A novel, essential trans-splicing protein connects the nematode SL1 snRNP to the CBC-ARS2 complex

Rotimi Yemi Fasimoye; Rosie Elizabeth Spencer; Eva Soto Martin; Peter Eijlers; Haitem Elmassoudi; Sarah Brivio; Carolina Mangana; Viktorija Sabele; Radoslava Rechtorikova; Marius Wenzel; Bernadette Connolly; Jonathan Pettitt; Berndt Marino Müller

doi:10.1093/nar/gkac534

A novel, essential trans-splicing protein connects the nematode SL1 snRNP to the CBC-ARS2 complex

Rotimi Yemi Fasimoye, Rosie Elizabeth Spencer, Eva Soto Martin, Peter Eijlers, Haitem Elmassoudi, Sarah Brivio, Carolina Mangana, Viktorija Sabele, Radoslava Rechtorikova, Marius Wenzel, Bernadette Connolly, Jonathan Pettitt^* (Corresponding Author), Berndt Marino Müller^* (Corresponding Author)

^*Corresponding author for this work

University of Aberdeen

Research output: Contribution to journal › Article › peer-review

3 Citations (Scopus)

11 Downloads (Pure)

Abstract

Spliced leader trans-splicing is essential for gene expression in many eukaryotes. To elucidate the molecular mechanism of this process, we characterise the molecules associated with the Caenorhabditis elegans major spliced leader snRNP (SL1 snRNP), which donates the spliced leader that replaces the 5′ untranslated region of most pre-mRNAs. Using a GFP-tagged version of the SL1 snRNP protein SNA-1 created by CRISPR-mediated genome engineering, we immunoprecipitate and identify RNAs and protein components by RIP-Seq and mass spectrometry. This reveals the composition of the SL1 snRNP and identifies associations with spliceosome components PRP-8 and PRP-19. Significantly, we identify a novel, nematode-specific protein required for SL1 trans-splicing, which we designate SNA-3. SNA-3 is an essential, nuclear protein with three NADAR domains whose function is unknown. Mutation of key residues in NADAR domains inactivates the protein, indicating that domain function is required for activity. SNA-3 interacts with the CBC-ARS2 complex and other factors involved in RNA metabolism, including SUT-1 protein, through RNA or protein-mediated contacts revealed by yeast two-hybrid assays, localisation studies and immunoprecipitations. Our data are compatible with a role for SNA-3 in coordinating trans-splicing with target pre-mRNA transcription or in the processing of the Y-branch product of the trans-splicing reaction.

Original language	English
Pages (from-to)	7591-7607
Number of pages	17
Journal	Nucleic Acids Research
Volume	50
Issue number	13
Early online date	23 Jun 2022
DOIs	https://doi.org/10.1093/nar/gkac534
Publication status	Published - 22 Jul 2022

Bibliographical note

Open Access via the OUP Agreement
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 University of Aberdeen Proteomics facility. We thank WormBase for providing the community resource that facilitated the interrogation of C. elegans molecular genetics used in this work.

FUNDING
Biotechnology and Biological Sciences Research Council [BB/T002859/1]; EASTBIO Biotechnology and Biological Sciences Research Council PhD Studentship [BB/M010996/1 to R.E.B.S.]; University of Aberdeen Elphinstone PhD studentship (to R.Y.F.); University of Aberdeen. Funding for open access charge: Read and publish Agreement Scottish Institutions (SHEDL affiliated).

Data Availability Statement

RIP-Seq data have been deposited in the ArrayExpress database at EMBL-EBI (www.ebi.ac.uk/arrayexpress) (57) under accession number E-MTAB-10289 (see also Supplemental File 2).

The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE (58) partner repository with the dataset identifier PXD024763 (see also Supplemental File 3).

Access to Document

10.1093/nar/gkac534Licence: CC BY

Fasimoye_etal_NAR_Novel_Essential_TRans_Splicing_VOR
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.
Final published version, 5.6 MBLicence: CC BY

Cite this

Fasimoye, R. Y., Spencer, R. E., Soto Martin, E., Eijlers, P., Elmassoudi, H., Brivio, S., Mangana, C., Sabele, V., Rechtorikova, R., Wenzel, M., Connolly, B., Pettitt, J., & Müller, B. M. (2022). A novel, essential trans-splicing protein connects the nematode SL1 snRNP to the CBC-ARS2 complex. Nucleic Acids Research, 50(13), 7591-7607. https://doi.org/10.1093/nar/gkac534

@article{2cee4001b2464b078fc58c910fe6ca1d,

title = "A novel, essential trans-splicing protein connects the nematode SL1 snRNP to the CBC-ARS2 complex",

abstract = "Spliced leader trans-splicing is essential for gene expression in many eukaryotes. To elucidate the molecular mechanism of this process, we characterise the molecules associated with the Caenorhabditis elegans major spliced leader snRNP (SL1 snRNP), which donates the spliced leader that replaces the 5′ untranslated region of most pre-mRNAs. Using a GFP-tagged version of the SL1 snRNP protein SNA-1 created by CRISPR-mediated genome engineering, we immunoprecipitate and identify RNAs and protein components by RIP-Seq and mass spectrometry. This reveals the composition of the SL1 snRNP and identifies associations with spliceosome components PRP-8 and PRP-19. Significantly, we identify a novel, nematode-specific protein required for SL1 trans-splicing, which we designate SNA-3. SNA-3 is an essential, nuclear protein with three NADAR domains whose function is unknown. Mutation of key residues in NADAR domains inactivates the protein, indicating that domain function is required for activity. SNA-3 interacts with the CBC-ARS2 complex and other factors involved in RNA metabolism, including SUT-1 protein, through RNA or protein-mediated contacts revealed by yeast two-hybrid assays, localisation studies and immunoprecipitations. Our data are compatible with a role for SNA-3 in coordinating trans-splicing with target pre-mRNA transcription or in the processing of the Y-branch product of the trans-splicing reaction.",

author = "Fasimoye, {Rotimi Yemi} and Spencer, {Rosie Elizabeth} and {Soto Martin}, Eva and Peter Eijlers and Haitem Elmassoudi and Sarah Brivio and Carolina Mangana and Viktorija Sabele and Radoslava Rechtorikova and Marius Wenzel and Bernadette Connolly and Jonathan Pettitt and M{\"u}ller, {Berndt Marino}",

note = "Open Access via the OUP Agreement 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 University of Aberdeen Proteomics facility. We thank WormBase for providing the community resource that facilitated the interrogation of C. elegans molecular genetics used in this work. FUNDING Biotechnology and Biological Sciences Research Council [BB/T002859/1]; EASTBIO Biotechnology and Biological Sciences Research Council PhD Studentship [BB/M010996/1 to R.E.B.S.]; University of Aberdeen Elphinstone PhD studentship (to R.Y.F.); University of Aberdeen. Funding for open access charge: Read and publish Agreement Scottish Institutions (SHEDL affiliated).",

year = "2022",

month = jul,

day = "22",

doi = "10.1093/nar/gkac534",

language = "English",

volume = "50",

pages = "7591--7607",

journal = "Nucleic Acids Research",

issn = "0305-1048",

publisher = "Oxford University Press",

number = "13",

}

TY - JOUR

T1 - A novel, essential trans-splicing protein connects the nematode SL1 snRNP to the CBC-ARS2 complex

AU - Fasimoye, Rotimi Yemi

AU - Spencer, Rosie Elizabeth

AU - Soto Martin, Eva

AU - Eijlers, Peter

AU - Elmassoudi, Haitem

AU - Brivio, Sarah

AU - Mangana, Carolina

AU - Sabele, Viktorija

AU - Rechtorikova, Radoslava

AU - Wenzel, Marius

AU - Connolly, Bernadette

AU - Pettitt, Jonathan

AU - Müller, Berndt Marino

N1 - Open Access via the OUP Agreement 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 University of Aberdeen Proteomics facility. We thank WormBase for providing the community resource that facilitated the interrogation of C. elegans molecular genetics used in this work. FUNDING Biotechnology and Biological Sciences Research Council [BB/T002859/1]; EASTBIO Biotechnology and Biological Sciences Research Council PhD Studentship [BB/M010996/1 to R.E.B.S.]; University of Aberdeen Elphinstone PhD studentship (to R.Y.F.); University of Aberdeen. Funding for open access charge: Read and publish Agreement Scottish Institutions (SHEDL affiliated).

PY - 2022/7/22

Y1 - 2022/7/22

N2 - Spliced leader trans-splicing is essential for gene expression in many eukaryotes. To elucidate the molecular mechanism of this process, we characterise the molecules associated with the Caenorhabditis elegans major spliced leader snRNP (SL1 snRNP), which donates the spliced leader that replaces the 5′ untranslated region of most pre-mRNAs. Using a GFP-tagged version of the SL1 snRNP protein SNA-1 created by CRISPR-mediated genome engineering, we immunoprecipitate and identify RNAs and protein components by RIP-Seq and mass spectrometry. This reveals the composition of the SL1 snRNP and identifies associations with spliceosome components PRP-8 and PRP-19. Significantly, we identify a novel, nematode-specific protein required for SL1 trans-splicing, which we designate SNA-3. SNA-3 is an essential, nuclear protein with three NADAR domains whose function is unknown. Mutation of key residues in NADAR domains inactivates the protein, indicating that domain function is required for activity. SNA-3 interacts with the CBC-ARS2 complex and other factors involved in RNA metabolism, including SUT-1 protein, through RNA or protein-mediated contacts revealed by yeast two-hybrid assays, localisation studies and immunoprecipitations. Our data are compatible with a role for SNA-3 in coordinating trans-splicing with target pre-mRNA transcription or in the processing of the Y-branch product of the trans-splicing reaction.

AB - Spliced leader trans-splicing is essential for gene expression in many eukaryotes. To elucidate the molecular mechanism of this process, we characterise the molecules associated with the Caenorhabditis elegans major spliced leader snRNP (SL1 snRNP), which donates the spliced leader that replaces the 5′ untranslated region of most pre-mRNAs. Using a GFP-tagged version of the SL1 snRNP protein SNA-1 created by CRISPR-mediated genome engineering, we immunoprecipitate and identify RNAs and protein components by RIP-Seq and mass spectrometry. This reveals the composition of the SL1 snRNP and identifies associations with spliceosome components PRP-8 and PRP-19. Significantly, we identify a novel, nematode-specific protein required for SL1 trans-splicing, which we designate SNA-3. SNA-3 is an essential, nuclear protein with three NADAR domains whose function is unknown. Mutation of key residues in NADAR domains inactivates the protein, indicating that domain function is required for activity. SNA-3 interacts with the CBC-ARS2 complex and other factors involved in RNA metabolism, including SUT-1 protein, through RNA or protein-mediated contacts revealed by yeast two-hybrid assays, localisation studies and immunoprecipitations. Our data are compatible with a role for SNA-3 in coordinating trans-splicing with target pre-mRNA transcription or in the processing of the Y-branch product of the trans-splicing reaction.

UR - https://abdn.elsevierpure.com/en/publications/2cee4001-b246-4b07-8fc5-8c910fe6ca1d

U2 - 10.1093/nar/gkac534

DO - 10.1093/nar/gkac534

M3 - Article

C2 - 35736244

SN - 0305-1048

VL - 50

SP - 7591

EP - 7607

JO - Nucleic Acids Research

JF - Nucleic Acids Research

IS - 13

ER -

A novel, essential trans-splicing protein connects the nematode SL1 snRNP to the CBC-ARS2 complex

Abstract

Bibliographical note

Data Availability Statement

Access to Document

Other files and links

Fingerprint

Centre for Genome-Enabled Biology and Medicine

Cite this

A novel, essential trans-splicing protein connects the nematode SL1 snRNP to the CBC-ARS2 complex

Abstract

Bibliographical note

Data Availability Statement

Access to Document

Other files and links

Fingerprint

Equipment

Centre for Genome-Enabled Biology and Medicine

Cite this