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. 1989 Jan;8(1):293–300. doi: 10.1002/j.1460-2075.1989.tb03376.x

Non-enzymatic excision of pre-tRNA introns?

H van Tol 1, H J Gross 1, H Beier 1
PMCID: PMC400802  PMID: 2714254

Abstract

We used human tRNA(Tyr) precursor as a substrate to study self-excision of a pre-tRNA intron. This RNA was synthesized in vitro in a HeLa cell extract. It contains a 5' leader, an intron of 20 nucleotides and a 3' trailer. Self-cleavage of pre-tRNA(Tyr) occurs in 100 mM NH4OAc at a pH ranging from 6 to 8.5 in the presence of spermine, MgCl2 and Triton X-100 under conditions very similar to enzymatic intron excision. The reaction is temperature-dependent, relatively fast as compared to the enzyme-catalysed reaction and leads to fragments which resist further degradation. The detailed structure of all major and minor cleavage products was established by fingerprint analyses. Non-enzymatic cleavage occurs predominantly at the 3' splice site and to a minor extent at the 5' splice site. Other minor cleavage sites are located within the intron and in the 3' trailer. Putative 5' and 3' tRNA halves resulting from pre-tRNA(Tyr) self-cleavage are substrates for wheat germ RNA ligase, suggesting that the cleavage reaction yields 2',3'-cyclic phosphate and 5'-hydroxyl termini. Pre-tRNA splicing endonuclease is believed to cleave both the 5' and the 3' splice site. However, on the basis of our results we propose that this enzyme may support the formation of a pre-tRNA tertiary structure favourable for autocatalytic intron excision and impair unspecific self-cleavage.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Attardi D. G., Margarit I., Tocchini-Valentini G. P. Structural alterations in mutant precursors of the yeast tRNALeu3 gene which behave as defective substrates for a highly purified splicing endoribonuclease. EMBO J. 1985 Dec 1;4(12):3289–3297. doi: 10.1002/j.1460-2075.1985.tb04079.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Baldi M. I., Mattoccia E., Ciafrè S., Attardi D. G., Tocchini-Valentini G. P. Binding and cleavage of pre-tRNA by the Xenopus splicing endonuclease: two separable steps of the intron excision reaction. Cell. 1986 Dec 26;47(6):965–971. doi: 10.1016/0092-8674(86)90811-1. [DOI] [PubMed] [Google Scholar]
  3. Brown R. S., Hingerty B. E., Dewan J. C., Klug A. Pb(II)-catalysed cleavage of the sugar-phosphate backbone of yeast tRNAPhe--implications for lead toxicity and self-splicing RNA. Nature. 1983 Jun 9;303(5917):543–546. doi: 10.1038/303543a0. [DOI] [PubMed] [Google Scholar]
  4. Cech T. R. The generality of self-splicing RNA: relationship to nuclear mRNA splicing. Cell. 1986 Jan 31;44(2):207–210. doi: 10.1016/0092-8674(86)90751-8. [DOI] [PubMed] [Google Scholar]
  5. Cech T. R., Zaug A. J., Grabowski P. J. In vitro splicing of the ribosomal RNA precursor of Tetrahymena: involvement of a guanosine nucleotide in the excision of the intervening sequence. Cell. 1981 Dec;27(3 Pt 2):487–496. doi: 10.1016/0092-8674(81)90390-1. [DOI] [PubMed] [Google Scholar]
  6. Choffat Y., Suter B., Behra R., Kubli E. Pseudouridine modification in the tRNA(Tyr) anticodon is dependent on the presence, but independent of the size and sequence, of the intron in eucaryotic tRNA(Tyr) genes. Mol Cell Biol. 1988 Aug;8(8):3332–3337. doi: 10.1128/mcb.8.8.3332. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Dignam J. D., Lebovitz R. M., Roeder R. G. Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucleic Acids Res. 1983 Mar 11;11(5):1475–1489. doi: 10.1093/nar/11.5.1475. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Domdey H., Jank P., Sänger L., Gross H. J. Studies on the primary and secondary structure of potato spindle tuber viroid: products of digestion with ribonuclease A and ribonuclease T1, and modification with bisulfite. Nucleic Acids Res. 1978 Apr;5(4):1221–1236. doi: 10.1093/nar/5.4.1221. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Epstein L. M., Gall J. G. Self-cleaving transcripts of satellite DNA from the newt. Cell. 1987 Feb 13;48(3):535–543. doi: 10.1016/0092-8674(87)90204-2. [DOI] [PubMed] [Google Scholar]
  10. Filipowicz W., Konarska M., Gross H. J., Shatkin A. J. RNA 3'-terminal phosphate cyclase activity and RNA ligation in HeLa cell extract. Nucleic Acids Res. 1983 Mar 11;11(5):1405–1418. doi: 10.1093/nar/11.5.1405. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Filipowicz W., Shatkin A. J. Origin of splice junction phosphate in tRNAs processed by HeLa cell extract. Cell. 1983 Feb;32(2):547–557. doi: 10.1016/0092-8674(83)90474-9. [DOI] [PubMed] [Google Scholar]
  12. Forster A. C., Symons R. H. Self-cleavage of plus and minus RNAs of a virusoid and a structural model for the active sites. Cell. 1987 Apr 24;49(2):211–220. doi: 10.1016/0092-8674(87)90562-9. [DOI] [PubMed] [Google Scholar]
  13. Garriga G., Lambowitz A. M. RNA splicing in neurospora mitochondria: self-splicing of a mitochondrial intron in vitro. Cell. 1984 Dec;39(3 Pt 2):631–641. doi: 10.1016/0092-8674(84)90470-7. [DOI] [PubMed] [Google Scholar]
  14. Gegenheimer P., Gabius H. J., Peebles C. L., Abelson J. An RNA ligase from wheat germ which participates in transfer RNA splicing in vitro. J Biol Chem. 1983 Jul 10;258(13):8365–8373. [PubMed] [Google Scholar]
  15. Gilbert W., Marchionni M., McKnight G. On the antiquity of introns. Cell. 1986 Jul 18;46(2):151–153. doi: 10.1016/0092-8674(86)90730-0. [DOI] [PubMed] [Google Scholar]
  16. Haseloff J., Gerlach W. L. Simple RNA enzymes with new and highly specific endoribonuclease activities. Nature. 1988 Aug 18;334(6183):585–591. doi: 10.1038/334585a0. [DOI] [PubMed] [Google Scholar]
  17. Hutchins C. J., Rathjen P. D., Forster A. C., Symons R. H. Self-cleavage of plus and minus RNA transcripts of avocado sunblotch viroid. Nucleic Acids Res. 1986 May 12;14(9):3627–3640. doi: 10.1093/nar/14.9.3627. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Kaine B. P., Gupta R., Woese C. R. Putative introns in tRNA genes of prokaryotes. Proc Natl Acad Sci U S A. 1983 Jun;80(11):3309–3312. doi: 10.1073/pnas.80.11.3309. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Konarska M., Filipowicz W., Domdey H., Gross H. J. Formation of a 2'-phosphomonoester, 3',5'-phosphodiester linkage by a novel RNA ligase in wheat germ. Nature. 1981 Sep 10;293(5828):112–116. doi: 10.1038/293112a0. [DOI] [PubMed] [Google Scholar]
  20. Lee M. C., Knapp G. Transfer RNA splicing in Saccharomyces cerevisiae. Secondary and tertiary structures of the substrates. J Biol Chem. 1985 Mar 10;260(5):3108–3115. [PubMed] [Google Scholar]
  21. Ma D. P., Doebley J. Nucleotide sequence of the split tRNAleu(UAA) gene from Sorghum bicolor chloroplasts. Gene. 1986;43(1-2):169–174. doi: 10.1016/0378-1119(86)90020-x. [DOI] [PubMed] [Google Scholar]
  22. MacPherson J. M., Roy K. L. Two human tyrosine tRNA genes contain introns. Gene. 1986;42(1):101–106. doi: 10.1016/0378-1119(86)90155-1. [DOI] [PubMed] [Google Scholar]
  23. Ogden R. C., Lee M. C., Knapp G. Transfer RNA splicing in Saccharomyces cerevisiae: defining the substrates. Nucleic Acids Res. 1984 Dec 21;12(24):9367–9382. doi: 10.1093/nar/12.24.9367. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Peebles C. L., 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. doi: 10.1016/0092-8674(83)90472-5. [DOI] [PubMed] [Google Scholar]
  25. Peebles C. L., Ogden R. C., Knapp G., Abelson J. Splicing of yeast tRNA precursors: a two-stage reaction. Cell. 1979 Sep;18(1):27–35. doi: 10.1016/0092-8674(79)90350-7. [DOI] [PubMed] [Google Scholar]
  26. Prody G. A., Bakos J. T., Buzayan J. M., Schneider I. R., Bruening G. Autolytic processing of dimeric plant virus satellite RNA. Science. 1986 Mar 28;231(4745):1577–1580. doi: 10.1126/science.231.4745.1577. [DOI] [PubMed] [Google Scholar]
  27. Quigley G. J., Teeter M. M., Rich A. Structural analysis of spermine and magnesium ion binding to yeast phenylalanine transfer RNA. Proc Natl Acad Sci U S A. 1978 Jan;75(1):64–68. doi: 10.1073/pnas.75.1.64. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Raba M., Limburg K., Burghagen M., Katze J. R., Simsek M., Heckman J. E., Rajbhandary U. L., Gross H. J. Nucleotide sequence of three isoaccepting lysine tRNAs from rabbit liver and SV40-transformed mouse fibroblasts. Eur J Biochem. 1979 Jun;97(1):305–318. doi: 10.1111/j.1432-1033.1979.tb13115.x. [DOI] [PubMed] [Google Scholar]
  29. Reich C., Olsen G. J., Pace B., Pace N. R. Role of the protein moiety of ribonuclease P, a ribonucleoprotein enzyme. Science. 1988 Jan 8;239(4836):178–181. doi: 10.1126/science.3122322. [DOI] [PubMed] [Google Scholar]
  30. Rich A., Kim S. H. The three-dimensional structure of transfer RNA. Sci Am. 1978 Jan;238(1):52–62. doi: 10.1038/scientificamerican0178-52. [DOI] [PubMed] [Google Scholar]
  31. Senapathy P. Origin of eukaryotic introns: a hypothesis, based on codon distribution statistics in genes, and its implications. Proc Natl Acad Sci U S A. 1986 Apr;83(7):2133–2137. doi: 10.1073/pnas.83.7.2133. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Silberklang M., Gillum A. M., RajBhandary U. L. Use of in vitro 32P labeling in the sequence analysis of nonradioactive tRNAs. Methods Enzymol. 1979;59:58–109. doi: 10.1016/0076-6879(79)59072-7. [DOI] [PubMed] [Google Scholar]
  33. Stange N., Beier H. A cell-free plant extract for accurate pre-tRNA processing, splicing and modification. EMBO J. 1987 Sep;6(9):2811–2818. doi: 10.1002/j.1460-2075.1987.tb02577.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Stange N., Gross H. J., Beier H. Wheat germ splicing endonuclease is highly specific for plant pre-tRNAs. EMBO J. 1988 Dec 1;7(12):3823–3828. doi: 10.1002/j.1460-2075.1988.tb03267.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Steger G., Hofmann H., Förtsch J., Gross H. J., Randles J. W., Sänger H. L., Riesner D. Conformational transitions in viroids and virusoids: comparison of results from energy minimization algorithm and from experimental data. J Biomol Struct Dyn. 1984 Dec;2(3):543–571. doi: 10.1080/07391102.1984.10507591. [DOI] [PubMed] [Google Scholar]
  36. Steinmetz A., Gubbins E. J., Bogorad L. The anticodon of the maize chloroplast gene for tRNA Leu UAA is split by a large intron. Nucleic Acids Res. 1982 May 25;10(10):3027–3037. doi: 10.1093/nar/10.10.3027. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Watson N., Gurevitz M., Ford J., Apirion D. Self cleavage of a precursor RNA from bacteriophage T4. J Mol Biol. 1984 Jan 25;172(3):301–323. doi: 10.1016/s0022-2836(84)80028-5. [DOI] [PubMed] [Google Scholar]
  38. Wintermeyer W., Zachau H. G. Mg 2+ -katalysierte, spezifische Spaltung von tRN. Biochim Biophys Acta. 1973 Feb 23;299(1):82–90. [PubMed] [Google Scholar]
  39. van Tol H., Stange N., Gross H. J., Beier H. A human and a plant intron-containing tRNATyr gene are both transcribed in a HeLa cell extract but spliced along different pathways. EMBO J. 1987 Jan;6(1):35–41. doi: 10.1002/j.1460-2075.1987.tb04715.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. van der Horst G., Tabak H. F. Self-splicing of yeast mitochondrial ribosomal and messenger RNA precursors. Cell. 1985 Apr;40(4):759–766. doi: 10.1016/0092-8674(85)90335-6. [DOI] [PubMed] [Google Scholar]

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