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. 1995 Jun;15(6):2933–2941. doi: 10.1128/mcb.15.6.2933

Guide RNA-mRNA chimeras, which are potential RNA editing intermediates, are formed by endonuclease and RNA ligase in a trypanosome mitochondrial extract.

L N Rusché 1, K J Piller 1, B Sollner-Webb 1
PMCID: PMC230524  PMID: 7760791

Abstract

RNA editing in kinetoplast mitochondrial transcripts involves the insertion and/or deletion of uridine residues and is directed by guide RNAs (gRNAs). It is thought to occur through a chimeric intermediate in which the 3' oligo(U) tail of the gRNA is covalently joined to the 3' portion of the mRNA at the site being edited. Chimeras have been proposed to be formed by a transesterification reaction but could also be formed by the known mitochondrial site-specific nuclease and RNA ligase. To distinguish between these models, we studied chimera formation in vitro directed by a trypanosome mitochondrial extract. This reaction was found to occur in two steps. First, the mRNA is cleaved in the 3' portion of the editing domain, and then the 3' fragment derived from this cleavage is ligated to the gRNA. The isolated mRNA 3' cleavage product is a more efficient substrate for chimera formation than is the intact mRNA, inconsistent with a transesterification mechanism but supporting a nuclease-ligase mechanism. Also, when normal mRNA cleavage is inhibited by the presence of a phosphorothioate, normal chimera formation no longer occurs. Rather, this phosphorothioate induces both cleavage and chimera formation at a novel site within the editing domain. Finally, levels of chimera-forming activity correlate with levels of mitochondrial RNA ligase activity when reactions are conducted under conditions which inhibit the ligase, including the lack of ATP containing a cleavable alpha-beta bond. These data show that chimera formation in the mitochondrial extract occurs by a nuclease-ligase mechanism rather than by transesterification.

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Selected References

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  1. Backus J. W., Smith H. C. Specific 3' sequences flanking a minimal apolipoprotein B (apoB) mRNA editing 'cassette' are critical for efficient editing in vitro. Biochim Biophys Acta. 1994 Jan 18;1217(1):65–73. [PubMed] [Google Scholar]
  2. Bakalara N., Simpson A. M., Simpson L. The Leishmania kinetoplast-mitochondrion contains terminal uridylyltransferase and RNA ligase activities. J Biol Chem. 1989 Nov 5;264(31):18679–18686. [PubMed] [Google Scholar]
  3. Benne R. RNA editing in mitochondria of Leishmania tarentolae and Crithidia fasciculata. Semin Cell Biol. 1993 Aug;4(4):241–249. doi: 10.1006/scel.1993.1029. [DOI] [PubMed] [Google Scholar]
  4. Blum B., Bakalara N., Simpson L. A model for RNA editing in kinetoplastid mitochondria: "guide" RNA molecules transcribed from maxicircle DNA provide the edited information. Cell. 1990 Jan 26;60(2):189–198. doi: 10.1016/0092-8674(90)90735-w. [DOI] [PubMed] [Google Scholar]
  5. Blum B., Simpson L. Formation of guide RNA/messenger RNA chimeric molecules in vitro, the initial step of RNA editing, is dependent on an anchor sequence. Proc Natl Acad Sci U S A. 1992 Dec 15;89(24):11944–11948. doi: 10.1073/pnas.89.24.11944. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Blum B., Simpson L. Guide RNAs in kinetoplastid mitochondria have a nonencoded 3' oligo(U) tail involved in recognition of the preedited region. Cell. 1990 Jul 27;62(2):391–397. doi: 10.1016/0092-8674(90)90375-o. [DOI] [PubMed] [Google Scholar]
  7. Blum B., Sturm N. R., Simpson A. M., Simpson L. Chimeric gRNA-mRNA molecules with oligo(U) tails covalently linked at sites of RNA editing suggest that U addition occurs by transesterification. Cell. 1991 May 17;65(4):543–550. doi: 10.1016/0092-8674(91)90087-f. [DOI] [PubMed] [Google Scholar]
  8. Bryant F. R., Benkovic S. J. Phosphorothioate substrates for T4 RNA ligase. Biochemistry. 1982 Nov 9;21(23):5877–5885. doi: 10.1021/bi00266a023. [DOI] [PubMed] [Google Scholar]
  9. Cech T. R. RNA editing: world's smallest introns? Cell. 1991 Feb 22;64(4):667–669. doi: 10.1016/0092-8674(91)90494-j. [DOI] [PubMed] [Google Scholar]
  10. Covello P. S., Gray M. W. On the evolution of RNA editing. Trends Genet. 1993 Aug;9(8):265–268. doi: 10.1016/0168-9525(93)90011-6. [DOI] [PubMed] [Google Scholar]
  11. Decker C. J., Sollner-Webb B. RNA editing involves indiscriminate U changes throughout precisely defined editing domains. Cell. 1990 Jun 15;61(6):1001–1011. doi: 10.1016/0092-8674(90)90065-m. [DOI] [PubMed] [Google Scholar]
  12. Eckstein F. Nucleoside phosphorothioates. Annu Rev Biochem. 1985;54:367–402. doi: 10.1146/annurev.bi.54.070185.002055. [DOI] [PubMed] [Google Scholar]
  13. Giannoni F., Bonen D. K., Funahashi T., Hadjiagapiou C., Burant C. F., Davidson N. O. Complementation of apolipoprotein B mRNA editing by human liver accompanied by secretion of apolipoprotein B48. J Biol Chem. 1994 Feb 25;269(8):5932–5936. [PubMed] [Google Scholar]
  14. Gott J. M., Visomirski L. M., Hunter J. L. Substitutional and insertional RNA editing of the cytochrome c oxidase subunit 1 mRNA of Physarum polycephalum. J Biol Chem. 1993 Dec 5;268(34):25483–25486. [PubMed] [Google Scholar]
  15. Gray M. W., Covello P. S. RNA editing in plant mitochondria and chloroplasts. FASEB J. 1993 Jan;7(1):64–71. doi: 10.1096/fasebj.7.1.8422976. [DOI] [PubMed] [Google Scholar]
  16. Griffiths A. D., Potter B. V., Eperon I. C. Stereospecificity of nucleases towards phosphorothioate-substituted RNA: stereochemistry of transcription by T7 RNA polymerase. Nucleic Acids Res. 1987 May 26;15(10):4145–4162. doi: 10.1093/nar/15.10.4145. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Hajduk S. L., Harris M. E., Pollard V. W. RNA editing in kinetoplastid mitochondria. FASEB J. 1993 Jan;7(1):54–63. doi: 10.1096/fasebj.7.1.8422975. [DOI] [PubMed] [Google Scholar]
  18. Harris M. E., Hajduk S. L. Kinetoplastid RNA editing: in vitro formation of cytochrome b gRNA-mRNA chimeras from synthetic substrate RNAs. Cell. 1992 Mar 20;68(6):1091–1099. doi: 10.1016/0092-8674(92)90080-v. [DOI] [PubMed] [Google Scholar]
  19. Harris M. E., Moore D. R., Hajduk S. L. Addition of uridines to edited RNAs in trypanosome mitochondria occurs independently of transcription. J Biol Chem. 1990 Jul 5;265(19):11368–11376. [PubMed] [Google Scholar]
  20. Harris M., Decker C., Sollner-Webb B., Hajduk S. Specific cleavage of pre-edited mRNAs in trypanosome mitochondrial extracts. Mol Cell Biol. 1992 Jun;12(6):2591–2598. doi: 10.1128/mcb.12.6.2591. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Heidenreich O., Pieken W., Eckstein F. Chemically modified RNA: approaches and applications. FASEB J. 1993 Jan;7(1):90–96. doi: 10.1096/fasebj.7.1.7678566. [DOI] [PubMed] [Google Scholar]
  22. Koslowsky D. J., Göringer H. U., Morales T. H., Stuart K. In vitro guide RNA/mRNA chimaera formation in Trypanosoma brucei RNA editing. Nature. 1992 Apr 30;356(6372):807–809. doi: 10.1038/356807a0. [DOI] [PubMed] [Google Scholar]
  23. Maschhoff K. L., Padgett R. A. The stereochemical course of the first step of pre-mRNA splicing. Nucleic Acids Res. 1993 Nov 25;21(23):5456–5462. doi: 10.1093/nar/21.23.5456. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. McSwiggen J. A., Cech T. R. Stereochemistry of RNA cleavage by the Tetrahymena ribozyme and evidence that the chemical step is not rate-limiting. Science. 1989 May 12;244(4905):679–683. doi: 10.1126/science.2470150. [DOI] [PubMed] [Google Scholar]
  25. Miller D., Mahendran R., Spottswood M., Costandy H., Wang S., Ling M. L., Yang N. Insertional editing in mitochondria of Physarum. Semin Cell Biol. 1993 Aug;4(4):261–266. doi: 10.1006/scel.1993.1031. [DOI] [PubMed] [Google Scholar]
  26. Mizuuchi K., Adzuma K. Inversion of the phosphate chirality at the target site of Mu DNA strand transfer: evidence for a one-step transesterification mechanism. Cell. 1991 Jul 12;66(1):129–140. doi: 10.1016/0092-8674(91)90145-o. [DOI] [PubMed] [Google Scholar]
  27. Moore M. J., Sharp P. A. Evidence for two active sites in the spliceosome provided by stereochemistry of pre-mRNA splicing. Nature. 1993 Sep 23;365(6444):364–368. doi: 10.1038/365364a0. [DOI] [PubMed] [Google Scholar]
  28. Pecoraro V. L., Hermes J. D., Cleland W. W. Stability constants of Mg2+ and Cd2+ complexes of adenine nucleotides and thionucleotides and rate constants for formation and dissociation of MgATP and MgADP. Biochemistry. 1984 Oct 23;23(22):5262–5271. doi: 10.1021/bi00317a026. [DOI] [PubMed] [Google Scholar]
  29. Peris M., Frech G. C., Simpson A. M., Bringaud F., Byrne E., Bakker A., Simpson L. Characterization of two classes of ribonucleoprotein complexes possibly involved in RNA editing from Leishmania tarentolae mitochondria. EMBO J. 1994 Apr 1;13(7):1664–1672. doi: 10.1002/j.1460-2075.1994.tb06430.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Phizicky E. M., Greer C. L. Pre-tRNA splicing: variation on a theme or exception to the rule? Trends Biochem Sci. 1993 Jan;18(1):31–34. doi: 10.1016/0968-0004(93)90085-2. [DOI] [PubMed] [Google Scholar]
  31. Piller K. J., Decker C. J., Rusché L. N., Harris M. E., Hajduk S. L., Sollner-Webb B. Editing domains of Trypanosoma brucei mitochondrial RNAs identified by secondary structure. Mol Cell Biol. 1995 Jun;15(6):2916–2924. doi: 10.1128/mcb.15.6.2916. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Piller K. J., Decker C. J., Rusché L. N., Sollner-Webb B. Trypanosoma brucei mitochondrial guide RNA-mRNA chimera-forming activity cofractionates with an editing-domain-specific endonuclease and RNA ligase and is mimicked by heterologous nuclease and RNA ligase. Mol Cell Biol. 1995 Jun;15(6):2925–2932. doi: 10.1128/mcb.15.6.2925. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Pollard V. W., Harris M. E., Hajduk S. L. Native mRNA editing complexes from Trypanosoma brucei mitochondria. EMBO J. 1992 Dec;11(12):4429–4438. doi: 10.1002/j.1460-2075.1992.tb05543.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Potter B. V., Connolly B. A., Eckstein F. Synthesis and configurational analysis of a dinucleoside phosphate isotopically chiral at phosphorus. Stereochemical course of Penicillium citrum nuclease P1 reaction. Biochemistry. 1983 Mar 15;22(6):1369–1377. doi: 10.1021/bi00275a008. [DOI] [PubMed] [Google Scholar]
  35. Read L. K., Corell R. A., Stuart K. Chimeric and truncated RNAs in Trypanosoma brucei suggest transesterifications at non-consecutive sites during RNA editing. Nucleic Acids Res. 1992 May 11;20(9):2341–2347. doi: 10.1093/nar/20.9.2341. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Riley G. R., Corell R. A., Stuart K. Multiple guide RNAs for identical editing of Trypanosoma brucei apocytochrome b mRNA have an unusual minicircle location and are developmentally regulated. J Biol Chem. 1994 Feb 25;269(8):6101–6108. [PubMed] [Google Scholar]
  37. Seiwert S. D., Stuart K. RNA editing: transfer of genetic information from gRNA to precursor mRNA in vitro. Science. 1994 Oct 7;266(5182):114–117. doi: 10.1126/science.7524149. [DOI] [PubMed] [Google Scholar]
  38. Simpson A. M., Bakalara N., Simpson L. A ribonuclease activity is activated by heparin or by digestion with proteinase K in mitochondrial extracts of Leishmania tarentolae. J Biol Chem. 1992 Apr 5;267(10):6782–6788. [PubMed] [Google Scholar]
  39. Sollner-Webb B. RNA editing. Curr Opin Cell Biol. 1991 Dec;3(6):1056–1061. doi: 10.1016/0955-0674(91)90129-m. [DOI] [PubMed] [Google Scholar]
  40. Stuart K. The RNA editing process in Trypanosoma brucei. Semin Cell Biol. 1993 Aug;4(4):251–260. doi: 10.1006/scel.1993.1030. [DOI] [PubMed] [Google Scholar]
  41. Sturm N. R., Maslov D. A., Blum B., Simpson L. Generation of unexpected editing patterns in Leishmania tarentolae mitochondrial mRNAs: misediting produced by misguiding. Cell. 1992 Aug 7;70(3):469–476. doi: 10.1016/0092-8674(92)90171-8. [DOI] [PubMed] [Google Scholar]

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