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

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.

Cooperative structure of the heterotrimeric pre-mRNA retention and splicing complex

“…In this extract, yeast B act∆prp2 spliceosomal complexes were assembled and purified as described in ref. 28 on site-specifically 32 P-labeled pre-mRNA. The pre-mRNA was produced by DNA splint-directed RNA ligation 74 of RNA fragments obtained by sitespecific DNA enzyme cleavage 75 of in vitro-transcribed actin pre-mRNA.…”

“…To this end, we generated a yeast strain carrying the prp2-1 mutation as well as a C-terminally tandem affinity purification (TAP)-tagged version of Snu17p. The prp2-1 mutation allows purification of B act∆prp2 complexes assembled on actin pre-mRNA 28 . Previous studies have indicated that the RES complex contacts U2 snRNP proteins 4,12 , suggesting that it might bind to the pre-mRNA in the vicinity of U2.…”

Dissection of the factor requirements for spliceosome disassembly and the elucidation of its dissociation products using a purified splicing system

“…In addition, the GTPase Snu114, which together with Brr2 is associated with the U5 snRNP, is also required for the activation step and is thought to regulate Brr2 activity (Small et al 2006). The transition from the B to the B act complex involves a significant change in the spliceosome's protein composition, with numerous polypeptides such as the U1 and U4/U6 proteins being displaced, while ;20 others, including those of the NTC (nineteen complex) are stably recruited to the B act complex (Chan et al 2003;Fabrizio et al 2009;Warkocki et al 2009). For catalytic activation of the spliceosome, the DEAH-box helicase Prp2 binds together with a cofactor, Spp2, and restructures the B act complex in an ATP-dependent manner, yielding the B* complex (Roy et al 1995;Silverman et al 2004); this leads to, among other things, destabilization and/or disruption of the binding of several spliceosomal proteins (Warkocki et al 2009;Lardelli et al 2010;Ohrt et al 2012).…”

“…The transition from the B to the B act complex involves a significant change in the spliceosome's protein composition, with numerous polypeptides such as the U1 and U4/U6 proteins being displaced, while ;20 others, including those of the NTC (nineteen complex) are stably recruited to the B act complex (Chan et al 2003;Fabrizio et al 2009;Warkocki et al 2009). For catalytic activation of the spliceosome, the DEAH-box helicase Prp2 binds together with a cofactor, Spp2, and restructures the B act complex in an ATP-dependent manner, yielding the B* complex (Roy et al 1995;Silverman et al 2004); this leads to, among other things, destabilization and/or disruption of the binding of several spliceosomal proteins (Warkocki et al 2009;Lardelli et al 2010;Ohrt et al 2012). Upon recruitment of the step 1 factor Cwc25 to the B* complex, step 1 catalysis occurs, whereby the adenosine at the BS attacks the 59 SS, generating the cleaved exon 1 and intron-lariat (IL)-39 exon intermediates (Chiu et al 2009;Warkocki et al 2009).…”

“…For catalytic activation of the spliceosome, the DEAH-box helicase Prp2 binds together with a cofactor, Spp2, and restructures the B act complex in an ATP-dependent manner, yielding the B* complex (Roy et al 1995;Silverman et al 2004); this leads to, among other things, destabilization and/or disruption of the binding of several spliceosomal proteins (Warkocki et al 2009;Lardelli et al 2010;Ohrt et al 2012). Upon recruitment of the step 1 factor Cwc25 to the B* complex, step 1 catalysis occurs, whereby the adenosine at the BS attacks the 59 SS, generating the cleaved exon 1 and intron-lariat (IL)-39 exon intermediates (Chiu et al 2009;Warkocki et al 2009). The catalytic center of the resulting C complex is remodeled by the DEAH-box helicase Prp16 as a prerequisite for the second step of splicing; i.e., exon ligation, a process that also requires the action of the step 2 factors Slu7 and Prp18 (Horowitz 2012).…”