Structural insights into how Prp5 proofreads the pre-mRNA branch site

Zhenwei Zhang, Norbert Rigo, Olexandr Dybkov, Jean-Baptiste Fourmann, Cindy L. Will, Vinay Kumar, Henning Urlaub, Holger Stark () and Reinhard Lührmann ()
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Zhenwei Zhang: MPI for Biophysical Chemistry
Norbert Rigo: Cellular Biochemistry, MPI for Biophysical Chemistry
Olexandr Dybkov: Cellular Biochemistry, MPI for Biophysical Chemistry
Jean-Baptiste Fourmann: Cellular Biochemistry, MPI for Biophysical Chemistry
Cindy L. Will: Cellular Biochemistry, MPI for Biophysical Chemistry
Vinay Kumar: Cellular Biochemistry, MPI for Biophysical Chemistry
Henning Urlaub: Bioanalytical Mass Spectrometry, MPI for Biophysical Chemistry
Holger Stark: MPI for Biophysical Chemistry
Reinhard Lührmann: Cellular Biochemistry, MPI for Biophysical Chemistry

Nature, 2021, vol. 596, issue 7871, 296-300

Abstract: Abstract During the splicing of introns from precursor messenger RNAs (pre-mRNAs), the U2 small nuclear ribonucleoprotein (snRNP) must undergo stable integration into the spliceosomal A complex—a poorly understood, multistep process that is facilitated by the DEAD-box helicase Prp5 (refs. 1–4). During this process, the U2 small nuclear RNA (snRNA) forms an RNA duplex with the pre-mRNA branch site (the U2–BS helix), which is proofread by Prp5 at this stage through an unclear mechanism5. Here, by deleting the branch-site adenosine (BS-A) or mutating the branch-site sequence of an actin pre-mRNA, we stall the assembly of spliceosomes in extracts from the yeast Saccharomyces cerevisiae directly before the A complex is formed. We then determine the three-dimensional structure of this newly identified assembly intermediate by cryo-electron microscopy. Our structure indicates that the U2–BS helix has formed in this pre-A complex, but is not yet clamped by the HEAT domain of the Hsh155 protein (Hsh155HEAT), which exhibits an open conformation. The structure further reveals a large-scale remodelling/repositioning of the U1 and U2 snRNPs during the formation of the A complex that is required to allow subsequent binding of the U4/U6.U5 tri-snRNP, but that this repositioning is blocked in the pre-A complex by the presence of Prp5. Our data suggest that binding of Hsh155HEAT to the bulged BS-A of the U2–BS helix triggers closure of Hsh155HEAT, which in turn destabilizes Prp5 binding. Thus, Prp5 proofreads the branch site indirectly, hindering spliceosome assembly if branch-site mutations prevent the remodelling of Hsh155HEAT. Our data provide structural insights into how a spliceosomal helicase enhances the fidelity of pre-mRNA splicing.

Date: 2021
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DOI: 10.1038/s41586-021-03789-5

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