The Evolutionarily Conserved Core Design of the Catalytic Activation Step of the Yeast Spliceosome

Abstract

Metazoan spliceosomes exhibit an elaborate protein composition required for canonical and alternative splicing. Thus, the minimal set of proteins essential for activation and catalysis remains elusive. We therefore purified in vitro assembled, precatalytic spliceosomal complex B, activated B(act), and step 1 complex C from the simple eukaryote Saccharomyces cerevisiae. Mass spectrometry revealed that yeast spliceosomes contain fewer proteins than metazoans and that each functional stage is very homogeneous. Dramatic compositional changes convert B to B(act), which is composed of approximately 40 evolutionarily conserved proteins that organize the catalytic core. Additional remodeling occurs concomitant with step 1, during which nine proteins are recruited to form complex C. The moderate number of proteins recruited to complex C will allow investigations of the chemical reactions in a fully defined system. Electron microscopy reveals high-quality images of yeast spliceosomes at defined functional stages, indicating that they are well-suited for three-dimensional structure analyses.

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

“…We therefore tested whether Snu17p interacts with pre-mRNA in the context of native spliceosomes. Because RES-complex proteins were previously shown to be most abundant and stably bound in activated yeast spliceosomes (B act ) 26,27 , we analyzed whether Snu17p contacts the pre-mRNA in purified B act complexes. 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.…”

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).…”

Structural and mechanistic insights into human splicing factor SF3b complex derived using an integrated approach guided by the cryo-EM density maps

“…43,109 Given the fact that yeast has a strong consensus sequence around the branch point adenosine when compared to that of metazoan introns, there is a special requirement for specific factors (p14) to maintain specificity for degenerate BPA recognition in metazoans. Moreover, p14 alone has been shown to lack specificity to bind the branch-point duplex (BPD).…”

“…[38][39][40][41][42][43][44][45][46][47][48][49][50][51] However, recent efforts to determine high resolution cryo-EM density maps, utilizing advances in detector technology 52 coupled with powerful statistical image processing algorithms 53,54 have yielded structures for the U4/U6.U5 trisnRNP complex from S. cerevisiae [55][56][57] as well as the U5.U2/U6 intron lariat spliceosome (ILS) complex from S.pombe. [58][59][60] However, in the yeast U5.U2/U6 structure, the U2 snRNP region has an overall resolution of 11 A and lacks the density for the SF3b subcomplex.…”