Structural insights into how Prp5 proofreads the pre-mRNA branch site.
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.
Mesh Terms:
Actins, Adenosine, Binding Sites, Cryoelectron Microscopy, DEAD-box RNA Helicases, Models, Molecular, Mutation, Protein Domains, RNA Precursors, RNA Splicing, Ribonucleoprotein, U1 Small Nuclear, Ribonucleoprotein, U2 Small Nuclear, Saccharomyces cerevisiae, Saccharomyces cerevisiae Proteins, Spliceosomes
Actins, Adenosine, Binding Sites, Cryoelectron Microscopy, DEAD-box RNA Helicases, Models, Molecular, Mutation, Protein Domains, RNA Precursors, RNA Splicing, Ribonucleoprotein, U1 Small Nuclear, Ribonucleoprotein, U2 Small Nuclear, Saccharomyces cerevisiae, Saccharomyces cerevisiae Proteins, Spliceosomes
Nature
Date: Dec. 01, 2020
PubMed ID: 34349264
View in: Pubmed Google Scholar
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