Intron removal requires assembly of the spliceosome on pre-mRNA and extensive remodelling to form the spliceosome’s catalytic centre. Here we report the cryo-electron microscopy structure of the yeast pre-catalytic B complex spliceosome at near-atomic resolution. The mobile U2 snRNP associates with U4/U6.U5 tri-snRNP through U2/U6 helix II and an interface between U4/U6 di-snRNP and the U2 snRNP SF3b-containing domain, which also transiently contacts the helicase Brr2. The U2 snRNP 3’ region is flexibly attached to the SF3b-containing domain and protrudes over the concave surface of tri-snRNP, where the U1 snRNP may reside before its release from the pre-mRNA 5’-splice site. The U6 ACAGAGA sequence forms a hairpin which weakly tethers the 5’-splice site. B complex proteins Prp38, Snu23, and Spp381 bind the Prp8 N-terminal domain and stabilise U6 ACAGAGA stem–pre-mRNA and Brr2–U4 snRNA interactions. The results thus provide important insights into events leading to active site formation.
The spliceosome catalyses the excision of introns from pre-mRNA in two steps, branching and exon ligation, and is assembled from five small nuclear ribonucleoprotein particles (snRNPs; U1, U2, U4, U5, U6) and numerous non-snRNP factors. For branching, the intron 5' splice site and the branch point sequence are selected and brought by the U1 and U2 snRNPs into the prespliceosome, which is a focal point for regulation by alternative splicing factors. The U4/U6.U5 tri-snRNP subsequently joins the prespliceosome to form the complete pre-catalytic spliceosome. Recent studies have revealed the structural basis of the branching and exon-ligation reactions, however, the structural basis of the early events in spliceosome assembly remains poorly understood. Here we report the cryo-electron microscopy structure of the yeast Saccharomyces cerevisiae prespliceosome at near-atomic resolution. The structure reveals an induced stabilization of the 5' splice site in the U1 snRNP, and provides structural insights into the functions of the human alternative splicing factors LUC7-like (yeast Luc7) and TIA-1 (yeast Nam8), both of which have been linked to human disease. In the prespliceosome, the U1 snRNP associates with the U2 snRNP through a stable contact with the U2 3' domain and a transient yeast-specific contact with the U2 SF3b-containing 5' region, leaving its tri-snRNP-binding interface fully exposed. The results suggest mechanisms for 5' splice site transfer to the U6 ACAGAGA region within the assembled spliceosome and for its subsequent conversion to the activation-competent B-complex spliceosome. Taken together, the data provide a working model to investigate the early steps of spliceosome assembly.
SF3a is an evolutionarily conserved heterotrimeric complex essential for pre-mRNA splicing. It functions in spliceosome assembly within the mature U2 snRNP (small nuclear ribonucleoprotein particle), and its displacement from the spliceosome initiates the first step of the splicing reaction. We have identified a core domain of the yeast SF3a complex required for complex assembly and determined its crystal structure. The structure shows a bifurcated assembly of three subunits, Prp9, Prp11 and Prp21, with Prp9 interacting with Prp21 via a bidentate-binding mode, and Prp21 wrapping around Prp11. Structureguided biochemical analysis also shows that Prp9 harbours a major binding site for stem-loop IIa of U2 snRNA. These findings provide mechanistic insights into the assembly of U2 snRNP.
HighlightsRecent advances in cryoEM are revolutionizing our understanding of how molecular machines function.The structure of Saccharomyces cerevisiae U4/U6.U5 tri-snRNP has been revealed.The structure of Schizosaccharomyces pombe U2.U6.U5 spliceosomal complex has been revealed.These structures greatly advanced our understanding of the mechanism of pre-mRNA splicing.
The removal of noncoding introns from pre-messenger RNA (pre-mRNA) is an essential step in eukaryotic gene expression and is catalyzed by a dynamic multi-megadalton ribonucleoprotein complex called the spliceosome. The spliceosome assembles on pre-mRNA substrates by the stepwise addition of small nuclear ribonucleoprotein particles and numerous protein factors. Extensive remodeling is required to form the RNA-based active site and to mediate the pre-mRNA branching and ligation reactions. In the past two years, cryo-electron microscopy (cryo-EM) structures of spliceosomes captured in different assembly and catalytic states have greatly advanced our understanding of its mechanism. This was made possible by long-standing efforts in the purification of spliceosome intermediates as well as recent developments in cryo-EM imaging and computational methodology. The resulting high-resolution densities allow for de novo model building in core regions of the complexes. In peripheral and less ordered regions, the combination of cross-linking, bioinformatics, biochemical, and genetic data is essential for accurate modeling. Here, we summarize these achievements and highlight the critical steps in obtaining near-atomic resolution structures of the spliceosome.
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