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. 1999 Jan;5(1):1–13. doi: 10.1017/s1355838299981244

Sequence specificity of in vivo reverse splicing of the Tetrahymena group I intron.

J Roman 1, M N Rubin 1, S A Woodson 1
PMCID: PMC1369735  PMID: 9917062

Abstract

Reverse splicing of group I introns is proposed to be a mechanism by which intron sequences are transferred to new genes. Integration of the Tetrahymena intron into the Escherichia coli 23S rRNA via reverse splicing depends on base pairing between the guide sequence of the intron and the target site. To investigate the substrate specificity of reverse splicing, the wild-type and 18 mutant introns with different guide sequences were expressed in E. coli. Amplification of intron-rRNA junctions by RT-PCR revealed partial reverse splicing at 69 sites and complete integration at one novel site in the 23S rRNA. Reverse splicing was not observed at some potential target sites, whereas other regions of the 23S rRNA were more reactive than expected. The results indicate that the frequency of reverse splicing is modulated by the structure of the rRNA. The intron is spliced 10-fold less efficiently in E. coli from a novel integration site (U2074) in domain V of the 23S rRNA than from a site homologous to the natural splice junction of the Tetrahymena 26S rRNA, suggesting that the forward reaction is less favored at this site.

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Selected References

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

  1. Barfod E. T., Cech T. R. The conserved U.G pair in the 5' splice site duplex of a group I intron is required in the first but not the second step of self-splicing. Mol Cell Biol. 1989 Sep;9(9):3657–3666. doi: 10.1128/mcb.9.9.3657. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Been M. D., Cech T. R. One binding site determines sequence specificity of Tetrahymena pre-rRNA self-splicing, trans-splicing, and RNA enzyme activity. Cell. 1986 Oct 24;47(2):207–216. doi: 10.1016/0092-8674(86)90443-5. [DOI] [PubMed] [Google Scholar]
  3. Been M. D., Cech T. R. Selection of circularization sites in a group I IVS RNA requires multiple alignments of an internal template-like sequence. Cell. 1987 Sep 11;50(6):951–961. doi: 10.1016/0092-8674(87)90522-8. [DOI] [PubMed] [Google Scholar]
  4. Belfort M., Perlman P. S. Mechanisms of intron mobility. J Biol Chem. 1995 Dec 22;270(51):30237–30240. doi: 10.1074/jbc.270.51.30237. [DOI] [PubMed] [Google Scholar]
  5. Belfort M., Roberts R. J. Homing endonucleases: keeping the house in order. Nucleic Acids Res. 1997 Sep 1;25(17):3379–3388. doi: 10.1093/nar/25.17.3379. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bevilacqua P. C., Kierzek R., Johnson K. A., Turner D. H. Dynamics of ribozyme binding of substrate revealed by fluorescence-detected stopped-flow methods. Science. 1992 Nov 20;258(5086):1355–1358. doi: 10.1126/science.1455230. [DOI] [PubMed] [Google Scholar]
  7. Bevilacqua P. C., Turner D. H. Comparison of binding of mixed ribose-deoxyribose analogues of CUCU to a ribozyme and to GGAGAA by equilibrium dialysis: evidence for ribozyme specific interactions with 2' OH groups. Biochemistry. 1991 Nov 5;30(44):10632–10640. doi: 10.1021/bi00108a005. [DOI] [PubMed] [Google Scholar]
  8. Campbell T. B., Cech T. R. Identification of ribozymes within a ribozyme library that efficiently cleave a long substrate RNA. RNA. 1995 Aug;1(6):598–609. [PMC free article] [PubMed] [Google Scholar]
  9. Cavalier-Smith T. Intron phylogeny: a new hypothesis. Trends Genet. 1991 May;7(5):145–148. [PubMed] [Google Scholar]
  10. Cech T. R. Self-splicing RNA: implications for evolution. Int Rev Cytol. 1985;93:3–22. doi: 10.1016/s0074-7696(08)61370-4. [DOI] [PubMed] [Google Scholar]
  11. Curcio M. J., Belfort M. Retrohoming: cDNA-mediated mobility of group II introns requires a catalytic RNA. Cell. 1996 Jan 12;84(1):9–12. doi: 10.1016/s0092-8674(00)80987-3. [DOI] [PubMed] [Google Scholar]
  12. Doudna J. A., Cormack B. P., Szostak J. W. RNA structure, not sequence, determines the 5' splice-site specificity of a group I intron. Proc Natl Acad Sci U S A. 1989 Oct;86(19):7402–7406. doi: 10.1073/pnas.86.19.7402. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Doudna J. A., Szostak J. W. RNA-catalysed synthesis of complementary-strand RNA. Nature. 1989 Jun 15;339(6225):519–522. doi: 10.1038/339519a0. [DOI] [PubMed] [Google Scholar]
  14. Dujon B. Group I introns as mobile genetic elements: facts and mechanistic speculations--a review. Gene. 1989 Oct 15;82(1):91–114. doi: 10.1016/0378-1119(89)90034-6. [DOI] [PubMed] [Google Scholar]
  15. Emerick V. L., Pan J., Woodson S. A. Analysis of rate-determining conformational changes during self-splicing of the Tetrahymena intron. Biochemistry. 1996 Oct 15;35(41):13469–13477. doi: 10.1021/bi960865i. [DOI] [PubMed] [Google Scholar]
  16. Eskes R., Yang J., Lambowitz A. M., Perlman P. S. Mobility of yeast mitochondrial group II introns: engineering a new site specificity and retrohoming via full reverse splicing. Cell. 1997 Mar 21;88(6):865–874. doi: 10.1016/s0092-8674(00)81932-7. [DOI] [PubMed] [Google Scholar]
  17. Grivell L. A. Transposition: mobile introns get into line. Curr Biol. 1996 Jan 1;6(1):48–51. doi: 10.1016/s0960-9822(02)00420-7. [DOI] [PubMed] [Google Scholar]
  18. Guo H., Zimmerly S., Perlman P. S., Lambowitz A. M. Group II intron endonucleases use both RNA and protein subunits for recognition of specific sequences in double-stranded DNA. EMBO J. 1997 Nov 17;16(22):6835–6848. doi: 10.1093/emboj/16.22.6835. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Herschlag D. Evidence for processivity and two-step binding of the RNA substrate from studies of J1/2 mutants of the Tetrahymena ribozyme. Biochemistry. 1992 Feb 11;31(5):1386–1399. doi: 10.1021/bi00120a015. [DOI] [PubMed] [Google Scholar]
  20. Jaeger J. A., Turner D. H., Zuker M. Predicting optimal and suboptimal secondary structure for RNA. Methods Enzymol. 1990;183:281–306. doi: 10.1016/0076-6879(90)83019-6. [DOI] [PubMed] [Google Scholar]
  21. Joseph S., Weiser B., Noller H. F. Mapping the inside of the ribosome with an RNA helical ruler. Science. 1997 Nov 7;278(5340):1093–1098. doi: 10.1126/science.278.5340.1093. [DOI] [PubMed] [Google Scholar]
  22. Leviev I., Levieva S., Garrett R. A. Role for the highly conserved region of domain IV of 23S-like rRNA in subunit-subunit interactions at the peptidyl transferase centre. Nucleic Acids Res. 1995 May 11;23(9):1512–1517. doi: 10.1093/nar/23.9.1512. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Maina C. V., Riggs P. D., Grandea A. G., 3rd, Slatko B. E., Moran L. S., Tagliamonte J. A., McReynolds L. A., Guan C. D. An Escherichia coli vector to express and purify foreign proteins by fusion to and separation from maltose-binding protein. Gene. 1988 Dec 30;74(2):365–373. doi: 10.1016/0378-1119(88)90170-9. [DOI] [PubMed] [Google Scholar]
  24. Michel F., Jacquier A., Dujon B. Comparison of fungal mitochondrial introns reveals extensive homologies in RNA secondary structure. Biochimie. 1982 Oct;64(10):867–881. doi: 10.1016/s0300-9084(82)80349-0. [DOI] [PubMed] [Google Scholar]
  25. Mitchell P., Osswald M., Brimacombe R. Identification of intermolecular RNA cross-links at the subunit interface of the Escherichia coli ribosome. Biochemistry. 1992 Mar 24;31(11):3004–3011. doi: 10.1021/bi00126a023. [DOI] [PubMed] [Google Scholar]
  26. Moazed D., Noller H. F. Interaction of tRNA with 23S rRNA in the ribosomal A, P, and E sites. Cell. 1989 May 19;57(4):585–597. doi: 10.1016/0092-8674(89)90128-1. [DOI] [PubMed] [Google Scholar]
  27. Moazed D., Robertson J. M., Noller H. F. Interaction of elongation factors EF-G and EF-Tu with a conserved loop in 23S RNA. Nature. 1988 Jul 28;334(6180):362–364. doi: 10.1038/334362a0. [DOI] [PubMed] [Google Scholar]
  28. Mueller M. W., Allmaier M., Eskes R., Schweyen R. J. Transposition of group II intron aI1 in yeast and invasion of mitochondrial genes at new locations. Nature. 1993 Nov 11;366(6451):174–176. doi: 10.1038/366174a0. [DOI] [PubMed] [Google Scholar]
  29. Munishkin A., Wool I. G. The ribosome-in-pieces: binding of elongation factor EF-G to oligoribonucleotides that mimic the sarcin/ricin and thiostrepton domains of 23S ribosomal RNA. Proc Natl Acad Sci U S A. 1997 Nov 11;94(23):12280–12284. doi: 10.1073/pnas.94.23.12280. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Muscarella D. E., Vogt V. M. A mobile group I intron from Physarum polycephalum can insert itself and induce point mutations in the nuclear ribosomal DNA of saccharomyces cerevisiae. Mol Cell Biol. 1993 Feb;13(2):1023–1033. doi: 10.1128/mcb.13.2.1023. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Nikolcheva T., Woodson S. A. Association of a group I intron with its splice junction in 50S ribosomes: implications for intron toxicity. RNA. 1997 Sep;3(9):1016–1027. [PMC free article] [PubMed] [Google Scholar]
  32. Noller H. F., Green R., Heilek G., Hoffarth V., Hüttenhofer A., Joseph S., Lee I., Lieberman K., Mankin A., Merryman C. Structure and function of ribosomal RNA. Biochem Cell Biol. 1995 Nov-Dec;73(11-12):997–1009. doi: 10.1139/o95-107. [DOI] [PubMed] [Google Scholar]
  33. Noller H. F., Kop J., Wheaton V., Brosius J., Gutell R. R., Kopylov A. M., Dohme F., Herr W., Stahl D. A., Gupta R. Secondary structure model for 23S ribosomal RNA. Nucleic Acids Res. 1981 Nov 25;9(22):6167–6189. doi: 10.1093/nar/9.22.6167. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Noller H. F. Ribosomal RNA and translation. Annu Rev Biochem. 1991;60:191–227. doi: 10.1146/annurev.bi.60.070191.001203. [DOI] [PubMed] [Google Scholar]
  35. Powers T., Noller H. F. Dominant lethal mutations in a conserved loop in 16S rRNA. Proc Natl Acad Sci U S A. 1990 Feb;87(3):1042–1046. doi: 10.1073/pnas.87.3.1042. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Pyle A. M., Cech T. R. Ribozyme recognition of RNA by tertiary interactions with specific ribose 2'-OH groups. Nature. 1991 Apr 18;350(6319):628–631. doi: 10.1038/350628a0. [DOI] [PubMed] [Google Scholar]
  37. Remaut E., Tsao H., Fiers W. Improved plasmid vectors with a thermoinducible expression and temperature-regulated runaway replication. Gene. 1983 Apr;22(1):103–113. doi: 10.1016/0378-1119(83)90069-0. [DOI] [PubMed] [Google Scholar]
  38. Richard G. F., Dujon B. Association of transcripts from a group-I intron-containing gene with high sedimentation coefficient particles. Curr Genet. 1997 Sep;32(3):175–181. doi: 10.1007/s002940050263. [DOI] [PubMed] [Google Scholar]
  39. Roman J., Woodson S. A. Integration of the Tetrahymena group I intron into bacterial rRNA by reverse splicing in vivo. Proc Natl Acad Sci U S A. 1998 Mar 3;95(5):2134–2139. doi: 10.1073/pnas.95.5.2134. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Roman J., Woodson S. A. Reverse splicing of the Tetrahymena IVS: evidence for multiple reaction sites in the 23S rRNA. RNA. 1995 Jul;1(5):478–490. [PMC free article] [PubMed] [Google Scholar]
  41. Sellem C. H., Lecellier G., Belcour L. Transposition of a group II intron. Nature. 1993 Nov 11;366(6451):176–178. doi: 10.1038/366176a0. [DOI] [PubMed] [Google Scholar]
  42. Semrad K., Schroeder R. A ribosomal function is necessary for efficient splicing of the T4 phage thymidylate synthase intron in vivo. Genes Dev. 1998 May 1;12(9):1327–1337. doi: 10.1101/gad.12.9.1327. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Sharp P. A. On the origin of RNA splicing and introns. Cell. 1985 Sep;42(2):397–400. doi: 10.1016/0092-8674(85)90092-3. [DOI] [PubMed] [Google Scholar]
  44. Strobel S. A., Cech T. R. Minor groove recognition of the conserved G.U pair at the Tetrahymena ribozyme reaction site. Science. 1995 Feb 3;267(5198):675–679. doi: 10.1126/science.7839142. [DOI] [PubMed] [Google Scholar]
  45. Strobel S. A., Cech T. R. Tertiary interactions with the internal guide sequence mediate docking of the P1 helix into the catalytic core of the Tetrahymena ribozyme. Biochemistry. 1993 Dec 14;32(49):13593–13604. doi: 10.1021/bi00212a027. [DOI] [PubMed] [Google Scholar]
  46. Strobel S. A., Cech T. R. Translocation of an RNA duplex on a ribozyme. Nat Struct Biol. 1994 Jan;1(1):13–17. doi: 10.1038/nsb0194-13. [DOI] [PubMed] [Google Scholar]
  47. Turmel M., Gutell R. R., Mercier J. P., Otis C., Lemieux C. Analysis of the chloroplast large subunit ribosomal RNA gene from 17 Chlamydomonas taxa. Three internal transcribed spacers and 12 group I intron insertion sites. J Mol Biol. 1993 Jul 20;232(2):446–467. doi: 10.1006/jmbi.1993.1402. [DOI] [PubMed] [Google Scholar]
  48. Walstrum S. A., Uhlenbeck O. C. The self-splicing RNA of Tetrahymena is trapped in a less active conformation by gel purification. Biochemistry. 1990 Nov 20;29(46):10573–10576. doi: 10.1021/bi00498a022. [DOI] [PubMed] [Google Scholar]
  49. Wilson K. S., Noller H. F. Mapping the position of translational elongation factor EF-G in the ribosome by directed hydroxyl radical probing. Cell. 1998 Jan 9;92(1):131–139. doi: 10.1016/s0092-8674(00)80905-8. [DOI] [PubMed] [Google Scholar]
  50. Woodson S. A., Cech T. R. Alternative secondary structures in the 5' exon affect both forward and reverse self-splicing of the Tetrahymena intervening sequence RNA. Biochemistry. 1991 Feb 26;30(8):2042–2050. doi: 10.1021/bi00222a006. [DOI] [PubMed] [Google Scholar]
  51. Woodson S. A., Cech T. R. Reverse self-splicing of the tetrahymena group I intron: implication for the directionality of splicing and for intron transposition. Cell. 1989 Apr 21;57(2):335–345. doi: 10.1016/0092-8674(89)90971-9. [DOI] [PubMed] [Google Scholar]
  52. Yang J., Zimmerly S., Perlman P. S., Lambowitz A. M. Efficient integration of an intron RNA into double-stranded DNA by reverse splicing. Nature. 1996 May 23;381(6580):332–335. doi: 10.1038/381332a0. [DOI] [PubMed] [Google Scholar]
  53. Young B., Herschlag D., Cech T. R. Mutations in a nonconserved sequence of the Tetrahymena ribozyme increase activity and specificity. Cell. 1991 Nov 29;67(5):1007–1019. doi: 10.1016/0092-8674(91)90373-7. [DOI] [PubMed] [Google Scholar]
  54. Zhang F., Ramsay E. S., Woodson S. A. In vivo facilitation of Tetrahymena group I intron splicing in Escherichia coli pre-ribosomal RNA. RNA. 1995 May;1(3):284–292. [PMC free article] [PubMed] [Google Scholar]
  55. Zimmerly S., Guo H., Perlman P. S., Lambowitz A. M. Group II intron mobility occurs by target DNA-primed reverse transcription. Cell. 1995 Aug 25;82(4):545–554. doi: 10.1016/0092-8674(95)90027-6. [DOI] [PubMed] [Google Scholar]

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