To investigate the mechanism of spliceosome assembly in vivo, we performed chromatin immunoprecipitation (ChIP) analysis of U1, U2, and U5 small nuclear ribonucleoprotein particles (snRNPs) to intron-containing yeast (S. cerevisiae) genes. The snRNPs display patterns that indicate a cotranscriptional assembly model: U1 first, then U2, and the U4/U6*U5 tri-snRNP followed by U1 destabilization. cis-splicing mutations also support a role of U2 and/or the tri-snRNP in U1 destabilization. Moreover, they indicate that splicing efficiency has a major impact on cotranscriptional snRNP recruitment and suggest that cotranscriptional recruitment of U2 or the tri-snRNP is required to commit the pre-mRNA to splicing. Branchpoint (BP) mutations had a major effect on the U1 pattern, whereas 5' splice site (5'ss) mutations had a stronger effect on the U2 pattern. A 5'ss-U1 snRNA complementation experiment suggests that pairing between U1 and the 5'ss occurs after U1 recruitment and contributes to a specific U1:substrate conformation required for efficient U2 and tri-snRNP recruitment.

• PMID: 15989965
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Journal Article | Research Support, N.I.H., Extramural | Research Support, U.S. Gov't, P.H.S.

Authors

Lacadie SA, Rosbash M

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