Evidence for complex dynamics during U2 snRNP selection of the intron branchpoint

• Published in: Nucleic acids research Vol. 49; no. 17; pp. 9965 - 9977
• Main Authors: Kao, Ching-Yang, Cao, En-Cih, Wai, Hsu Lei, Cheng, Soo-Chen
• Format: Journal Article
• Language: English
• Published: England Oxford University Press 27.09.2021
• Subjects: Actins - genetics; Adenosine Triphosphatases - genetics; DEAD-box RNA Helicases - genetics; Humans; Ribonucleoprotein, U2 Small Nuclear - metabolism; RNA and RNA-protein complexes; RNA Splicing - genetics; RNA Splicing Factors - metabolism; RNA, Messenger - genetics; Saccharomyces cerevisiae - genetics; Saccharomyces cerevisiae - metabolism; Saccharomyces cerevisiae Proteins - genetics; Saccharomyces cerevisiae Proteins - metabolism; Spliceosomes - genetics; Splicing Factor U2AF - metabolism
• ISSN: 0305-1048; 1362-4962
• DOI: 10.1093/nar/gkab695

Abstract Splicing of pre-mRNA is initiated by binding of U1 to the 5′ splice site and of Msl5-Mud2 heterodimer to the branch site (BS). Subsequent binding of U2 displaces Msl5-Mud2 from the BS to form the prespliceosome, a step governing branchpoint selection and hence 3′ splice site choice, and linking splicing to myelodysplasia and many cancers in human. Two DEAD-box proteins, Prp5 and Sub2, are required for this step, but neither is stably associated with the pre-mRNA during the reaction. Using BS-mutated ACT1 pre-mRNA, we previously identified a splicing intermediate complex, FIC, which contains U2 and Prp5, but cannot bind the tri-snRNP. We show here that Msl5 remains associated with the upstream cryptic branch site (CBS) in the FIC, with U2 binding a few bases downstream of the BS. U2 mutants that restore U2-BS base pairing enable dissociation of Prp5 and allows splicing to proceed. The CBS is required for splicing rescue by compensatory U2 mutants, and for formation of FIC, demonstrating a role for Msl5 in directing U2 to the BS, and of U2-BS base pairing for release of Prp5 and Msl5-Mud2 to form the prespliceosome. Our results provide insights into how the prespliceosome may form in normal splicing reaction.