A Role for the C. Elegans Argonaute Protein CSR-1 in Small Nuclear RNA 3’ Processing
Brandon M. Waddell, Cheng-Wei Wu
Abstract
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.
Introduction
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].
Materials & methods
Genome-wide RNAi screen and RNAi experiments
RNAi screen was performed using a protocol previously described in detail [43]. Briefly, synchronized L1 MWU3 larvae obtained from hypochlorite treatment were grown in liquid nematode growth media (NGM) and fed with dsRNA producing HT115(DE3) bacteria for 3 days, followed by manual screening for snRNA misprocessing reporter GFP activation using an Olympus SZX61 stereomicroscope. The MRC genomic RNAi feeding library (Geneservice, Cambridge, UK) and the ORFeome RNAi feeding library (Open Biosystems, Huntsville, AL) were used totaling approximately 19,000 clones screened. Clones that activated the snRNA misprocessing reporter from the primary screen were rescreened three additional times using solid NGM agar plates for confirmation. NGM agar RNAi plates were prepared with 50 μg mL-1 carbenicillin and 100 μg mL-1 of isopropyl β-D-thiogalactopyranoside (IPTG) and seeded with HT115(DE3) E. coli expressing the corresponding target dsRNA clone or expressing the pPD129.36(L4440) plasmid that serve as the empty vector (EV) control.
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].
Discussion
The Integrator is a metazoan specific multi-protein complex that was initially discovered as the elusive termination machinery that facilitates the 3’ processing of U-rich snRNAs [6]. Human mutations to the Integrator complex are characterized by increased levels of misprocessed U-rich snRNA transcripts that are accompanied by disruptions to gene expression and RNA processing [15]. While recent studies have expanded on the core functions of the Integrator beyond snRNA processing including cleavage of nascent mRNAs during RNA polymerase pause-release [14], additional factors that regulate the Integrator complex or influence snRNA 3’ processing remain underexplored. In this study through the use of an in vivo snRNA misprocessing reporter in the C. elegans system, we identified several genes, including the Argonaute encoding csr-1, that when knocked down result in the misprocessing of snRNA transcripts. We propose that csr-1 is required for the germline expression of Integrator subunit proteins, and that loss of csr-1 contributes to snRNA misprocessing by altering the abundance of Integrator complex subunits (Fig 6C). Additionally, given that CSR-1 also binds to 22G-RNA targeting snRNA transcripts, it is possible that CSR-1 can directly cleave snRNA molecules given its catalytic slicing activity, a function that has been proposed for the 3’ processing of histone transcripts in C. elegans [24,27,28,30].
Conclusions
Overall, we demonstrate in this study a positive role for the csr-1 gene encoding the only essential Argonaute protein in C. elegans as a regulator of snRNA processing, through a mechanism where csr-1 is required for the translation and expression of Integrator subunit genes within the germline. Beyond csr-1, the genome-wide RNAi screen presented in this study has also identified several yet to be characterized regulators of snRNA processing including those encoding nuclear protein complex as well as members of the endogenous siRNA pathway. Given the recent expansion of a wide-ranging role for the Integrator complex in gene expression control beyond snRNA processing, and its emerging implication in human diseases [9,15,16], it will ultimately be of interest to determine whether these novel regulators may also influence snRNA independent functions of the Integrator in contributing to transcriptome stability.
Acknowledgments
Some strains were provided by the Caenorhabditis Genetic Centre (University of Minnesota, Minneapolis, MN) which is supported by the NIH Office of Research Infrastructure Program (P40 OD010440). We thank Dr. Carolyn M. Phillips (University of Southern California) for sharing the USC1258 csr-1a(cmp135) worm strain and Dr. Carlos Carvalho (University of Saskatchewan) for assistance with the DeltaVision system. BMW was supported by a USask Devolved Scholarship.
Citation: Waddell BM, Wu C-W (2024) A role for the C. elegans Argonaute protein CSR-1 in small nuclear RNA 3’ processing. PLoS Genet 20(5): e1011284. https://doi.org/10.1371/journal.pgen.1011284
Editor: John Isaac Murray, University of Pennsylvania School of Medicine, UNITED STATES
Received: October 11, 2023; Accepted: May 2, 2024; Published: May 14, 2024
Copyright: © 2024 Waddell, Wu. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All datasets supporting this manuscript are publicly available and found within the article and the supporting information. All numerical data are presented in S4 Table. RNA-sequencing data generated from this study are publicly available on the NCBI GEO depository GSE243495 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE243495).
Funding: This work is supported by a Natural Sciences and Engineering Research Council of Canada Discovery Grant to CWW(04486). The funders had no roles in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
https://journals.plos.org/plosgenetics/article?id=10.1371/journal.pgen.1011284#abstract1
