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A role for the C. elegans Argonaute protein CSR-1 in small nuclear RNA 3’ processing [1]

['Brandon M. Waddell', 'Department Of Veterinary Biomedical Sciences', 'Western College Of Veterinary Medicine', 'University Of Saskatchewan', 'Saskatoon', 'Saskatchewan', 'Cheng-Wei Wu', 'Toxicology Centre', 'Department Of Biochemistry', 'Microbiology']

Date: 2024-05

The Integrator is a multi-subunit protein complex that catalyzes the maturation of snRNA transcripts via 3’ cleavage, a step required for snRNA incorporation with snRNP for spliceosome biogenesis. Here we developed a GFP based in vivo snRNA misprocessing reporter as a readout of Integrator function and performed a genome-wide RNAi screen for Integrator regulators. We found that loss of the Argonaute encoding csr-1 gene resulted in widespread 3’ misprocessing of snRNA transcripts that is accompanied by a significant increase in alternative splicing. Loss of the csr-1 gene down-regulates the germline expression of Integrator subunits 4 and 6 and is accompanied by a reduced protein translation efficiency of multiple Integrator catalytic and non-catalytic subunits. Through isoform and motif mutant analysis, we determined that CSR-1’s effect on snRNA processing is dependent on its catalytic slicer activity but does not involve the CSR-1a isoform. Moreover, mRNA-sequencing revealed high similarity in the transcriptome profile between csr-1 and Integrator subunit knockdown via RNAi. Together, our findings reveal CSR-1 as a new regulator of the Integrator complex and implicate a novel role of this Argonaute protein in snRNA 3’ processing.

Small nuclear RNA molecules are an important structural component of the spliceosome, which is the cellular machinery that performs RNA splicing. Proper RNA splicing is important for ensuring proteins are accurately produced within the cell, and this process is broadly important for contributing to basic physiological processes such as development. Inside the cell, small nuclear RNA is processed by a protein complex called the Integrator, and mutations to the Integrator affect RNA splicing and can lead to neurodevelopmental defects. Here, we used the roundworm Caenorhabditis elegans to describe how an Argonaute encoding gene called csr-1 is required for maintaining Integrator protein expression and that loss of csr-1 gene expression contributes to the dysregulation of small nuclear RNA processing. These results provide new insights into our understanding of fundamental factors that regulate small nuclear RNA processing in cells, which are directly important in RNA splicing control.

In this study, we utilize the genetic model C. elegans to develop a GFP based in vivo snRNA misprocessing reporter as a readout for Integrator malfunction and performed a genome-wide RNAi screen to identify potential Integrator regulators. We identified a novel role for the csr-1 (Chromsome-Segregation and RNAi deficient) gene encoding an essential Argonaute protein as a regulator of the Integrator complex in snRNA processing [ 20 ]. CSR-1 is well characterized for its core role in the protection of germline gene expression, this is achieved through a tethered interaction with target transcripts that is mediated by interfacing with small RNAs (22G-RNAs) that are antisense to the targeted gene [ 21 , 22 ]. More recently, a role for CSR-1 in the embryo cleavage of maternal mRNAs to facilitate clearance and removal have also been demonstrated [ 23 ]. Deletion of csr-1 results in sterility that is accompanied by loss of P-granule formation, defects in chromosome segregation, and mis-expression of replication dependent histone proteins [ 20 , 24 ]. Here, we show that loss of csr-1 results in an aberrant increase in snRNA 3’ misprocessing and the alternative splicing of ~400 transcripts across the transcriptome. Mechanistically, our results show that loss of csr-1 down-regulate expression of Integrator subunit proteins in the germline that is supported by Ribo-Seq analysis indicating a reduced translation efficiency of Integrator subunits functioning in both the catalytic and non-catalytic domains. Together, this study provides new insights into csr-1 as a regulator of the Integrator complex that can influence snRNA 3’ processing in C. elegans.

Beyond snRNA 3’ processing, recent evidence has elucidated a broader role for the Integrator in contributing to transcriptional homeostasis; these functions include the 3’ processing of non-coding Piwi-interacting RNAs as well as cleavage of nascent mRNAs at RNA polymerase II paused sites to facilitate either gene transcription activation or repression [ 12 – 14 ]. Phenotypically, human mutations to the Integrator complex have been linked to severe neurodevelopmental syndrome and developmental ciliopathies resulting in oral-facial digital syndromes [ 9 , 15 , 16 ]. Analysis of the Cancer Genome Atlas has also revealed an increase in non-synonymous mutations to the Integrator subunits in primary tumour samples [ 13 , 17 ]. In model organisms, the knockdown of Integrator causes developmental arrest and results in a shortened lifespan of C. elegans, and depletion of Integrator in mouse results in cortical neuron migration defects leading to neurological disorders [ 11 , 18 , 19 ]. To date, while the core functions of the Integrator are well developed, regulators of the Integrator complex itself are less understood. Identifying mechanisms of Integrator regulation is of interest given its diverse influence on the transcriptome and its emergence in various human diseases.

Eukaryotic RNA splicing is catalyzed by the spliceosome that removes noncoding intron segments from pre-mRNA transcripts to produce a mature mRNA for protein translation [ 1 ]. A core component of the spliceosome is the uridylate-rich small nuclear RNA (snRNA) molecules U1, U2, U4, U5, and U6 that are incorporated within small nuclear ribonucleoprotein (snRNP) complexes that serve to facilitate splice site recognition for intron removal [ 1 , 2 ]. The biosynthesis of snRNA transcripts begins with transcription by RNA polymerase II to yield a pre-snRNA transcript with an extended 3’ precursor [ 3 , 4 ]. Post transcription, the pre-snRNA transcripts are processed and cleaved by the Integrator complex at the 3’ end to yield mature snRNA transcripts that are then incorporated with snRNP towards spliceosome biogenesis [ 5 , 6 ]. The Integrator is a metazoan conserved protein complex that is composed of at least 15 distinct subunits in humans and was discovered in 2005 as the elusive molecular machinery for snRNA 3’ processing [ 6 , 7 ]. The Integrator catalytic module includes subunits 4, 9, and 11 that are directly involved in snRNA cleavage, while the functions of non-catalytic subunits are not well defined [ 8 , 9 ]. Knockdown of both catalytic and non-catalytic subunits of the Integrator leads to RNA polymerase II termination failure and 3’ misprocessing of the snRNA transcripts. This results in transcriptional read-through errors that can lead to the aberrant polyadenylation of snRNA, or the synthesis of a long chimeric RNA that is composed of the snRNA transcript and its unprocessed 3’ end tethered to its downstream mRNA gene [ 10 , 11 ].

Results

Identification of novel snRNA processing regulators The Integrator complex serves as the principle regulator of snRNA processing in eukaryotes that catalyzes 3’ post-transcriptional cleavage required for snRNA maturation [6]. Disruption of the Integrator complex has been shown to impair C. elegans development, and can mimic a transcriptome profile similar to cadmium exposure [11,18]. To identify novel regulators of the Integrator or snRNA processing, we developed a visual biomarker of snRNA misprocessing in C. elegans by adapting the strategy previously employed in the Drosophila S2 cells [25]. We chose to design the snRNA misprocessing reporter using the C47F8.9 transcript encoding the U2 snRNA as we previously showed that the knockdown of Integrator subunits by RNAi results in the misprocessing and increased aberrant polyadenylation of this transcript [18]. A PCR amplified genomic fragment of C47F8.9 containing the promoter, transcript, and a potential 3’ motif for cleavage recognition was cloned in frame with GFP (Fig 1A). C. elegans lack a conserved 3’ box sequence 9–19 nucleotides downstream of the coding region that is found in other metazoans serving as a cleavage signal for the Integrator [5,26]. As such, we cloned approximately 75 base pairs downstream of the C47F8.9 transcript which contains a potential 3’ motif that is conserved across U2 snRNA transcripts. Under normal conditions, the GFP signal is absent as snRNA transcripts are cleaved by the Integrator complex resulting in the loss of GFP transcript; however, RNAi knockdown of ints-4 encoding a catalytic subunit of the Integrator results in transcriptional read-through that strongly activates GFP expression (Fig 1B). PPT PowerPoint slide

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TIFF original image Download: Fig 1. Genome-wide screening of snRNA 3’ processing factors. a) Schematic of U2 snRNA misprocessing reporter. An 883bp fragment containing the U2 snRNA (C47F8.9 gene), transcript, and 3’ sequence was cloned and fused to GFP and stably integrated into the C. elegans genome to create an in vivo snRNA misprocessing reporter. b) Representative fluorescent micrograph and brightfield image showing basal expression of the snRNA misprocessing reporter and GFP activation after Integrator disruption via ints-4 (RNAi). The yellow signal near the pharynx represents the myo-3p::tdTomato co-injection makers. c) Outline of the genome-wide RNAi screen to identify gene knockdowns that activate the snRNA misprocessing reporter. d) Enrichment analysis of 47 genes that when knocked down activate the snRNA misprocessing reporter. e) Representative fluorescent micrograph of the snRNA misprocessing reporter fed with EV, csr-1, npp-1, or npp-6 RNAi. The scale bar is 100 μm. f) Primer design to measure total and misprocessed transcripts of the U2 snRNA. g) Relative levels of misprocessed and total U2 snRNA in worms fed with EV, csr-1, npp-1, npp-6 as determined via qPCR. *P<0.05, **P<0.01 as determined by student t-test. https://doi.org/10.1371/journal.pgen.1011284.g001 Next, we performed a genome-wide RNAi screen to identify genes that when knocked down via RNAi result in the activation of the snRNA misprocessing reporter. We screened ~19,000 genes and verified 47 genes that when silenced via RNAi result in GFP activation (Fig 1C). These include genes encoding additional subunits of the Integrator, those involved in nuclear membrane and transport, and regulators of siRNA processing machinery (Fig 1D and S1 Table). We focused on 3 genes that showed the strongest GFP activation that did not encode a known subunit of the Integrator complex, which were csr-1, npp-1 (Nuclear Pore complex Protein), and npp-6 (Fig 1E). To verify that the increase in GFP reporter fluorescence reflects the misprocessing of the endogenous snRNA transcript, we designed a pair of primers that measure the total and misprocessed levels of C47F8.9 (Fig 1F). We then knocked down csr-1, npp-1, and npp-6 in N2 wildtype worms via RNAi and found that the knockdown of all three genes resulted in a significant increase in misprocessed levels of C47F8.9 without affecting the total transcript levels. To confirm that the processing of other snRNA transcripts was also regulated by these three genes, we employed the same qPCR strategy and found that the knockdown of csr-1 and npp-6 led to increased misprocessing of the U4 snRNA transcript K03B8.10 (S1A Fig). Overall, the results here identified several novel regulators of snRNA processing and verified a role for csr-1 and npp-6 as a requirement of the 3’ processing of U2 and U4 snRNA transcripts.

Isoform and domain requirements for CSR-1 in snRNA processing We decided to focus on characterizing csr-1 as the endogenous levels of snRNA misprocessing were the greatest compared to the npp-6. Since csr-1 RNAi is predicted to knockdown both csr-1a and csr-1b isoforms given that the dsRNA targets a shared coding region, we first utilized a csr-1a null mutant (cmp135) that removes 20 bp of the coding sequence in exon 1 that is exclusively expressed by the csr-1a isoform (Fig 2A). Compared to wildtype, the csr-1a(cmp135) mutants do not show increased levels of misprocessed U2 or U4 snRNA transcripts (Figs 2B and S1B). We next tested the csr-1(tm892) mutant that contains a 400 bp deletion to both csr-1 isoforms and found that compared to wildtype, csr-1(tm892) mutants showed a significantly increased level of misprocessed U2 and U4 snRNA, with the level of misprocessing comparable to those observed via csr-1 RNAi (Figs 2C and S1C). We then examined a hypomorphic allele where csr-1 is partially rescued in the germline and found that the levels of misprocessed U2 and U4 snRNA were also significantly elevated, albeit not to the same degree as the csr-1(tm892) mutant (Fig 2D). Together, these results suggest that the csr-1b isoform, but not the csr-1a isoform, is required for snRNA processing. However, given that csr-1(tm892) deletes both isoforms, we cannot rule out the possibility that snRNA misprocessing observed in this mutant is caused by the loss of both isoforms. PPT PowerPoint slide

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TIFF original image Download: Fig 2. Isoform analysis of csr-1 function in snRNA processing. a) Diagrammatic illustration of two CSR-1 isoforms, their range of nucleotide deletion in loss of function mutants, and sites of modification to RNAi resistant single copy insertion strains. Relative levels of misprocessed and total U2 snRNA in N2 wildtype (WT) and b) csr-1a(cmp135) mutant, c) csr-1(tm892) mutant, and d) csr-1(tm892) mutant with germline csr-1 rescue. e) Effects of inserting a single copy of RNAi resistant wildtype csr-1WT or slicing-inactive (SIN) csr-1SIN on levels of misprocessed and total U2 snRNA after feeding with csr-1 dsRNA targeting the re-encoded region. All bar graphs indicate mean ± standard error, **P<0.01 and ***P<0.001 as determined by student’s t-test in c and d, and by two-way ANOVA in e. https://doi.org/10.1371/journal.pgen.1011284.g002 The CSR-1 protein encodes a Piwi domain that is catalytically active with RNA slicing activity [27]. To determine if the slicing activity of CSR-1 is required for snRNA misprocessing, we utilized two worm strains that express either a single copy insertion of wildtype (csr-1WT) or slicing inactive (csr-1SIN; D606A, D681A mutations) variant of csr-1b [28]. The two variants of the single copy csr-1b also contain a re-encoded region in exon 6 that renders resistance to a csr-1 RNAi targeting 420 bp within exon 6 that is only effective against the endogenous csr-1 gene (Fig 2A) [28]. Wildtype worms fed with dsRNA targeting the csr-1 re-encoded region resulted in a significant increase in U2 and U4 snRNA misprocessing (Figs 2E and S1E). In the csr-1WT strain, RNAi against the re-encoded region did not cause an increase in U2 or U4 snRNA misprocessing, suggesting that single copy addition of RNAi resistant wildtype csr-1b is sufficient to compensate for the knockdown of endogenous csr-1 (Figs 2E and S1E). In contrast, RNAi against the re-encoded region in worms expressing csr-1SIN resulted in the misprocessing of U2 and U4 snRNA to a similar extent observed in the wildtype strain (Figs 2E and S1E). This suggested that the single copy addition of a slicing inactive csr-1b variant does not rescue snRNA misprocessing caused by the knockdown of the endogenous csr-1 gene (Figs 2E and S1E). Overall, the results here support the requirement of the CSR-1’s enzymatic slicing activity for snRNA processing.

csr-1 knockdown affects transcriptome-wide snRNA processing To investigate the role csr-1 has on the transcriptome, we knocked down csr-1 using RNAi and performed mRNA-sequencing of oligo(dT) enriched transcripts. An increased accumulation of snRNA transcripts beyond their 3’ end is observed in the csr-1 knocked down worms indicating transcriptional read-through as evidence for 3’ misprocessing (Fig 3A). We found that expression for 38 snRNA transcripts can be detected in the EV control sample, and knockdown of csr-1 resulted in a 2-fold increase in 27/38 of these transcripts (S2A Fig). Furthermore, 18 snRNA transcripts were detected in the csr-1 RNAi knocked down worms that were not detected in the EV control fed worms (S2B Fig). Given that snRNA transcripts are only polyadenylated as a consequence of misprocessing that results in transcriptional read-through [10,18], an increased expression of snRNA abundance in oligo(dT) enriched samples also reflects an increase in snRNA misprocessing. To account for any potential developmental differences caused by csr-1 (RNAi) in the transcriptomic data, we performed real-age prediction using transcriptome staging (RAPToR) [29]. We found that EV and csr-1 (RNAi) samples have a predicted age of 71.82 ± 0.15 and 71.74 ± 0.16 hours, respectively, which suggest that gene expression variance between the two conditions were unlikely to be caused by potential developmental differences (S2C Fig). However, it should be noted that loss of csr-1 has been shown to delay the onset of oocyte production which can lead to potential alternation of germline gene expression [30]. PPT PowerPoint slide

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TIFF original image Download: Fig 3. The transcriptome effect of csr-1 knockdown resembles Integrator disruption. a) RNA sequencing reads in EV or csr-1 (RNAi) fed worms aligned to the C. elegans genome in the region of different snRNA genes visualized using the Integrated Genomics Viewer. b) Linear regression analysis on log 2 fold change of snRNA downstream genes between ints-4 (RNAi) and csr-1 (RNAi), ***P<0.001 as determined by the F-test. N = 72 snRNA downstream genes are plotted. c) Number of transcripts alternatively spliced by csr-1 (RNAi) compared to EV and d) Gene Ontology (GO) terms enriched by transcripts that undergo significant alternative splicing events. Enrichment analysis of GO terms by mRNA transcripts that were e) up-regulated or f) down-regulated by >2-fold in csr-1 (RNAi) compared to EV. g) Clustered heat map of log 2 fold change in gene expression changes caused by csr-1 (RNAi) / EV compared to RNAi knockdown of different Integrator complex subunit genes / EV that function within the catalytic or holder class. Pearson’s correlation (R) value for each gene knockdown compared to csr-1 (RNAi) is shown below. RNA-sequencing expression data for Integrator subunit knockdown were retrieved from [11] for analysis. N = 1353 genes up or down-regulated by >2-fold after csr-1(RNAi) are plotted. https://doi.org/10.1371/journal.pgen.1011284.g003 A recent study has shown that the transcriptional read-through effect of Integrator malfunction results in the up-regulation of genes that are located downstream of the misprocessed snRNA [11]. We compared the fold change of genes that are located directly downstream of each snRNA transcript after Integrator subunit-4 (ints-4) RNAi knockdown to the fold change observed for the same genes after csr-1 RNAi knockdown and found that the expression of snRNA downstream genes is highly correlated (R = 0.626) between ints-4 and csr-1 knockdown (Fig 3B). We then performed the same analysis using RNA-seq data of the csr-1(tm892) mutant recently reported by Singh et al. and found a similar result where expressions of snRNA downstream genes were significantly correlated between csr-1(tm892) mutants and ints-4 (RNAi) (S2D Fig) [30]. This indicated that knockdown or loss of csr-1 also results in similar up-regulation of snRNA downstream genes observed after ints-4 depletion as a consequence of transcriptional read-through. Given that csr-1 knockdown resulted in snRNA misprocessing, we next determined the effects it has on alternative splicing. Analysis of the RNA sequencing data showed that 6,808 alternative splicing events were detected in csr-1 knocked down worms relative to EV, with 391 of these events found to be statistically significant and composed of primarily exon skipping events (Fig 3C). Enrichment analysis of these 391 significant events reveals clustering to a wide range of cellular processes including calcium transport, muscle functions, and response to stimulus (Fig 3D). However, these enriched processes of alternatively spliced transcripts were largely distinct from the enriched cellular processes of genes that were up or down-regulated by csr-1 RNAi (Fig 3E and 3F). Of the transcripts that were alternatively spliced by csr-1 (RNAi), 60% (232/391) were differentially expressed at the mRNA levels (139 up-regulated, 93 down-regulated), which is ~2-fold higher than the global effect csr-1 (RNAi) has on the whole transcriptome where 31% of the genes were differentially regulated (FDR<0.05, no fold change cut off). While this suggests that transcripts alternatively spliced in response to csr-1 depletion have increased rates of differential expression, the functional significance of these alterations remains to be experimentally determined. To further compare the transcriptome profile between csr-1 and the Integrator, we generated a clustered heat map of expression changes for genes that were up or down-regulated by csr-1 RNAi by > 2-fold in comparison to expression changes for these same genes after RNAi knockdown of genes encoding the catalytic and holder class of the Integrator subunit [11]. We observed a striking similarity across the 9 different Integrator subunit genes which when knocked down by RNAi exhibit a correlation value of 0.59–0.69 in the expression of genes that were differentially regulated by csr-1 RNAi by > 2-fold (Fig 3G). A similar correlation in expression patterns was also observed between Integrator subunit knockdown and the previously published csr-1(tm892) mutant transcriptome obtained from RNA-seq and microarray studies (S2E Fig) [20,30]. We did not compare the gene expression changes to the auxiliary class (ints-3, ints-10, ints-12, and ints-13) as RNAi knockdown of these subunits resulted in minimal changes in gene expression compared to the EV control [11]. Given that we are comparing transcriptomic data between studies that employed different growth condition that likely introduces batch variation in the RNA samples, we next compared the differentially regulated gene profile of csr-1 knockdown to another study that measured the effects of ints-4 RNAi on the transcriptome [18]. Via linear regression analysis, we observed that the gene expression changes caused by csr-1 knockdown were significantly correlated to those induced by ints-4 RNAi from two independent studies and that the correlation values between the two studies were comparable at R = 0.49 and R = 0.59 (S2F Fig). Together, the RNA-sequencing data presented here show that knockdown of csr-1 by RNAi results in the aberrant expression of snRNA transcripts, an increase in alternatively spliced transcripts, and exhibits a high similarity to the transcriptome profile induced by knockdown of the Integrator complex.

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