Reconstitution of both steps of Saccharomyces cerevisiae splicing with purified spliceosomal components.

Nature Structural & Molecular Biology
Zbigniew WarkockiReinhard Lührmann

Abstract

The spliceosome is a ribonucleoprotein machine that removes introns from pre-mRNA in a two-step reaction. To investigate the catalytic steps of splicing, we established an in vitro splicing complementation system. Spliceosomes stalled before step 1 of this process were purified to near-homogeneity from a temperature-sensitive mutant of the RNA helicase Prp2, compositionally defined, and shown to catalyze efficient step 1 when supplemented with recombinant Prp2, Spp2 and Cwc25, thereby demonstrating that Cwc25 has a previously unknown role in promoting step 1. Step 2 catalysis additionally required Prp16, Slu7, Prp18 and Prp22. Our data further suggest that Prp2 facilitates catalytic activation by remodeling the spliceosome, including destabilizing the SF3a and SF3b proteins, likely exposing the branch site before step 1. Remodeling by Prp2 was confirmed by negative stain EM and image processing. This system allows future mechanistic analyses of spliceosome activation and catalysis.

References

Jan 1, 1990·Molecular and Cellular Biology·U Vijayraghavan, J Abelson
Dec 1, 1989·Genes & Development·P FabrizioJ Abelson
Feb 1, 1993·Genes & Development·D S Horowitz, J Abelson
Mar 1, 1996·Analytical Chemistry·A ShevchenkoM Mann
Jul 22, 1998·Molecular and Cellular Biology·O GozaniR Reed
Feb 13, 2001·Trends in Cell Biology·P BurkhardS V Strelkov
Sep 13, 2003·Science·Shih-Peng ChanSoo-Chen Cheng
Oct 24, 2003·Journal of Structural Biology·B SanderH Stark
Nov 25, 2003·BioEssays : News and Reviews in Molecular, Cellular and Developmental Biology·Timothy W Nilsen
Apr 20, 2005·Journal of Mass Spectrometry : JMS·Qizhi HuR Graham Cooks
May 26, 2005·Protein Expression and Purification·F William Studier
Dec 25, 2007·Nature Methods·Berthold KastnerHolger Stark
Mar 7, 2008·Nature·Sergey BessonovReinhard Lührmann
Jun 25, 2008·Annual Review of Biophysics·Anna Marie Pyle
Jun 28, 2008·Science·Chi-Kang Tseng, Soo-Chen Cheng
Feb 26, 2009·Cell·Markus C WahlReinhard Lührmann
Aug 26, 2009·Molecular and Cellular Biology·Ying-Fang ChiuSoo-Chen Cheng

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Citations

Nov 6, 2012·Nature Reviews. Drug Discovery·Sophie BonnalJuan Valcárcel
Nov 19, 2013·Nature Structural & Molecular Biology·Ramya KrishnanNils G Walter
Apr 10, 2013·Proceedings of the National Academy of Sciences of the United States of America·Daniel J CrawfordMelissa J Moore
Jan 31, 2012·Nucleic Acids Research·Jonathan ChangStewart Shuman
Mar 29, 2011·Cold Spring Harbor Perspectives in Biology·Cindy L Will, Reinhard Lührmann
Nov 21, 2012·Molecular and Cellular Biology·Hsin-Chou ChenSoo-Chen Cheng
Oct 17, 2012·Molecular and Cellular Biology·Hsueh-Lien Liu, Soo-Chen Cheng
Nov 9, 2011·Molecular and Cellular Biology·Wei ShaoCharles C Query
Jan 25, 2013·RNA·Janine O IlaganMelissa S Jurica
Mar 19, 2014·Annual Review of Biochemistry·Inga Jarmoskaite, Rick Russell
Oct 9, 2014·Nucleic Acids Research·Hsin-Chou ChenSoo-Chen Cheng
Feb 15, 2016·Wiley Interdisciplinary Reviews. RNA·Inessa DeVladimir Pena
Jan 20, 2016·Biochemistry·William PereaNancy L Greenbaum
Oct 20, 2015·RNA Biology·Piotr Wysoczanski, Markus Zweckstetter
Apr 7, 2012·Trends in Biochemical Sciences·Aaron A Hoskins, Melissa J Moore
Nov 8, 2011·Current Opinion in Chemical Biology·Aaron A HoskinsMelissa J Moore
Oct 15, 2011·Parasitology International·Renu Tuteja
Sep 29, 2015·Nature Methods·Mario R BlancoNils G Walter
Oct 21, 2011·Wiley Interdisciplinary Reviews. RNA·David S Horowitz
Apr 18, 2013·Protein Science : a Publication of the Protein Society·Lingdi ZhangRui Zhao
Nov 30, 2010·The EMBO Journal·Gert WeberMarkus C Wahl
Feb 19, 2015·BioMed Research International·Julien Robert-PaganinNicolas Leulliot
Apr 3, 2012·Current Opinion in Cell Biology·Olivier CordinJean D Beggs
Mar 5, 2013·Biochimica Et Biophysica Acta·Tien-Hsien ChangShang-Lin Chang
Aug 12, 2014·Biochemical Society Transactions·Chandani Warnasooriya, David Rueda

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