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.
Subject(s)
RNA, Fungal/isolation & purification , RNA, Fungal/metabolism , Saccharomyces cerevisiae Proteins/isolation & purification , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Spliceosomes/metabolism , Adenosine Triphosphatases/isolation & purification , Adenosine Triphosphatases/metabolism , DEAD-box RNA Helicases/isolation & purification , DEAD-box RNA Helicases/metabolism , Image Processing, Computer-Assisted , Microscopy, Electron/methods , Models, Biological , RNA Helicases/isolation & purification , RNA Helicases/metabolism , RNA Splicing Factors , Ribonucleoprotein, U2 Small Nuclear/isolation & purification , Ribonucleoprotein, U2 Small Nuclear/metabolism , Ribonucleoprotein, U5 Small Nuclear/isolation & purification , Ribonucleoprotein, U5 Small Nuclear/metabolism , Ribonucleoproteins, Small Nuclear/isolation & purification , Ribonucleoproteins, Small Nuclear/metabolism , Spliceosomes/ultrastructureABSTRACT
Reductive S-S bond cleavage of a disulfide precursor obtained from a pyridine-catalyzed Peterson-type reaction starting from camphorpyrazole , thionyl chloride and 2-methyl-2-(methyldithio)propionaldehyde yields 2,2'-bis(camphorpyrazol-1-yl)-2-methylpropane-2-thiol (HSiprbpm3cam, ); the first zinc complexes bearing this ligand exhibit kappa3 coordination of the ligand.
