Arrested Yeast Splicing Complexes Indicate Stepwise SnRNP Recruitment During in Vivo Spliceosome Assembly
Overview
Authors
Affiliations
Pre-mRNA splicing is catalyzed by the spliceosome, a macromolecular machine dedicated to intron removal and exon ligation. Despite an abundance of in vitro information and a small number of in vivo studies, the pathway of yeast (Saccharomyces cerevisiae) in vivo spliceosome assembly remains uncertain. To address this situation, we combined in vivo depletions of U1, U2, or U5 snRNAs with chromatin immunoprecipitation (ChIP) analysis of other splicing snRNPs along an intron-containing gene. The data indicate that snRNP recruitment to nascent pre-mRNA predominantly proceeds via the canonical three-step assembly pathway: first U1, then U2, and finally the U4/U6*U5 tri-snRNP. Tandem affinity purification (TAP) using a U2 snRNP-tagged protein allowed the characterization of in vivo assembled higher-order splicing complexes. Consistent with an independent snRNP assembly pathway, we observed high levels of U1-U2 prespliceosomes under U5-depletion conditions, and we observed significant levels of a U2/U5/U6/Prp19-complex mature splicing complex under wild-type conditions. These complexes have implications for the steady-state distribution of snRNPs within nuclei and also reinforce the stepwise recruitment of U1, U2, and the tri-snRNP during in vivo spliceosome assembly.
Co-transcriptional gene regulation in eukaryotes and prokaryotes.
Shine M, Gordon J, Scharfen L, Zigackova D, Herzel L, Neugebauer K Nat Rev Mol Cell Biol. 2024; 25(7):534-554.
PMID: 38509203 PMC: 11199108. DOI: 10.1038/s41580-024-00706-2.
Seltzer S, Giannopoulos P, Bismar T, Trifiro M, Paliouras M Asian J Androl. 2022; 25(3):296-308.
PMID: 36259569 PMC: 10226513. DOI: 10.4103/aja202263.
Keeping up with the condensates: The retention, gain, and loss of nuclear membrane-less organelles.
Lacroix E, Audas T Front Mol Biosci. 2022; 9:998363.
PMID: 36203874 PMC: 9530788. DOI: 10.3389/fmolb.2022.998363.
Chromatin-associated microprocessor assembly is regulated by the U1 snRNP auxiliary protein PRP40.
Stepien A, Dolata J, Gulanicz T, Bielewicz D, Bajczyk M, Smolinski D Plant Cell. 2022; 34(12):4920-4935.
PMID: 36087009 PMC: 9709975. DOI: 10.1093/plcell/koac278.
Maudlin I, Beggs J RNA Biol. 2021; 18(sup2):782-793.
PMID: 34705599 PMC: 8782173. DOI: 10.1080/15476286.2021.1991673.