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EMBO J. 1999 Jun 1; 18(11): 3119–3132.
PMCID: PMC1171393

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


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.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Abelson J, Trotta CR, Li H. tRNA splicing. J Biol Chem. 1998 May 22;273(21):12685–12688. [PubMed]
  • Belford HG, Westaway SK, Abelson J, Greer CL. Multiple nucleotide cofactor use by yeast ligase in tRNA splicing. Evidence for independent ATP- and GTP-binding sites. J Biol Chem. 1993 Feb 5;268(4):2444–2450. [PubMed]
  • Bork P, Sander C. A hybrid protein kinase-RNase in an interferon-induced pathway? FEBS Lett. 1993 Nov 15;334(2):149–152. [PubMed]
  • Cameron V, Uhlenbeck OC. 3'-Phosphatase activity in T4 polynucleotide kinase. Biochemistry. 1977 Nov 15;16(23):5120–5126. [PubMed]
  • Cameron V, Soltis D, Uhlenbeck OC. Polynucleotide kinase from a T4 mutant which lacks the 3' phosphatase activity. Nucleic Acids Res. 1978 Mar;5(3):825–833. [PMC free article] [PubMed]
  • Chapman RE, Walter P. Translational attenuation mediated by an mRNA intron. Curr Biol. 1997 Nov 1;7(11):850–859. [PubMed]
  • Chapman R, Sidrauski C, Walter P. Intracellular signaling from the endoplasmic reticulum to the nucleus. Annu Rev Cell Dev Biol. 1998;14:459–485. [PubMed]
  • Cox JS, Walter P. A novel mechanism for regulating activity of a transcription factor that controls the unfolded protein response. Cell. 1996 Nov 1;87(3):391–404. [PubMed]
  • Cox JS, Shamu CE, Walter P. Transcriptional induction of genes encoding endoplasmic reticulum resident proteins requires a transmembrane protein kinase. Cell. 1993 Jun 18;73(6):1197–1206. [PubMed]
  • Culver GM, McCraith SM, Zillmann M, Kierzek R, Michaud N, LaReau RD, Turner DH, Phizicky EM. An NAD derivative produced during transfer RNA splicing: ADP-ribose 1"-2" cyclic phosphate. Science. 1993 Jul 9;261(5118):206–208. [PubMed]
  • Culver GM, McCraith SM, Consaul SA, Stanford DR, Phizicky EM. A 2'-phosphotransferase implicated in tRNA splicing is essential in Saccharomyces cerevisiae. J Biol Chem. 1997 May 16;272(20):13203–13210. [PubMed]
  • Di Nicola Negri E, Fabbri S, Bufardeci E, Baldi MI, Gandini Attardi D, Mattoccia E, Tocchini-Valentini GP. The eucaryal tRNA splicing endonuclease recognizes a tripartite set of RNA elements. Cell. 1997 Jun 13;89(6):859–866. [PubMed]
  • Dong B, Silverman RH. A bipartite model of 2-5A-dependent RNase L. J Biol Chem. 1997 Aug 29;272(35):22236–22242. [PubMed]
  • Dong B, Xu L, Zhou A, Hassel BA, Lee X, Torrence PF, Silverman RH. Intrinsic molecular activities of the interferon-induced 2-5A-dependent RNase. J Biol Chem. 1994 May 13;269(19):14153–14158. [PubMed]
  • Fabbri S, Fruscoloni P, Bufardeci E, Di Nicola Negri E, Baldi MI, Attardi DG, Mattoccia E, Tocchini-Valentini GP. Conservation of substrate recognition mechanisms by tRNA splicing endonucleases. Science. 1998 Apr 10;280(5361):284–286. [PubMed]
  • Gething MJ, Sambrook J. Protein folding in the cell. Nature. 1992 Jan 2;355(6355):33–45. [PubMed]
  • Greer CL, Peebles CL, Gegenheimer P, Abelson J. Mechanism of action of a yeast RNA ligase in tRNA splicing. Cell. 1983 Feb;32(2):537–546. [PubMed]
  • Greer CL, Söll D, Willis I. Substrate recognition and identification of splice sites by the tRNA-splicing endonuclease and ligase from Saccharomyces cerevisiae. Mol Cell Biol. 1987 Jan;7(1):76–84. [PMC free article] [PubMed]
  • Hammond C, Helenius A. Quality control in the secretory pathway. Curr Opin Cell Biol. 1995 Aug;7(4):523–529. [PubMed]
  • Hassel BA, Zhou A, Sotomayor C, Maran A, Silverman RH. A dominant negative mutant of 2-5A-dependent RNase suppresses antiproliferative and antiviral effects of interferon. EMBO J. 1993 Aug;12(8):3297–3304. [PMC free article] [PubMed]
  • Kawahara T, Yanagi H, Yura T, Mori K. Endoplasmic reticulum stress-induced mRNA splicing permits synthesis of transcription factor Hac1p/Ern4p that activates the unfolded protein response. Mol Biol Cell. 1997 Oct;8(10):1845–1862. [PMC free article] [PubMed]
  • Kawahara T, Yanagi H, Yura T, Mori K. Unconventional splicing of HAC1/ERN4 mRNA required for the unfolded protein response. Sequence-specific and non-sequential cleavage of the splice sites. J Biol Chem. 1998 Jan 16;273(3):1802–1807. [PubMed]
  • Kleman-Leyer K, Armbruster DW, Daniels CJ. Properties of H. volcanii tRNA intron endonuclease reveal a relationship between the archaeal and eucaryal tRNA intron processing systems. Cell. 1997 Jun 13;89(6):839–847. [PubMed]
  • Knapp G, Ogden RC, Peebles CL, Abelson J. Splicing of yeast tRNA precursors: structure of the reaction intermediates. Cell. 1979 Sep;18(1):37–45. [PubMed]
  • Kohno K, Normington K, Sambrook J, Gething MJ, Mori K. The promoter region of the yeast KAR2 (BiP) gene contains a regulatory domain that responds to the presence of unfolded proteins in the endoplasmic reticulum. Mol Cell Biol. 1993 Feb;13(2):877–890. [PMC free article] [PubMed]
  • Kuznetsov G, Nigam SK. Folding of secretory and membrane proteins. N Engl J Med. 1998 Dec 3;339(23):1688–1695. [PubMed]
  • Lykke-Andersen J, Garrett RA. Structural characteristics of the stable RNA introns of archaeal hyperthermophiles and their splicing junctions. J Mol Biol. 1994 Nov 11;243(5):846–855. [PubMed]
  • Lykke-Andersen J, Garrett RA. RNA-protein interactions of an archaeal homotetrameric splicing endoribonuclease with an exceptional evolutionary history. EMBO J. 1997 Oct 15;16(20):6290–6300. [PMC free article] [PubMed]
  • Mattoccia E, Baldi IM, Gandini-Attardi D, Ciafrè S, Tocchini-Valentini GP. Site selection by the tRNA splicing endonuclease of Xenopus laevis. Cell. 1988 Nov 18;55(4):731–738. [PubMed]
  • McCraith SM, Phizicky EM. A highly specific phosphatase from Saccharomyces cerevisiae implicated in tRNA splicing. Mol Cell Biol. 1990 Mar;10(3):1049–1055. [PMC free article] [PubMed]
  • Miao F, Abelson J. Yeast tRNA-splicing endonuclease cleaves precursor tRNA in a random pathway. J Biol Chem. 1993 Jan 5;268(1):672–677. [PubMed]
  • Milligan JF, Groebe DR, Witherell GW, Uhlenbeck OC. Oligoribonucleotide synthesis using T7 RNA polymerase and synthetic DNA templates. Nucleic Acids Res. 1987 Nov 11;15(21):8783–8798. [PMC free article] [PubMed]
  • Mori K, Sant A, Kohno K, Normington K, Gething MJ, Sambrook JF. A 22 bp cis-acting element is necessary and sufficient for the induction of the yeast KAR2 (BiP) gene by unfolded proteins. EMBO J. 1992 Jul;11(7):2583–2593. [PMC free article] [PubMed]
  • Mori K, Ma W, Gething MJ, Sambrook J. A transmembrane protein with a cdc2+/CDC28-related kinase activity is required for signaling from the ER to the nucleus. Cell. 1993 Aug 27;74(4):743–756. [PubMed]
  • Mori K, Kawahara T, Yoshida H, Yanagi H, Yura T. Signalling from endoplasmic reticulum to nucleus: transcription factor with a basic-leucine zipper motif is required for the unfolded protein-response pathway. Genes Cells. 1996 Sep;1(9):803–817. [PubMed]
  • Nikawa J, Akiyoshi M, Hirata S, Fukuda T. Saccharomyces cerevisiae IRE2/HAC1 is involved in IRE1-mediated KAR2 expression. Nucleic Acids Res. 1996 Nov 1;24(21):4222–4226. [PMC free article] [PubMed]
  • Peebles CL, Gegenheimer P, Abelson J. Precise excision of intervening sequences from precursor tRNAs by a membrane-associated yeast endonuclease. Cell. 1983 Feb;32(2):525–536. [PubMed]
  • Phizicky EM, Consaul SA, Nehrke KW, Abelson J. Yeast tRNA ligase mutants are nonviable and accumulate tRNA splicing intermediates. J Biol Chem. 1992 Mar 5;267(7):4577–4582. [PubMed]
  • Reyes VM, Abelson J. A synthetic substrate for tRNA splicing. Anal Biochem. 1987 Oct;166(1):90–106. [PubMed]
  • Reyes VM, Abelson J. Substrate recognition and splice site determination in yeast tRNA splicing. Cell. 1988 Nov 18;55(4):719–730. [PubMed]
  • Shamu CE, Walter P. Oligomerization and phosphorylation of the Ire1p kinase during intracellular signaling from the endoplasmic reticulum to the nucleus. EMBO J. 1996 Jun 17;15(12):3028–3039. [PMC free article] [PubMed]
  • Shamu CE, Cox JS, Walter P. The unfolded-protein-response pathway in yeast. Trends Cell Biol. 1994 Feb;4(2):56–60. [PubMed]
  • Lindahl T. Facts and artifacts of ancient DNA. Cell. 1997 Jul 11;90(1):1–3. [PubMed]
  • Sidrauski C, Cox JS, Walter P. tRNA ligase is required for regulated mRNA splicing in the unfolded protein response. Cell. 1996 Nov 1;87(3):405–413. [PubMed]
  • Sidrauski C, Chapman R, Walter P. The unfolded protein response: an intracellular signalling pathway with many surprising features. Trends Cell Biol. 1998 Jun;8(6):245–249. [PubMed]
  • Spinelli SL, Consaul SA, Phizicky EM. A conditional lethal yeast phosphotransferase (tpt1) mutant accumulates tRNAs with a 2'-phosphate and an undermodified base at the splice junction. RNA. 1997 Dec;3(12):1388–1400. [PMC free article] [PubMed]
  • Thompson LD, Daniels CJ. Recognition of exon-intron boundaries by the Halobacterium volcanii tRNA intron endonuclease. J Biol Chem. 1990 Oct 25;265(30):18104–18111. [PubMed]
  • Tirasophon W, Welihinda AA, Kaufman RJ. A stress response pathway from the endoplasmic reticulum to the nucleus requires a novel bifunctional protein kinase/endoribonuclease (Ire1p) in mammalian cells. Genes Dev. 1998 Jun 15;12(12):1812–1824. [PMC free article] [PubMed]
  • Trotta CR, Miao F, Arn EA, Stevens SW, Ho CK, Rauhut R, Abelson JN. The yeast tRNA splicing endonuclease: a tetrameric enzyme with two active site subunits homologous to the archaeal tRNA endonucleases. Cell. 1997 Jun 13;89(6):849–858. [PubMed]
  • Valenzuela P, Venegas A, Weinberg F, Bishop R, Rutter WJ. Structure of yeast phenylalanine-tRNA genes: an intervening DNA segment within the region coding for the tRNA. Proc Natl Acad Sci U S A. 1978 Jan;75(1):190–194. [PMC free article] [PubMed]
  • Wang XZ, Harding HP, Zhang Y, Jolicoeur EM, Kuroda M, Ron D. Cloning of mammalian Ire1 reveals diversity in the ER stress responses. EMBO J. 1998 Oct 1;17(19):5708–5717. [PMC free article] [PubMed]
  • Welihinda AA, Kaufman RJ. The unfolded protein response pathway in Saccharomyces cerevisiae. Oligomerization and trans-phosphorylation of Ire1p (Ern1p) are required for kinase activation. J Biol Chem. 1996 Jul 26;271(30):18181–18187. [PubMed]
  • Welihinda AA, Tirasophon W, Green SR, Kaufman RJ. Protein serine/threonine phosphatase Ptc2p negatively regulates the unfolded-protein response by dephosphorylating Ire1p kinase. Mol Cell Biol. 1998 Apr;18(4):1967–1977. [PMC free article] [PubMed]
  • Xu Q, Teplow D, Lee TD, Abelson J. Domain structure in yeast tRNA ligase. Biochemistry. 1990 Jul 3;29(26):6132–6138. [PubMed]
  • Zhou A, Paranjape J, Brown TL, Nie H, Naik S, Dong B, Chang A, Trapp B, Fairchild R, Colmenares C, et al. Interferon action and apoptosis are defective in mice devoid of 2',5'-oligoadenylate-dependent RNase L. EMBO J. 1997 Nov 3;16(21):6355–6363. [PMC free article] [PubMed]

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