Journal Article

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

Odenwälder, Peter, Platzmann, Florian, Fabrizio, Patrizia, Ficner, Ralf, Warkocki, Zbigniew, Urlaub, Henning, Stark, Holger, Schmitzová, Jana, Lührmann, Reinhard

2009

Overview

Pre-mRNA splicing is catalyzed by the spliceosome in a two-step reaction. Both catalytic steps have now been reconstituted using purified, defined components. This system identifies a role for Cwc25 in the first step of splicing and allows future detailed mechanistic analyses of splicing. 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.

• Publisher: Nature Publishing Group US,Nature Publishing Group
• Subject: Adenosine Triphosphatases - isolation & purification / Adenosine Triphosphatases - metabolism / Biochemistry / Biological Microscopy / Biomedical and Life Sciences / Brewer's yeast / Catalysis / DEAD-box RNA Helicases - isolation & purification / DEAD-box RNA Helicases - metabolism / Genetic aspects / Health aspects / Homogeneity / Image Processing, Computer-Assisted / Life Sciences / Membrane Biology / Microscopy, Electron - methods / Models, Biological / Molecular biology / Molecular structure / Mutation / Physiological aspects / Protein Structure / Proteins / Ribonucleoprotein, U2 Small Nuclear - isolation & purification / Ribonucleoprotein, U2 Small Nuclear - metabolism / Ribonucleoprotein, U5 Small Nuclear - isolation & purification / Ribonucleoprotein, U5 Small Nuclear - metabolism / Ribonucleoproteins / Ribonucleoproteins, Small Nuclear - isolation & purification / Ribonucleoproteins, Small Nuclear - metabolism / RNA Helicases - isolation & purification / RNA Helicases - metabolism / RNA splicing / RNA Splicing Factors / RNA, Fungal - isolation & purification / RNA, Fungal - metabolism / Saccharomyces cerevisiae / Saccharomyces cerevisiae - metabolism / Saccharomyces cerevisiae Proteins - isolation & purification / Saccharomyces cerevisiae Proteins - metabolism / Spliceosomes - metabolism / Spliceosomes - ultrastructure / Structure