A nematode-specific ribonucleoprotein complex mediates interactions between the major nematode spliced leader snRNP and its target pre-mRNAs

Peter Eijlers; Mohammed Ismael Al-Khafaji; Eva Soto Martin; Rotimi Yemi Fasimoye; David Stead; Marius Wenzel; Berndt Muller; Jonathan Pettitt

doi:10.1093/nar/gkae321

A nematode-specific ribonucleoprotein complex mediates interactions between the major nematode spliced leader snRNP and its target pre-mRNAs

Peter Eijlers, Mohammed Ismael Al-Khafaji, Eva Soto Martin, Rotimi Yemi Fasimoye, David Stead, Marius Wenzel^* (Corresponding Author), Berndt Muller^* (Corresponding Author), Jonathan Pettitt^* (Corresponding Author)

^*Corresponding author for this work

University of Aberdeen

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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
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	https://doi.org/10.1093/nar/gkae321
Publication status	Published - 8 Jul 2024

Bibliographical note

Acknowledgements
We 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

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Elijlers_etal_NAR_A_nematode_specific_VoR
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Cite this

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title = "A nematode-specific ribonucleoprotein complex mediates interactions between the major nematode spliced leader snRNP and its target pre-mRNAs",

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.",

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",

author = "Peter Eijlers and Al-Khafaji, {Mohammed Ismael} and {Soto Martin}, Eva and Fasimoye, {Rotimi Yemi} and David Stead and Marius Wenzel and Berndt Muller and Jonathan Pettitt",

note = "Acknowledgements We 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.",

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T1 - A nematode-specific ribonucleoprotein complex mediates interactions between the major nematode spliced leader snRNP and its target pre-mRNAs

AU - Eijlers, Peter

AU - Al-Khafaji, Mohammed Ismael

AU - Soto Martin, Eva

AU - Fasimoye, Rotimi Yemi

AU - Stead, David

AU - Wenzel, Marius

AU - Muller, Berndt

AU - Pettitt, Jonathan

N1 - Acknowledgements We 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.

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N2 - 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.

AB - 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.

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KW - Ribonucleoproteins, Small Nuclear/metabolism

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KW - Caenorhabditis elegans/genetics

KW - Caenorhabditis elegans Proteins/metabolism

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A nematode-specific ribonucleoprotein complex mediates interactions between the major nematode spliced leader snRNP and its target pre-mRNAs

Abstract

Bibliographical note

Data Availability Statement

Keywords

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