Content area
Full Text
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 A, 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 Â 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.
The spliceosome catalyses pre-mRNA splicing and is assembled by recruitment of U1 and U2 small nuclear (sn)RNPs to the 5' splice site and branch site, respectively, of the pre-mRNA intron, followed by integration of the preformed U4/U6.U5 tri-snRNP1. This generates the spliceosomal B complex, and after extensive RNP rearrangements, the pre-catalytic Bact complex is formed (Extended Data Fig. 1a). The latter is converted into the catalytically active B* complex by RNA helicase PRP2 (refs 2,3). During activation, a catalytic RNA-RNA network that resembles the catalytic core of group II self-splicing introns4-6, as well as the spliceosome's active site, are established. The B* complex catalyses step one of splicing, in which the branch site adenosine (BS-A) carries out a nucleophilic attack at the 5' splice site. This yields the cleaved 5' exon and the lariat-3' exon, in which the 5' end of the intron is covalently attached to the BS-A, forming a branched intron structure. Concomitantly the spliceosomal C complex is formed. The branched intron region must be displaced from the catalytic centre of the spliceosome after step one to allow juxtapositioning ofthe step-two reactants, the 3'-OH of the 5' exon (the nucleophile for step two) and the 3' splice site. This remodelling is catalysed by RNA helicase PRP16 and leads to the...