TRNA ligase structure reveals kinetic competition between non-conventional mRNA splicing and mRNA decay.

Jirka Peschek,Peter Walter

doi:10.7554/elife.44199

Abstract

Yeast tRNA ligase (Trl1) is an essential trifunctional enzyme that catalyzes exon-exon ligation during tRNA biogenesis and the non-conventional splicing of HAC1 mRNA during the unfolded protein response (UPR). The UPR regulates the protein folding capacity of the endoplasmic reticulum (ER). ER stress activates Ire1, an ER-resident kinase/RNase, which excises an intron from HAC1 mRNA followed by exon-exon ligation by Trl1. The spliced product encodes for a potent transcription factor that drives the UPR. Here we report the crystal structure of Trl1 RNA ligase domain from Chaetomium thermophilum at 1.9 Å resolution. Structure-based mutational analyses uncovered kinetic competition between RNA ligation and degradation during HAC1 mRNA splicing. Incompletely processed HAC1 mRNA is degraded by Xrn1 and the Ski/exosome complex. We establish cleaved HAC1 mRNA as endogenous substrate for ribosome-associated quality control. We conclude that mRNA decay and surveillance mechanisms collaborate in achieving fidelity of non-conventional mRNA splicing during the UPR.

Highlights

RNA ligases are found in all domains of life
A yeast genetic screen in Saccharomyces cerevisiae revealed a single His(148)Tyr point mutation lying within Trl1-LIG that in vivo abolished the unfolded protein response (UPR) signaling yet did not impair tRNA splicing
While our efforts failed using S. cerevisiae-derived protein, we were successful using protein derived from Chaetomium thermophilum, a thermophilic fungus that previously proved invaluable for structural studies (Amlacher et al, 2011; Bock et al, 2014). ctTrl1 has the same tripartite domain structure as scTrl1 (Figure 1A) with 39% overall sequence identity (Figure 1—figure supplement 1)

Summary

Introduction

RNA ligases are found in all domains of life. They catalyze the ligation of RNA molecules via phosphodiester bonds during different RNA processing events, such as repair, editing and splicing (Popow et al, 2012). The CPD activity opens the 2’,3’ cyclic phosphate by hydrolysis to form a 3’-OH/2’ phosphate terminus, and, second, the KIN activity phosphorylates the 5’-OH in an NTP-dependent reaction – preferring GTP over ATP These first two steps ‘heal’ (i.e., modify) the RNA termini in preparation for the ligation reaction. Trl ligates tRNA halves after intron excision by the tRNA splicing endonuclease (SEN) complex (Greer et al, 1983; Peebles et al, 1983) It is a key component of the UPR, a major intracellular stress signaling pathway (Sidrauski et al, 1996). The evolutionarily most conserved – and in fungi sole – branch of the UPR signals via a unique, nonconventional mRNA splicing reaction: Protein folding stress in the ER activates the cytosolic endonuclease domain of the transmembrane stress sensor Ire (Cox et al, 1993; Mori et al, 1993). We further establish cleaved HAC1 mRNA as an endogenous substrate for ribosome-associated mRNA quality control

Results

Discussion

Materials and methods

Funding Funder