A Snu114-GTP-Prp8 module forms a relay station for efficient splicing in yeast.

Bernhard Loll,Claudia Alings,Eva Absmeier,Florian Heyd,Junqiao Jia,Marco Preußner,Markus C Wahl,Oleg M Ganichkin

doi:10.1093/nar/gkaa182

Abstract

The single G protein of the spliceosome, Snu114, has been proposed to facilitate splicing as a molecular motor or as a regulatory G protein. However, available structures of spliceosomal complexes show Snu114 in the same GTP-bound state, and presently no Snu114 GTPase-regulatory protein is known. We determined a crystal structure of Snu114 with a Snu114-binding region of the Prp8 protein, in which Snu114 again adopts the same GTP-bound conformation seen in spliceosomes. Snu114 and the Snu114–Prp8 complex co-purified with endogenous GTP. Snu114 exhibited weak, intrinsic GTPase activity that was abolished by the Prp8 Snu114-binding region. Exchange of GTP-contacting residues in Snu114, or of Prp8 residues lining the Snu114 GTP-binding pocket, led to temperature-sensitive yeast growth and affected the same set of splicing events in vivo. Consistent with dynamic Snu114-mediated protein interactions during splicing, our results suggest that the Snu114–GTP–Prp8 module serves as a relay station during spliceosome activation and disassembly, but that GTPase activity may be dispensable for splicing.

Highlights

Precursor messenger RNA splicing entails the removal of non-coding introns and the ligation of neighboring coding exons and represents a key co-/posttranscriptional gene expression and gene regulatory process in eukaryotes
As Snu114 bears close resemblance to the ribosomal translocases, EF-G/eEF2 (Figure 1A), which undergo large-scale conformational changes upon binding to ribosomes, and as the Snu114 GTPase has been implicated in U5 snRNP or U4/U6U5 tri-snRNP assembly [12,13], we wondered whether a wider conformational spectrum may be accessible to the protein outside its spliceosomal environments
Prp8SBR is largely congruent with the Nterminal three quarters of a Prp8 element previously characterized as a Snu114/Cwc21-interacting domain (SCwid; Prp8 residues 253–543) [33]

Summary

Introduction

Precursor messenger RNA (pre-mRNA) splicing entails the removal of non-coding introns and the ligation of neighboring coding exons and represents a key co-/posttranscriptional gene expression and gene regulatory process in eukaryotes. Splicing is mediated by the spliceosome, an elaborate RNA-protein (RNP) molecular machine that encompasses five small nuclear (sn) RNPs (U1, U2, U4, U5 and U6 in the case of the major spliceosome) and many non-snRNP factors [1,2]. Almost all of the comparatively small number of introncontaining genes in yeast harbor a single intron, and the resulting pre-mRNAs are spliced constitutively [1]. Most genes in higher eukaryotes contain more than one intron and their pre-mRNAs can be spliced in a flexible manner, giving rise to different mature mRNAs that contain different combinations of exons (alternative splicing) [4]

Methods

Results

Conclusion