Structures of the fully assembled Saccharomyces cerevisiae spliceosome before activation

Abstract

The precatalytic spliceosome (B complex) is preceded by the pre-B complex. Here we report the cryo-electron microscopy structures of the pre-B and B complexes at average resolutions of 3.3 to 4.6 and 3.9 angstroms, respectively. In the pre-B complex, the duplex between the 5' splice site (5'SS) and U1 small nuclear RNA (snRNA) is recognized by Yhc1, Luc7, and the Sm ring. In the B complex, U1 small nuclear ribonucleoprotein is dissociated, the 5'-exon-5'SS sequences are translocated near U6 snRNA, and three B-specific proteins may orient the precursor messenger RNA. In both complexes, U6 snRNA is anchored to loop I of U5 snRNA, and the duplex between the branch point sequence and U2 snRNA is recognized by the SF3b complex. Structural analysis reveals the mechanism of assembly and activation for the yeast spliceosome.

PsiCLIP reveals dynamic RNA binding by DEAH-box helicases before and after exon ligation

“…Upon examining individual snRNAs, there is strong psiCLIP crosslinking located within and adjacent to the Sm sites, known to bind SmB (Figure 2g). The three-dimensional structure for the pre-catalytic spliceosomal complex pre-B (Bai et al, 2018) shows that the SmB protein is in close spatial proximity to the U1 snRNA nucleotides detected using psiCLIP ( Figure 2h). The 5' end of U1 snRNA extends into the direction of the SmB protein providing an explanation as to why the crosslinks are mainly found at the beginning of the Sm site motif.…”

“…Additionally, the crosslinking upstream of the Sm site identified by psiCLIP (G535, A536) are shown in the structure to interact with the long tail of the SmB-protein ( Figure 2h). This part of SmB could so far only be modelled into the U1 snRNP in the cryoEM structure of the yeast pre-B complex, due to its high flexibility (Bai et al, 2018). The psiCLIP data suggest that a similar configuration of RNA and SmB protein will be present in the other snRNPs ( Figure 2g).…”

“…The U2 snRNA region highlighted in purple is shown in G in the crystal structure. h) Region containing the U1 snRNP Sm ring of the spliceosomal complex pre-B (Bai et al, 2018) with the SmB protein in salmon, the nucleotides of U1 snRNA in gold, with those showing the highest psiCLIP signal in purple, corresponding to the purple boxes in g. Blue denotes the Sm site, as in g.…”

RNAs in the spliceosome: Insight from cryoEM structures

“…The first high‐resolution cryoEM structure of the spliceosome was the Schizosaccharomyces pombe ILS complex determined by the Shi lab in 2015 (Yan et al, ). In the following years, high‐resolution structures of the yeast U1 snRNP (Li et al, ), tri‐snRNP (Nguyen et al, ; Wan et al, ), A (Plaschka, Lin, Charenton, & Nagai, ), pre‐B (Bai, Wan, Yan, Lei, & Shi, ), B (Bai, Wan, Yan, Lei, & Shi, 2018; Plaschka, Lin, & Nagai, ), B act (Yan, Wan, Bai, Huang, & Shi, ), C (Galej et al, ; Wan, Yan, Bai, Huang, & Shi, ), C* (Fica et al, ; Yan, Wan, Bai, Huang, & Shi, ), P (Bai, Yan, Wan, Lei, & Shi, ; Liu et al, ; Wilkinson et al, ), and ILS (Wan, Yan, Bai, Lei, & Shi, ) complexes, as well as the human B (Bertram et al, ), B act (Haselbach et al, ; Zhang et al, ), C (Zhan, Yan, Zhang, Lei, & Shi, ), and C* (Bertram et al, ; Zhang et al, ) complexes were determined. These structures provided unprecedented details on the organization of RNA and protein components in the spliceosome and their functions in the splicing reaction.…”

Blocking late stages of splicing quickly limits pre-spliceosome assembly in vivo

“…Thanks to extensive biochemical and genetic studies (reviewed in [4]), together with high-resolution structures obtained by cryo-electron microscopy [5][6][7][8][9][10][11][12][13], we now have a comprehensive mechanistic understanding of splicing. However, until relatively recently, pre-mRNA splicing was studied mainly as an isolated process whereas, within the context of the cell, splicing functionally interacts with other cellular systems such as transcription, chromatin and RNA processing (reviewed in [14]).…”