent WT + Dicer OE?

(6) Fig. 6E: why the bars are broken?

(7) Fig. 6F: representative images of the MOV10 KO and FMR1 KO neurons with or without Dicer OE should be shown.

(8) Fig. 6F: if Dicer OE can reverse the dendrite phenotypes of MOV10 Het hippocampal neurons, the authors need to examine whether Dicer expression is indeed reduced in the Het neurons compared to WT.

(9) Does OE Dicer restore the reduction of soma size in MOV10 Het neurons?

(10) P.3, second paragraph: the sentence is too long and should be divided into separate sentences.

(11) I think dendrite width is seldom measured. The authors should discuss the significance of a change in dendrite width observed in their study.

(12) P.19: Kennedy et al. 2020 is not found in the reference list

(13) P.23: “suggest a mechanism for regulating local DICER expression when MOV10 and FMRP are present”. I don’t think the authors have provided evidence showing a local dendritic (but not global) reduction of DICER.

(14) P.23: “there is also a large number of RNAs that are unique to FMRP and MOV10 and it is likely that misregulation of these mRNAs cause the unique spine features.” As mentioned in previous comment, it is not appropriate to conclude “unique spine features” from the available data because the findings on MOV10 and FMR1 KO were not done side-by-side and the ages or even populations of neurons examined are different.

Reviewer #2: FMRP and MOV10 are two RNA binding proteins functionally connected and playing a role in miRNA-mediated translation regulation. In this manuscript, the authors aim to demonstrate that FMRP and MOV10 regulate the expression of the DICER protein, an endonuclease responsible for miRNA maturation, through the miRNA mediated regulation of its mRNA. This regulation would be important for proper neuronal architecture. In this purpose, the authors used a combination of model impaired in MOV10 or FMRP expression to analyze dendrites and spines morphology, miRNA expression and their association with AGO2 and the expression of the endogenous DICER protein or a reporter protein produced from a construct presenting the DICER 3’UTR region.

The question raised in this study is undoubtedly interesting. However, several inaccuracies or inconsistencies make it difficult to properly appreciate the present work. Moreover, to be fully convincing, the experimental data presented here should be reinforced by additional controls and complementary experiments.

In the present manuscript, the authors first showed an altered dendritic arborization in cultured hippocampal neurons from heterozygote Mov10 mice or Fmr1-KO mice (Figure 1, Figure 2) and in Mov10 Het brain sections (Figure 2), confirmed previously published improper neurite development in the murine neuroblastoma cell line Neuro2A Mov10-KO (Sup Figure 1), observed a perturbed mature/immature spines ratio in brain sections from 14 PND Mov10 Het mice compare to WT (Figure 2) and a smaller soma in cultured hippocampal neurons from heterozygote Mov10 mice compare to WT but not in Fmr1-KO (Figure 3 and Sup Figure 2).

Major points:

- Figure 1 A: images and in particular the morphology of the selected Mov10 Het neuron do not reflect the quantitative characterization presented in B. At least 3 images of representative neurons from each condition should be presented.

- Figure1B-E: Statistical tests should be detailed for each experiments. In particular, the number of different neuronal cultures should be specified.

- Figure 3B and Supplemental Fig 2: the number of different neuronal cultures used for statistical analysis should be specified. Could the individual measurements of soma size area be provided?

Next, to connect these morphological alterations to molecular mechanisms, the authors hypothesized that miRNA biogenesis may be affected by the loss of Mov10 and FMRP. They present results showing a two-fold in miRNA association with Ago2 (Figure 4) in Fmr1-KO P0 brain compare to WT. However, no difference could be detected in the global levels of mature or immature miRNA (Sup Figure 4).

Major points:

- Figure 4 A: It seems that there are inconsistencies between the legend of the figure and the text. Is it GO enrichment for FMRP bound mRNA (legend) or differentially Ago2-associated mRNA in WT vs Fmr1-KO (text)? Were they identified by iCLIP or eCLIP? Where is the list available? The text refers to Kenny et al., 2020 and the references section mentions Kenny et al., 2019.

- Figure 4 B/ Sup table 1: legends are not accurate: what are full line, dashed line? How statistical analysis were performed?

- To rule out any indirect effect, the level of the Ago2 protein in WT vs Fmr1-KO P0 brains should be checked.

The Dicer mRNA being a target of FMRP, MOV10 and Ago2, the authors analyzed the expression of the DICER protein and showed that DICER protein expression is decreased in Mov10-KO N2A cells or P2 Fmr1-KO brain extracts compare to WT (Figure 5A-B).

Major points:

To fully support the hypothesis that FMRP and MOV10 modulate the Ago2-miRNA mediated regulation of the Dicer mRNA, additional experiments are required:

- What are the levels of Dicer mRNA in these different models?

- What is the impact of the absence of FMRP on the association of the Dicer mRNA with Mov10? and conversely?

- What is the impact of the absence of FMRP or Mov10 on the association of the Dicer mRNA with Ago2?

Minor point:

- What is the purpose of Figure 5C here?

As MOV10 and FMRP bind murine Dicer mRNA and human DICER mRNA respectively in the 3’UTR, the authors used a DICER1 3’UTR luciferase construct to dissect the potential role of FMRP and MOV10 in the modulation of DICER1 via the 3’UTR. They showed a decreased luciferase activity in the absence of FMRP or MOV10 proteins (Figure 6C).

Major points:

- Figure 6: Both legend and Material and Methods sections poorly explain the luciferase assay and in particular how long and short constructs participate in the calculation of the Relative Luciferase Actvity (Could the authors explain: “Mov10 KO and Fmr1 KD samples were normalized to WT Dicer long 3’UTR and Dicer short 3’UTR values were subtracted for final graph”?)

Minor point:

- Many steps in mRNA biogenesis could affect the reporter protein expression. qPCR to evaluate the expression of the reporter mRNA in the different conditions would strengthen the authors hypothesis.

By CLIP-seq data comparison and miRNA recognition elements (MRE) analysis, the authors determined potential MSE the in close proximity of FMRP and MOV10 binding sites in the DICER 3’UTR (Sup Figure 5).

Minor points:

- Are mouse and human Dicer mRNA 3’UTR (FMRP binding sites, Mov10 binding sites, predicted MSE ) conserved?

- Sup Figure 5: B and C should be harmonized to facilitate the reading.

MOV10-KO negative effect is enhanced miR103-3p over-expression (but not miR-195-3p or miR-206 over-expression) and is abolished by the deletion of the miR103-3p sites on the luciferase construct (Figure 6 E and Sup Figure 5).

MYC-tagged human DICER1 over-expression restores neurites length to WT levels in N2A MOV10-KO and improved dendritic arborization in Mov10 Het cells neurons but not in Fmr1-KO neurons.

Major points:

- Figure 6 D: Incomplete legend

- Figure 6 E: Inconsistencies between text and legend.

- Figure 6 F left:

o Are Mov10 Het cells cultured hippocampal neurons? 7 DIV? 14 DIV? Were analyzed neurons detected for MYC-DICER expression? What is the level of over-expression in these neurons? Is heterologous expression homogenous?

o How does the over-expression of DICER impact dendritic arborization in WT neurons?

o Can the authors comment the difference in the numbers of intersections between WT (Figure 1), Mov10 Het over-expressing negative CT and Mov10 Het over-expressing DICER (Figure 6F)?

- Considering the cooperative model proposed by the authors, what is the effects of FMRP over-expression on the arborization of Mov10 Het neurons?

- Figure 6F right:

o Same questions as above regarding the control of MYC-DICER over-expression in analyzed neurons.

o Why is the negative control for FMR1-KO neurons different from the one used for Mov10 Het neurons?

o Can the authors comment the difference in the numbers of intersections between WT (Figure 1), Fmr1-KO over-expressing negative CT and Fmr1-KO t over-expressing DICER (Figure 6F)?

To conclude, the authors discuss the potential role of FMRP in facilitating the loading of miRNA on AGO2 (Figure 7A). In parallel, they propose a model in which MOV10 alone unwinds secondary structures containing MREs and thus facilitates AGO2 binding whereas in association with FMRP, the complex is stabilized and AGO2 association is blocked (Figure 7B).

Minor point:

Could the authors discuss these antagonistic effects of FMRP on the regulation of the DICER mRNA, on one hand by facilitating the miRNA machinery assembly but on the other hand protecting DICER mRNA, in collaboration with Mov10, by inhibiting its recognition by the Ago2 complex?

In FMRP-KO brain, DICER protein levels are decreased but the levels of miRNA remain intact. To explain this apparent contradiction, the authors propose a role of FMRP, and Mov10, on the local expression of DICER protein in dendrites, leading consequently to local downstream expression of many genes.

Reviewer #3: This paper investigates the functional interaction between FMRP, MOV10 and Dicer and their role in dendritic development.

The authors report that: 1. Dendritic branching of cultured hippocampal neurons from Mov10 heterozygous and Fmr1 KO is similarly reduced compared with WT neurons; 2. Dendritic spines in cortical neurons of Mov Het at DIV 14 have more mature spines than WT (differently than Fmr1 KO); 3. Cell soma is smaller in Mov Het hippocampal cultured neurons compared WT neurons. However, cell soma of Fmr1 KO was not different than WT neurons. 4. A global reduction of miRNAs associated with AGO in the absence of FMRP 5. reduced DICER expression in the absence of both FMRP and AGO, however no substantial change in the expression of miRNAs in Fmr1 KO; 6. Overexpression of DICER restores dendritic arborization in MOV10 heterozygous, but not in Fmr1 KO.

The authors conclude that the MOV10-Fmrp-AGO2 complex regulates DiCER expression, which in turn affects dendritic development.

This is a good paper exploring an interesting aspect related to the functional interaction between MOV10 and FMRP.

There are a few concerns that need to be addressed.

1. Methods are clearly described with useful details. At page 7 the differentiation methods used for N2A cells should be described.

2. Abnormal morphology of dendrites in cultured hippocampal neurons from Mov10 Het and Fmr1 KO mice. The authors confirmed their previously published results (Skariah et al., 2017). They should also mention other papers addressing the same issue and obtaining similar (Braun and Segal, 2000) and different results in cultured Fmr1 KO hippocampal neurons (Jacob and Doering, 2007). An increased branching of Fmr1 KO hippocampal neurons has been ascribed to the presence of astrocytes. Did the authors consider the contribution of glia?

3. Figure 1. Legend of figure 1 (see also results) indicates that data are from 56, 94 and 58 neurons. However, they do not indicate how many dishes in different cultures were used to gain these results. This is important to verify how reproducible are the data in different dishes, and more importantly in different cultures.

4. Density of dendritic spines, page 14 Why the authors examined cortical neurons in the brain instead of hippocampal neurons, considering that they want to compare in vivo and in vitro conditions? Spines could be examined in vitro as well. In addition, the region/layer of cortex examined should be indicated. They also measured the width of dendritic branches. Which part of neuron dendrite was used to measure these different parameters, width of dendrites and density and morphology of spines?

5. Reduced soma size of Mov10 Het cultured hippocampal neurons. I suggest moving this part before the ex-vivo analysis of dendritic spines, to complete the in vitro examination of hippocampal morphology first.

6. Other papers have addressed the question whether miRNAs are differentially expressed in the Fmr1KO mice. They should be cited and discussed considering that the authors found no change in the whole brain at PO. Example:

Zhang M, Li X, Xiao D, Lu T, Qin B, Zheng Z, Zhang Y, Liu Y, Yan T, Han X. Identification of differentially expressed microRNAs and their target genes in the hippocampal tissues of Fmr1 knockout mice. Am J Transl Res. 2020 Mar 15;12(3):813-824. PMID: 32269714; PMCID: PMC7137065.

Liu T, Wan RP, Tang LJ, Liu SJ, Li HJ, Zhao QH, Liao WP, Sun XF, Yi YH, Long YS. A MicroRNA Profile in Fmr1 Knockout Mice Reveals MicroRNA Expression Alterations with Possible Roles in Fragile X Syndrome. Mol Neurobiol. 2015;51(3):1053-63. doi: 10.1007/s12035-014-8770-1. Epub 2014 Jun 7. PMID: 24906954.

7. References are not always properly cited (ex. Bolduc et al., 2008 does not refer to abnormal dendritic spines in Drosophila, but excessive protein synthesis; similarly, Kelleher and Bear, 2008 and Liu-Yesucevitz refer to mRNA translation rather than spine/dendritic dysmorphogenesis). Similarly, Batish et al., 2012 is a study addressing the mRNA travel along dendrites as single particle rather than complexes of RBPs and Contractor et al., 2015 does not address the dendritic maturation and neurite extension but the altered neuronal excitability in FXS.

8. The authors suggest that DICER reduction in Fmr1 KO and Mov10 Het may affect miRNAs locally instead than leading to a global reduction of miRNA production. The authors should discuss the opposite effect on dendritic spines in Mov10 and Fmr1 KO.

Darnell 2011 is cited twice.

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PLoS One. 2021 Nov 30;16(11):e0260005. doi: 10.1371/journal.pone.0260005.r002

Author response to Decision Letter 0

31 Aug 2021

Response to Reviewers

Thank you for this opportunity to fix mistakes and make this a stronger manuscript.

Reviewer #1:

1.Is it possible that MOV10 deficiency in the Het KO reduces Dicer expression at P14, which can potentially explain the spine and dendrite phenotypes?

To address this question, we performed Dicer western blots on P14 whole brains from WT and Mov10 Het and found no difference (see Supplementary Fig 3B). In addition, we performed Dicer immunostain on DIV14 cultured hippocampal neurons from WT and Mov10 Het and found no difference in overall intensity in these neurons. Thus, we hypothesize that DICER expression is controlled locally in the dendrite and spine. Unfortunately, more precise measurements of DICER localization in dendrites and spines are beyond the scope of this study.

2.Likewise, Fig. 1 examines dendrite defects in cultured hippocampal neurons while Fig. 2 examines spine defects in cortical neuron in vivo using Golgi stain. It would be much better if they correlate dendrite and spine defects in the same group of neurons (either hippocampal or cortical, in vitro or in vivo).

To expand our study of the cultured hippocampal neurons in Figure 1, we examined spines in the somatosensory cortex by Golgi staining. To address the reviewer’s concern, we re-examined the Golgi-stained hippocampal regions in those brain sections but unfortunately, it was over-saturated.

3. Furthermore, the authors claim that the spine defects in MOV10 KO are different from FMRP KO, but the spines from MOV10 KO are at P14 while the spine defects in FMR1 KO mice are implicated from studies by other labs done at different ages (4- wk old and adult, p.14). Without side-by-side comparison in the same experiments, same age and same group of neurons it Is hard to draw this conclusion.

We appreciated the reviewer’s suggestion and analyzed the brains from P14 Fmr1 ko mice and found that they were similar to WT and that both were distinctly different from the Mov10 Het. We now include the measurements from the age-matched Fmr1 ko mice in Figure 3C.

4.In Fig. 6C the MOV10 KO are N2A cells and FMR1 KD is done in 293T cells. They should not be put together in the same graph for comparison.

Thank you for pointing this out: we have now made 2 figures in Figure 6 (C and D).

Other comments:

5.Fig. 1: how many independent experiments have been performed?

Neuron culture was performed on at least 3 litters from each genotype and the N was compiled from the three experiments. We have now added this information to the figure 1 legend and in the methods.

6.Fig. 2C: is there statistical significance between genotypes for mature spines and immature spines?

We performed ANOVA analysis on WT mature vs. Mov10 Het mature and did not find a significant difference between these genotypes.

7.Fig. 4B: does MOV10 KO brain show changes in the amount of Ago2-associated miRNAs compared to WT? And what do the two lines in the plot refer to?

Unfortunately, we have not evaluated the MOV10 ko brain animal because we are still developing that animal. The solid black line is the best fit of the line for a perfect correlation and the dashed blue line is the actual fit of the data. We have added this information to the Figure 4 legend.

8.Fig. 6D upper panel: Does the green signal represent MYC-Dicer OE cells? It should be stated clearly in the figure. The authors should also show the images for all three experimental conditions that match the quantification in the lower panel.

Yes, the green signal represents immunoreactivity to MYC epitope, which we have now added to the figure. We have created a new Figure 7 showing all of the experimental conditions with Dicer over-expression in both Neuro2A and in neurons.

9.Fig. 6D: Top left image of the upper panel indicates WT + Dicer OE but there is no such condition in the histogram of the lower panel. Does the blue bar represent WT + Dicer OE?

We have now included WT + Dicer OE in the new Figure 7A

10.Fig. 6E: why the bars are broken?

We were trying to plot all of the data on one graph. In response to the reviewer, we replotted the data on the same scale for the Y axis (Figure 6E).

11.Fig. 6F: representative images of the MOV10 KO and FMR1 KO neurons with or without Dicer OE should be shown.

We have now added representative images in Figure 7B and C.

12.Fig. 6F: if Dicer OE can reverse the dendrite phenotypes of MOV10 Het hippocampal neurons, the authors need to examine whether Dicer expression is indeed reduced in the Het neurons compared to WT.

We examined DICER expression in WT and MOV10 Het brain at P14 (Supplemental Fig 3B) and performed DICER immunostain on DIV14 cultured hippocampal neurons from WT and Mov10 Het and found no difference in intensity in these neurons (data not shown). Thus, we hypothesize that DICER expression is controlled locally in the dendrite and spine, which is not visualizable by any of these methods.

13.Does OE Dicer restore the reduction of soma size in MOV10 Het neurons?

We did this reanalysis but did not obtain a large enough sample size of transfected neurons to reject the null hypothesis.

14.P.3, second paragraph: the sentence is too long and should be divided into separate sentences.

We have done this.

15.I think dendrite width is seldom measured. The authors should discuss the significance of a change in dendrite width observed in their study.

This is an interesting question. We did this experiment because of the effect of MOV10 loss inNeuro2A on neurite width (Supplemental fig 1). The width reflects the maturation process as we now add the following description on the top of pg.17:

“Recent work shows that dendritic widths may be shaped by intracellular transport and forces from the cytoskeleton and the area proportionality accords with a requirement for microtubules to transport materials and nutrients for dendrite tip growth (Liao et al., 2021). Thus, reduced MOV10 levels likely perturb dendrite formation because MOV10 binds cytoskeletal mRNAs (Skariah et al, 2017).”

16.P.19: Kennedy et al. 2020 is not found in the reference list

We have fixed this.

17.P.23: “suggest a mechanism for regulating local DICER expression when MOV10 and FMRP are present”. I don’t think the authors have provided evidence showing a local dendritic (but not global) reduction of DICER.

The reviewer is correct. This is our proposed model.

18.P.23: “there is also a large number of RNAs that are unique to FMRP and MOV10 and it is likely that misregulation of these mRNAs cause the unique spine features.” As mentioned in previous comment, it is not appropriate to conclude “unique spine features” from the available data because the findings on MOV10 and FMR1 KO were not done side-by-side and the ages or even populations of neurons examined are different.

We have now done this and provided the analysis in Figure 3C.

Reviewer #2:

Major points:

1.Figure 1 A: images and in particular the morphology of the selected Mov10 Het neuron do not reflect the quantitative characterization presented in B. At least 3 images of representative neurons from each condition should be presented.

We have added these representative images to Figure 1.

2.Figure1B-E: Statistical tests should be detailed for each experiments. In particular, the number of different neuronal cultures should be specified.

Fig. 1: how many independent experiments have been performed?

We prepared neurons from 3 independent litters of each genotype and thus, 3 independent cultures. The total number, N, was compiled from the three biological experiments. We have now added this information to the Figure 1 legend and in the methods.

3.Figure 3B and Supplemental Fig 2: the number of different neuronal cultures used for statistical analysis should be specified. Could the individual measurements of soma size area be provided?

We used the neuron cultures described in Figure 1 for soma size analysis and have now included this information as Supplemental table 2.

Major points:

4.Figure 4 A: It seems that there are inconsistencies between the legend of the figure and the text. Is it GO enrichment for FMRP bound mRNA (legend) or differentially Ago2-associated mRNA in WT vs Fmr1-KO (text)? Were they identified by iCLIP or eCLIP? Where is the list available? The text refers to Kenny et al., 2020 and the references section mentions Kenny et al., 2019.

Thank you for pointing these issues out. The GO enrichment is for the 29 shared brain MOV10- and FMRP- and AGO-CLIP targets, as we now describe in the Figure 4 legend and in the Results. The MOV10 iCLIP list was published in Skariah et al 2017, the AGO2 eCLIP list was published in Kenny et al 2020 (not 2019, which we have now corrected) and the FMRP iCLIP list was published by Darnell 2011.

5.Figure 4 B/ Sup table 1: legends are not accurate: what are full line, dashed line?

We now describe the full and dashed lines in the Figure 4 legend.

The Supplementary Table 1.1 is of the significantly changed AGO2-bound miRNAs.

6.How statistical analysis were performed?

They were performed by ECLIPSE: They set a significance threshold of -log10(P-value) ≥ 3 and a log2 fold change ≥ 3, which is now added to the eCLIP methods.

7.To rule out any indirect effect, the level of the Ago2 protein in WT vs Fmr1-KO P0 brains should be checked.

We agree: the AGO2 levels are the same in Fmr1 knockout and WT brains. This was published in Supplemental Figure S6 of Kenny 2020. We now state this on the bottom of page 18.

8.The Dicer mRNA being a target of FMRP, MOV10 and Ago2, the authors analyzed the expression of the DICER protein and showed that DICER protein expression is decreased in Mov10-KO N2A cells or P2 Fmr1-KO brain extracts compare to WT (Figure 5A-B).

Major points:

To fully support the hypothesis that FMRP and MOV10 modulate the Ago2-miRNA mediated regulation of the Dicer mRNA, additional experiments are required:

What are the levels of Dicer mRNA in these different models?

Dicer 1 mRNA levels are likely to be unchanged because Dicer1 mRNA is not among the mRNAs changed in the brains of Fmr1 ko mice compared to WT (Korb et al., 2017; Darnell et al 2011) nor in the MOV10 knockout Neuro2A cell line (Skariah et al., 2017). We believe regulation is at the level of translation. We address this on pg. 20, line 399

9.What is the impact of the absence of FMRP on the association of the Dicer mRNA with Mov10? and conversely?

Based on our earlier work showing that FMRP facilitates loading of mRNAs onto MOV10 (Kenny et al, 2014), we would predict that in the absence of FMRP, MOV10 binds less Dicer mRNA. We look forward to exploring this question when we have the Mov10 ko mouse in-hand.

10.What is the impact of the absence of FMRP or Mov10 on the association of the Dicer mRNA with Ago2?

We examined AGO-association with Dicer1 in the absence of FMRP in the Kenny 2020 study and found that it was reduced in the absence of FMRP but not significantly.

11.What is the purpose of Figure 5C here?

We have moved this figure to the supplementary data. It is S3.

12.As MOV10 and FMRP bind murine Dicer mRNA and human DICER mRNA respectively in the 3’UTR, the authors used a DICER1 3’UTR luciferase construct to dissect the potential role of FMRP and MOV10 in the modulation of DICER1 via the 3’UTR. They showed a decreased luciferase activity in the absence of FMRP or MOV10 proteins (Figure 6C).

Major points:

13.Figure 6: Both legend and Material and Methods sections poorly explain the luciferase assay and in particular how long and short constructs participate in the calculation of the Relative Luciferase Actvity (Could the authors explain: “Mov10 KO and Fmr1 KD samples were normalized to WT Dicer long 3’UTR and Dicer short 3’UTR values were subtracted for final graph”?)

We apologize for not clearly stating our methodology: Dicer short 3’UTR contains the luciferase reporter only and allows us to examine the effect of FMRP or MOV10 loss on expression of the luciferase coding sequence itself. This is relevant as others have reported that FMRP binds luciferase coding sequence and affects expression (Chen et al. Mol Cell 2014). Dicer long 3’UTR allows us to determine the effects of the absence of MOV10 and FMRP on the entire 3’UTR. The final results had the background expression of Dicer short 3’UTR luciferase vector subtracted from the long results to account for effects on the reporter itself. Finally, we normalized to the WT expression of the Dicer luciferase vector which was set to 1. We have now added a clear description to the Methods.

14. Many steps in mRNA biogenesis could affect the reporter protein expression. qPCR to evaluate the expression of the reporter mRNA in the different conditions would strengthen the authors hypothesis.

Thank you for this suggestion. Our goal here was to report the effect on reporter expression in the absence of both MOV10 and FMRP. We do think the reduction is at the level of RNA transcription or degradation.

15.By CLIP-seq data comparison and miRNA recognition elements (MRE) analysis, the authors determined potential MSE the in close proximity of FMRP and MOV10 binding sites in the DICER 3’UTR (Sup Figure 5).

16. Minor points:

Are mouse and human Dicer mRNA 3’UTR (FMRP binding sites, Mov10 binding sites, predicted MSE ) conserved?

Thank you for asking. Yes! They are conserved and we have included this information in Supplemental figure 5C

17.Sup Figure 5: B and C should be harmonized to facilitate the reading.

We modified Supplemental Figure 5 B and C

18.Major points:

1. Figure 6 D: Incomplete legend

We have added information about how the analysis was performed.

2. Figure 6 E: Inconsistencies between text and legend.

We have revised the figure legend to reflect the constructs introduced.

3. Figure 6 F left:

Are Mov10 Het cells cultured hippocampal neurons? 7 DIV? 14 DIV?

Yes, they are DIV7 in Figure 7

19.Were analyzed neurons detected for MYC-DICER expression? What is the level of over-expression in these neurons?

Yes. We do not know the level of over-expression because we did not have enough material to immunoblot for DICER

20.Is heterologous expression homogenous?

We looked by immunostain but it was difficult to tell. It is probably heterogeneous because it is a transfection experiment

21.How does the over-expression of DICER impact dendritic arborization in WT neurons?

We did not examine WT neurons because we did not observe a difference when Dicer was over-expressed in Neuro2A (Fig 7A)

22.Can the authors comment the difference in the numbers of intersections between WT (Figure 1), Mov10 Het over- expressing negative CT and Mov10 Het over-expressing DICER (Figure 6F)?

We believe that the differences are due to a differential experimental treatment and visualization. For example, Figure 1 was Map2 staining to maximize visualization of dendrites of DIV 14 cultures while Figure 7 was DIV 7 cultures along with exposure to Lipofectamine and plasmid DNA followed by immunostaining

23.Considering the cooperative model proposed by the authors, what is the effects of FMRP over-expression on the arborization of Mov10 Het neurons?

That is in interesting question. We have not tested FMRP over-expression; however, despite working together to regulate a subset of mRNAs, MOV10 and FMRP each regulate a large number of independent mRNAs.

24.Figure 6F right:

o Same questions as above regarding the control of MYC-DICER over-expression in analyzed neurons.Why is the negative control for FMR1-KO neurons different from the one used for Mov10 Het neurons?

The negative controls were both empty expression vectors except for the tags.

25.Can the authors comment the difference in the numbers of intersections between WT (Figure 1), Fmr1-KO over-expressing negative CT and Fmr1-KO t over-expressing DICER (Figure 6F)?

Similar to what we wrote before, we believe that the differences are due to different experimental treatments, culture times and visualization. For example, Figure 1 was Map2 staining to maximize visualization of dendrites of DIV 14 cultures while Figure 7 was DIV 7 cultures along with exposure to Lipofectamine and plasmid DNA followed by immunostain.

26. To conclude, the authors discuss the potential role of FMRP in facilitating the loading of miRNA on AGO2 (Figure 7A). In parallel, they propose a model in which MOV10 alone unwinds secondary structures containing MREs and thus facilitates AGO2 binding whereas in association with FMRP, the complex is stabilized and AGO2 association is blocked (Figure 7B). Minor point:

Thank you for this thought-provoking question. We believe that FMRP, like most RNA binding proteins, has many different functions in the cell, based on its phosphorylation state, its binding partners and its location in the cell. We might envision that in the cell body, DICER translation is facilitated by the association of FMRP and MOV10 with its mRNA. When the DICER-AGO-MOV10-FMRP complex is transported in the dendrite to the synapse, it awaits stimulation which activates DICER to produce miRNAs locally, which associate with AGO2 through its association with FMRP. Presumably translation is occurring of FMRP-MOV10 bound mRNAs because this complex blocks AGO association. However, translation must eventually stop. Perhaps methylation of the FMRP’s RGG box releases the FMRP-MOV10 complex from the 3’UTR of synaptically localized mRNAs and AGO now associates with the mRNAs to block translation. We have now added this paragraph to the Discussion.

Reviewer #3:

This is a good paper exploring an interesting aspect related to the functional interaction between MOV10 and FMRP. There are a few concerns that need to be addressed.

1. Methods are clearly described with useful details. At page 7 the differentiation methods used for N2A cells should be described.

We added the specific details for differentiation to the Methods.

2. Abnormal morphology of dendrites in cultured hippocampal neurons from Mov10 Het and Fmr1 KO mice. The authors confirmed their previously published results (Skariah et al., 2017). They should also mention other papers addressing the same issue and obtaining similar (Braun and Segal, 2000) and different results in cultured Fmr1 KO hippocampal neurons (Jacob and Doering, 2007). Did the authors consider the contribution of glia?.

We appreciate the reviewer pointing out these references. We have now added them to the manuscript. We did consider the role of glia. We do not culture on an astrocyte feeder layer, like was often done in the past. This may be why we see defects in the Fmr1 ko neurons. Others are now culturing without feeders and see a result similar to ours, i.e., impaired arborization of Fmr1 ko neurons, which we cite.

We performed 3 independent experiments from at least 3 liters per genotype. N was compiled from the three experiments. We have now added this information to the figure 1 legend and in the methods. Neurons were cultured in 24 well plates from 3 litters from each genotype.

To expand our study, we examined spines in the somatosensory cortex in vivo, which we have now indicated in the methods, while examining dendrite morphology in hippocampal culture in vitro, as others have done (Galvez and Greenough, 2005). To address the reviewer’s concern, we re-examined the Golgi-stained hippocampal regions but unfortunately, it was over-saturated.

With regard to the second question, for the width measurements, it was the width of the primary branches and for the density and morphology of spines it included a 10 micron region from each level of dendrite. We have also added this to the methods

Thank you for this suggestion. We have done this.

PMID: 32269714; PMCID: PMC7137065.Liu T, Wan RP, Tang LJ, Liu SJ, Li HJ, Zhao QH, Liao WP, Sun XF, Yi YH, Long YS. A MicroRNA Profile in Fmr1 Knockout Mice Reveals MicroRNA Expression Alterations with Possible Roles in Fragile X Syndrome. Mol Neurobiol. 2015;51(3):1053-63. doi: 10.1007/s12035-014-8770-1. Epub 2014 Jun 7. PMID: 24906954.

Thank you for this suggestion. We have added this information to our manuscript.

We apologize for these errors and appreciate the opportunity to fix them.

Thinking about the reviewer’s question, it would seem like FMRP and MOV10 are working together in dendrite formation in cultured neurons but have discrete functions at P14 spines. We would hypothesize that the mRNAs that regulate dendritic arborization are distinct in time and perhaps place in the brain than those regulating spine morphology.

9.Darnell 2011 is cited twice.

Thank you for pointing this out. We have removed this.

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PLoS One. doi: 10.1371/journal.pone.0260005.r003

Decision Letter 1

Barbara Bardoni

12 Oct 2021

PONE-D-21-14401R1FMRP and MOV10 regulate Dicer1 expression and dendrite developmentPLOS ONE

Dear Dr. Ceman,

Please consider the criticism of the reviewer 2 to be able to improve your manuscript

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Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: (No Response)

Reviewer #3: All comments have been addressed

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2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: (No Response)

Reviewer #2: Partly

Reviewer #3: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: (No Response)

Reviewer #2: No

Reviewer #3: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #1: (No Response)

Reviewer #2: Yes

Reviewer #3: Yes

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5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #1: (No Response)

Reviewer #2: Yes

Reviewer #3: Yes

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6. Review Comments to the Author

Reviewer #1: (No Response)

Reviewer #2: The authors greatly improved the manuscript, detailing some experimental procedures, adding complementary controls and reinforcing the discussion section. However, it still remains few points that need to be clarified:

1- Reduced expression of Mov10 leads to smaller soma size

New figure 2, supplementary figure 2 and Supplementary table 1-2

There is a discrepancy between the individual measurements of soma size area provided in the Supplementary table 1-2 and the figures.

Figure 2: Some extreme values do not appear on the scatter plot. Is it due to an error in the graph scaling or were the values discarded as outliers? In this latter case, the mean and the sem should be revised.

According to the scatter plot, the values in the WT condition in Figure 2 and Supplementary Figure 2 are different (different mean and/or sem). However, the Supplementary table 1-2 presents a single serie of WT values.

Could the statistical options for the Student t-test be detailed? Is there a Welsh correction? Is the test one or two tailed?

2- Global reduction of AGO2-associated miRNAs in the absence of FMRP

Thank you to the authors for these further details. However, I still have a question:

Lane 358-359: Using this method, we found that the significantly enriched peaks fell within 279 miRNAs (p < .001, Fig 4A, Supplementary Table 1-1).

According to the supplementary table "Ago2 eCLIP-miRNA Table" and statistical analysis provided for IP1 WT , 531 hits present an IP1 vs Input1 -log10(P-value) ≥ 3, including 523 hits with an IP1 vs. Input1 log2 Fold Change ≥ 3. How were the 279 miRNAs mentioned in the text selected? Could they be highlighted in the supplementary table?

3- Dicer1 3’UTR regulation by MOV10 and FMRP

Response to the reviewer: Finally, we normalized to the WT expression of the Dicer luciferase vector which was set to 1. We have now added a clear description to the Methods.

Thank you to the authors for these further details. Regarding these precisions, I still have a question: Figure 6 C, D, E and Supplementary Figure S5 A: Relative Luciferase Activity: If the values were normalized to the WT values, how a sem can be calculated for the WT conditions?

4- Additional point:

Student's t test is not recommended as a pots-hoc test after a significant one way anova test.

Reviewer #3: The authors have answered to all my concerns.

There are still a few typos.

1. Pag 14 Correct Fig 1E

2. Page 19 erase ?

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7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

PLoS One. 2021 Nov 30;16(11):e0260005. doi: 10.1371/journal.pone.0260005.r004

Author response to Decision Letter 1

29 Oct 2021

Thank you for this opportunity to make corrections on the manuscript. We appreciate the opportunity to respond to the comments below. Our answers are in red.

1- Reduced expression of Mov10 leads to smaller soma size

New figure 2, supplementary figure 2 and Supplementary table 1-2

There is a discrepancy between the individual measurements of soma size area provided in the Supplementary table 1-2 and the figures.

Could the statistical options for the Student t-test be detailed? Is there a Welsh correction? Is the test one or two tailed?

Response: We looked into this and it appears to be a scaling issue when the graph is generated in our graphing software, Graphpad Prism. The software is automatically optimizing the y-axis for all data points to create a more cohesive image when extreme values are detected. In the case of Figure 2, the software excluded three outlier values and for Supplementary Figure 2 it excluded two outlier values. Each soma measurement was performed from the neurons imaged and analyzed for dendrite morphology in Figure 1, therefore only a single series of values for each genotype analyzed. We included an early version of Supplementary Figure 2 which was used for our power analysis prior to measuring all the values. We regret not catching this sooner. We have updated Figure 2B and Supplementary Figure 2. For soma size measurements, we performed a one-tailed t-test with Welch's correction. We have now specified this information clearly for all figures where that test was performed.

2- Global reduction of AGO2-associated miRNAs in the absence of FMRP

Thank you to the authors for these further details. However, I still have a question:

Lane 358-359: Using this method, we found that the significantly enriched peaks fell within 279 miRNAs (p < .001, Fig 4A, Supplementary Table 1-1).

Response: We thank the reviewer for this question. Briefly, we de-duplicated the genes in Supplementary Table 1-1 and meant to report the number of unique miRNAs. To get this value, we selected all of the hits with -log(P-value)>3 and deduplicated the gene name list. “279” was a mistake—we meant to write “261”. The names of the 261 significantly enriched miRNAs can be found on the second sheet of Supplementary Table 1-1 named “Sig. enriched miRNAs”. We corrected 279 enriched peaks to 261

3- Dicer1 3’UTR regulation by MOV10 and FMRP

Response to the reviewer: Finally, we normalized to the WT expression of the Dicer luciferase vector which was set to 1. We have now added a clear description to the Methods.

Response: Thank you for pointing out our mistake: we should not have had an error bar denoting the sem for the WT value. The error bar corresponded to the pre normalization value and was incorrectly copied when the graph was remade. We have now corrected this on all relevant figures.

4- Additional point:

Student's t test is not recommended as a pots-hoc test after a significant one way anova test.

Response: We have removed the incorrect information from the manuscript.

Reviewer #3: The authors have answered to all my concerns.

There are still a few typos.

1. Pag 14 Correct Fig 1E

2. Page 19 erase ?

Response: We have addressed these.

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PLoS One. doi: 10.1371/journal.pone.0260005.r005

Decision Letter 2

Barbara Bardoni

2 Nov 2021

FMRP and MOV10 regulate Dicer1 expression and dendrite development

PONE-D-21-14401R2

Dear Dr. Ceman,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Kind regards,

Barbara Bardoni

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

PLoS One. doi: 10.1371/journal.pone.0260005.r006

Acceptance letter

Barbara Bardoni

16 Nov 2021

PONE-D-21-14401R2

FMRP and MOV10 regulate Dicer1 expression and dendrite development

Dear Dr. Ceman:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Barbara Bardoni

Academic Editor

PLOS ONE

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 Fig. Mov10 KO Neuro2A cells have shorter and wider neurites compared to WT.

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S2 Fig. No change in total soma area in vitro between WT and Fmr1 KO neurons.

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S3 Fig. DICER protein levels do not change in brain when Mov10 is reduced.

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S4 Fig. miRNAseq reveals no difference in the level of mature vs. immature microRNAs in the absence of FMRP.

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S5 Fig. Overexpression of miR-195-3p and miR-206 does not significantly decrease Dicer 3’UTR expression in the absence of Mov10.

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S1 Table. AGO2 eCLIP miRNAs.

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S2 Table. Soma size area in WT, Mov10 Het and Fmr1 KO neurons.

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S1 Raw image

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Attachment

Submitted filename: Response to reviewers.docx

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Submitted filename: Response to Reviewers.docx

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Data Availability Statement

eCLIP data files are available from the NCBI Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo/) under the accession numbers GSE129885 (AGOeCLIP-SEQ).

[pone.0260005.ref001] 1.Ashley CT, Sutcliffe JS, Kunst CB, Leiner HA, Eichler EE, Nelson DL, et al. (1993) Human and murine FMR-1: alternative splicing and translational initiation downstream of the CGG-repeat. Nat Genet 4:244–251. doi: 10.1038/ng0793-244 [DOI] [PubMed] [Google Scholar]

[pone.0260005.ref002] 2.Irwin SA, Christmon CA, Grossman AW, Galvez R, Kim SH, DeGrush BJ, et al. (2005) Fragile X mental retardation protein levels increase following complex environment exposure in rat brain regions undergoing active synaptogenesis. Neurobiol Learn Mem 83:180–187. doi: 10.1016/j.nlm.2004.11.004 [DOI] [PubMed] [Google Scholar]

[pone.0260005.ref003] 3.Doers ME, Musser MT, Nichol R, Berndt ER, Baker M, Gomez TM, et al. (2014) iPSC-derived forebrain neurons from FXS individuals show defects in initial neurite outgrowth. Stem Cells Dev 23:1777–1787. doi: 10.1089/scd.2014.0030 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0260005.ref004] 4.Guo W, Polich ED, Su J, Gao Y, Christopher DM, Allan AM, et al. (2015) Fragile X Proteins FMRP and FXR2P Control Synaptic GluA1 Expression and Neuronal Maturation via Distinct Mechanisms. Cell reports 11:1651–1666. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0260005.ref005] 5.Telias M, Kuznitsov-Yanovsky L, Segal M, Ben-Yosef D (2015) Functional Deficiencies in Fragile X Neurons Derived from Human Embryonic Stem Cells. The Journal of neuroscience 35:15295–15306. doi: 10.1523/JNEUROSCI.0317-15.2015 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0260005.ref006] 6.Telias M, Segal M, Ben-Yosef D (2016) Immature Responses to GABA in Fragile X Neurons Derived from Human Embryonic Stem Cells. Frontiers in cellular neuroscience 10:121. doi: 10.3389/fncel.2016.00121 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0260005.ref007] 7.Shen M, Wang F, Li M, Sah N, Stockton ME, Tidei JJ, et al. (2019) Reduced mitochondrial fusion and Huntingtin levels contribute to impaired dendritic maturation and behavioral deficits in Fmr1-mutant mice. Nat Neurosci 22:386–400. doi: 10.1038/s41593-019-0338-y [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0260005.ref008] 8.Ascano M, Mukherjee N, Bandaru P, Miller JB, Nusbaum JD, Corcoran DL, et al. (2012) FMRP targets distinct mRNA sequence elements to regulate protein expression. Nature 492:382–386. doi: 10.1038/nature11737 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0260005.ref009] 9.Tran SS, Jun HI, Bahn JH, Azghadi A, Ramaswami G, Van Nostrand EL, et al. (2019) Widespread RNA editing dysregulation in brains from autistic individuals. Nat Neurosci 22:25–36. doi: 10.1038/s41593-018-0287-x [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0260005.ref010] 10.Skariah G, Seimetz J, Norsworthy M, Lannom MC, Kenny PJ, Elrakhawy M, Forsthoefel C, Drnevich J, Kalsotra A, Ceman S (2017) Mov10 suppresses retroelements and regulates neuronal development and function in the developing brain. BMC Biology 15:54. doi: 10.1186/s12915-017-0387-1 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0260005.ref011] 11.Brown V, Jin P, Ceman S, Darnell JC, O’Donnell WT, Tenenbaum SA, et al. (2001) Microarray identification of FMRP-associated brain mRNAs and altered mRNA translational profiles in fragile X syndrome. Cell 107:477–487. doi: 10.1016/s0092-8674(01)00568-2 [DOI] [PubMed] [Google Scholar]

[pone.0260005.ref012] 12.Miyashiro KY, Beckel-Mitchener A, Purk TP, Becker KG, Barret T, Liu L, et al. (2003) RNA cargoes associating with FMRP reveal deficits in cellular functioning in Fmr1 null mice. Neuron 37:417–431. doi: 10.1016/s0896-6273(03)00034-5 [DOI] [PubMed] [Google Scholar]

[pone.0260005.ref013] 13.Kenny PJ, Zhou H, Kim M, Skariah G, Khetani RS, Drnevich J, et al. (2014) MOV10 and FMRP regulate AGO2 association with microRNA recognition elements. Cell reports 9:1729–1741. doi: 10.1016/j.celrep.2014.10.054 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0260005.ref014] 14.Meister G, Landthaler M, Peters L, Chen PY, Urlaub H, Luhrmann R, et al. (2005) Identification of novel argonaute-associated proteins. Curr Biol 15:2149–2155. doi: 10.1016/j.cub.2005.10.048 [DOI] [PubMed] [Google Scholar]

[pone.0260005.ref015] 15.Banerjee S, Neveu P, Kosik KS (2009) A Coordinated Local Translational Control Point at the Synapse Involving Relief from Silencing and MOV10 Degradation. Neuron 64:871. doi: 10.1016/j.neuron.2009.11.023 [DOI] [PubMed] [Google Scholar]

[pone.0260005.ref016] 16.Skariah G, Perry KJ, Drnevich J, Henry JJ, Ceman S (2018) RNA helicase Mov10 is essential for gastrulation and central nervous system development. Dev Dyn 247:660–671. doi: 10.1002/dvdy.24615 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0260005.ref017] 17.Zafarullah M, Tassone F (2019) Molecular Biomarkers in Fragile X Syndrome. Brain Sci 9. doi: 10.3390/brainsci9050096 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0260005.ref018] 18.Kurzynska-Kokorniak A, Koralewska N, Pokornowska M, Urbanowicz A, Tworak A, Mickiewicz A, et al. (2015) The many faces of Dicer: the complexity of the mechanisms regulating Dicer gene expression and enzyme activities. Nucleic acids research 43:4365–4380. doi: 10.1093/nar/gkv328 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0260005.ref019] 19.Chendrimada TP, Finn KJ, Ji X, Baillat D, Gregory RI, Liebhaber SA, et al. (2007) MicroRNA silencing through RISC recruitment of eIF6. Nature 447:823–8. doi: 10.1038/nature05841 [DOI] [PubMed] [Google Scholar]

[pone.0260005.ref020] 20.DeMarco B, Stefanovic S, Williams A, Moss KR, Anderson BR, Bassell GJ, et al. (2019) FMRP-G-quadruplex mRNA–miR-125a interactions: Implications for miR-125a mediated translational regulation of PSD-95 mRNA. Plos One 14: e0217275. doi: 10.1371/journal.pone.0217275 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0260005.ref021] 21.Beaudoin GM 3rd, Lee SH, Singh D, Yuan Y, Ng YG, Reichardt, et al. (2012) Culturing pyramidal neurons from the early postnatal mouse hippocampus and cortex. Nat Protoc 7:1741–1754. doi: 10.1038/nprot.2012.099 [DOI] [PubMed] [Google Scholar]

[pone.0260005.ref022] 22.Ferreira TA, Iacono LL, Gross CT (2010) Serotonin receptor 1A modulates actin dynamics and restricts dendritic growth in hippocampal neurons. Eur J Neurosci 32:18–26. doi: 10.1111/j.1460-9568.2010.07283.x [DOI] [PubMed] [Google Scholar]

[pone.0260005.ref023] 23.Glaser EM, Van der Loos H (1981) Analysis of thick brain sections by obverse- reverse computer microscopy: application of a new, high clarity Golgi-Nissl stain. J Neurosci Methods 4:117–125. doi: 10.1016/0165-0270(81)90045-5 [DOI] [PubMed] [Google Scholar]

[pone.0260005.ref024] 24.Irwin SA, Idupulapati M, Gilbert ME, Harris JB, Chakravarti AB, Rogers EJ, et al. (2002) Dendritic spine and dendritic field characteristics of layer V pyramidal neurons in the visual cortex of fragile-X knockout mice. Am J Med Genet 111:140–146. doi: 10.1002/ajmg.10500 [DOI] [PubMed] [Google Scholar]

[pone.0260005.ref025] 25.Van Nostrand EL, Pratt GA, Shishkin AA, Gelboin-Burkhart C, Fang MY, Sundararaman B, et al. (2016) Robust transcriptome-wide discovery of RNA-binding protein binding sites with enhanced CLIP (eCLIP). Nat Methods 13:508–514. doi: 10.1038/nmeth.3810 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0260005.ref026] 26.Wulczyn FG, Smirnova L, Rybak A, Brandt C, Kwidzinski E, Ninnemann O, et al. (2007) Post-transcriptional regulation of the let-7 microRNA during neural cell specification. The FASEB Journal 21:415–426. doi: 10.1096/fj.06-6130com [DOI] [PubMed] [Google Scholar]

[pone.0260005.ref027] 27.Guo W, Allan AM, Zong R, Zhang L, Johnson EB, Schaller EG, et al. (2011) Ablation of Fmrp in adult neural stem cells disrupts hippocampus-dependent learning. Nat Med 17:559–565. doi: 10.1038/nm.2336 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0260005.ref028] 28.Rangasamy S OS, Gerald B, Hilbert A, Svejda S, Narayanan V. (2016) Reduced neuronal size and mTOR pathway activity in the Mecp2 A140V Rett syndrome mouse model. F1000 Res 5:2269. doi: 10.12688/f1000research.8156.1 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0260005.ref029] 29.Rajkowska G SL, Goldman-Rakic PS. (1998) Neuronal and Glial Somal Size in the Prefrontal Cortex: A Postmortem Morphometric Study of Schizophrenia and Huntington Disease. Arch Gen Psychiatry 55:215–224. doi: 10.1001/archpsyc.55.3.215 [DOI] [PubMed] [Google Scholar]

[pone.0260005.ref030] 30.Marchetto MC CC, Acab A, Yu D, Yeo GW, Mu Y, Chen G, et al. (2010) A model for neural development and treatment of Rett syndrome using human induced pluripotent stem cells. Cell 143:527–539. doi: 10.1016/j.cell.2010.10.016 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0260005.ref031] 31.Chailangkarn T AA, Muotri AR. (2012) Modeling neurodevelopmental disorders using human neurons. Current Opinion Neurobiol 22:785–790. doi: 10.1016/j.conb.2012.04.004 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0260005.ref032] 32.Jentarra GM, Olfers SL, Rice SG, Srivastava N, Homanics GE, Blue M, et al. (2010) Abnormalities of cell packing density and dendritic complexity in the MeCP2 A140V mouse model of Rett syndrome/X-linked mental retardation. BMC Neurosci 11:19. doi: 10.1186/1471-2202-11-19 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0260005.ref033] 33.Galvez R, Greenough WT (2005) Sequence of abnormal dendritic spine development in primary somatosensory cortex of a mouse model of the fragile X mental retardation syndrome. Am J Med Genet A 135:155–160. doi: 10.1002/ajmg.a.30709 [DOI] [PubMed] [Google Scholar]

[pone.0260005.ref034] 34.Greenough WT, Klintsova AY, Irwin SA, Galvez R, Bates KE, Weiler IJ (2001) Synaptic regulation of protein synthesis and the fragile X protein. Proc Natl Acad Sci U S A 98:7101–7106. doi: 10.1073/pnas.141145998 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0260005.ref035] 35.Comery TA, Harris JB, Willems PJ, Oostra BA, Irwin SA, Weiler IJ, et al. (1997) Abnormal dendritic spines in fragile X knockout mice: maturation and pruning deficits. Proc Natl Acad Sci U S A 94:5401–5404. doi: 10.1073/pnas.94.10.5401 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0260005.ref036] 36.Nimchinsky EA, Oberlander AM, Svoboda K (2001) Abnormal development of dendritic spines in FMR1 knock-out mice. The Journal of neuroscience 21:5139–5146. doi: 10.1523/JNEUROSCI.21-14-05139.2001 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0260005.ref037] 37.Hong J, Zhang H, Kawase-Koga Y, Sun T (2013) MicroRNA function is required for neurite outgrowth of mature neurons in the mouse postnatal cerebral cortex. Frontiers in cellular neuroscience 7:151. doi: 10.3389/fncel.2013.00151 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0260005.ref038] 38.McKinney BC, Grossman AW, Elisseou NM, Greenough WT (2005) Dendritic spine abnormalities in the occipital cortex of C57BL/6 Fmr1 knockout mice. Am J Med Genet B Neuropsychiatr Genet 136B:98–102. doi: 10.1002/ajmg.b.30183 [DOI] [PubMed] [Google Scholar]

[pone.0260005.ref039] 39.Grossman AW, Elisseou NM, McKinney BC, Greenough WT (2006) Hippocampal pyramidal cells in adult Fmr1 knockout mice exhibit an immature-appearing profile of dendritic spines. Brain Res 1084:158–164. doi: 10.1016/j.brainres.2006.02.044 [DOI] [PubMed] [Google Scholar]

[pone.0260005.ref040] 40.Levenga J, de Vrij FM, Buijsen RA, Li T, Nieuwenhuizen IM, Pop A, et al. (2011) Subregion-specific dendritic spine abnormalities in the hippocampus of Fmr1 KO mice. Neurobiol Learn Mem 95:467–472. doi: 10.1016/j.nlm.2011.02.009 [DOI] [PubMed] [Google Scholar]

[pone.0260005.ref041] 41.Liao M, Liang X, Howard J (2021) The narrowing of dendrite branches across nodes follows a well-defined scaling law. PNAS 118: e2022395118. doi: 10.1073/pnas.2022395118 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0260005.ref042] 42.Thomas Treiber NT, Uwe Plessmann, Simone Harlander, Julia-Lisa Daiß, Norbert Eichner, Gerhard Lehmann, et al. (2017) A Compendium of RNA-Binding Proteins that Regulate MicroRNA Biogenesis. Molecular cell 66:270–284.e213. doi: 10.1016/j.molcel.2017.03.014 [DOI] [PubMed] [Google Scholar]

[pone.0260005.ref043] 43.Michlewski G CJ (2019) Post-transcriptional control of miRNA biogenesis. Rna 25:1–16. doi: 10.1261/rna.068692.118 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0260005.ref044] 44.Ramirez-Moya J, Wert-Lamas L, Riesco-Eizaguirre G, Santisteban P (2019) Impaired microRNA processing by DICER1 downregulation endows thyroid cancer with increased aggressiveness. Oncogene 38:5486–5499. doi: 10.1038/s41388-019-0804-8 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0260005.ref045] 45.Lee RC, Feinbaum RL, Ambros V (1993) The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14. Cell 75:843. doi: 10.1016/0092-8674(93)90529-y [DOI] [PubMed] [Google Scholar]

[pone.0260005.ref046] 46.Lee Y, Jeon K, Lee JT, Kim S, Kim VN (2002) MicroRNA maturation: stepwise processing and subcellular localization. EMBO J 21:4663–4670. doi: 10.1093/emboj/cdf476 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0260005.ref047] 47.Lee Y, Ahn C, Han J, Choi H, Kim J, Yim J, et al. (2003. b) The nuclear RNase III Drosha initiates microRNA processing. Nature 425:415–419. doi: 10.1038/nature01957 [DOI] [PubMed] [Google Scholar]

[pone.0260005.ref048] 48.Denli A, Tops B, Plasterk R, Ketting R, Hannon G (2004) Processing of primary microRNAs by the microprocessor complex. Nature 432:231–235. doi: 10.1038/nature03049 [DOI] [PubMed] [Google Scholar]

[pone.0260005.ref049] 49.Gregory R, Yan K, Amuthan G, Chendrimada T, Doratoaj B, Cooch N, et al. (2004) The microprocessor complex mediates the genesis of microRNAs. Nature 432:235–240. doi: 10.1038/nature03120 [DOI] [PubMed] [Google Scholar]

[pone.0260005.ref050] 50.Han J, Lee Y, Yeom KH, Nam JW, Heo I, Rhee JK, et al. (2006) Molecular basis for the recognition of primary microRNAs by the Drosha- DGCR8 complex. Cell 125:887–901. doi: 10.1016/j.cell.2006.03.043 [DOI] [PubMed] [Google Scholar]

[pone.0260005.ref051] 51.Adams-Cioaba MA, Guo Y, Bian C, Amaya MF, Lam R, Wasney GA, et al. (2010) Structural Studies of the Tandem Tudor Domains of Fragile X Mental Retardation Related Proteins FXR1 and FXR2. PloS one 5:e13559. doi: 10.1371/journal.pone.0013559 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0260005.ref052] 52.Caudy AA, Myers M, Hannon GJ, Hammond SM (2002) Fragile X-related protein and VIG associate with the RNA interference machinery. Genes & development 16:2491–2496. doi: 10.1101/gad.1025202 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0260005.ref053] 53.Ishizuka A, Siomi MC, Siomi H (2002) A Drosophila fragile X protein interacts with components of RNAi and ribosomal proteins. Genes & development 16:2497–2508. doi: 10.1101/gad.1022002 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0260005.ref054] 54.Jin P, Alisch RS, Warren ST (2004) RNA and microRNAs in fragile X mental retardation. Nat Cell Biol 6:1048–1053. doi: 10.1038/ncb1104-1048 [DOI] [PubMed] [Google Scholar]

[pone.0260005.ref055] 55.Edbauer D, Neilson JR, Foster KA, Wang C-F, Seeburg DP, Batterton MN, et al. (2010) Regulation of Synaptic Structure and Function by FMRP-Associated MicroRNAs miR-125b and miR-132. Neuron 65:373. doi: 10.1016/j.neuron.2010.01.005 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0260005.ref056] 56.Kenny PJ, Kim M, Skariah G, Nielsen J, Lannom MC, Ceman S (2020) The FMRP- MOV10 complex: a translational regulatory switch modulated by G-Quadruplexes. Nucleic acids research 48:862–878. doi: 10.1093/nar/gkz1092 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0260005.ref057] 57.Lee EK, Kim HH, Kuwano Y, Abdelmohsen K, Srikantan S, Subaran SS, et al. (2010) hnRNP C promotes APP translation by competing with FMRP for APP mRNA recruitment to P bodies. Nature structural & molecular biology 17:732–739. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0260005.ref058] 58.Darnell JC, Van Driesche SJ, Zhang C, Hung KYS, Mele A, Fraser CE, Stone EF, Chen C, Fak JJ, Chi SW, Licatalosi DD, Richter JD, Darnell RB (2011) FMRP Stalls Ribosomal Translocation on mRNAs Linked to Synaptic Function and Autism. Cell 146:247–261. doi: 10.1016/j.cell.2011.06.013 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0260005.ref059] 59.Kenny P, Ceman S (2016) RNA Secondary Structure Modulates FMRP’s Bi- Functional Role in the MicroRNA Pathway. International Journal of Molecular Sciences 17:985. doi: 10.3390/ijms17060985 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0260005.ref060] 60.Chi SW, Zang JB, Mele A, Darnell RB (2009) Argonaute HITS-CLIP decodes microRNA-mRNA interaction maps. Nature 460:479. doi: 10.1038/nature08170 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0260005.ref061] 61.Korb E, Herre M, Zucker-Scharff I, Gresack J, Allis CD, Darnell RB (2017) Excess translation of epigenetic regulators contributes to Fragile X syndrome and is alleviated by BRD4 inhibition. Cell 170:1209–1223. doi: 10.1016/j.cell.2017.07.033 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0260005.ref062] 62.Liu T., Wan RP., Tang LJ. (2015) A MicroRNA Profile in Fmr1 Knockout Mice Reveals MicroRNA Expression Alterations with Possible Roles in Fragile X Syndrome. Mol Neurobiol 51:1053–1063. doi: 10.1007/s12035-014-8770-1 [DOI] [PubMed] [Google Scholar]

[pone.0260005.ref063] 63.Zhang M, Li X, Xiao D, Lu T, Qin B, Zheng Z, et al. (2020) Identification of differentially expressed microRNAsand their target genes in the hippocampal tissues of Fmr1 knockout mice. Am J Transl Res 12:813–824. [PMC free article] [PubMed] [Google Scholar]

[pone.0260005.ref064] 64.Mayr C, Bartel DP (2009) Widespread shortening of 3’UTRS by alternative cleavage and polyadenylation activates oncogenes in cancer cells. Cell 138: 673–684. doi: 10.1016/j.cell.2009.06.016 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0260005.ref065] 65.Gregersen LH, Schueler M, Munschauer M, Mastrobuoni G, Chen W, Kempa S, et al. (2014) MOV10 Is a 5’ to 3’ RNA helicase contributing to UPF1 mRNA target degradation by translocation along 3’ UTRs. Molecular cell 54:573–585. doi: 10.1016/j.molcel.2014.03.017 [DOI] [PubMed] [Google Scholar]

[pone.0260005.ref066] 66.Agarwal V, Bell GW, Nam JW, Bartel DP (2015) Predicting effective microRNA target sites in mammalian mRNAs. Elife 4. doi: 10.7554/eLife.05005 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0260005.ref067] 67.Sambandan S, Akbalik G, Kochen L, Rinne J, Kahlstatt J, Glock C, et al. (2017) Activity-dependent spatially localized miRNA maturation in neuronal dendrites. Science 355:634–637. doi: 10.1126/science.aaf8995 [DOI] [PubMed] [Google Scholar]

[pone.0260005.ref068] 68.Pilotte J, Dupont-Versteegden EE, Vanderklish PW (2011) Widespread regulation of miRNA biogenesis at the Dicer step by the cold-inducible RNA-binding protein, RBM3. PloS one 6:e28446. doi: 10.1371/journal.pone.0028446 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0260005.ref069] 69.Daugaard I, Hansen TB (2017) Biogenesis and Function of Ago-Associated RNAs. Trends Genet 33:208–219. doi: 10.1016/j.tig.2017.01.003 [DOI] [PubMed] [Google Scholar]

[pone.0260005.ref070] 70.O’Brien J, Hayder H, Zayed Y, Peng C (2018) Overview of MicroRNA Biogenesis, Mechanisms of Actions, and Circulation. Front Endocrinol 9:402. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0260005.ref071] 71.Kiebler MA, Bassell GJ (2006) Neuronal RNA granules: movers and makers. Neuron 51:685–690. doi: 10.1016/j.neuron.2006.08.021 [DOI] [PubMed] [Google Scholar]

[pone.0260005.ref072] 72.Mikl M, Vendra G, Doyle M, Kiebler MA (2010) RNA localization in neurite morphogenesis and synaptic regulation: current evidence and novel approaches. J Comp Physiol A Neuroethol Sens Neural Behav Physiol 196:321–334. doi: 10.1007/s00359-010-0520-x [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0260005.ref073] 73.Bicker S, Khudayberdiev S, Weiss K, Zocher K, Baumeister S, Schratt G (2013) The DEAH-box helicase DHX36 mediates dendritic localization of the neuronal precursor- microRNA-134. Genes & development 27:991–996. doi: 10.1101/gad.211243.112 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0260005.ref074] 74.Fritzsche R et al. (2013) Interactome of two diverse RNA granules links mRNA localization to translational repression in neurons. Cell reports 5:1749–1762. doi: 10.1016/j.celrep.2013.11.023 [DOI] [PubMed] [Google Scholar]

[pone.0260005.ref075] 75.Fu K, Tian S, Tan H, Wang C, Wang H, Wang M, et al. (2019) Biological and RNA regulatory function of MOV10 in mammalian germ cells. BMC Biol 17:39. doi: 10.1186/s12915-019-0659-z [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0260005.ref076] 76.Darnell JC, Klann E (2013) The translation of translational control by FMRP: therapeutic targets for FXS. Nat Neurosci 16:1530–1536. doi: 10.1038/nn.3379 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0260005.ref077] 77.Bagni C, Tassone F, Neri G, Hagerman R (2012) Fragile X syndrome: causes, diagnosis, mechanisms, and therapeutics. The Journal of clinical investigation 122:4314–4322. doi: 10.1172/JCI63141 [DOI] [PMC free article] [PubMed] [Google Scholar]

PERMALINK

FMRP and MOV10 regulate Dicer1 expression and dendrite development

Monica C Lannom

Joshua Nielsen

Aatiqa Nawaz

Temirlan Shilikbay

Stephanie Ceman

Roles

Abstract

Introduction

Materials and methods

Animals

Hippocampal neuron culture

Immunofluorescence and microscopy of cultured neurons

Neuronal transfection

Sholl analysis

Neurite outgrowth

Golgi staining

Western blot

Luciferase reporter assays

eCLIP of P0 WT and Fmr1 KO brain

miRNAseq of P0 WT and Fmr1 KO brain

Results

Both Mov10 Heterozygote (Het) and Fmr1 Knockout (KO) cultured hippocampal neurons show abnormal morphology

Fig 1. Mov10 Het hippocampal neurons show an abnormal morphology similar to Fmr1 KO neurons.

Reduced expression of Mov10 leads to smaller soma size

Fig 2. Mov10 Het neurons have a reduction in total soma area in vitro.

Mov10 Het mice have reduced density of immature dendritic spines compared to WT

Fig 3. Mov10 Het mice have fewer immature spines and wider apical oblique and basilar (secondary and tertiary) branches.

Global reduction of AGO2-associated miRNAs in the absence of FMRP

Fig 4. Global miRNA reduction in brain in the absence of FMRP.

Reduced DICER expression in the absence of FMRP or MOV10

Fig 5. DICER protein expression is significantly decreased in the absence of MOV10 and FMRP.

Dicer1 3’UTR regulation by MOV10 and FMRP

Fig 6.

Overexpression of MYC-Dicer1 rescues impaired neuronal phenotype

Fig 7. Overexpression of MYC-Dicer1 rescues impaired neuronal phenotype.

Discussion

Fig 8. MOV10 and FMRP interact with AGO2 to regulate Dicer1 mRNA expression.

Conclusions

Supporting information

Acknowledgments

Data Availability

Funding Statement

References

Decision Letter 0

Barbara Bardoni

Roles

Author response to Decision Letter 0

Decision Letter 1

Barbara Bardoni

Roles

Author response to Decision Letter 1

Decision Letter 2

Barbara Bardoni

Roles

Acceptance letter

Barbara Bardoni

Roles

Associated Data

Supplementary Materials

Data Availability Statement

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