Cryo-EM structure of a human spliceosome activated for step 2 of splicing

Abstract

Spliceosome rearrangements facilitated by RNA helicase PRP16 before catalytic step two of splicing are poorly understood. Here we report a 3D cryo-electron microscopy structure of the human spliceosomal C complex stalled directly after PRP16 action (C*). The architecture of the catalytic U2-U6 ribonucleoprotein (RNP) core of the human C* spliceosome is very similar to that of the yeast pre-Prp16 C complex. However, in C* the branched intron region is separated from the catalytic centre by approximately 20 Å, and its position close to the U6 small nuclear RNA ACAGA box is stabilized by interactions with the PRP8 RNase H-like and PRP17 WD40 domains. RNA helicase PRP22 is located about 100 Å from the catalytic centre, suggesting that it destabilizes the spliced mRNA after step two from a distance. Comparison of the structure of the yeast C and human C* complexes reveals numerous RNP rearrangements that are likely to be facilitated by PRP16, including a large-scale movement of the U2 small nuclear RNP.

A sequence-based, deep learning model accurately predicts RNA splicing branchpoints

“…This extended base pairing is observed in a recent cryo-EM structure of the human spliceosome 11 and is consistent with an early biochemical study showing that "SAP 145, together with four other SF3a/SF3b subunits, UV cross-links to pre-mRNA in a 20-nucleotide region upstream of the BPS" 19 . This region of U2 snRNA shares 100% sequence identity with U2 snRNA in budding yeast, albeit in humans 4 of these bases are modified to form psuedouridines, while in yeast only 2 have this modification (Fig.…”

“…We conclude that branchpoint strength plays a role, similar to that of 3'ss strength, in alternative splicing. We identify a novel upstream recognition element, which is consistent with a recent cryo-EM model of the spliceosome depicting the relevant bases in duplex with U2 snRNA 11 . Finally, we show that LaBranchoR predictions overlap with more pathogenic variants than previous computational predictions, as well as the raw data itself.…”

“…To the best of our knowledge, this sequence motif has not been previously observed in association with branchpoints and perhaps represents a novel sequence feature aiding in branchpoint recognition. A recent cryo-EM spliceosome structure shows these bases in duplex with U2 snRNA 11 (Fig. 4e).…”

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.…”

Mobile Group II Introns as Ancestral Eukaryotic Elements

“…It is a highly dynamic RNP complex that comprises five small nuclear RNAs (snRNAs) and a multitude of proteins forming a tight interacting network of molecules [64]. In the past few years, cryo-EM has led to profound structural insights of a number of spliceosomes at different stages of splicing [70–73]. Although the origin and evolution of spliceosomal introns and spliceosomes can be a polarizing question [74, 75], structural and functional comparison between the spliceosomal and group II intron complexes make their close relationship irrefutable.…”