A role for the kinetochore protein, NUF2, in ribosome biogenesis

ty j brown; Jennifer Pichurin; Carlos Ramirez Parrado; Lilian Kabeche; Susan J Baserga

doi:10.1091/mbc.E24-08-0337

. 2025 Jan 28;36(2):ar16. doi: 10.1091/mbc.E24-08-0337

A role for the kinetochore protein, NUF2, in ribosome biogenesis

ty j brown ^a, Jennifer Pichurin ^b, Carlos Ramirez Parrado ^b, Lilian Kabeche ^b,^c, Susan J Baserga ^a,^b,^d,^*

Editor: Kerry Bloom^e

PMCID: PMC11809303 PMID: 39705402

Abstract

Ribosome biogenesis (RB) is an intricate and evolutionarily conserved process that takes place mainly in the nucleolus and is required for eukaryotic cells to maintain homeostasis, grow in size, and divide. Our laboratory has identified the NUF2 protein, part of the mitotic kinetochore, in a genome-wide siRNA screen for proteins required for making ribosomes in MCF10A human breast epithelial cells. After rigorous validation and using several biochemical and cell-based assays, we find a role for NUF2 in pre-rRNA transcription, the primary and rate-limiting step of RB. siRNA depletion of other components of the NUF2 kinetochore sub-complex, NDC80, SPC24, and SPC25, also reduce pre-rRNA transcription. Interestingly, essential protein components for pre-rRNA transcription, including the largest subunit of RNA polymerase I, POLR1A, are reduced upon siRNA depletion of NUF2 and its protein partners. Their reduced levels are a likely mechanism for the decrease in pre-rRNA transcription. siRNA depletion of NUF2 and NDC80 also cause increased TP53 and CDKN1A (p21) mRNA levels, which can be restored by codepletion of RPL5, indicating activation of the nucleolar stress pathway (NSP). These results reveal a new connection between proteins with a known role in mitosis to the function of the nucleolus in RB during interphase.

Nucleoli reform after every mitosis in growing mammalian cells to carry out RB. What governs postmitotic nucleolar reformation and function is poorly understood.
In a high throughput genome-wide siRNA screen for nucleolar number and function in human breast epithelial cells, the authors identified the mitotic kinetochore protein, NUF2. Furthermore, NUF2 and its kinetochore complex partners were shown to be required for pre-rRNA transcription, the first step in RB, thereby activating the NSP.
These findings connect faithful mitosis to the resumption of nucleolar function in interphase with implications for the underlying pathophysiology of human genetic disease.

INTRODUCTION

Ribosome biogenesis (RB) is a complex, essential process that takes place primarily in the eukaryotic cell nucleolus and that results in the production of ribosomes for protein synthesis (Baßler and Hurt, 2019; Bohnsack and Bohnsack, 2019; Cerezo et al., 2019; Dorner et al., 2023). RB is conserved from single-celled to multicellular organisms and its genetic impairment in humans can lead to diseases known as ribosomopathies (Aubert et al., 2018; Warren, 2018; Farley-Barnes et al., 2019; Da Costa et al., 2020; McFadden and Baserga, 2022). Furthermore, the upregulation of RB is often associated with cancer because cancer cells require increased protein synthesis to meet the demands of rapid growth and cell division (Pelletier et al., 2018; Aspesi and Ellis, 2019; Penzo et al., 2019; Ferreira et al., 2020; Kampen et al., 2020). In addition, the number and size of tumor nucleoli serve as a prognostic marker for pathologists (Derenzini et al., 2009). Given the energy cost of producing ribosomes, both the physiological needs of the cell must be met and unnecessary energy must be conserved. The physiological needs of the cells are intrinsically tied to the cell cycle, and thus RB is regulated throughout the cell cycle (Bernstein and Baserga, 2004; Thapa et al., 2013; Armistead et al., 2014; Cao et al., 2022). As the mammalian nuclear membrane breaks down at the beginning of mitosis, RB is completely inhibited until telophase as the daughter cells reform their nuclear envelopes (Güttinger et al., 2009; Hernandez-Verdun, 2011; Delgado-Román and Muñoz-Centeno, 2021; Pham et al., 2024).

The NUF2 protein (nuclear filamentous 2) plays a conserved, critical role in mitosis as a component of the heterotetrameric NDC80 complex of the kinetochore (McKinley and Cheeseman, 2016; Musacchio and Desai, 2017; Hara and Fukagawa, 2020). Fully assembled at the centromere upon entry into mitosis (Navarro and Cheeseman, 2021; Dong and Li, 2022), a properly formed kinetochore, including the NDC80 subcomplex, is vital for cells to equally divide their chromosomes into two daughter cells. The NDC80 subcomplex consists of four protein subunits that in 1:1:1:1 stoichiometry constitute two dimers: NUF2 and NDC80 (aka Hec1), which attach to microtubules, and SPC24 and SPC25 (spindle pole component), which connect the complex to inner kinetochore proteins (Ciferri et al., 2005; Wei et al., 2005; Wei et al., 2007). The two dimers interact via long coiled-coil domains and are part of a larger kinetochore complex that consists of hundreds of proteins. NUF2 siRNA depletion in HeLa cells causes accumulation of roughly 30% of the cells in mitosis/G2 followed by cell death, though the mitotic block is not to the same degree as cells exposed to the microtubule inhibitor vinblastine, or depleted of SPC24 (Deluca et al., 2002; Subramonian et al., 2021; Kim et al., 2024). NUF2 depletion or inhibition in yeast, vertebrate cells, or Xenopus egg extracts results in defective kinetochore assembly and lack of mitotic progression (Nabetani et al., 2001; Deluca et al., 2002; Hori et al., 2003; McCleland et al., 2003; Deluca et al., 2005). Interestingly, the levels of the four NDC80 subcomplex proteins can be interdependent as experiments using degron depletion of SPC24 in HeLa cells led to a reduction in the levels of the three others (Kim et al., 2024). The interphase, non-mitotic function of NUF2 has not yet been elucidated, although the human protein atlas (proteinatlas.org) reveals that NUF2 localizes to the nucleoplasm in several human tissue culture cell lines.

Because of NUF2’s known role in mitosis, we were surprised to obtain it as a hit in our genome-wide siRNA screen for proteins required for nucleolar function (Farley-Barnes et al., 2018). The RNAi screening platform that we developed uses changes in nucleolar number as a phenotypic readout and is supported by excellent screening statistics. So far, we have used it as a starting point to identify and then define the function of several human proteins in RB (Farley-Barnes et al., 2020; Ogawa et al., 2021; McCool et al., 2023a; McCool et al., 2023b). We have also used it successfully to define microRNAs that target nucleolar proteins and reduce RB, showing for the first time a direct interaction between the MIR28 family of microRNAs and the ribosomal protein, RPS28 (Bryant et al., 2024). This unbiased approach to discover regulators of RB in human cells has the potential to reveal unforeseen connections among diverse cellular processes.

Here we explore the molecular basis for how the NUF2 protein regulates RB in MCF10A cells, a human breast epithelial cell line. We first validated it as a hit in the genome-wide siRNA screen with several methodologies, including a rescreen with higher fidelity siRNAs and subsequent oligonucleotide deconvolution. Using a battery of approaches that assess pre-rRNA transcription, we find that NUF2 is required for this initial step in RB. The reduced pre-rRNA transcription observed upon NUF2 siRNA depletion is likely due to lower levels of proteins required for transcription, including the largest subunit of RNA polymerase I, POLR1A. This results in fewer ribosomes being made, as demonstrated by a severe reduction in global protein synthesis and the activation of a TP53-dependent nucleolar stress pathway (NSP). Taken together, we provide evidence that NUF2, a protein with a canonical role in mitosis, is required for RB in the nucleolus of human cells.

RESULTS

NUF2 is required for maintenance of typical nucleolar number and viability in MCF10A cells

We have previously demonstrated that depletion of essential nucleolar proteins required for RB causes a decrease from the 2–3 nucleoli typically present in breast epithelial MCF10A cells to one nucleolus (Freed et al., 2012). We exploited this connection between the number of nucleoli and nucleolar function in RB by performing a genome-wide RNAi screen to identify novel regulators of RB (Farley-Barnes et al., 2018; Ogawa et al., 2021). In short, hits were classified as whether their depletion resulted in a significant shift in the percentage of cells that bear just one nucleolus per nucleus. The percentage of cells depleted of each mRNA with one nucleolus was calculated and referred to as the “Percent effect” and reported relative to the percent effect of the negative control (siGFP, set to 0%) and the positive control (siUTP4, set to 100%).

In our genome-wide siRNA screen we unexpectedly found that depletion of NUF2 caused a significant increase in the proportion of cells with one nucleolus. NUF2 is a mitotic kinetochore protein whose role in chromosome segregation is well defined (Nabetani et al., 2001; Deluca et al., 2002; Hori et al., 2003; McCleland et al., 2003; Deluca et al., 2005; Subramonian et al., 2021; Kim et al., 2024). siRNA depletion of NUF2 resulted in a higher percent effect than the positive control, with almost 60% of depleted cells bearing just one nucleolus (Percent effect = 107%; Figure 1A). To validate this finding with NUF2 siRNAs designed to have fewer off-target effects, we rescreened the hits from the primary screen using siON-TARGET pools (siONT; Horizon Discovery). For the siONT validation, we used a nontargeting pool of siRNAs (siNT) as the negative control (Percent effect = 0%) and siNOL11 as the positive control (Percent effect = 100%; Figure 1B). Similarly, NUF2 siRNA depletion led to a significant shift toward a higher percentage of cells with one nucleolus (Percent effect = 123%; Figures 1, B and D pool). Because we have previously confirmed the link between changes in nucleolar number and nucleolar function (Farley-Barnes et al., 2018; Farley-Barnes et al., 2020; Ogawa et al., 2021; McCool et al., 2023a; McCool et al., 2023b) these findings implicate NUF2 as a potential regulator of nucleolar function.

FIGURE 1: — NUF2 regulates nucleolar number and viability in MCF10A human breast epithelial cells. (A, left) Genome-wide siRNA campaign (Farley-Barnes *et al.*, 2018) reveals that NUF2 depletion reduces nucleolar number in MCF10A cells. Representative immunofluorescent images of MCF10A cells treated with siGENOME siRNA pools targeting GFP (negative control), UTP4 (positive control), or NUF2. The cells were treated with the indicated siRNAs for 72 h and then fixed and stained with Hoechst dye (blue) to label nuclei and an α-fibrillarin antibody (FBL; green; 72B9) to detect nucleoli. The contrast between the colors was increased uniformly in the ImageJ software (Schindelin *et al.*, 2012) to increase clarity. (A, right) Quantitation of A, left. Histograms indicate the relative frequencies of nucleolar number (nucleoli/nucleus) in the images. The light gray bars represent the negative control (siGFP). Black bars represent the positive control (siUTP4) or siNUF2. The dark gray bars represent the overlap of the light gray and black bars. N = total number of cells analyzed; percent effect is the one nucleolus percent effect. Scale bar, 20 µm. (B) Validation that siNUF2 reduces nucleolar number in MCF10A cells using siON-TARGETplus (siONT) SMARTpool RNAs. As above in A, except cells were treated with the NUF2 siONT RNAs. A nontargeting siRNA (siNT) was used as the negative control) and siNOL11 was the positive control. Scale bar, 20 µm. (C) Deconvolution of the four individual NUF2 siONT SMARTpool RNAs reveals that ¾ of them reduce the nucleolar number (si-1, si-3, si-4). Representative images and the corresponding histograms from the siRNA oligonucleotide deconvolution of the siONT SMARTpool. Bar colors as in B. Scale bar, 20 µm. (D) Quantitation of the results in B and C reveals that three of the four siONT RNAs (si-1, si-3, si-4) reduce the nucleolar number. Bar graph summary of the percent effect of the siONT SMARTpool or the four individual siRNAs. Each dot represents a separate biological replicate. Bars indicate the mean percent effect across replicates ± standard error of the mean (SEM). Dashed lines indicate the percent effect of positive control (siNOL11 set to 100%) and negative control (siNT set to 0%). (E) Treatment of MCF10A cells with the pool or the four individual siONT NUF2 siRNAs for 72 h reduces cell viability. A nontargeting siRNA (siNT) was used as the negative control and set to 100% (dashed line). Bars indicate the mean cell viability across replicates (dots) per treatment. Error bars represent SEM. (F) Treatment of MCF10A cells with a pool or the three individual siONT NUF2 siRNAs (si-1, si-3, si-4) for 72 h reduces NUF2 protein levels. (Left) Western blot with anti-NUF2 antibody of lysates from cells treated for 72 h with siNT, the indicated individual NUF2 siONT RNAs (si-1, si-3, or si-4), and a pool of all three in equimolar concentrations (pool). (Right) NUF2 levels were quantitated using ImageJ software and normalized to the total protein lane from each sample and set relative to siNT. siNT is set to 1. Dots represent biological replicates (n = 3–6). Data were analyzed by ordinary one-way ANOVA with Holm–Šídák's multiple comparisons test, with single-pooled variance, where (****) P ≤ 0.0001 (G) Treatment of MCF10A cells with a pool or the three individual siONT NUF2 siRNAs (si-1, si-3, si-4) for 72 h reduces *NUF2* mRNA levels. qRT-PCR was used to assess the extent of *NUF2* knockdown in MCF10A cells. cDNA was synthesized from total RNA extracted from MCF10A cells treated with the indicated siRNAs and probed using primers against *NUF2. NUF2* mRNA levels were normalized to a 7SL internal control and the 2^−ΔΔCt values are reported relative to levels in negative control cells (siNT; values from each replicate set to 1; represented by dashed line). Each biological replicate (indicated by dots; N = 3) includes 3–6 technical replicates. The average between biological replicates for each condition is indicated by the bars ± SEM. Data were analyzed by ordinary one-way ANOVA with Holm–Šídák's multiple comparisons test, with single-pooled variance, where (****) P ≤ 0.0001. (H) Table summary of results in C–G comparing the four individual siRNAs targeting NUF2. N.D. is not determined.

To provide further confidence that the observed reduction in nucleolar number was due to NUF2 siRNA depletion, as opposed to an off-target effect, we performed oligonucleotide deconvolution of the individual siRNAs that comprise the siONT pool of four siRNAs. Whether at least two of the four individual siRNAs cause a reduction in nucleolar number, then it is more likely to be a result of NUF2 siRNA depletion, and not the result of an off-target effect. Here, three of the four siRNAs targeting NUF2 (si-1, si-3, si-4) significantly reduced nucleolar number (Figures 1, C and D), providing support for NUF2 as a bona fide hit in our screening campaign.

In each of these assays, viability was also scored relative to the negative control. For the three individuals siONT RNAs or the siONT pool that resulted in a reduction in the number of nucleoli/nucleus, there was a concomitant significant reduction in viability (Figure 1E). This is consistent with NUF2 having a role in RB, which is an essential process.

We tested the extent to which siRNAs targeting NUF2 resulted in its depletion at the protein (Western blot) and mRNA (qRT-PCR) levels. We observed that treating MCF10A cells with any of the three validated siONT RNAs against NUF2, or an equimolar siONT RNA pool, significantly reduced NUF2 protein (Figure 1F) and mRNA (Figure 1G) compared with the nontargeting control (siNT). Together, the findings (summarized in Figure 1H) bolster our hypothesis that depletion of NUF2 via RNAi results in decreased nucleolar number in MCF10A cells. This implies that NUF2 plays a novel role in ribosome synthesis.

NUF2 is required for pre-rRNA transcription

Transcription at the rDNA loci by RNA polymerase I (RNAPI) yields the multicistronic 47S pre-rRNA precursor (Figure 2A). The 47S pre-rRNA transcript undergoes many modifications and is processed to yield three of the four mature rRNA molecules in the mature ribosome (Baßler and Hurt, 2019; Bohnsack and Bohnsack, 2019; Cerezo et al., 2019; Dorner et al., 2023). In humans, these are the 28S and 5.8S large subunit rRNAs and the 18S in the small ribosomal subunit (Lafontaine, 2015).

Given that transcription of the primary 47S transcript is the initial and rate-limiting step of RB, we hypothesized that NUF2 could regulate the accumulation or stability of that pre-rRNA. One way we sought to investigate this possibility was with a 5-ethynyl uridine (5-EU) incorporation assay, which was developed for high-throughput nucleolar rRNA synthesis in our laboratory (Bryant et al., 2022). For this assay, cells depleted of NUF2 were treated with 5-EU for an hour, followed by the addition of a fluorophore via click chemistry to the newly incorporated 5-EU to distinguish nascent RNA transcripts. We fixed and stained their DNA to label nuclei and costained them with an anti-fibrillarin (FBL) antibody to detect nucleoli. We then utilized an imaging platform to visualize these objects and measured the 5-EU signal restricted to the nucleolus, allowing us to ascertain the levels of nascent rRNA transcripts specifically (Figure 2B). Once measured, rRNA levels are reported in terms of “percent inhibition,” meaning that the higher the percentage inhibition, the lower the 5-EU signal in the nucleoli of those cells. For negative control cells treated with a nontargeting siRNA (siNT), this value was set to 0%; the positive control cells treated with siRNAs targeting RNAPI's catalytic subunit (POLR1A) were set to 100%. NUF2 siRNA depletion led to reduced nucleolar rRNA biogenesis similar to that in the positive control cells with a percent inhibition of 104% (Figure 2C, summarized in Figure 2F).

We tested whether NUF2 has a role in regulating pre-rRNA transcription and/or accumulation by measuring the abundance of the 47S and 45S pre-rRNA precursors using qRT-PCR. The 47S precursor rapidly undergoes cleavage at the 5′ end (A’), yielding the 45S pre-rRNA species (Bowman, 1987; Henras et al., 2008; Freed et al., 2012; Aubert et al., 2018). This is easily detected with qRT-PCR and is indistinguishable from the 47S transcript (Figure 2D, top). While this assay only reports steady-state levels of the processed pre-rRNAs, it has long been used as an indicator of pre-rRNA transcription (Bywater et al., 2012; Sorino et al., 2020; McCool et al., 2023a). We applied qRT-PCR to cells siRNA depleted of NUF2, with the positive control, siNOL11, and with the negative control, siNT. We quantitated the 2^−ΔΔCt values for primary transcript levels normalized to an internal 7SL control and reported them relative to transcript levels in the negative control cells (siNT) (Figure 2D, bottom). We observed an ∼80% decrease in primary transcript levels when NUF2 is depleted with siRNAs, a more drastic reduction than the ∼70% decrease in primary transcript levels when positive control NOL11 is depleted. These results support our hypothesis that NUF2 is involved in the RNAPI transcription step of RB.

We asked whether the observed decrease in the abundance of pre-rRNA upon siRNA depletion of NUF2 is a result of decreased RNAPI activity at the rDNA promoter using a dual-luciferase reporter assay (Ghoshal et al., 2004; Farley-Barnes et al., 2018; Ogawa et al., 2021; McCool et al., 2023a; Bryant et al., 2024). This involves two plasmids, one bearing the firefly luciferase under the rDNA promoter (−410 to + 327), which reports RNAPI activity, and one bearing a Renilla luciferase under a constitutive reporter (Figure 2E, bottom), which serves as a transfection control. We measured the luminescence of each luciferase, reporting the ratio of firefly to Renilla relative to the ratio in negative control cells treated with siNT. Consistent with our previous findings in Figures 2, C and D, we observed a significant decrease in RNAPI promoter activity in MCF10A cells siRNA depleted of NUF2, with activity at ∼60% of that observed in negative control cells (siNT; Figure 2E). Positive control cells were treated with 1 µM BMH-21 for 24 h before being assayed. BMH-21 is a small-molecule DNA intercalator that severely inhibits RNAPI transcription (Jacobs et al., 2022), and, in our system, reduces RNAPI transcriptional activity at the rDNA promoter to ∼20% versus the negative control (siNT). These results, along with those from the 5-EU incorporation assay and 45S/47S transcript levels (Figures 2, C and D), support our hypothesis that NUF2 is required for optimal pre-rRNA transcription by RNAPI.

NUF2 depletion decreases the levels of the RNAPI transcription machinery

Because our results strongly argue that NUF2 has a role in pre-rRNA transcription, we hypothesized that siRNA depletion of NUF2 may lead to reduced protein levels of RNAPI subunits and its associated factors. We tested this by Western blot analyses on cell lysates depleted of NUF2 using siRNAs. We measured protein levels of several key factors involved in pre-rRNA transcription, including POLR1A, the catalytic subunit of RNAPI (Lafontaine et al., 2021; Misiaszek et al., 2021), as well as RNAPI-specific transcription factors RRN3 and UBTF (Miller et al., 2001; Hori et al., 2023). We also measured levels of FBL, a nucleolar protein with a role in pre-rRNA processing and in promoting transcription as a methyl group donor (Tycowski et al., 1996; Iyer-Bierhoff et al., 2018), and the MYC protein, which regulates RB both directly and indirectly (Grandori et al., 2005; Grewal et al., 2005). We observed that depletion of NUF2 over 72 h causes a significant progressive decrease in POLR1A levels to ∼30% (24 h), 40% (48 h), and 50% (72 h) relative to levels in siNT-treated negative control cells (Figure 3A). These reductions are comparable to those observed in the positive control cells treated with siPOLR1A at the respective time points. We also observed decreased protein levels of RRN3 (Figure 3B) and FBL (Figure 3C) to slightly more than 50% in both cases when NUF2 is depleted. Interestingly, treatment with positive control siPOLR1A did not decrease RRN3 levels (Figure 3B) but did decrease FBL levels (Figure 3C). In contrast, neither POLR1A nor NUF2 depletion caused any significant changes in the levels of UBTF (Figure 3D) or of MYC (Figure 3E). The reduction in the levels of several proteins required for transcription (POLR1A, RRN3, and UBTF) upon siRNA depletion of NUF2 are consistent with the reduced pre-rRNA transcription that we observe (Figure 2).

FIGURE 3: — siRNA depletion of NUF2 in MCF10A cells causes decreased protein synthesis and decreased levels of the RNAPI transcription apparatus and its associated factors. (A) NUF2 depletion reduces the levels of RNAPI's catalytic subunit, POLR1A. (B, left) Representative Western blot images. Cells were treated with indicated siRNAs for 24, 48, or 72 h before total protein was harvested and 25 μg was run on a 10% SDS–PAGE. Protein levels were detected using an α-POLR1A antibody and normalized to total protein. siNT (nontargeting siRNA) is the negative control and siPOLR1A is the positive control. (B, right) Quantitation of POLR1A levels after 24, 48, and 72 h of siRNA treatment. Bars represent the mean of biological replicates ± SEM (N = 3-4). Data were analyzed by ordinary one-way ANOVA with Holm–Šídák's multiple comparisons test, with single-pooled variance, where (****) P ≤ 0.0001, (***) P ≤ 0.0005, (**) P ≤ 0.005, and (*) P ≤ 0.05. (B) siNUF2 depletion reduces the levels of the RNAPI transcription factor, RRN3. (B, top) Representative Western blot images. Cells were treated with indicated siRNAs for 72 h, including the siPOLR1A positive control and the siNT (nontargeting) negative control. Total protein was harvested and 25 μ g was run on a 10% SDS–PAGE. RRN3 was detected at a molecular weight of 74 kDa using an α-RRN3 antibody and normalized to the total protein signal. (B, bottom) Quantitation of protein band intensity normalized to total protein and reported relative to siNT (N = 3). Bars represent the mean of biological replicates ± SEM. Data were analyzed by ordinary one-way ANOVA with Holm–Šídák's multiple comparisons test, with single-pooled variance, where (*) P ≤ 0.05 and (n.s.) is not significant. (C) siNUF2 depletion reduces the levels of methyl group donor FBL. (C, top) Representative Western blot images. Cells were treated with the indicated siRNAs for 72 h where siPOLR1A is the positive control and siNT (nontargeting) is the negative control. Total protein was harvested and 25 μ g was run on a 10% SDS–PAGE. FBL was detected at a molecular weight of 34 kDa using an α-FBL antibody and normalized to the total protein signal. (C, bottom) Quantitation of protein band intensity normalized to total protein and reported relative to siNT (N = 3). Bars represent the mean of biological replicates ± SEM. Data were analyzed by ordinary one-way ANOVA with Holm-Šídák's multiple comparisons test, with single-pooled variance, where (*) P ≤ 0.05. (D) Levels of the RNAPI transcription factor UBTF remain unchanged when NUF2 is depleted. (D, top) Representative Western blot images. Cells were treated with the indicated siRNAs where siPOLR1A is the positive control and siNT (nontargeting) is the negative control. Total protein was harvested and 25 μ g was run on a 10% SDS–PAGE. UBTF was detected at a molecular weight of 89 kDa using an α-UBTF antibody and normalized to the total protein signal. (D, bottom) Quantitation of protein band intensity normalized to total protein and reported relative to siNT (N = 3). Bars represent the mean of biological replicates ± SEM. Data were analyzed by ordinary one-way ANOVA with Holm–Šídák's multiple comparisons test, with single-pooled variance, where (n.s.) is not significant. (E) NUF2 depletion does not significantly affect levels of the RNAPI transcription factor, MYC. (E, top) Representative Western blot images. Cells were treated with indicated siRNAs for 72 h where siPOLR1A is the positive control and siNT (nontargeting) is the negative control. The total protein harvested was 25 μ g, and it run on a 10% SDS–PAGE. MYC was detected at a molecular weight of 50 kDa using an α-MYC antibody and normalized to the total protein signal. (E, bottom) Quantitation of protein band intensity normalized to total protein and reported relative to siNT (N = 3). Bars represent the mean of biological replicates ± SEM. Data were analyzed by ordinary one-way ANOVA with Holm–Šídák's multiple comparisons test, with single-pooled variance, where (n.s.) is not significant. (F) NUF2 is required for protein synthesis. (F, left) Representative Western blot images from the puromycin incorporation assay (Schmidt *et al.* 2009). Cells were treated with the indicated siRNAs for 48 or 72 h. siRPL4 is the positive control and siNT (nontargeting) is the negative control. One hour before protein harvest, cells were treated with 1 μM puromycin. Total protein was harvested and 25 μ g was run on a 10% SDS–PAGE. Nascent proteins were detected using an α-puromycin antibody and normalized to the total protein signal. (F, right) Quantitation of puromycin levels normalized to total protein levels and reported relative to siNT (N = 3). Bars represent the mean of biological replicates ± SEM. Data were analyzed by ordinary one-way ANOVA with Holm–Šídák's multiple comparisons test, with single-pooled variance, where (****) P ≤ 0.0001 and (***) P ≤ 0.0005.

NUF2 is required for global protein synthesis

Given NUF2’s role in rDNA transcription, we hypothesized that its depletion would lead to reduced protein synthesis. To assess this, we performed a puromycin incorporation assay (Schmidt et al., 2009) on MCF10A cells depleted of NUF2. Puromycin, an aminoacyl-tRNA analogue, is stably incorporated into nascent polypeptide chains as a chain terminator and allows us to measure the amount of protein synthesis. After 48 or 72 h of siRNA treatment, 1 µM puromycin was added for an hour. Protein synthesis was analyzed by SDS–PAGE and Western blotting with anti-puromycin antibodies. NUF2 depletion results in a dramatic decrease in global protein synthesis (Figure 3F), similar to that observed after depletion of the RPL4 (ribosomal protein L4, positive control). siNT (nontargeting siRNA) was used as a negative control, and set at 1. Reduced protein synthesis upon NUF2 depletion is likely a result of the reduced pre-rRNA transcription that we observe.

Depletion of other subunits of the kinetochore complex, NDC80, SPC24, and SPC25, also reduces pre-rRNA transcription

We tested whether depletion of the other three kinetochore complex subunits (NDC80 [Hec1], SPC24, and SPC25; [Ciferri et al., 2005; Wei et al., 2005; Wei et al., 2007]) would also reduce pre-rRNA transcription as we observed following NUF2 depletion (Figure 2D). We measured the levels of the 47S and 45S pre-rRNA precursors using qRT-PCR in MCF10A cells depleted of NDC80, SPC24, and SPC25 by siRNA for 72 h. We found that depletion of each of the kinetochore proteins drastically reduced the levels of the 47S/45S pre-rRNAs, consistent with a role for them in pre-rRNA transcription (Figure 4A; 90%, 75%, and 90%, respectively). Therefore, all four of these kinetochore proteins, NUF2, NDC80, SPC24, and SPC25, play a role in pre-rRNA transcription.

NDC80, SPC24, and SPC25 are all required for maintaining normal levels of the RNAPI transcription apparatus as well as for global protein synthesis

Because the other members of the NDC80 complex are required for pre-rRNA transcription, we asked whether their depletion would decrease the levels of POLR1A or other RNAPI-associated transcription-promoting factors. Consistent with what we observed after the depletion of NUF2 (Figure 3A), the depletion of NDC80, SPC24, and SPC25 all led to reduced POLR1A levels (Figure 4B; 45%, 50%, and 60%, respectively). Likewise, we observe a significant decrease in RNAPI transcription factor RRN3 levels upon depletion of NDC80, SPC24, or SPC25 to between 40 and 50% compared with its levels in siNT cells (Figure 4C). We also see a significant reduction in FBL abundance by 45 and 50% in cells treated with siSPC24 or siSPC25, respectively (Figure 4D). Strikingly, FBL levels did not change significantly in siNDC80-treated cells (Figure 4D). Furthermore, as was the case when NUF2 was depleted, we did not observe a significant change in the levels of either UBTF (Figure 4E) or MYC (Figure 4F) upon siRNA depletion of NDC80, SPC24, or SPC25.

FIGURE 4: — Three other kinetochore complex proteins, NDC80, SPC24, or SPC25, are also required for ribosome biogenesis in MCF10A cells. (A) Depletion of NDC80, SPC24, or SPC25 leads to decreased levels of the 47S/45S primary rRNA transcript. qRT-PCR was performed on cDNA that was synthesized from total RNA harvested from cells treated with the indicated siRNAs for 72 h. The 2^−ΔΔCt values for the 47S/45S transcript in each condition were normalized to a 7SL internal control and reported relative to values in negative control cells (siNT; nontargeting; dotted line). siNOL11 is the positive control. Data were analyzed by ordinary one-way ANOVA with Holm–Šídák's multiple comparisons test, with single-pooled variance, where (****) P ≤ 0.0001. (B) Depletion of NDC80, SPC24, or SPC25 reduces levels of RNAPI's catalytic subunit, POLR1A. (B, left) Representative Western blot images. Cells were treated with the indicated siRNAs for 72 h before total protein was harvested. siPOLR1A is the positive control and siNT (nontargeting) is the negative control. Twenty-five μg of protein from each sample was run on a 10% SDS–PAGE. Protein levels were detected using an α-POLR1A antibody and normalized to total protein. (B, right) Quantitation of POLR1A levels. Bars represent the mean of biological replicates ± SEM (N = 3–4). Data were analyzed by ordinary one-way ANOVA with Holm–Šídák's multiple comparisons test, with single-pooled variance, where (***) P ≤ 0.0005 and (**) P ≤ 0.005. (C) Depletion of NDC80, SPC24 or SPC25 reduces the levels of the RNAPI transcription factor, RRN3. (C, left) Representative Western blot images. Cells were treated with indicated siRNAs for 72 h. siPOLR1A is the positive control and siNT (nontargeting) is the negative control. Total protein was harvested and 25 μ g was run on a 10% SDS–PAGE. RRN3 was detected at a molecular weight of 74 kDa using an α-RRN3 antibody and normalized to the total protein signal. (B, right) Quantitation of protein band intensity normalized to total protein and reported relative to siNT (N = 3). Bars represent the mean of biological replicates ± SEM. Data were analyzed by ordinary one-way ANOVA with Holm–Šídák's multiple comparisons test, with single-pooled variance, where (**) P ≤ 0.005, (*) P ≤ 0.05, and (n.s.) is not significant. (D) Levels of the methyl group donor FBL are reduced upon depletion of SPC24 or SPC25, but not NDC80. (D, top) Representative Western blot images. Cells were treated with indicated siRNAs for 72 h. siPOLR1A is the positive control and siNT (nontargeting) is the negative control. Total protein was harvested and 25 μ g was run on a 10% SDS–PAGE gel. FBL was detected at a molecular weight of 34 kDa using an α-FBL antibody and normalized to the total protein signal. (D, bottom) Quantitation of protein band intensity normalized to total protein and reported relative to siNT (N = 3). Bars represent the mean of biological replicates ± SEM. Data were analyzed by ordinary one-way ANOVA with Holm–Šídák's multiple comparisons test, with single-pooled variance, where (**) P ≤ 0.005, (*) P ≤ 0.05, and (n.s.) is not significant. (E) Levels of RNAPI transcription factor, UBTF, remain unchanged when NDC80, SPC24 or SPC25 are depleted. (E, top) Representative Western blot images. Cells were treated with the indicated siRNAs for 72 h. siPOLR1A is the positive control and siNT (nontargeting) is the negative control. The total protein was harvested and 25 μ g was run on a 10% SDS–PAGE. UBTF was detected at a molecular weight of 89 kDa using an α-UBTF antibody and normalized to the total protein signal. (E, bottom) Quantitation of protein band intensity normalized to total protein and reported relative to siNT (N = 3). Bars represent the mean of biological replicates ± SEM. Data were analyzed by ordinary one-way ANOVA with Holm–Šídák's multiple comparisons test, with single-pooled variance, where (n.s.) is not significant. (F) Depletion of NDC80, SPC24, or SPC25 does not significantly alter levels of the transcription factor MYC. (F, top) Representative Western blot images. Cells were treated with indicated siRNAs for 72 h. siPOLR1A is the positive control and siNT (nontargeting) is the negative control. Total protein was harvested and 25 μ g was run on a 10% SDS–PAGE gel. MYC was detected at a molecular weight of 50 kDa using an α-MYC antibody and normalized to the total protein signal. (F, bottom) Quantitation of protein band intensity normalized to total protein and reported relative to siNT (N = 3). Bars represent the mean of biological replicates ± SEM. Data were analyzed by ordinary one-way ANOVA with Holm–Šídák's multiple comparisons test, with single-pooled variance, where (n.s.) is not significant. (G) NDC80, SPC24, or SPC25 are required for protein synthesis at 72 h of siRNA depletion. (G, top) Representative Western blot images. Cells were treated with indicated siRNAs for 48 or 72 h. siRPL4 is the positive control and siNT (nontargeting) is the negative control. The growth medium was supplemented with puromycin (final concentration: 1 µM) for 1 h before lysing cells and harvesting protein. Twenty-five µg of protein was run on a 10% SDS–PAGE gel. Nascent proteins were detected using an α-puromycin antibody and normalized to total protein signal. (G, bottom) Quantitation of puromycin levels normalized to total protein levels and reported relative to siNT (N = 3). Bars represent the mean of biological replicates ± SEM. Data were analyzed by ordinary one-way ANOVA with Holm–Šídák's multiple comparisons test, with single-pooled variance, where (****) P ≤ 0.0001 and (***) P ≤ 0.0005. (H) Table summarizing results for siRNA depletion of NUF2 (Figures D, 2 and 3), NDC80, SPC24, or SPC25 (Figure 4). Cells colored black have a P value P ≤ 0.0001. Cells colored dark gray have a P value ≤ 0.0005. Cells colored medium gray have a P value P ≤ 0.005. Cells colored light gray have a P value ≤ 0.05. Uncolored cells are not significantly different. (I) siRNA depletion of NDC80, SPC24, or SPC25 reduces the levels of the NUF2 protein. (I, left) Representative Western blot images. Cells were treated with the indicated siRNAs for 24, 48, or 72 h before being lysed to extract their proteins. Twenty-five μ g of protein was run on a 10% SDS–PAGE. NUF2 protein was detected at 54 kDa using an α-NUF2 antibody. (I, right) Quantitation of NUF2 band intensities was normalized to GAPDH. Bars represent the mean of three biological replicates ± SEM.

Given that other NDC80 complex proteins regulate the RB pathway similarly to the observed effects with siNUF2, we postulated that depleting them would also lead to a decrease in global protein synthesis. To test this, we used puromycin incorporation to assay protein synthesis (Schmidt et al., 2009) on cells depleted of NDC80, SPC24, or SPC25 for either 48 or 72 h. After 48 h of treatment with siNDC80, there was a similar reduction in protein synthesis as was observed in cells treated with siRPL4, the positive control, or siNUF2, to ∼50% (Figure 4G). Notably, at this same time point, there were no significant changes in protein synthesis detected in cells treated with siSPC24 or siSPC25. We did, however, observe reduced protein synthesis when any of the three (NDC80, SPC24, and SPC25) were depleted for 72 h, resulting in about a 50% decrease in protein synthesis relative to siNT-treated cells. Taken together, the four kinetochore proteins NUF2, NDC80, SPC24, and SPC25 are required for optimal pre-rRNA transcriptional and protein synthesis, though with some variability in the extent (summarized in Figure 4H).

Dependence of NUF2 levels on the presence of the NDC80, SPC24, and SPC25 kinetochore proteins

Prior studies have found that the protein levels of the four kinetochore proteins, NUF2, NDC80, SPC24, and SPC25 are interdependent (Kim et al., 2024). We tested whether NUF2 levels were dependent on the presence of NDC80, SPC24, and SPC25 by depleting each of them using siRNA in MCF10A cells. We assessed NUF2 levels via Western blot at 24, 48, and 72 h after depletion. We found that depletion of each protein results in reduced NUF2 levels beginning at 24 h and becoming more pronounced at 48 and 72 h when compared with the negative control (Figure 4I; siNT, nontargeting). NUF2 protein levels are therefore, dependent on the presence of usual levels of its kinetochore binding partners, NDC80, SPC24, and SPC25 in this cell line.

Defects in ribosome biogenesis upon depletion of NUF2 or NDC80 trigger stabilization of TP53 via the NSP

Because we have established a role for NUF2 and its complex members in RB, we hypothesized that its depletion would activate the NSP. Under normal growth conditions, levels of the TP53 protein are regulated by the E3 ubiquitin ligase, MDM2. MDM2 ubiquitinates TP53, targeting it for degradation (Figure 5A, top). When ribosome biogenesis is interrupted, the 5S ribonucleoprotein (RNP), composed of RPL5, RPL11, and the 5S rRNA, binds and sequesters MDM2 leading to the stabilization of TP53 (Figure 5B, bottom) (Rubbi and Milner, 2003; Bywater et al., 2012; Donati et al., 2013; Sloan et al., 2013; James et al., 2014; Hannan et al., 2022). Thus, an increase in TP53 protein levels is one of the measures of the NSP. Because the TP53 protein activates CDKN1A (p21) transcription, mRNA levels of CDKN1A are a downstream measure of the NSP (Boisvert et al., 2007; James et al., 2014; McCool et al., 2023a). CDKN1A is a regulator of the cell cycle whose increased synthesis is associated with cell cycle arrest and apoptosis.

FIGURE 5: — siRNA depletion of NUF2 or NDC80 activates the NSP in MCF10A cells. (A, top) Schematic of TP53 regulation by MDM2 under normal conditions. (A, bottom) Schematic of TP53 accumulation upon nucleolar stress and MDM2 sequestration by the 5S RNP, a complex that includes RPL5, RPL11, and the 5S rRNA. (B) siRNA depletion of NUF2 depletion increases TP53 protein levels. (B, left) Representative Western blot images. Cells were treated with the indicated siRNAs for 72 h. siRPL4 is the positive control and siNT (nontargeting) is the negative control. Total protein was harvested and 25 μ g was run on a 10% SDS–PAGE. TP53 levels were detected with an α-TP53 antibody and normalized to the total protein signal. (B, right) Quantitation of TP53 levels normalized to total protein levels and reported relative to siNT (N = 3). Bars represent the mean of biological replicates SEM. Data were analyzed by ordinary one-way ANOVA with Holm–Šídák's multiple comparisons test, with single-pooled variance, where (****) P ≤ 0.0001. (C) *CDKN1A* (p21) levels are increased when NUF2 is depleted. qRT-PCR was performed on cDNA synthesized from total RNA, which was harvested from cells treated with the indicated siRNAs for 72 h. Primers for *CDKN1A* (p21) were used for amplification and levels were normalized to a 7SL internal control. The 2^−ΔΔCt values of *CDKN1A* (p21) are reported relative to levels in negative control cells (siNT; nontargeting). siNOL11 is the positive control. Data were analyzed by ordinary one-way ANOVA with Holm–Šídák's multiple comparisons test, with single-pooled variance, where (****) P ≤ 0.0001. (D) Codepleting RPL5 prevents activation of the NSP when NUF2 or NDC80 are depleted by siRNA. (D, left) Representative Western blot images. Cells were treated with indicated siRNAs for 72 h. siRPL4 is the positive control and siNT (nontargeting) is the negative control. Total protein was harvested and 25 μ g was run on a 10% SDS–PAGE. TP53 levels were detected with an α-TP53 antibody and normalized to the total protein signal. (D, right) Quantitation of TP53 levels normalized to total protein levels where the log₂ of each value is reported relative to the log2 of siNT values (N = 3−4). Bars represent the mean of biological replicates ± SEM.

We tested whether NUF2 depletion would activate the NSP by measuring TP53 protein levels by Western blot. When comparing TP53 levels in cells depleted of NUF2 to levels in the negative control cells (siNT), we observed a significant increase of ∼75% (Figure 5B). This effect was higher than the 50% increase in TP53 levels in the positive control cells (siRPL4), which we expected to trigger the NSP with increased levels of TP53 (He et al., 2016) (Figure 5B). We hypothesized that there would be a concomitant increase in CDKN1A transcript levels in NUF2-depleted cells which we tested using qRT-PCR. Indeed, we observed a significant increase in CDKN1A transcript levels in MCF10A cells treated with siNUF2 and the positive control, siNOL11, both yielding 2^-ΔΔCt values ∼8-fold higher than in siNT-treated cells (Figure 5C).

Based on the mechanism that underlies the NSP, the elevated TP53 protein levels rely on an abundance of the 5S RNP (5S, RPL5, and RPL11) (Rubbi and Milner, 2003; Bywater et al., 2012; Sloan et al., 2013; James et al., 2014; Hannan et al., 2022). We investigated the degree to which we could lower TP53 levels in cells lacking NUF2 or its dimer partner, NDC80, by inhibiting the NSP through RPL5 codepletion (Hannan et al., 2022). Indeed, while TP53 protein levels increased after treatment with either siNUF2 or siNDC80, TP53 levels were reduced by cotransfection of siRPL5 (Figure 5D). Together, these data suggest that depleting NUF2 and NDC80 both trigger elevated TP53 levels through the NSP.

Lack of correlation between the presence of one nucleolus and micronuclei upon knockdown of members of the NDC80 complex

NUF2 has a well-defined role within the kinetochore as part of the NDC80 complex, which connects kinetochore microtubules to the chromosome, and thus is necessary for faithful chromosome segregation (McKinley and Cheeseman, 2016; Musacchio and Desai, 2017; Hara and Fukagawa, 2020). Chromosome missegregation can lead to mitotic arrest, aneuploidy, and cell death (Santaguida and Amon, 2015; Mazzagatti et al., 2024). Missegregation events can also lead to the formation of micronuclei (MN), which are smaller nuclei (1/100^th–1/3^rd the size of the primary nucleus [PN]) formed around the DNA from a lagging chromosome or parts of chromosomes (Santaguida and Amon, 2015; Guscott et al., 2022; Di Bona and Bakhoum, 2024). The presence of MN can therefore be used as a marker of chromosome missegregation events (Hoffelder et al., 2004; Thompson and Compton, 2011; Huang et al., 2012; Fonseca et al., 2019; Cohen-Sharir et al., 2021). Due to defective nuclear envelope assembly, MN rupture can exacerbate genome instability (Kwon et al., 2020; Maciejowski and Hatch, 2020).

While cell cycle distribution of cervical cancer–derived HeLa cells depleted of NUF2 by siRNA has previously been determined (Deluca et al., 2002), the effect of NUF2 depletion on the cell cycle has not yet been explored in the near-normal breast epithelial cell line, MCF10A. We performed flow cytometry on MCF10A cells depleted of NUF2 or NDC80 for 72 h by siRNA and measured their DNA content. We compared them to cells treated similarly with siNT (negative control) or the microtubule-destabilizing drug, nocodazole (positive control; 24 h). NUF2-depleted cells (siNUF2; Figure 6, C and E) revealed 45% cells with a 2N DNA content (G1) and 25% with a 4N DNA content (G2/M). This is a shift in the percentage of cells from G1 to G2/M when compared with the negative control siNT (∼70% 2N; ∼6% 4N; Figure 6, A and E). As expected, the nocodazole treated MCF10A cells revealed ∼50% of cells with a 4N DNA content, consistent with mitotic arrest (Figure 6, B and E). Surprisingly, we observed only small changes in cell cycle distribution when cells were siRNA depleted of NUF2’s binding partner, NDC80 (siNDC80; ∼70% 2N; ∼11% 4N; Figure 6, D and E). Our results are therefore similar to what has been observed previously for NUF2 depletion in HeLa cells (Deluca et al., 2002), with an increase in the percentage of cells in G2/M in MCF10A cells siRNA depleted of NUF2.

FIGURE 6: — NUF2 and NDC80 are required for progression through mitosis in MCF10A cells. (A–D) Flow cytometry to measure the DNA content of MCF10A cells siRNA depleted of NUF2 or NDC80 reveals that their depletion leads to accumulation in mitosis. FlowJo analysis classified cells by DNA content, with 2N representing cells in G1 that have not yet replicated their DNA. 4N represents cells in the G2/M phase that have twice the amount of DNA. (A) Results from cell cycle analysis by flow cytometry. DNA content of cells treated with siNT (nontargeting) for 72 h and 1% DMSO overnight as a negative control and as a vehicle control. (B) Results from cell cycle analysis by flow cytometry for the positive control cells. The DNA content of cells that were treated with siNT (nontargeting) for 72 h, and then supplemented with nocodazole at a concentration of 100 ng/ml for the last overnight incubation. (C and D) DNA contents of MCF10A cells treated with siRNAs targeting NUF2 or NDC80 for 72 h before fixing and staining for flow cytometry. (E) Bar graph comparison of the cell cycle analysis described in A. Bars are split according to the percentage of cells in each phase of the cell cycle. (F) Depleting kinetochore components NUF2, NDC80, SPC24, and SPC25 leads to MN formation in MCF10A cells. Cells were treated with the indicated siRNAs for 24, 48, and 72 h and subsequently fixed and stained with Hoechst to detect primary nuclei and associated micronuclei. The percentage of micronuclei containing cells (primary nuclei that had an associated micronuclei) were reported as a percentage of total cells and represented by bars of different shades at the 24, 48, and 72 h time points ± SEM; N = over 200 cells per condition per replicate, three biological replicates. (G) siRNA depletion of NUF2 and NDC80 increases the percentage of cells with one nucleolus but is not dependent on micronucleus formation. Cells were treated with the indicated siRNAs for 24, 48, and 72 h before being fixed and costained with Hoechst to detect PN and associated micronuclei (MN), and anti-UBTF to detect nucleoli. Cells were binned into MN positive (MN+) and negative (MN−), and the number of UBTF foci was subsequently quantitated in the PN of these populations. The percentage of PN containing only one nucleolus was quantitated. Bars of different shades of gray represent 24, 48, and 72 h after depletion ± SEM; N = over 100 cells per condition per replicate, three biological replicates.

We reasoned that failed segregation of sister chromatids during anaphase could lead to changes in gene expression of the rDNA and therefore to defective RB. We sought to determine whether there was a correlation between chromosome missegregation and the presence of an increased percentage of cells with one nucleolus in NDC80 complex-depleted MCF10A cells. We used the presence of MN as a marker of chromosome missegregation, scoring the percentage of MN-bearing cells by immunofluorescence microscopy. As expected, we observed an increase in the percentage of MN-bearing cells after NUF2, NDC80, SPC24, and SPC25 depletion compared with the nontargeting siRNA control (siNT), even as early as 24 h (Figure 6F). However, there was overall slightly greater MN formation in the NUF2/NDC80 versus the SPC24/SPC25-depleted cells (∼20 and 10%, respectively), and therefore slightly greater chromosome missegregation when either NUF2 or NDC80 were depleted.

We further sought to quantify the percentage of primary nuclei with one nucleolus in cells with and without MN after NDC80-complex depletion over 72 h in MCF10A cells. We expected that cells without MN (MN−) did not undergo a chromosome missegregation error; likewise, we expected that cells with MN (MN+) did have a missegregation error. We observed a significant increase in the percentage of cells that contained one nucleolus in either NUF2 or NDC80-depleted cells compared with siNT beginning at 48 h (Figure 6G). In contrast, siRNA depletion of either SPC24 or SPC25 led to a less drastic increase in the percentage of cells with one nucleolus even after 72 h of depletion (Figure 6G). Strikingly, in all conditions, irrespective of the presence of MN, we observed a similar percentage of cells with one nucleolus (Figure 6G). Taken together, these results demonstrate a lack of correlation between the increased percentage of cells with one nucleolus and the presence of MN upon siRNA depletion of the NDC80 subcomplex members. Together, they suggest that the effects of NDC80 subcomplex depletion on RB are not strictly correlated with chromosome missegregation.

DISCUSSION

Here we report a novel role for the NUF2 protein and its kinetochore partners, NDC80, SPC24, and SPC25, in RB in human cells. NUF2 was originally identified as a hit in our genome-wide RNAi screen for nucleolar function (Farley-Barnes et al., 2018). We validated it as a hit by re-screening with siONT pools and testing their deconvolution. Three biochemical assays (5-EU incorporation, rDNA promoter-reporter gene, and 47S/45S qRT-PCR) revealed that NUF2 is required in the RB pathway for RNAPI transcription. This is due to decreased levels of proteins essential for RNAPI transcription, including POLR1A, RRN3, and FBL. As is expected for a positive regulator of rDNA transcription, cells depleted of NUF2 demonstrated reduced protein synthesis, likely due to reduced levels of ribosomes. Similarly, although they were not hits in our screen, NUF2 kinetochore protein partners NDC80, SPC24, and SPC25 were also required for transcription of the 47S/45S primary pre-rRNA transcript. Furthermore, their siRNA depletion led to reduced levels of POLR1A, RRN3, and protein synthesis. siRNA depletion of both NUF2 and its interacting protein, NDC80, caused an increase in TP53 protein and CDKN1A mRNA levels, hallmarks of the NSP. Codepletion of RPL5 restored TP53 levels, indicating that the increased TP53 protein was caused by an increase in free 5S RNP. Taken together, our results point to a connection between kinetochore proteins, known to be involved in the mechanics of cell division during mitosis, and the interphase function of making ribosomes in the cell nucleolus.

We hypothesize that NUF2 and other kinetochore proteins may exert their positive effect on RB in the nucleolus through their known role in mitosis. The nucleolus in metazoans dissembles early in mitosis, and RB is halted (Güttinger et al., 2009; Hernandez-Verdun, 2011; Delgado-Román and Muñoz-Centeno, 2021; Pham et al., 2024). Upon nucleolar reassembly at the end of mitosis, pre-rRNA transcription and RB then resume in interphase. We postulate that reduced nucleolar re-assembly and nucleolar function in NUF2-depleted cells occur because of defective mitosis. So far there is no evidence of a direct effect of NUF2 on the interphase nucleolus in multiple extant datasets. NUF2 is not present in two different datasets of proteins from biochemically purified human nucleoli (Ahmad et al., 2009; Jarboui et al., 2011). NUF2 is not localized to the nucleolus in the experiments presented in the human protein atlas (proteinatlas.org) (Uhlén et al., 2015), and we have confirmed this by costaining for the nucleolar protein UBTF in interphase cells stained for NUF2 (Supplemental Figure S1). Results from the Biogrid further argue against a nucleolar localization for NUF2 because it does not coimmunoprecipitate nucleolar proteins (Oughtred et al., 2021). There is thus extensive evidence that NUF2 is not a nucleolar protein in multiple independently derived datasets and individual experiments. Our results, together with these results from others, support the conclusion that faithful mitosis is required for the resumption of RB in interphase with the start of pre-rRNA transcription.

While the two processes, mitosis, and RB, occur sequentially during the cell cycle and thus are usually considered to be distinct, there are emerging critical links between them that suggest cross-talk. Several nucleolar proteins have a defined role in RB in the interphase cell that also play a role in mitosis. Among these are the HCA66 protein (ortholog of S. cerevisiae Utp6), a protein subunit of the SSU processome (Dragon et al., 2002), that is independently required during mitosis for centriole duplication in human tissue culture cells (Bonnart et al., 2012). In the yeast S. cerevisiae, Utp7, another subunit of the SSU processome (Dragon et al., 2002), functions in kinetochores (Jwa et al., 2008). In addition, both the mature rRNAs and the pre-rRNAs cover the condensed chromosomes during mitosis, binding to the Ki-67 protein (Fan and Penman, 1971; Carron et al., 2012; Sirri et al., 2016; Ma et al., 2022). Similarly, in Drosophila and C. elegans, NUF2 and its kinetochore partners have been shown to play a postmitotic role in dendrite growth and extension (Cheerambathur et al., 2019; Hertzler et al., 2020). In addition to our results presented here, diverse connections between the kinetochore proteins and ribosome biogenesis and cell growth have been previously observed.

The budding yeast orthologues of NUF2, NDC80, and SPC25 have been previously connected to the nucleolus in a high-throughput screen with a different readout. Temperature-sensitive alleles of essential genes revealed that these three kinetochore proteins led to increased nucleolar size at the nonpermissive temperature (Neumüller et al., 2013). This is consistent with the effect of conditional alleles of other spindle pole body proteins in this particular screen. However, it is the opposite of what was seen with temperature-sensitive nucleolar proteins with a known function in RB, which led to reduced nucleolar size at the nonpermissive temperature. Still, the results show a clear connection between kinetochore proteins and nucleolar size. Perhaps the difference in the effect on the nucleolus between our results and theirs is because budding yeast has a closed mitosis, while the MCF10A tissue culture cells we used do not. Or perhaps kinetochore proteins have diverged between yeast to humans so that they no longer perform all the same functions, a possibility supported by their lack of complementation (Ólafsson et al., 2023).

Like with nucleolar proteins required for making ribosomes, we found that depletion of NUF2 in MCF10A cells caused stabilization of TP53 and increased transcription of CDKN1A (p21). We confirmed that this occurs via the NSP by codepleting RPL5, a protein component of the 5S RNP, which restored levels of TP53. A recent phenotypic unbiased screen for TP53 stabilization in A549 human lung adenocarcinoma cells revealed the same result and extended it to TP53 stabilization upon the depletion of the kinetochore proteins NDC80 and SKA1 (Hannan et al., 2022) (Supplemental Table S3). In contrast, the depletion of the SPC24 and SPC25 kinetochore proteins in this study did not significantly stabilize the levels of TP53 (Supplemental Table S3). One important conclusion from this screen was that the NSP is not only activated by depletion of nucleolar proteins, as might be expected. Depletion of a number of cell cycle proteins, including ones that are part of the mitotic machinery, also activates the NSP as we have found here for NUF2. The results from this powerful unbiased screen support our findings that there are unforeseen connections between proteins with a defined cell cycle function and the NSP.

Two variants in the NUF2 gene have recently been found to be likely pathogenic for a developmental disorder in two unrelated children. Interestingly, their clinical presentation is similar to that of some ribosomopathies, the human diseases of making ribosomes. One of them (p.Ile124Ser in one allele of NUF2) presented with microcephaly, short stature, and borderline cognitive development (Uehara et al., 2021). The other one (p.Leu303Arg in one allele of NUF2) presented with microcephaly, short stature, and bone marrow failure and died of respiratory infection at age 2.5 years (Vial et al., 2022). Probing the nature of the effects of each variant in patient's cells on cell division and viability, both were found to result in cell cycle defects, consistent with NUF2’s role in mitosis. Deficiencies in neurodevelopment like microcephaly and cognitive impairment are often also present with recessive ribosomopathies, like alopecia neuroendocrine syndrome (Nousbeck et al., 2008; Bryant et al., 2021), resulting from pathogenic variants in the nucleolar RBM28 protein. In contrast, ribososomopathies due to haploinsufficiency, like Diamond Blackfan anemia, present primarily with bone marrow failure and often result from deletion or mutation in ribosomal proteins (Liu and Karlsson, 2024; Wlodarski et al., 2024). Given our results, which indicate a role for NUF2 in RB, it will be critical to determine the extent to which expression of these two NUF2 variants also reduces pre-rRNA transcription. Whether they do, the underlying pathophysiology of their clinical presentation may also be due to defective RB, as we and others have suggested previously for some proteins implicated in Fanconi anemia (Sondalle et al., 2019; Gueiderikh et al., 2021).

MATERIALS AND METHODS

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Cell culture and media

MCF10A cells (ATCC CRL-10317) were subcultured no more than 14 times in DMEM/nutrient mixture F-12 (Gibco 11330032) containing horse serum (Gibco 16050130), 10 µg/ml insulin (Sigma I1882), 0.5 µg/ml hydrocortisone (Sigma H0135), 100 ng/ml cholera toxin (Sigma C8052), and 20 ng/ml epidermal growth factor (Peprotech AF-100-15). These cells were kept in an incubator with a controlled, humidified atmosphere at 37°C and 5% CO₂.

RNAi

siRNAs were purchased from Horizon Discovery Biosciences (Supplemental Tables S1 and S2). For the original screen (Farley-Barnes et al., 2018; Ogawa et al., 2021), siGENOME SMARTpool siRNAs were used. For the 5-EU secondary screen (Figure 2B), ON-TARGETplus pools (siONT) were used (Bryant et al., 2022). For the NOL11 positive control throughout we used the siGENOME SMARTpool siRNAs. For the oligonucleotide deconvolution validation of siNUF2 (Figure 1C), the four individual siONT RNAs that comprise the pool were used. An equimolar custom pool of these three siONT RNAs (si-1, si-3, and si-4) that passed the deconvolution validation was used to deplete NUF2 for the remaining functional assays. For the siNT−, siRPL4−, or siPOLR1A-treated cells, siONT pools were used.

siRNA transfection was performed using Lipofectamine RNAiMAX Transfection Reagent (Invitrogen 13778150) as described in (Ogawa et al., 2021). For the high throughput screens (Farley-Barnes et al., 2018; Bryant et al., 2022), ∼3000 MCF10A cells were reverse-transfected into 384-well plates on day 0. Cells were fixed and stained on day 3 posttransfection. For other functional assays, 6- or 12-well plates were seeded with ∼1 × 10⁵ or ∼5 × 10⁴ MCF10A cells, respectively, on day −1. Twenty-four hours later (day 0), cells were treated using the indicated siRNAs at a final concentration of 33 nM, unless otherwise stated. Cells were assayed on days 1, 2, or 3 posttransfection. For siONT deconvolution, the individual siRNAs passed validation whether they were able to significantly decrease nucleolar number, with a mean one-nucleolus percent effect greater than or equal to +3 SD above the siNT mean, using the siNT SD.

5-EU incorporation assay to quantitate nucleolar rRNA biogenesis

MCF10A cells were treated with siRNAs for 72 h as described above and supplemented with 1 mM 5-EU (Click Chemistry Tools 1261) for the last hour to allow nascent RNA to incorporate the uridine analogue. Cells were then washed, fixed, permeabilized, and blocked before nucleoli were stained using the 72B9 primary α-fibrillarin (FBL) antibody (Reimer et al., 1987) and DNA was stained with Hoechst 33342 Dye. The incorporation of 5-EU in the nucleolus was visualized and measured after the click-chemistry addition of a fluorophore analogue labeling nascent transcripts. See (Bryant et al., 2022) for the detailed protocol.

qRT-PCR analysis

RNA was harvested using TRIzol (Life Technologies 5596018) per the manufacturer and precipitated in 75% ethyl alcohol. Concentration and purity of RNA were measured with NanoDrop (Thermo Fisher Scientific, ND2000CLAPTOP); all samples had an A_260/230 value above 1.7. cDNA preparation was performed with 1 µg of RNA using the iScript gDNA Clear cDNA Synthesis Kit (Bio-Rad 1725035), and qPCR was performed using iTaq Universal SYBR Green Supermix (Bio-Rad 1725121). We used primers for NUF2 (5′-AAC GAT AGT GCT GCA AGA GG-3′; 5′-TCT CCA CCA CTT CTT GTC TG-3′) to amplify and detect mRNA levels. To measure pre-rRNA transcript levels, we used primers against both the 47S and 45S pre-rRNAs (5′-GAA CGG TGG TGT GTC GTT C-3′; 5′-CGT CTC GTC TCG TCT CAC TC-3′). For both experiments, we also used primers against 7SL RNA (5′-ATC GGG TGT CCG CAC TAA GTT-3′; 5′-CAG CAA CGG GAG TTT TGA CCT-3′) as an internal control to which the NUF2 mRNA or 47/45S transcript levels were normalized. Normalized transcript levels from experimental samples are reported relative to mRNA levels observed in the nontargeting (siNT) negative control sample of the same experiment. Forty rounds of amplification were completed after 30 s at 95°C for initial denaturation. The amplification parameters were 95°C for 15 s and 60°C for 30 s, followed by melt-curve analysis to ensure a single product was amplified, with parameters as follows: 95°C for 15 s then gradual (0.3°C/15 s) increase from 65°C to 94°C. The ^ΔΔC_T method was used to quantitate the amplification to compare NUF2 mRNA levels or 47S/45S rRNA levels across samples.

Western blots

After 72 h of siRNA depletion, cells were scraped from 6-well plates and lysed in AZ lysis buffer (50 mM Tris pH 7.5, 250 mM NaCl, 1% Igepal, 0.1% SDS, 5 mM EDTA pH 8.0 with 1X complete protease inhibitors (cOmplete Protease Inhibitor Cocktail Roche 11697498001). Total protein was harvested and prepared as in (Farley-Barnes et al., 2018). The Bradford assay was used to quantitate total protein from each sample and 25 µg of total protein was separated by SDS–PAGE containing 0.5% trichloroethanol (Acros Organics 139441000) for stain-free imaging and transferred to a polyvinylidene difluoride (PVDF) membrane (Bio-Rad 1620177). After transfer, membranes were blocked in 5% milk for 1 h rocking at room temperature and incubated overnight with the specified primary antibodies in 5% milk at 4°C. Total protein was measured using the Bio-Rad ChemiDoc stainfree imaging protocol. Proteins were detected with the following antibodies: α-POLR1A (dilution 1:1000; Santa Cruz Biotechnology sc-48385), α-p53 (dilution 1:5000; Santa Cruz Biotechnology sc-126), α-NUF2 (dilution 1:1000; Abcam ab176556), α-NDC80 (1:1000 Abcam ab9G3), α-SPC24 (dilution 1:1000; Novus Biologicals NBP2-47264), α-UBTF (dilution 1:500; Santa Cruz Biotechnology sc-13125), α-RRN3 (dilution 1:1000; Proteintech 25918-1-AP), α-MYC (1:1000 dilution; Invitrogen MA1-880), and α-FBL (1:1000 dilution; Invitrogen MA3-16771). The Western blots were developed with enhanced chemiluminescence reagents (Thermo Fisher Scientific 34096). Images were acquired by digital imaging using the Bio-Rad ChemiDoc Imaging System. Quantitation of protein levels was done using ImageJ software to measure the integrated intensity of the band corresponding to the protein of interest's molecular weight. Band intensity was normalized to the integrated intensity of the corresponding lane's total protein. For POLR1A, both bands were quantified.

Dual-luciferase assay for RNAPI activity at the rDNA promoter

MCF10A cells were treated with siRNAs as described above. Forty-eight hours after siRNAs were added, cells were cotransfected with 1000 ng of pHrD-IRES-Luc (Ghoshal et al., 2004) and 0.1 ng of a Renilla internal control plasmid using Lipofectamine 3000 (Thermo Fisher Scientific L3000015). For the positive control, cells were treated with the small molecule BMH-21, which inhibits RNAPI transcription, at 48 h after siRNA transfection. Twenty-four hours after the addition of plasmids and BMH-21, cells were harvested and luminescence was measured using the Dual-luciferase Reporter Assay System (Promega E1910) following the manufacturer's instructions with a GloMax 20/20 luminometer (Promega). The ratio of pHrD-IRES-luciferase/Renilla activity was calculated (control for transfection efficiency) and displayed relative to the nontargeting control (siNT).

Protein synthesis

Puromycin labeling of nascent polypeptide chains as in (Schmidt et al., 2009) was performed to assess global protein synthesis. Total cellular extracts, treated as described, were analyzed by Western blot using an α-puromycin antibody (Kerafast EQ0001) at a 1:10,000 dilution. Quantitation of puromycin-conjugated proteins was done using ImageJ software (Schindelin et al., 2012)to measure the integrated intensity of each lane. The puromycin signal was normalized to the integrated intensity of the corresponding lane's total protein.

Cell cycle analysis

MCF10A cells were treated with siRNAs for 72 h as described above. Medium was supplemented with nocodazole (100 ng/ml) or DMSO (vehicle; 1% final concentration) for the final 24 h. Cells were fixed using 4% paraformaldehyde and their DNA stained with propidium iodide. The DNA content of the stained cells was measured by the Cytoflex LX flow cytometer. Results from the flow cytometer were analyzed by FlowJo v10.8 Software (BD Life Sciences), which grouped cells into the phases of the cell cycle based on their DNA content. This experiment and analyses were performed with assistance from Yale's Flow Cytometry Facility.

Immunofluorescence for micronuclei (MN)

For quantitative assessments of MN frequency and nuclei number in MN positive or negative cells, unsynchronized cells growing on glass coverslips were treated with the indicated siRNAs for 24, 48, and 72 h and subsequently fixed with 3.5% paraformaldehyde for 15 min, washed with PBS + 0.5% Triton X-100 for 5 min, and washed with PBS + 1% BSA for 10 min. Antibodies were diluted in PBS + 1% BSA, and cells on coverslips were incubated with anti-UBTF antibody at a dilution of 1:1000 (Santa Cruz Biotechnology sc-13125) for 12–16 h at 4°C. Cells were then washed with PBS for 5 min. The secondary antibody (Alexa Fluor 488 anti-Mouse; Jackson ImmunoResearch 715-545-150) was diluted in PBS + 1% BSA, with DAPI (1:5000; Thermo Fisher Scientific 62248), and cells were incubated with secondary antibodies for 1 h at room temperature (∼25°C). Images were acquired on a Nikon Ti2 system with Nikon Large FOV Monochrome CMOS Camera. Images were analyzed using ImageJ (Schindelin et al., 2012). For quantitative analysis of MN frequency, MN were defined as discrete DNA aggregates separate from the PN, but within 5 µm of the PN, in cells where interphase primary nuclear morphology was normal. Cells with an apoptotic appearance or containing more than four MN were excluded. For quantitative analysis of the number of nucleoli in primary nuclei, primary nuclei were binned as having an associated micronucleus (MN+) or not (MN−) as described above. Distinct and nonoverlapping UBTF foci were counted in each PN.

NUF2 subcellular localization (Supplemental Figure S1)

For staining, fixation, and imaging of mitotic NUF2, UBTF, and ACA, MCF10A cells were grown onto glass coverslips until ∼70% confluent. The medium was carefully removed, and cells were washed with room temperature PBS. Pre-extraction buffer (0.5% Triton X-100 diluted in cytoskeleton buffer (20 mM HEPES, 100 mM NaCl, 300 mM sucrose, and 3 mM MgCl₂ at pH 7.4) was added for 10 min. Afterward, the cells were washed once with PBS gently and fixed using 3.5% paraformaldehyde as described for “Immunofluorescence for MN.”

To quantify the abundance of NUF2 signal that was present in the nucleolus, nucleus or cytosol (Supplemental Figure S1D), we used ImageJ (Schindelin et al., 2012) to measure a small circle that was ∼2 µM in diameter and measured NUF2 signal at one location that overlapped with UBTF signal (termed nucleolus), one location that overlapped with DAPI signal but did not overlap with UBTF signal (termed nucleus) and one location that did not overlap with either DAPI nor UBTF signal and was adjacent to DAPI signal (termed cytosol). The ratio of these three measurements was calculated per cell.

For (Supplemental Figure S1E), the images were analyzed using the Plot Profile function on ImageJ. The signal intensity for all three channels was overlayed using the same line.

Supplementary Material

mbc-36-ar16-s001.pdf^{(1.9MB, pdf)}

ACKNOWLEDGMENTS

We thank Yulia Surovtseva and Laura Abriola at the Yale Center for Molecular Discovery for their expertise in high throughput screening and for the use of their screening facilities. We thank Emily Sutton PhD in the Baserga laboratory for careful reading of the manuscript. This work was supported by the following grants from the National Institutes of Health (NIH): R35GM131687 (S.J.B.) and R35GM150648 (L.K.).

Abbreviations used:

5EU: 5 ethynyl uridine
FBL: fibrillarin
MN: micronuclei
siNT: nontargeting siRNA
NUF2: nuclear filamentous 2
NSP: nucleolar stress pathway
PVDF: Polyvinylidene difluoride
PN: primary nucleus
RB: ribosome biogenesis
RNAPI: RNA polymerase I
rRNA: ribosomal RNA
SDS‐PAGE: SDS polyacrylamide gel electrophoresis
siONT: siON‐TARGETplus SMARTpool siRNAs
SEM: standard error of the mean.

Footnotes

This article was published online ahead of print in MBoC in Press (http://www.molbiolcell.org/cgi/doi/10.1091/mbc.E24-08-0337) on December 20, 2024.

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PERMALINK

A role for the kinetochore protein, NUF2, in ribosome biogenesis

ty j brown

Jennifer Pichurin

Carlos Ramirez Parrado

Lilian Kabeche

Susan J Baserga

Roles

Abstract

INTRODUCTION

RESULTS

NUF2 is required for maintenance of typical nucleolar number and viability in MCF10A cells

FIGURE 1:

NUF2 is required for pre-rRNA transcription

FIGURE 2:

NUF2 depletion decreases the levels of the RNAPI transcription machinery

FIGURE 3:

NUF2 is required for global protein synthesis

Depletion of other subunits of the kinetochore complex, NDC80, SPC24, and SPC25, also reduces pre-rRNA transcription

NDC80, SPC24, and SPC25 are all required for maintaining normal levels of the RNAPI transcription apparatus as well as for global protein synthesis

FIGURE 4:

Dependence of NUF2 levels on the presence of the NDC80, SPC24, and SPC25 kinetochore proteins

Defects in ribosome biogenesis upon depletion of NUF2 or NDC80 trigger stabilization of TP53 via the NSP

FIGURE 5:

Lack of correlation between the presence of one nucleolus and micronuclei upon knockdown of members of the NDC80 complex

FIGURE 6:

DISCUSSION

MATERIALS AND METHODS

Cell culture and media

RNAi

5-EU incorporation assay to quantitate nucleolar rRNA biogenesis

qRT-PCR analysis

Western blots

Dual-luciferase assay for RNAPI activity at the rDNA promoter

Protein synthesis

Cell cycle analysis

Immunofluorescence for micronuclei (MN)

NUF2 subcellular localization (Supplemental Figure S1)

Supplementary Material

ACKNOWLEDGMENTS

Abbreviations used:

Footnotes

REFERENCES

Reviewer Report

Associated Data

Supplementary Materials

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