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Author(s): Zhenwei Zhang [sup.1] , Norbert Rigo [sup.2] , Olexandr Dybkov [sup.2] , Jean-Baptiste Fourmann [sup.2] , Cindy L. Will [sup.2] , Vinay Kumar [sup.2] , Henning Urlaub [sup.3] [sup.4] [...], 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. .sup.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 mechanism.sup.5. 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 (Hsh155.sup.HEAT), 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 Hsh155.sup.HEAT to the bulged BS-A of the U2-BS helix triggers closure of Hsh155.sup.HEAT, which in turn destabilizes Prp5 binding. Thus, Prp5 proofreads the branch site indirectly, hindering spliceosome assembly if branch-site mutations prevent the remodelling of Hsh155.sup.HEAT. Our data provide structural insights into how a spliceosomal helicase enhances the fidelity of pre-mRNA splicing. The cryo-electron microscopy structure of a newly identified, early spliceosomal complex reveals the mechanism by which the RNA helicase Prp5 enhances the fidelity of the excision of introns from precursor mRNAs.