title: A novel mechanism for Prp5 function in prespliceosome formation and proofreading the branch site sequence.
abstract: The DEAD-box RNA helicase Prp5 is required for the formation of the prespliceosome through an ATP-dependent function to remodel U2 small nuclear ribonucleoprotein particles (snRNPs) and an ATP-independent function of unknown mechanism. Prp5 has also been implicated in proofreading the branch site sequence, but the molecular mechanism has not been well characterized. Using actin precursor mRNA (pre-mRNA) carrying branch site mutations, we identified a Prp5-containing prespliceosome with Prp5 directly bound to U2 small nuclear RNA (snRNA). Prp5 is in contact with U2 in regions on and near the branchpoint-interacting stem-loop (BSL), suggesting that Prp5 may function in stabilizing the BSL. Regardless of its ATPase activity, Prp5 mutants that suppress branch site mutations associate with the spliceosome less tightly and allow more tri-snRNP binding for the reaction to proceed. Our results suggest a novel mechanism for how Prp5 functions in prespliceosome formation and proofreading of the branch site sequence. Prp5 binds to the spliceosome in association with U2 by interacting with the BSL and is released upon the base-pairing of U2 with the branch site to allow the recruitment of the tri-snRNP. Mutations impairing U2-branch site base-pairing retard Prp5 release and impede tri-snRNP association. Prp5 mutations that destabilize the Prp5-U2 interaction suppress branch site mutations by allowing progression of the pathway.
authors.affiliation: Institute of Molecular Biology, Academia Sinica, Taipei 115, Taiwan; Institute of Microbiology and Immunology, National Yang-Ming University, Taipei 112, Taiwan.
authors.forename: Wen-Wei
authors.initials: WW
authors.lastname: Liang
authors.affiliation: Institute of Molecular Biology, Academia Sinica, Taipei 115, Taiwan;
authors.forename: Soo-Chen
authors.initials: SC
authors.lastname: Cheng
mesh_terms: D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism; D064447:Checkpoint Kinase 2 / Q000235:genetics / Q000378:metabolism; D053487:DEAD-box RNA Helicases / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D011485:Protein Binding; D012322:RNA Precursors / Q000378:metabolism; D012326:RNA Splicing; D017411:Ribonucleoproteins, Small Nuclear / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D017381:Spliceosomes / Q000378:metabolism*
chemical_list: D018797:Cell Cycle Proteins; D012322:RNA Precursors; D017411:Ribonucleoproteins, Small Nuclear; D029701:Saccharomyces cerevisiae Proteins; D064447:Checkpoint Kinase 2; C067022:RAD53 protein, S cerevisiae; C064447:PRP5 protein, S cerevisiae; D053487:DEAD-box RNA Helicases
keywords: Prp5; U2; branch site; proofreading; splicing