Key result
In vivo depletions of U1, U2, or U5 snRNAs indicate that snRNP recruitment to nascent pre-mRNA predominantly proceeds via a canonical three-step assembly pathway.
The study demonstrates that in vivo spliceosome assembly in yeast follows a stepwise recruitment of U1, U2, and the tri-snRNP.
No immediate clinical implications; hypothesis-generating for spliceosome assembly mechanisms across species.
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.
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Tardiff et al. (2006) studied Yeast (Saccharomyces cerevisiae) spliceosome assembly. In vivo depletions of U1, U2, or U5 snRNAs vs. Wild-type conditions was evaluated on snRNP recruitment to nascent pre-mRNA. In vivo depletions of U1, U2, or U5 snRNAs indicate that snRNP recruitment to nascent pre-mRNA predominantly proceeds via a canonical three-step assembly pathway.
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