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  Substrate-assisted mechanism of RNP disruption by the spliceosomal Brr2 RNA helicase.

Theuser, M., Höbartner, C., Wahl, M. C., & Santos, K. F. (2017). Substrate-assisted mechanism of RNP disruption by the spliceosomal Brr2 RNA helicase. Proceedings of the National Academy of Sciences of the United States of America, 113(28), 7798-7803. doi:10.1073/pnas.1524616113.

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Item Permalink: http://hdl.handle.net/11858/00-001M-0000-002B-193A-3 Version Permalink: http://hdl.handle.net/11858/00-001M-0000-002C-7EB0-9
Genre: Journal Article

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http://www.pnas.org/content/113/28/7798.full (Publisher version)
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 Creators:
Theuser, M., Author
Höbartner, C.1, Author              
Wahl, M. C., Author
Santos, K. F., Author
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1Research Group of Nucleic Acid Chemistry, MPI for biophysical chemistry, Max Planck Society, ou_578605              

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Free keywords: pre-mRNA splicing; RNA helicase; RNP remodeling; small nuclear ribonucleoprotein particle; spliceosome catalytic activation
 Abstract: The Brr2 RNA helicase disrupts the U4/U6 di-small nuclear RNA-protein complex (di-snRNP) during spliceosome activation via ATP-driven translocation on the U4 snRNA strand. However, it is unclear how bound proteins influence U4/U6 unwinding, which regions of the U4/U6 duplex the helicase actively unwinds, and whether U4/U6 components are released as individual molecules or as sub-complexes. Here, we set up a recombinant Brr2-mediated U4/U6 di-snRNP disruption system, showing that sequential addition of the U4/U6 proteins small nuclear ribonucleoprotein-associated protein 1 (Snu13), pre-mRNA processing factor 31 (Prp31), and Prp3 to U4/U6 di-snRNA leads to a stepwise decrease of Brr2-mediated U4/U6 unwinding, but that unwinding is largely restored by a Brr2 cofactor, the C-terminal Jab1/MPN domain of the Prp8 protein. Brr2-mediated U4/U6 unwinding was strongly inhibited by mutations in U4/U6 di-snRNAs that diminish the ability of U6 snRNA to adopt an alternative conformation but leave the number and kind of U4/U6 base pairs unchanged. Irrespective of the presence of the cofactor, the helicase segregated a Prp3-Prp31-Snu13-U4/U6 RNP into an intact Prp31-Snu13-U4 snRNA particle, free Prp3, and free U6 snRNA. Together, these observations suggest that Brr2 translocates only a limited distance on the U4 snRNA strand and does not actively release RNA-bound proteins. Unwinding is then completed by the partially displaced U6 snRNA adopting an alternative conformation, which leads to dismantling of the Prp3-binding site on U4/U6 di-snRNA but leaves the Prp31- and Snu13-binding sites on U4 snRNA unaffected. In this fashion, Brr2 can activate the spliceosome by stripping U6 snRNA of all precatalytic binding partners, while minimizing logistic requirements for U4/U6 di-snRNP reassembly after splicing.

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Language(s): eng - English
 Dates: 2017-07-12
 Publication Status: Published online
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 Rev. Method: Peer
 Identifiers: DOI: 10.1073/pnas.1524616113
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Title: Proceedings of the National Academy of Sciences of the United States of America
Source Genre: Journal
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Pages: - Volume / Issue: 113 (28) Sequence Number: - Start / End Page: 7798 - 7803 Identifier: -