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

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      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 subcomplexes. 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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      Affiliations
      [1 ] Laboratory of Structural Biochemistry, Freie Universität Berlin, D-14195 Berlin, Germany;
      [2 ] Max Planck Research Group Nucleic Acid Chemistry, Max Planck Institute for Biophysical Chemistry, 37077 Goettingen, Germany; Institute for Organic and Biomolecular Chemistry, Georg-August-University Göttingen, 37077 Goettingen, Germany.
      [3 ] Laboratory of Structural Biochemistry, Freie Universität Berlin, D-14195 Berlin, Germany; mwahl@zedat.fu-berlin.de karine.santos@fu-berlin.de.
      Journal
      Proc. Natl. Acad. Sci. U.S.A.
      Proceedings of the National Academy of Sciences of the United States of America
      Proceedings of the National Academy of Sciences
      1091-6490
      0027-8424
      Jul 12 2016
      : 113
      : 28
      27354531
      1524616113
      10.1073/pnas.1524616113
      4948317

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