Multiple decay events target HAC1 mRNA during splicing to regulate the unfolded protein response

Abstract

In the unfolded protein response (UPR), protein-folding stress in the endoplasmic reticulum (ER) activates a large transcriptional program to increase ER folding capacity. During the budding yeast UPR, the trans-ER-membrane kinase-endoribonuclease Ire1 excises an intron from the HAC1 mRNA and the exon cleavage products are ligated and translated to a transcription factor that induces hundreds of stress-response genes. HAC1 cleavage by Ire1 is thought to be the rate limiting step of its processing. Using cells with mutations in RNA repair and decay enzymes, we show that phosphorylation of two different HAC1 splicing intermediates by Trl1 RNA 5′-kinase is required for their degradation by the 5′→3′ exonuclease Xrn1 to enact opposing effects on the UPR. Kinase-mediated decay (KMD) of cleaved HAC1 3′-exon competes with its ligation to limit productive splicing and suppress the UPR, whereas KMD of the excised intron activates HAC1 translation, likely by relieving an inhibitory base-pairing interaction between the intron and 5′-untranslated region. We also found that ligated but 2′-phosphorylated HAC1 mRNA is endonucleolytically cleaved, yielding a KMD intermediate with both 5′-and 2′-phosphates at its 5′-end that inhibit 5′→3′ decay, and suggesting that Ire1 initiates the degradation of incompletely processed HAC1 s to proofread ligation or attenuate the UPR. These multiple decay events expand the scope of RNA-based regulation in the budding yeast UPR and may have implications for the control of the metazoan UPR by mRNA processing.

Reconstitution of the Human tRNA Splicing Endonuclease Complex: insight into the regulation of pre-tRNA cleavage

“…Therefore, we propose that CLP1 acts as a critical negative regulator of tricRNA biogenesis and tRNA maturation. Beyond tRNA splicing CLP1 may also function as a negative regulator of other RNA processing pathways dependent upon RtcB ligation such as the unfolded protein response (68)(69)(70)(71).…”

No-Go Decay mRNA cleavage in the ribosome exit tunnel produces 5′-OH ends phosphorylated by Trl1

“…We therefore asked whether the 3′-NGD B4 RNA fragments were substrates of Trl1. Since TRL1 is an essential gene, we used trl1Δ strains expressing pre-spliced intron-less versions of the 10 intron-containing tRNAs that restore viability 33,34 . If Trl1 were required for B4 degradation, loss of Trl1 function should increase the amount of B4 RNA.…”

tRNA introns: Presence, processing, and purpose

“…Due to its small size, the fate of the metazoan XBP1 intron is unknown. However, a yeast strain that replaces Rlg1/Trl1 with RtcB was found to circularize the HAC1 mRNA intron (Cherry et al, ). These findings provide further credence to the notion that RtcB is the critical RNA circularization factor, as yeast with endogenous Rlg1/Trl1 do not generate circular HAC1 introns (Cherry et al, ).…”

“…The cleaved exons are then joined by the tRNA ligase Rlg1/Trl1. Upon translation, this unconventionally-spliced mRNA encodes a transcription factor that activates stress response genes (Cherry, Peach, & Hesselberth, 2019). The situation is similar in metazoa: a close homolog of Ire1 cleaves XBP1 mRNA, and RtcB ligates the exons (Kosmaczewski et al, 2014).…”