Structure of the yeast spliceosomal postcatalytic P complex

Abstract

The spliceosome undergoes dramatic changes in a splicing cycle. Structures of B, Bact, C, C*, and ILS complexes revealed mechanisms of 5′ splice site (ss) recognition, branching, and intron release, but lacked information on 3′ ss recognition, exon ligation and release. Here, we report a cryoEM structure of the post-catalytic P complex at 3.3Å resolution, revealing that 3′ ss is mainly recognized through non-Watson-Crick basepairing with the 5′ ss and branch point. Furthermore, an unidentified protein becomes stably associated with the P complex, securing the 3′ exon and potentially regulating Prp22 activity. Prp22 binds nucleotides 15–21 in the 3′ exon, enabling it to pull the intron-exon or ligated exon in a 3′ to 5′ direction to achieve 3′ ss proofreading or exon release, respectively.

Cus2 enforces the first ATP-dependent step of splicing by binding to yeast SF3b1 through a UHM-ULM interaction

“…During the RNA rearrangements associated with the first catalytic step of splicing, U2 snRNA is remodeled to disrupt stem IIa and form stem IIc (Hilliker et al 2007;Perriman and Ares 2007;Galej et al 2016;Rauhut et al 2016;Wan et al 2016;Yan et al 2016;Bai et al 2017;Liu et al 2017;Plaschka et al 2017;Wan et al 2017;Wilkinson et al 2017;Yan et al 2017;Plaschka et al 2018). However, before it can be reused in a subsequent round of splicing U2 snRNA must be refolded into the stem IIa form, which makes critical contacts with SF3a and SF3b proteins for pre-spliceosome assembly (Liu et al 2017;Wilkinson et al 2017;Plaschka et al 2018).…”

“…These changes lead to release of some proteins and recruitment of others to the spliceosome, ensuring the fidelity and directionality of spliceosome assembly and catalysis (Koodathingal and Staley 2013). Recent cryo-electron microscopy (cryo-EM) models of spliceosomes at distinct steps (tri-snRNP, the A, B, B act , C, and P complexes) have brilliantly illuminated the composition and conformation of the spliceosome before and after each transition Yan et al 2015;Galej et al 2016;Rauhut et al 2016;Wan et al 2016;Yan et al 2016;Bai et al 2017;Bertram et al 2017a;Bertram et al 2017b;Liu et al 2017;Plaschka et al 2017;Wan et al 2017;Wilkinson et al 2017;Yan et al 2017;Zhang et al 2017;Plaschka et al 2018;Zhan et al 2018b;Zhan et al 2018a;Zhang et al 2018). A challenge now is to understand the distinct mechanism of action of each DExD/H protein in catalyzing its specific transition.…”

Structures of the Human Spliceosomes Before and After Release of the Ligated Exon

“…Recognition of the 3'SS is reminiscent of that in S. cerevisiae [33][34][35] . In particular, the guanine base of the 3'SS pairs up with the guanine (G1) at the 5'-end of the 5'SS through hydrogen bonds (H-bonds) 36,37 and stacks against an adenine from nucleotide A45 of U6 snRNA ( Fig.…”

Termination of pre-mRNA splicing requires that the ATPase and RNA unwindase Prp43 acts on the catalytic snRNA U6

“…1A) (Madhani and Guthrie 1994;Hang et al 2015). Following release of U4 and a suite of proteins, the nineteen complex (NTC) and other factors bind, and then catalytic core of the spliceosome engages the substrate and catalyzes the splicing reactions (Galej et al 2016;Liu et al 2017;Fabrizio et al 2009;Bai et al 2017;Wilkinson et al 2017;Wan et al 2019;Yan et al 2017;Wan et al 2016). Finally, the mRNA is released from the spliceosome, and then the excised intron is released for degradation in a manner coupled to spliceosome disassembly (Company et al 1991;Wagner et al 1998;Schwer and Gross 1998;Arenas and Abelson 1997;Martin et al 2002).…”

“…Of the eight SF2 ATPases that play a conserved role in splicing, the three DEAD box ATPases function early in assembly; the Ski2-like DExH ATPase functions during spliceosome activation; and the four DEAH ATPases function at the catalytic stage of splicing, suggesting distinct activities are required at different stages of splicing (Cordin and Beggs 2013). All four DEAH ATPases impact the interaction of the pre-mRNA substrate with the spliceosome: after spliceosome activation, Prp2 enables positioning of the branch site for branching (Wlodaver and Staley 2014;Warkocki et al 2015;Krishnan et al 2013;Liu and Cheng 2012;Rauhut et al 2016;Yan et al 2016); after branching, Prp16 promotes repositioning of the branch site to enable 3' splice site docking for exon ligation (Ohrt et al 2013;Galej et al 2016;Wan et al 2016;Semlow et al 2016;Schwer and Guthrie 1992); after exon ligation, Prp22p triggers release of the mRNA (Company et al 1991;Wilkinson et al 2017;Bai et al 2017;Liu et al 2017;Wagner et al 1998;Schwer and Gross 1998); and subsequently Prp43, which also functions in ribosome biogenesis (Leeds et al 2006;Combs et al 2006;Lebaron et al 2005), releases the excised intron for decay and coordinately disassembles the spliceosome for recycling (Arenas and Abelson 1997;Martin et al 2002;Wan et al 2017;Zhang et al 2019).…”