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.
U2 - 10.1093/nar/gkac534
DO - 10.1093/nar/gkac534
M3 - Article
C2 - 35736244
VL - 50
SP - 7591
EP - 7607
JO - Nucleic Acids Research
JF - Nucleic Acids Research
SN - 0305-1048
IS - 13
ER -