Reconstitution of both steps of Saccharomyces cerevisiae splicing with purified spliceosomal components.
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.
Warkocki
Zbigniew
Z
Department of Cellular Biochemistry, Max Planck Institute of Biophysical Chemistry, Göttingen, Germany.
Odenwälder
Peter
P
Schmitzová
Jana
J
Platzmann
Florian
F
Stark
Holger
H
Urlaub
Henning
H
Ficner
Ralf
R
Fabrizio
Patrizia
P
Lührmann
Reinhard
R
D000251:Adenosine Triphosphatases / Q000302:isolation & purification / Q000378:metabolism; D053487:DEAD-box RNA Helicases / Q000302:isolation & purification / Q000378:metabolism; D007091:Image Processing, Computer-Assisted; D008854:Microscopy, Electron / Q000379:methods; D008954:Models, Biological; D020365:RNA Helicases / Q000302:isolation & purification / Q000378:metabolism; D000072260:RNA Splicing Factors; D012331:RNA, Fungal / Q000302:isolation & purification* / Q000378:metabolism*; D017413:Ribonucleoprotein, U2 Small Nuclear / Q000302:isolation & purification / Q000378:metabolism; D017415:Ribonucleoprotein, U5 Small Nuclear / Q000302:isolation & purification / Q000378:metabolism; D017411:Ribonucleoproteins, Small Nuclear / Q000302:isolation & purification / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000302:isolation & purification* / Q000378:metabolism*; D017381:Spliceosomes / Q000378:metabolism* / Q000648:ultrastructure
C544745:Cwc25 protein, S cerevisiae; C080779:PRP18 protein, S cerevisiae; D000072260:RNA Splicing Factors; D012331:RNA, Fungal; D017413:Ribonucleoprotein, U2 Small Nuclear; D017415:Ribonucleoprotein, U5 Small Nuclear; D017411:Ribonucleoproteins, Small Nuclear; C095479:SLU7 protein, S cerevisiae; C096994:SPP2 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; C507323:YSF3 protein, S cerevisiae; D000251:Adenosine Triphosphatases; C075669:PRP16 protein, S cerevisiae; C066490:PRP2 protein, S cerevisiae; D053487:DEAD-box RNA Helicases; D020365:RNA Helicases