title: Mechanism of non-spliceosomal mRNA splicing in the unfolded protein response pathway.
abstract: The unfolded protein response is an intracellular signaling pathway that, in response to accumulation of misfolded proteins in the lumen of the endoplasmic reticulum (ER), upregulates transcription of ER resident chaperones. A key step in this pathway is the non-conventional, regulated splicing of the mRNA encoding the positive transcriptional regulator Hac1p. In the yeast Saccharomyces cerevisiae, the bifunctional transmembrane kinase/endoribonuclease Ire1p cleaves HAC1 mRNA at both splice junctions and tRNA ligase joins the two exons together. We have reconstituted HAC1 mRNA splicing in an efficient in vitro reaction and show that, in many ways, the mechanism of HAC1 mRNA splicing resembles that of pre-tRNA splicing. In particular, Ire1p endonucleolytic cleavage leaves 2', 3'-cyclic phosphates, the excised exons remain associated by base pairing, and exon ligation by tRNA ligase follows the same chemical steps as for pre-tRNA splicing. To date, this mechanism of RNA processing is unprecedented for a messenger RNA. In contrast to the striking similarities to tRNA splicing, the structural features of the splice junctions recognized by Ire1p differ from those recognized by tRNA endonuclease. We show that small stem-loop structures predicted to form at both splice junctions of HAC1 mRNA are required and sufficient for Ire1p cleavage.
authors.affiliation: Howard Hughes Medical Institute and Department of Biochemistry and Biophysics, University of California at San Francisco, San Francisco, CA 94143-0448, USA.
authors.forename: T N
authors.initials: TN
authors.lastname: Gonzalez
authors.forename: C
authors.initials: C
authors.lastname: Sidrauski
authors.forename: S
authors.initials: S
authors.lastname: Dörfler
authors.forename: P
authors.initials: P
authors.lastname: Walter
mesh_terms: D020029:Base Pairing; D001483:Base Sequence; D050976:Basic-Leucine Zipper Transcription Factors; D005091:Exons / Q000235:genetics; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism; D006151:Guanosine / Q000235:genetics / Q000378:metabolism; D006868:Hydrolysis; D007438:Introns / Q000235:genetics; D008562:Membrane Glycoproteins / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D009690:Nucleic Acid Conformation; D009711:Nucleotides / Q000235:genetics / Q000378:metabolism; D010710:Phosphates / Q000378:metabolism; D017510:Protein Folding; D017346:Protein Serine-Threonine Kinases; D011130:RNA Ligase (ATP) / Q000378:metabolism; D012326:RNA Splicing / Q000235:genetics*; D012333:RNA, Messenger / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012343:RNA, Transfer / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012045:Regulatory Sequences, Nucleic Acid / Q000235:genetics; D012097:Repressor Proteins / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D017381:Spliceosomes / Q000502:physiology; D013379:Substrate Specificity; D014157:Transcription Factors
chemical_list: D050976:Basic-Leucine Zipper Transcription Factors; D005656:Fungal Proteins; C091341:HAC1 protein, S cerevisiae; D008562:Membrane Glycoproteins; D009711:Nucleotides; D010710:Phosphates; D012333:RNA, Messenger; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D006151:Guanosine; D012343:RNA, Transfer; C082863:IRE1 protein, S cerevisiae; D017346:Protein Serine-Threonine Kinases; D011130:RNA Ligase (ATP)