Abstract
Spliced leader trans-splicing of pre-mRNAs is a critical step in the gene expression of many eukaryotes. How the spliced leader RNA and its target transcripts are brought together to form the trans-spliceosome remains an important unanswered question. Using immunoprecipitation followed by protein analysis via mass spectrometry and RIP-Seq, we show that the nematode-specific proteins, SNA-3 and SUT-1, form a complex with a set of enigmatic non-coding RNAs, the SmY RNAs. Our work redefines the SmY snRNP and shows for the first time that it is essential for nematode viability and is involved in spliced leader trans-splicing. SNA-3 and SUT-1 are associated with the 5′ ends of most, if not all, nascent capped RNA polymerase II transcripts, and they also interact with components of the major nematode spliced leader (SL1) snRNP. We show that depletion of SNA-3 impairs the co-immunoprecipitation between one of the SL1 snRNP components, SNA-2, and several core spliceosomal proteins. We thus propose that the SmY snRNP recruits the SL1 snRNP to the 5′ ends of nascent pre-mRNAs, an instrumental step in the assembly of the trans-spliceosome.
Original language | English |
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Article number | gkae321 |
Pages (from-to) | 7245-7260 |
Number of pages | 16 |
Journal | Nucleic Acids Research |
Volume | 52 |
Issue number | 12 |
Early online date | 27 Apr 2024 |
DOIs | |
Publication status | Published - 8 Jul 2024 |
Bibliographical note
AcknowledgementsWe thank Maheshika Kurukulasuriya for her assistance with immunoprecipitations from embryo extracts. Some strains were provided by the Caenorhabditis Genetics Center, which is funded by NIH Office of Research Infrastructure Programs (P40 OD010440). Sequencing was performed by the Centre for Genome-Enabled Biology and Medicine of the University of Aberdeen, and proteomics analysis by the Aberdeen proteomics facility. We thank Kate Burgoyne and Craig Pattinson (Aberdeen Proteomics) for technical support. We thank WormBase for providing the community resource that facilitated the interrogation of C. elegans molecular genetics used in this work .
Author contribution: P.E., M.A., E.S.-M., R.F., M.W., B.M. and J.P. contributed experiments. J.P. and B.M. conceived the research and managed and coordinated the research activity; J.P. and B.M. acquired funding for the project; D.S. guided the proteomics analysis, M.W., B.M. and J.P. designed and implemented the computational analysis; M.W., B.M. and J.P. wrote the manuscript and prepared figures and tables.
Data Availability Statement
The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD040352 (see also Supplementary Table S2). RIP-Seq data have been deposited in the ArrayExpress database at EMBL-EBI (www.ebi.ac.uk/arrayexpress) under accession number E-MTAB-13193 (see also Supplementary Table S3).Keywords
- Animals
- RNA, Spliced Leader/metabolism
- RNA Precursors/metabolism
- Ribonucleoproteins, Small Nuclear/metabolism
- Spliceosomes/metabolism
- Caenorhabditis elegans/genetics
- Caenorhabditis elegans Proteins/metabolism
- Trans-Splicing
- Protein Binding