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ATP requirement for Prp5p function is determined by Cus2p and the structure of U2 small nuclear RNA

Perriman, Rhonda and Barta, Imre and Voeltz, Gia K. and Abelson, John and Ares, Manuel, Jr. (2003) ATP requirement for Prp5p function is determined by Cus2p and the structure of U2 small nuclear RNA. Proceedings of the National Academy of Sciences of the United States of America, 100 (24). pp. 13857-13862. ISSN 0027-8424. PMCID PMC283511. doi:10.1073/pnas.2036312100.

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Stable addition of U2 small nuclear ribonucleoprotein (snRNP) to form the prespliceosome is the first ATP-dependent step in splicing, and it requires the DEXD/H box ATPase Prp5p. However, prespliceosome formation occurs without ATP in extracts lacking the U2 snRNP protein Cus2p. Here we show that Prp5p is required for the ATP-independent prespliceosome assembly that occurs in the absence of Cus2p. Addition of recombinant Cus2p can restore the ATIP dependence of prespliceosome assembly, but only if it is added before Prp5p. Prp5p with an altered ATP-binding domain (Prp5-GNTp) can support growth in vivo, but only in a cus2 deletion strain, mirroring the in vitro results. Other Prp5 ATP-binding domain substitutions are lethal, even in the cus2 deletion strain, but can be suppressed by U2 small nuclear RNA mutations that hyper-stabilize U2 stem IIa. We infer that the presence of Cus2p and stem IIa-destabilized forms of U2 small nuclear RNA places high demands on the ATP-driven function of Prp5p. Because Prp5p is not dispensable in vitro even in the absence of ATP, we propose that the core Prp5p function in bringing U2 to the branchpoint is not directly ATP-dependent. The positive role of Cus2p in rescuing mutant U2 can be reconciled with its antagonistic effect on Prp5 function in a model whereby Cus2p first helps Prp5p to activate the U2 snRNP for prespliceosome formation but then is displaced by Prp5p before or during the stabilization of U2 at the branchpoint.

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Additional Information:© 2003 by the National Academy of Sciences. Contributed by John Abelson, September 30, 2003. We thank Andrey Balakin for making U2-CC, Carrie Davis and Stephanie Ruby for critical reading of the manuscript, and the reviewers for constructive comments. This work was supported by National Institutes of Health Grants GM47408 (to M.A.) and GM32637 (to J.A.) and an American Cancer Society Postdoctoral Fellowship (to R.P.).
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American Cancer SocietyUNSPECIFIED
Subject Keywords:pre-mRNA splicing, branch site, RNA helicase, commitment complex, mRNA
Issue or Number:24
PubMed Central ID:PMC283511
Record Number:CaltechAUTHORS:PERpnas03
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Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:1334
Deposited By: Tony Diaz
Deposited On:11 Jan 2006
Last Modified:08 Nov 2021 19:09

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