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. Author manuscript; available in PMC: 2018 Feb 27.
Published in final edited form as: Dev Biol. 2017 Jun 30;429(1):240–248. doi: 10.1016/j.ydbio.2017.06.022

Distinct cis elements in the 3′ UTR of the C. elegans cebp-1 mRNA mediate its regulation in neuronal development

Panid Sharifnia a, Kyung Won Kim a, Zilu Wu b, Yishi Jin a,b,*
PMCID: PMC5828015  NIHMSID: NIHMS943467  PMID: 28673818

Abstract

The 3′ untranslated regions (3′ UTRs) of mRNAs mediate post-transcriptional regulation of genes in many biological processes. Cis elements in 3′ UTRs can interact with RNA-binding factors in sequence-specific or structure-dependent manners, enabling regulation of mRNA stability, translation, and localization. Caenorhabditis elegans CEBP-1 is a conserved transcription factor of the C/EBP family, and functions in diverse contexts, from neuronal development and axon regeneration to organismal growth. Previous studies revealed that the levels of cebp-1 mRNA in neurons depend on its 3′ UTR and are also negatively regulated by the E3 ubiquitin ligase RPM-1. Here, by systematically dissecting cebp-1’s 3′ UTR, we test the roles of specific cis elements in cebp-1 expression and function in neurons. We present evidence for a putative stem-loop in the cebp-1 3′ UTR that contributes to basal expression levels of mRNA and to negative regulation by rpm-1. Mutant animals lacking the endogenous cebp-1 3′ UTR showed a noticeable increased expression of cebp-1 mRNA and enhanced the neuronal developmental phenotypes of rpm-1 mutants. Our data reveal multiple cis elements within cebp-1’s 3′ UTR that help to optimize CEBP-1 expression levels in neuronal development.

Keywords: rpm-1, Mechanosensory neurons, Axon regeneration, mRNA secondary structure, Stem-loop, Translation regulation, mRNA stability

1. Introduction

Numerous mechanisms involving post-transcriptional regulation of mRNA contribute to temporal and spatial control of neuronal responses to a variety of stimuli (Holt and Schuman, 2013). For example, local translation of mRNA in presynaptic terminals is induced by neuronal activity, and in turn regulates synaptic plasticity in Aplysia (Martin et al., 1997). Alternative splicing is a key step in the production of specific isoforms of the synaptic membrane protein neurexin, enabling different receptor interactions in mammalian hippocampal synapses (Traunmuller et al., 2016). The importance of understanding mRNA regulation in the nervous system is cemented by the notion that several neurological diseases are linked to RNA-binding proteins, such as TDP43 and FMRP (Ibrahim et al., 2012; Liu-Yesucevitz et al., 2011).

Many studies have shown that cis elements in 5′ or 3′ untranslated regions (UTRs) of mRNA regulate mRNA stability, splicing, translation and localization (Mignone et al., 2002). microRNA mediated regulation is prevalent in temporal and spatial control of mRNA translation and stability, and generally acts through matching sequences in the 3′ UTR (Fabian et al., 2010). Other types of cis elements can function in a sequence- or structure-dependent manner. For example, the “zipcode” in the 3′ UTR of β-actin consists of ~ 50 nucleotides that form a stem-loop structure, which is recognized by the Zipcode Binding Proteins (ZBPs) to regulate the localization and translation of β-actin mRNA (Kislauskis et al., 1994; Ross et al., 1997). In yeast, subcellular localization of ASH1 mRNA also depends on a stem-loop structure in its 3′ UTR (Chartrand et al., 1999; Gonzalez et al., 1999). In bacteria, stem-loops in the 3′ terminal ends of puf mRNA regulate its stability (Belasco and Chen, 1988; Chen et al., 1988). In Aplysia neurons, unique cis elements in the 5′ or 3′ UTR of mRNAs of the neuropeptide sensorin mediate differential control of its mRNA localization and translation (Meer et al., 2012). A long 3′ UTR in the importin β mRNA is necessary for axonal retrograde signaling in response to injury in mice (Perry et al., 2012).

In C. elegans, cebp-1 encodes a conserved transcription factor of the C/EBP family, and is a key downstream target of the DLK-1 and p38 MAP kinase cascade (Yan et al., 2009). This kinase cascade plays important roles in neuronal development and stress response, and is under the negative control of the E3 ubiquitin ligase RPM-1 (Nakata et al., 2005). We previously showed that regulation of mRNA stability and translation of cebp-1 involves its 3′ UTR (Yan et al., 2009). Here, we address the function of specific cis regulatory elements in the cebp-1 3′ UTR. We show that multiple cis elements in cebp-1 3′ UTR contribute to its expression level in neurons. Our data suggest that an RNA secondary structure based mechanism regulates the efficiency of translation of cebp-1 mRNA, dependent on the activity of RPM-1.

2. Results

2.1. Identification of cis elements in the 3′ UTR of cebp-1

To gain clues for cis elements that regulate cebp-1 mRNA, we compared the 3′ UTR sequence of C. elegans cebp-1 to those of C. brenneri cebp-1 and mouse C/EBPδ and identified several regions with conserved sequences (Fig. S1A). miRNA prediction programs also suggest possible seed sequences within nucleotides 79–98 (seed element A) of cebp-1 3′ UTR, for example, CATTCC, matching those for mir-1 (Materials and Methods). To assess the roles of these predicted elements in regulating cebp-1 transcripts, we generated a series of deletions of the 3′ UTR and placed these downstream of a destabilized GFP reporter (DsGFP), a sensitive readout for dynamic expression (Frand et al., 2005; Li et al., 1998) (Fig. S1B). As cebp-1 is expressed in many tissues (Kim et al., 2016), to address 3′ UTR-mediated regulation specifically in the nervous system, we used the pan-neuronal rgef-1 promoter to drive transgene expression. We initially analyzed transgenic lines generated as high-copy extra-chromosomal arrays. While we observed that removal of three elements affected DsGFP expression by visual inspection of fluorescence intensity (Fig. S1C), we also observed highly variable expression among different lines generated with the same construct, likely due to the variable nature of extra-chromosomal arrays.

To precisely compare expression levels involving specific sequence elements in cebp-1 3′ UTR, we next generated stably integrated single-copy transgenes on chromosome II using MosSCI methodology (Frokjaer-Jensen et al., 2008). We used GFP as a reporter to facilitate visual assessment in this series of reporters (Fig. 1A). We quantitatively measured GFP fluorescent intensity in the nerve ring of animals at the 4th larval stage (L4) to discern changes in transgene expression. As a control for 3′ UTR specificity, we examined a similar reporter with the 3′ UTR of unc-54, which is commonly used in C. elegans transgenic expression (Fire et al., 1990). In wild type animals, deletion of the predicted microRNA seed element A did not affect GFP expression, compared to the full 3′ UTR reporter (Fig. 1C). Deletion of cis element D caused increased GFP expression (Fig. 1C). Removal of cis element E, which consists of stretches of repetitive A/Ts near the 3′ end of 3′ UTR, resulted in the largest increase in GFP intensity (Fig. 1C). The latter effect was not observed from the multi-copy transgenic lines (Fig. S1C), likely due to variable baseline expression of those high-copy number transgenes.

Fig. 1.

Fig. 1

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Identification of two cis elements in cebp-1 3′ UTR regulating reporter gene expression. A) Illustration of cebp-1 3′ UTR and transgene expression constructs. Top illustration shows full length 3′ UTR (1–600 nucleotides), with color labeling of cis elements examined in this study. Color scheme continues into other figures. cebp-1 3′ UTR reporter constructs GFP driven by Prgef-1 pan-neuronal promoter; and a control (CTRL) construct is with unc-54 3′ UTR. Prgef-1 and GFP are not drawn to scale. All transgenes are inserted at ttTi5606 on Chromosome II. FL represents full length cebp-1 3′ UTR, and Δ followed by a letter to denote the cis element deleted in the expression construct. B) Representative projection images of confocal Z-stack of GFP expression in the nerve ring for cebp-1 3′ UTR FL and ΔE reporters, and the control 3′ UTR of unc-54. Dotted red line through cebp-1 3′ UTR shows example of line scan used for quantification of GFP using ImageJ. Scale bar, 20 μm. C) Quantification of GFP intensity in the nerve ring of L4 stage animals, n ≥ 10 for each genotype. AU: artificial unit; Error bars represent standard error of the mean (SEM). Statistics, One-way ANOVA with Tukey’s post hoc tests; ns, not significant; **** p < 0.0001. D) qRT-PCR for GFP mRNA in L4 animals, ribosomal gene rps-25 used as a reference. 3 biological replicates for each strain. Error bars, SEM; ns, not significant, * p < 0.5 (One-way ANOVA with Tukey’s post hoc tests).

With the integrated single-copy reporters, we were able to quantitatively measure steady-state mRNA levels of GFP using qRT-PCR (Materials and Methods). The mRNA levels of GFP from the transgene with the full length cebp-1 3′ UTR were significantly lower than those with the transgene with the unc-54 3′ UTR. Interestingly, even though transgenes in which element D or E was deleted displayed higher levels of GFP fluorescence compared to the full-length 3′ UTR reporter, we observed no significant changes in GFP mRNA abundance by qRT-PCR (Fig. 1D). As these transgenes express GFP in the same set of neurons, these results suggest that the elements D and E individually may not directly affect mRNA stability, and that multiple elements in cebp-1’s 3′ UTR may regulate mRNA stability and translation in the nervous system.

2.2. Function of cis element D may involve a putative stem-loop structure

Secondary RNA structures regulate mRNA stability and translation (Wan et al., 2011). We therefore searched for possible secondary RNA structures in the cebp-1 3′ UTR, using the algorithms RNApromo (Rabani et al., 2008) and RNA fold (Hofacker et al., 1994; McCaskill, 1990; Zuker and Stiegler, 1981), both of which predicted similar stem loop structures within element D (Fig. S2A). To test this prediction we generated single-copy expression transgenes in which we deleted portions of element D (Fig. 2A, labeled as ΔD1 and ΔD2). Such deletions would likely disrupt the predicted RNA structure (Fig. S2B). Removal of element D2, but not D1, increased GFP fluorescence intensity, compared to the reporters for full-length 3′ UTR or ΔD (Fig. 2C). To further test if the predicted stem-loop structure was important for cebp-1’s 3′ UTR transgene expression, we mutated nucleotides ACTGG within the predicted stem to TTAAT (ΔD3 in Figs. 2A, S2C). This mutant construct showed up-regulation of GFP, similar to the ΔD2 transgene (Fig. 2B,C). Unlike other well-known hairpins (Kislauskis et al., 1994; Chartrand et al., 1999), the predicted hairpin stem length in element D of cebp-1 3′ UTR is relatively short (labeled stem loop #2 in Fig. S2A). To test if this putative stem-loop could function independent of nucleotide sequence identity, we constructed a reporter that reversed the bases of the stem but retained the predicted stem-loop structure (Dsr in Fig. S2D). We found that GFP intensity from the Dsr transgene was similar to that of the full length 3′ UTR (Fig. 2C), supporting a conclusion that element D may form a secondary stem-loop structure.

Fig. 2.

Fig. 2

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cis element D likely has a putative stem-loop secondary structure and may mediate translation of GFP reporter. A) Schematic of expression constructs for ΔD, ΔD1-3 and Dsr cebp-1 mutant 3′ UTR reporters. Prediction for presence (+) or absence (0) for RNA secondary or hairpin structure in each 3′ UTR mutant is shown on the right column. B) Sample z-stack images of the nerve ring of cebp-1 3′ UTR (FL) and ΔD3 reporter in wild type background. Scale bar, 20 μm. C) Quantification of GFP in the nerve ring of L4 stage animals. n ≥ 10 for each genotype. D) qRT-PCR for GFP mRNA in L4 animals; rps-25 used as a reference. 3 biological replicates for each strain. C-D: Error bars, SEM. Statistics, One-way ANOVA with Tukey’s post hoc tests, **** p < 0.0001; ns, not significant.

We next assessed if changes of GFP fluorescence from the 3′ UTR transgenes with mutant cis element D affected mRNA stability. By qRT-PCR analysis, we found no statistically significant difference in GFP mRNA levels among animals expressing distinct cebp-1 3′ UTR deletion transgenes or mutated stem loop transgenes, compared to the full-length reporter (Fig. 2D). We also cultured animals in the presence of α-amanitin, an inhibitor of transcription, and found that levels of GFP mRNA from these transgenes remained similar to those of full-length cebp-1 3′ UTR controls (Fig. S3). Together, these findings suggest that cis element D may regulate the translation of mRNA via a stem-loop based mechanism.

2.3. rpm-1 negatively regulates cebp-1 3′ UTR transgene expression through two separate cis elements

The E3 ubiquitin ligase rpm-1 negatively regulates the DLK-1 MAP kinase cascade, acting upstream of cebp-1 (Yan et al., 2009). Loss of rpm-1 function causes an increase in cebp-1 mRNA levels. We next examined which of the cis elements in cebp-1 3′ UTR were necessary for rpm-1-dependent regulation. We generated strains expressing each single-copy transgene of cebp-1 3′ UTR in rpm-1(lf) mutants, and compared GFP fluorescence intensity from transgenic animals relative to those in the wild type background (Materials and Methods). Consistent with our previous findings (Yan et al., 2009), GFP intensity from the cebp-1 full length 3′ UTR reporter showed significant increase in rpm-1(lf), compared to that in wild type animals (Fig. 3A,B). Moreover, GFP mRNA abundance, measured by qRT-PCR, from the cebp-1 full length 3′ UTR transgene, but not that from the transgene reporter with unc-54 3′ UTR, showed a similar increase in rpm-1(lf) (Fig. 3C).

Fig. 3.

Fig. 3

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rpm-1 regulates cebp-1 3′ UTR reporters through cis elements C and D2. A) Sample z-stack images of GFP expression in nerve ring from cebp-1 3′ UTR (FL) and cebp-1 3′ UTR(ΔE) reporters in wild type and rpm-1(lf) background. Scale bar, 20 μm. B) Quantification of GFP in the nerve ring of L4 stage animals. Strains grown at 25 °C. n ≥ 10 for each genotype. Error bars, SEM. **** p < 0.0001 (One-way ANOVA with Tukey’s post hoc tests). C) qRT-PCR for GFP mRNA in L4 animals; rps-25 used as a reference gene. 3 biological replicates for each strain, except that data for CTRL; rpm-1(lf) were from 2 biological replicates. Error bars, SEM. ns, not significant, *** p < 0.001 (One-way ANOVA with Tukey’s post hoc tests).

Similar to the transgenic animals expressing reporters with the full-length 3′ UTR of cebp-1, the cebp-1 3′ UTR deletion transgenes with ΔA, ΔB or ΔE all showed increased GFP fluorescence in rpm-1(lf) (Fig. 3B). In contrast, 3′ UTR transgenes with ΔC or ΔD did not show detectable changes in rpm-1(lf). Similar results were also observed using extra-chromosomal arrays, arguing against expression from these reporters being strongly dependent on insertion position in the genome (Fig. S1C). Moreover, quantitative comparisons of GFP mRNA levels showed no differences from the ΔC or ΔD transgene (Fig. 3C), suggesting that both elements may mediate the regulation of mRNA stability by rpm-1.

Since our data above support a role of RNA secondary structure in element D, we further examined if this structural element could be involved in rpm-1 mediated regulation. Expression from the ΔD1 transgene showed dependency on rpm-1, similar to the full-length cebp-1 3′ UTR. In contrast, the ΔD2 transgene showed no change in fluorescence intensity, nor in mRNA levels, of GFP in rpm-1(lf), compared to wild type (Fig. 3B,C). Interestingly, the ΔD2 transgene showed even higher levels of GFP expression, relative to all other transgenes. rpm-1 may negatively regulate mRNA stability through element C, and mRNA translation through the putative stem-loop structure in element D.

Lastly, we wanted to investigate the role of the cis element D with single cell resolution. We expressed the Dendra fluorescent reporter with either full-length or ΔD2 cebp-1 3′ UTR, driven by the mec-4 promoter for mechanosensory neurons, in multiple extra-chromosomal arrays (Materials and Methods). We analyzed fluorescence intensity in the somas of posterior lateral microtubule (PLM) neurons. In wild type backgrounds, average fluorescence intensity from ΔD2 transgene was similar to that from full-length 3′ UTR transgene. However, in rpm-1(lf) animals, ΔD2 transgenic reporters did not show any increase in fluorescence intensity, whereas fluorescence intensity from transgenes of full-length cebp-1 3′ UTR was increased, as expected (Fig. S4B), and consistent with the conclusion that element D plays a key role in regulating cebp-1 mRNA depending on the type of neuron.

2.4. Deletion of 3′ UTR cis elements causes partial impairment of cebp-1 function in neuronal development and axon regeneration

A key question is whether the 3′ UTR cis elements are required for normal CEBP-1 function in vivo. To address this, we undertook functional rescue experiments of cebp-1’s function in PLM neuronal development and axon regeneration.

In wild type animals, the PLM axon extends from its cell body and terminates before the cell body of the anterior lateral microtubule (ALM) mechanosensory neuron, and also extends a branch to the ventral nerve cord to form synapses (synaptic branch) (Fig. 4A,B). In rpm-1(lf), the PLM axon pattern is disrupted, with approximately 40% of animals displaying axon overextension and loss of synaptic branch (Fig. 4A,D). cebp-1(0) strongly suppresses this rpm-1(lf) developmental phenotype (Yan et al., 2009). To examine the impact of cis elements in cebp-1 3′ UTR on cebp-1 function, we generated single-copy transgenes expressing full-length CEBP-1 protein tagged to GFP with different 3′ UTRs, driven by the pan-neuronal rgef-1 promoter (Fig. 4C). In cebp-1(0) background, these transgenes did not affect PLM neuronal morphology (Fig. S5A,B). Expression of transgenes with full length 3′ UTR of cebp-1 (juSi127) fully rescued the suppression of rpm-1(lf) by cebp-1(0) (Fig. 4D). However, the other transgenes, containing either cebp-1 3′ UTR ΔD2 (juSi220) or ΔE (juSi222), or the unc-54 3′ UTR (juSi126), failed to rescue (Fig. 4D). As PLM developmental phenotype in rpm-1(lf) is associated with an increase in cebp-1 mRNA, we next measured cebp-1 mRNA produced by these transgenes. Compared to the transgene with full cebp-1 3′ UTR, the other three transgenes all showed noticeable increase, with juSi220 showing a statistically significant difference (Fig. 4E). These observations support a conclusion that cis elements within cebp-1 3′ UTR are critical for optimizing cebp-1 activity in PLM neuronal development.

Fig. 4.

Fig. 4

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cis elements D and E in 3′ UTR are required for proper function of CEBP-1 in mechanosensory neuron development. A) Sample z-stack images of PLM neuron axon morphology in wild type, rpm-1(lf), cebp-1(0), rpm-1(lf); cebp-1(0). Anterior is to the left, arrow denotes the “hook” phenotype (Schaefer et al., 2000), red asterix denotes the synaptic branch from the PLM to the nerve cord, and yellow asterix denotes the ALM cell body. Scale bar, 20 μm. B) Schematic of ALM and PLM mechanosensory neuron in wild type animals. Red asterix denotes the synaptic branch from PLM to nerve cord (labeled in black) C) Schematic of pan-neural expression of CEBP-1 protein with full-length 3′ UTR or deletion of cis elements D2 or E. Single-copy transgene is inserted on Chromosome II. D) Percentage of synaptic branch loss in strains expressing various MosSci transgenes of CEBP-1 in rpm-1(lf); cebp-1(0). WT represents wild-type. Quantification was from 3 biological replicates, total number of animals are shown in each column. E) qRT-PCR for cebp-1 mRNA from L4 animals of each single-copy CEBP-1 transgene in the rpm-1(lf); cebp-1(0). rps-25 used as a reference gene for qRT-PCR. 3 biological replicates for each strain used. D-E: Error bars, SEM. ns, not significant, **** p < 0.0001 * p < 0.05 (One-way ANOVA with Dunn’s multiple comparisons test).

In adult C. elegans, laser axotomy to PLM axons triggers a robust regrowth response that is abrogated in cebp-1(0) mutants (Yan et al., 2009). We tested how the cis elements of cebp-1 3′ UTR might affect cebp-1 function in PLM regeneration. We found that in cebp-1(0) animals, cebp-1 expressed with its full 3′ UTR (juSi127) or with ΔD2 (juSi220) or ΔE deletion (juSi222) or with unc-54 3′ UTR rescued the failed axon regeneration phenotype to different degrees, with juSi220 being the most efficient in rescuing regeneration defects of cebp-1(0) (Fig. S5C, Supplementary Table 1). These data suggest that 3′ UTR mediated regulation of cebp-1 activity is highly sensitive to biological contexts.

2.5. CRISPR deletion of the endogenous cebp-1 3′ UTR enhances neuronal defects in rpm-1(lf)

To directly address the effect of the cis elements on endogenous cebp-1 function, we next used CRISPR-Cas9 genome editing technology to delete regions of the 3′ UTR from the cebp-1 locus (Materials and Methods). Limited by the choice of effective sgRNAs, we obtained two deletion mutations, ju1451 and ju1452, removing 462 and 454 bp, of the 3′ UTR, respectively (Fig. 5A). cebp-1 mRNA levels in these two mutants showed a consistent trend of increased cebp-1 mRNA compared to wild type, though this was not statistically significant (Fig. S6). Homozygous mutants for deletions in the 3′ UTR were viable and healthy, and did not significantly affect PLM axon development (Fig. 5B,D), nor had effects on nerve regeneration (Fig. 5E, Supplementary Table 2). However, in the rpm-1(lf) background, we found ju1452 significantly enhanced the loss of PLM synaptic branch, compared to rpm-1(lf) alone (Fig. 5C). These results show that these cis elements within cebp-1’s 3′ UTR play functionally relevant roles in neuronal development in a manner depending on signaling strength.

Fig. 5.

Fig. 5

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Deletion of endogenous cebp-1 3′ UTR results in enhanced touch neuron developmental phenotypes of rpm-1(lf). A) Genome alignment of cebp-1 with black arrow showing direction of transcription/translation. Deletions ju1451 and ju1452 shown in relation to cebp-1 3′ UTR with dotted lines. B) Sample z-stack images of PLM axon in wild type, cebp-1(ju1452), and rpm-1(lf); cebp-1(ju1452). Anterior is on the left, red asterix denotes the synaptic branch from the PLM to the nerve cord, yellow asterix denotes the ALM cell body, and white hashtag represents the AVM. Scale bar, 20 μm. C) Percentage of synaptic branch loss in different strains. Experiments from 3 biological replicates with total number of animals are shown for each genotype. D) Quantification of PLM overextension in wild type, cebp-1(ju1451), cebp-1(ju1452). Experiments from 3 biological replicates with total number of animals are shown for each genotype. E) Normalized regrowth 24 h after axotomy in L4 worms, n > 25 worm each group. C-E: Error bars, SEM. Statistics, One-way ANOVA with Tukey’s post hoc tests; ns, not significant; * p < 0.05, *** p < 0.001.

3. Discussion

The C/EBP family of transcription factors has been studied for its many roles in different tissues, including the nervous system (Ramji and Foka, 2002). Mammalian genomes encode six members of this family, and C. elegans CEBP-1 is most similar to the mammalian C/EBPδ. We first identified cebp-1 as necessary for axon regeneration, and this function is also reported for C/EBPδ in a mammalian axon regeneration model (Lopez de Heredia and Magoulas, 2013; Yan et al., 2009). The activity of cebp-1 can be regulated at transcriptional and post-transcriptional levels (Kim et al., 2016; Yan et al., 2009). Here, we focused on post-transcriptional regulation involving the 3′ UTR of cebp-1, and our data reveal several cis elements contributing to context-dependent regulation of cebp-1 function in the nervous system.

We have identified two cis elements D and E, in cebp-1’s 3′ UTR, that mediate its baseline expression. Our analysis on the expression levels of GFP reporters containing deletions and base-pair changes in element D further provides evidence that element D likely contains a stem-loop structure. Loss of rpm-1 function results in increased stability of the full-length 3′ UTR reporter, but not the stability of the reporters with deletions in element D. These results suggest this putative stem-loop could be involved in the negative control of cebp-1 during two different settings, one at baseline and another in response to rpm-1 signaling. It is possible that this secondary structure serves as a scaffold for different trans factors to control cebp-1 mRNA. Future efforts will be required to identify interacting proteins.

Our data show that elements C and D2 in cebp-1 3′ UTR are involved in the regulation of cebp-1 by rpm-1. Since deletion of element C results in no difference in GFP fluorescence and mRNA levels in wild type animals, it suggests that this element is specifically important for mRNA stability regulation by rpm-1. However, with the removal of element D2, we observed increased GFP fluorescence but not mRNA levels, suggesting that element D2 may affect translation. Thus, different cis elements in the 3′ UTR of cebp-1 regulate the stability and translation of this transcription factor (Fig. 6). Interestingly a cebp-1 homolog C/EBPδ is reported to be post-transcriptionally stabilized by a p38 MAPK cascade in response to ultra-violet radiation (Li et al., 2008), suggesting functional conservation of this regulation. Consistent with the moderate changes in expression levels from the 3′ UTR reporters, we find that these cis elements made modest contribution to cebp-1 function in a context dependent manner. CEBP-1 protein expressed from transgenes with the cebp-1 3′ UTR deletion, or with a heterologous 3′ UTR of unc-54, did not rescue the touch neuron developmental defects in the rpm-1(lf); cebp-1(0). On the other hand, only CEBP-1 expressed from ΔD2 transgene fully rescued the axon regeneration defect. Previous observations have revealed that cebp-1 acts in other dosage-dependent processes influencing larval development and axon regeneration (Kim et al., 2016, data not shown). These results indicate that the ability of the animal to tolerate changes in cebp-1 levels is minimal, and that precise control over cebp-1 levels may be accomplished by using multiple cis regulatory elements.

Fig. 6.

Fig. 6

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Model of cebp-1 3′ UTR’s role. A) In wild type background or with rpm-1 signaling, cebp-1 levels are kept low under negative regulation by RPM-1, which partly depends on its 3′ UTR (labeled in orange) through a putative stem-loop and element C. B) In rpm-1(lf) background, negative regulation is absent, leading to increased cebp-1 mRNA levels and abnormal neural development. C) rpm-1(lf), in conjunction with deletion of cebp-1’s 3′ UTR, leads to a further increase in cebp-1 mRNA leading to enhancement of abnormal neural development.

We note that cebp-1 3′ UTR mutations or single-copy transgenic animals in wild type background did not cause any defects even if the cebp-1 mRNA levels are slightly increased. However, a previous study showed that overexpression of CEBP-1 using high-copy extrachromosomal transgenes in wild type background caused PLM axon termination defects (Yan et al., 2009). Such differences might reflect the dosage-sensitivity of PLM development to the CEBP-1 levels. Alternatively, the nature of transgenes might cause secondary effects on gene expression – for example, the copy number of the promoter or enhancer elements might induce competition of regulatory factors on endogenous genes in PLM development. As studies using single-copy transgene expression are made possible only recently, we foresee future work from C. elegans field would likely reveal mechanistic understanding.

In our experimental design, we used the heterologous pan-neuronal promoter, rgef-1 to drive transgene expression. This allows precise assessment of the specific effects of individual cis elements in the 3′ UTR, but does not address whether cis elements function combinatorially with transcriptional regulation to affect CEBP-1 function. We addressed this caveat by generating deletions in the endogenous locus of cebp-1 3′ UTR. Due to technical constraints by sgRNA, the deletions were larger than those using single copy insertions. We found that these mutations in the 3′ UTR caused a trend of increased cebp-1 mRNA. Although the deletions did not affect the development of the mechanosensory neurons or their regeneration, in conjunction with the rpm-1(lf) mutation, mutants lacking cebp-1 3′ UTR enhanced the loss of the PLM synaptic branch, suggesting cebp-1’s 3′ UTR is important for the formation of the synapse, similar to results observed using the single-copy transgenes. The difference in phenotype for axon regeneration and neural development suggests the dosage of cebp-1 is more important during development than in the adult animal. With the deletion of its endogenous 3′ UTR the expression of cebp-1 is sufficient for a regenerative response. However, during development cebp-1 mRNA may be more precisely regulated through its 3′ UTR. Thus, when the 3′ UTR deletion mutations are combined with rpm-1(lf), which already increases cebp-1 mRNA, the combinatorial increase in cebp-1 mRNA disrupts neuronal development (Fig. 6). Since cebp-1 has been found to function in other tissues as well as neurons, the cebp-1 3′ UTR may allow tissue and temporal specific regulation through different cis elements (Kim et al., 2016; McEwan et al., 2016). Overall, our findings on the cebp-1 3′ UTR are consistent with studies on the roles of post-transcriptional regulation mediated by 3′ UTR in other neuronal models. For example, removing regulatory elements in 3′ UTR of the Cam Kinase II reduce the amount of CamKII in postsynaptic density, and has modest effects on learning and memory behavior (Miller et al., 2002). Removal of the long 3′ UTR of importin β delays, but does not eliminate, retrograde signaling in axon injury response (Perry et al., 2012). Our data support the importance of cis elements in 3′ UTRs in post-transcriptional regulation in the nervous system. Future studies may reveal how other tissues employ similar mechanisms to regulate cebp-1 activity.

Supplementary Material

supplemental materials

Acknowledgments

We thank M. Andrusiak, S. Blazie, K. Noma, Y. Sun and A. D. Chisholm for comments on the manuscript, and our lab members for discussion. We thank J. Ewbank and N. Pujol and their lab members for their hospitality during P.S.’s study under her Chateaubriand Fellowship. P.S. was partly supported by NIH predoctoral training grant T32 GM008666; K.W.K by American Heart Association postdoctoral fellowship 13POST14800057. This work was supported by NIH R01 035546 and HHMI to Y.J.

Appendix A. Supporting information

Supplementary data associated with this article can be found in the online version at doi:10.1016/j.ydbio.2017.06.022.

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