title: The large N-terminal region of the Brr2 RNA helicase guides productive spliceosome activation.
abstract: The Brr2 helicase provides the key remodeling activity for spliceosome catalytic activation, during which it disrupts the U4/U6 di-snRNP (small nuclear RNA protein), and its activity has to be tightly regulated. Brr2 exhibits an unusual architecture, including an ∼ 500-residue N-terminal region, whose functions and molecular mechanisms are presently unknown, followed by a tandem array of structurally similar helicase units (cassettes), only the first of which is catalytically active. Here, we show by crystal structure analysis of full-length Brr2 in complex with a regulatory Jab1/MPN domain of the Prp8 protein and by cross-linking/mass spectrometry of isolated Brr2 that the Brr2 N-terminal region encompasses two folded domains and adjacent linear elements that clamp and interconnect the helicase cassettes. Stepwise N-terminal truncations led to yeast growth and splicing defects, reduced Brr2 association with U4/U6•U5 tri-snRNPs, and increased ATP-dependent disruption of the tri-snRNP, yielding U4/U6 di-snRNP and U5 snRNP. Trends in the RNA-binding, ATPase, and helicase activities of the Brr2 truncation variants are fully rationalized by the crystal structure, demonstrating that the N-terminal region autoinhibits Brr2 via substrate competition and conformational clamping. Our results reveal molecular mechanisms that prevent premature and unproductive tri-snRNP disruption and suggest novel principles of Brr2-dependent splicing regulation.
authors.affiliation: Laboratory of Structural Biochemistry, Freie Universität Berlin, D-14195 Berlin, Germany;
authors.forename: Eva
authors.initials: E
authors.lastname: Absmeier
authors.affiliation: Laboratory of Structural Biochemistry, Freie Universität Berlin, D-14195 Berlin, Germany;
authors.forename: Jan
authors.initials: J
authors.lastname: Wollenhaupt
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.affiliation: Laboratory of Structural Biochemistry, Freie Universität Berlin, D-14195 Berlin, Germany;
authors.forename: Christian
authors.initials: C
authors.lastname: Becke
authors.affiliation: Research Group Bioanalytical Mass Spectrometry, Max Planck Institute for Biophysical Chemistry, D-37077 Göttingen, Germany; Bioanalytics Group, Institute for Clinical Chemistry, University Medical Center Göttingen, Georg-August-Universität, D-37099 Göttingen, Germany;
authors.forename: Chung-Tien
authors.initials: CT
authors.lastname: Lee
authors.affiliation: Laboratory of RNA Biochemistry, Freie Universität Berlin, D-14195 Berlin, Germany.
authors.forename: Marco
authors.initials: M
authors.lastname: Preussner
authors.affiliation: Laboratory of RNA Biochemistry, Freie Universität Berlin, D-14195 Berlin, Germany.
authors.forename: Florian
authors.initials: F
authors.lastname: Heyd
authors.affiliation: Research Group Bioanalytical Mass Spectrometry, Max Planck Institute for Biophysical Chemistry, D-37077 Göttingen, Germany; Bioanalytics Group, Institute for Clinical Chemistry, University Medical Center Göttingen, Georg-August-Universität, D-37099 Göttingen, Germany;
authors.forename: Henning
authors.initials: H
authors.lastname: Urlaub
authors.affiliation: Department of Cellular Biochemistry, Max Planck Institute for Biophysical Chemistry, D-37077 Göttingen, Germany;
authors.forename: Reinhard
authors.initials: R
authors.lastname: Lührmann
authors.affiliation: Laboratory of Structural Biochemistry, Freie Universität Berlin, D-14195 Berlin, Germany;
authors.forename: Karine F
authors.initials: KF
authors.lastname: Santos
authors.affiliation: Laboratory of Structural Biochemistry, Freie Universität Berlin, D-14195 Berlin, Germany;
authors.forename: Markus C
authors.initials: MC
authors.lastname: Wahl
mesh_terms: D000251:Adenosine Triphosphatases / Q000378:metabolism; D002597:Chaetomium / Q000201:enzymology / Q000235:genetics; D003460:Crystallization; D006801:Humans; D008958:Models, Molecular; D011485:Protein Binding; D017510:Protein Folding; D019154:Protein Splicing; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D020365:RNA Helicases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism; D017414:Ribonucleoprotein, U4-U6 Small Nuclear / Q000737:chemistry / Q000378:metabolism; D017415:Ribonucleoprotein, U5 Small Nuclear / Q000737:chemistry / Q000378:metabolism; D017411:Ribonucleoproteins, Small Nuclear / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics / Q000254:growth & development; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017381:Spliceosomes / Q000201:enzymology* / Q000235:genetics
chemical_list: C077018:PRP8 protein, S cerevisiae; C115374:PRPF8 protein, human; D016601:RNA-Binding Proteins; D017414:Ribonucleoprotein, U4-U6 Small Nuclear; D017415:Ribonucleoprotein, U5 Small Nuclear; D017411:Ribonucleoproteins, Small Nuclear; C545711:SNRNP200 protein, human; D029701:Saccharomyces cerevisiae Proteins; D000251:Adenosine Triphosphatases; C104158:BRR2 protein, S cerevisiae; D020365:RNA Helicases
keywords: RNA helicase structure and function; X-ray crystallography; pre-mRNA splicing; remodeling of RNA–protein complexes; spliceosome catalytic activation