Structure of a spliceosome remodelled for exon ligation

Abstract

The spliceosome excises introns from pre-mRNAs in two sequential transesterifications – branching and exon ligation1 – catalysed at a single catalytic metal site in U6 snRNA2,3. The recent structures of the spliceosomal C complex4,5 with the cleaved 5’-exon and lariat—3’-exon bound to the catalytic centre revealed that branching-specific factors such as Cwc25 lock the branch helix into position for nucleophilic attack of the branch adenosine at the 5’-splice site. Furthermore, the ATPase Prp16 is positioned to bind and translocate the intron downstream of the branch point to destabilize branching-specific factors and release the branch helix from the active site4. Here we present the 3.8Å cryo-EM structure of a Saccharomyces cerevisiae spliceosome stalled after Prp16-mediated remodelling but prior to exon ligation. While the U6 snRNA catalytic core remains firmly held in the active site cavity of Prp8 by proteins common to both steps, the branch helix has rotated by 75 degrees compared to complex C and is stabilized into a new position by Prp17, Cef1, and the reoriented Prp8 RNaseH domain. This rotation of the branch helix removes the branch adenosine from the catalytic core, creates a space for 3’-exon docking, and restructures the pairing of the 5’-splice site with the U6 snRNA ACAGAGA region. Slu7 and Prp18, which promote exon ligation, bind together to the Prp8 RNaseH domain. The ATPase Prp22, bound to Prp8 in place of Prp16, could interact with the 3’-exon, suggesting a possible basis for mRNA release after exon ligation6,7. Together with the C complex structure4, our new C* complex structure reveals the two major conformations of the spliceosome during the catalytic stages of splicing.

Splicing Factor Mutations in Myelodysplasias: Insights from Spliceosome Structures

“…Second, the global conformations of the tandem ZnK1 and ZnK2 differ between the two structures: the MBNL1 ZnK1 and ZnK2 each bind separate RNA strands whereas the TIS11d ZnK1 and ZnK2 bind adjacent sites of a contiguous oligonucleotide. The new spliceosome structures now reveal two examples of CCCH-type ZnK’s bound to RNA: CWC24 bound to the 5′ exon of the B ACT complex [43] and CWC2 bound to U6 snRNA in the B ACT , C, C*, and ILS complexes [43, 44, 70–75]. Moreover, the recent SpU2AF MIN structure [46] contains the full length fission yeast U2AF1 subunit, which shares 55% sequence identity with human U2AF1 and as such provides a reliable starting point for modeling putative RNA interactions with the mutated ZnK1/ZnK2 domains.…”

Crystal structure of U2 snRNP SF3b components: Hsh49p in complex with Cus1p-binding domain

“…At the early stage of its assembly, U1 and U2 snRNPs recognize the 5 ′ splice site and the branch point sequence (BPS), respectively, and initiate the assembly of the spliceosome. The recent structures of an activated B-complex spliceosome (B act ) (Rauhut et al 2016;Yan et al 2016), C-complex (Galej et al 2016;Wan et al 2016), and C * -complex (Bertram et al 2017;Fica et al 2017;Yan et al 2017) have provided important insights into the mechanism of pre-mRNA splicing. The branch helix is formed when the pre-mRNA BPS pairs with U2 snRNA in U2 snRNP and is escorted into the active site of the spliceosome.…”

Electron Bio-Imaging Centre (eBIC): the UK national research facility for biological electron microscopy

“…The data collected at eBIC have, at the time of writing, generated ten research publications (Hospenthal et al, 2016;Serna et al, 2016;Joseph et al, 2016;Wilkinson et al, 2016;Iadanza et al, 2016;Ramsay et al, 2016;Fica et al, 2017;Swuec et al, 2017;Ilangovan et al, 2017;Boland et al, 2017). We have also received a number of personal communications from inhouse and external users reporting reconstructions at better than 4 Å resolution, with a few extending beyond 3 Å .…”