Mechanism of non-spliceosomal mRNA splicing in the unfolded protein response pathway

The unfolded protein response is an intracellular signaling pathway that, i n response to accumulation of misfolded proteins in the lumen of the endopl asmic reticulum (ER), upregulates transcription of ER resident chaperones. A key step in this pathway is the nonconventional, regulated splicing of th e mRNA encoding the positive transcriptional regulator Hac1p, In the yeast Saccharomyces cerevisiae, the bifunctional transmembrane kinase/endoribonuc lease Ire1p cleaves HAC1 mRNA at both splice junctions and tRNA ligase join s the two exons together. We have reconstituted HAC1 mRNA splicing in an ef ficient in vitro reaction and show that, in many ways, the mechanism of HAC 1 mRNA splicing resembles that of pre-tRNA splicing. In particular, Ire1p e ndonucleolytic cleavage leaves 2',3'-cyclic phosphates, the excised exons r emain associated by base pairing, and exon ligation by tRNA ligase follows the same chemical steps as for pre-tRNA splicing. To date, this mechanism o f RNA processing is unprecedented for a messenger RNA. In contrast to the s triking similarities to tRNA splicing, the structural features of the splic e junctions recognized by Ire1p differ from those recognized by tRNA endonu clease. We show that small stem-loop structures predicted to form at both s plice junctions of HAC1 mRNA are required and sufficient for Ire1p cleavage .