title: The Prp8 RNase H-like domain inhibits Brr2-mediated U4/U6 snRNA unwinding by blocking Brr2 loading onto the U4 snRNA.
abstract: The spliceosomal RNA helicase Brr2 catalyzes unwinding of the U4/U6 snRNA duplex, an essential step for spliceosome catalytic activation. Brr2 is regulated in part by the spliceosomal Prp8 protein by an unknown mechanism. We demonstrate that the RNase H (RH) domain of yeast Prp8 binds U4/U6 small nuclear RNA (snRNA) with the single-stranded regions of U4 and U6 preceding U4/U6 stem I, contributing to its binding. Via cross-linking coupled with mass spectrometry, we identify RH domain residues that contact the U4/U6 snRNA. We further demonstrate that the same single-stranded region of U4 preceding U4/U6 stem I is recognized by Brr2, indicating that it translocates along U4 and first unwinds stem I of the U4/U6 duplex. Finally, we show that the RH domain of Prp8 interferes with U4/U6 unwinding by blocking Brr2's interaction with the U4 snRNA. Our data reveal a novel mechanism whereby Prp8 negatively regulates Brr2 and potentially prevents premature U4/U6 unwinding during splicing. They also support the idea that the RH domain acts as a platform for the exchange of U6 snRNA for U1 at the 5' splice site. Our results provide insights into the mechanism whereby Brr2 unwinds U4/U6 and show how this activity is potentially regulated prior to spliceosome activation.
authors.affiliation: Department of Cellular Biochemistry, Max Planck Institute for Biophysical Chemistry, D-37077 Göttingen, Germany.
authors.forename: Sina
authors.initials: S
authors.lastname: Mozaffari-Jovin
authors.forename: Karine F
authors.initials: KF
authors.lastname: Santos
authors.forename: He-Hsuan
authors.initials: HH
authors.lastname: Hsiao
authors.forename: Cindy L
authors.initials: CL
authors.lastname: Will
authors.forename: Henning
authors.initials: H
authors.lastname: Urlaub
authors.forename: Markus C
authors.initials: MC
authors.lastname: Wahl
authors.forename: Reinhard
authors.initials: R
authors.lastname: Lührmann
mesh_terms: D008958:Models, Molecular; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D020365:RNA Helicases / Q000378:metabolism*; D012342:RNA, Small Nuclear / Q000378:metabolism*; D017414:Ribonucleoprotein, U4-U6 Small Nuclear / Q000235:genetics / Q000378:metabolism*; D017415:Ribonucleoprotein, U5 Small Nuclear / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: C077018:PRP8 protein, S cerevisiae; D012342:RNA, Small Nuclear; D017414:Ribonucleoprotein, U4-U6 Small Nuclear; D017415:Ribonucleoprotein, U5 Small Nuclear; D029701:Saccharomyces cerevisiae Proteins; C405482:U4 small nuclear RNA; C405486:U6 small nuclear RNA; C104158:BRR2 protein, S cerevisiae; D020365:RNA Helicases