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. 1997 Jul 1;16(13):4069–4081. doi: 10.1093/emboj/16.13.4069

Purification of a functional enzymatic editing complex from Trypanosoma brucei mitochondria.

L N Rusché 1, J Cruz-Reyes 1, K J Piller 1, B Sollner-Webb 1
PMCID: PMC1170030  PMID: 9233816

Abstract

Kinetoplastid mitochondrial RNA editing, the insertion and deletion of U residues, is catalyzed by sequential cleavage, U addition or removal, and ligation reactions and is directed by complementary guide RNAs. We have purified a approximately 20S enzymatic complex from Trypanosoma brucei mitochondria that catalyzes a complete editing reaction in vitro. This complex possesses all four activities predicted to catalyze RNA editing: gRNA-directed endonuclease, terminal uridylyl transferase, 3' U-specific exonuclease, and RNA ligase. However, it does not contain other putative editing complex components: gRNA-independent endonuclease, RNA helicase, endogenous gRNAs or pre-mRNAs, or a 25 kDa gRNA-binding protein. The complex is composed of eight major polypeptides, three of which represent RNA ligase. These findings identify polypeptides representing catalytic editing factors, reveal the nature of this approximately 20S editing complex, and suggest a new model of editosome assembly.

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Selected References

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  1. 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]
  2. Byrne E. M., Connell G. J., Simpson L. Guide RNA-directed uridine insertion RNA editing in vitro. EMBO J. 1996 Dec 2;15(23):6758–6765. [PMC free article] [PubMed] [Google Scholar]
  3. 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]
  4. Corell R. A., Read L. K., Riley G. R., Nellissery J. K., Allen T. E., Kable M. L., Wachal M. D., Seiwert S. D., Myler P. J., Stuart K. D. Complexes from Trypanosoma brucei that exhibit deletion editing and other editing-associated properties. Mol Cell Biol. 1996 Apr;16(4):1410–1418. doi: 10.1128/mcb.16.4.1410. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Cruz-Reyes J., Sollner-Webb B. Trypanosome U-deletional RNA editing involves guide RNA-directed endonuclease cleavage, terminal U exonuclease, and RNA ligase activities. Proc Natl Acad Sci U S A. 1996 Aug 20;93(17):8901–8906. doi: 10.1073/pnas.93.17.8901. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. 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]
  7. Frech G. C., Bakalara N., Simpson L., Simpson A. M. In vitro RNA editing-like activity in a mitochondrial extract from Leishmania tarentolae. EMBO J. 1995 Jan 3;14(1):178–187. doi: 10.1002/j.1460-2075.1995.tb06988.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Göringer H. U., Koslowsky D. J., Morales T. H., Stuart K. The formation of mitochondrial ribonucleoprotein complexes involving guide RNA molecules in Trypanosoma brucei. Proc Natl Acad Sci U S A. 1994 Mar 1;91(5):1776–1780. doi: 10.1073/pnas.91.5.1776. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. 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]
  10. Kable M. L., Seiwert S. D., Heidmann S., Stuart K. RNA editing: a mechanism for gRNA-specified uridylate insertion into precursor mRNA. Science. 1996 Aug 30;273(5279):1189–1195. doi: 10.1126/science.273.5279.1189. [DOI] [PubMed] [Google Scholar]
  11. Köller J., Nörskau G., Paul A. S., Stuart K., Göringer H. U. Different Trypanosoma brucei guide RNA molecules associate with an identical complement of mitochondrial proteins in vitro. Nucleic Acids Res. 1994 Jun 11;22(11):1988–1995. doi: 10.1093/nar/22.11.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Leegwater P., Speijer D., Benne R. Identification by UV cross-linking of oligo(U)-binding proteins in mitochondria of the insect trypanosomatid Crithidia fasciculata. Eur J Biochem. 1995 Feb 1;227(3):780–786. doi: 10.1111/j.1432-1033.1995.tb20201.x. [DOI] [PubMed] [Google Scholar]
  13. Missel A., Göringer H. U. Trypanosoma brucei mitochondria contain RNA helicase activity. Nucleic Acids Res. 1994 Oct 11;22(20):4050–4056. doi: 10.1093/nar/22.20.4050. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. 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]
  15. 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]
  16. 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]
  17. Piller K. J., Rusché L. N., Cruz-Reyes J., Sollner-Webb B. Resolution of the RNA editing gRNA-directed endonuclease from two other endonucleases of Trypanosoma brucei mitochondria. RNA. 1997 Mar;3(3):279–290. [PMC free article] [PubMed] [Google Scholar]
  18. Piller K. J., Rusché L. N., Sollner-Webb B. Trypanosoma brucei RNA editing. A full round of uridylate insertional editing in vitro mediated by endonuclease and RNA ligase. J Biol Chem. 1996 Mar 1;271(9):4613–4619. doi: 10.1074/jbc.271.9.4613. [DOI] [PubMed] [Google Scholar]
  19. Pollard V. W., Rohrer S. P., Michelotti E. F., Hancock K., Hajduk S. L. Organization of minicircle genes for guide RNAs in Trypanosoma brucei. Cell. 1990 Nov 16;63(4):783–790. doi: 10.1016/0092-8674(90)90144-4. [DOI] [PubMed] [Google Scholar]
  20. Rusché L. N., Piller K. J., Sollner-Webb B. Guide RNA-mRNA chimeras, which are potential RNA editing intermediates, are formed by endonuclease and RNA ligase in a trypanosome mitochondrial extract. Mol Cell Biol. 1995 Jun;15(6):2933–2941. doi: 10.1128/mcb.15.6.2933. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Sabatini R., Hajduk S. L. RNA ligase and its involvement in guide RNA/mRNA chimera formation. Evidence for a cleavage-ligation mechanism of Trypanosoma brucei mRNA editing. J Biol Chem. 1995 Mar 31;270(13):7233–7240. doi: 10.1074/jbc.270.13.7233. [DOI] [PubMed] [Google Scholar]
  22. Seiwert S. D., Heidmann S., Stuart K. Direct visualization of uridylate deletion in vitro suggests a mechanism for kinetoplastid RNA editing. Cell. 1996 Mar 22;84(6):831–841. doi: 10.1016/s0092-8674(00)81062-4. [DOI] [PubMed] [Google Scholar]
  23. 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]
  24. Sharp P. A. Split genes and RNA splicing. Cell. 1994 Jun 17;77(6):805–815. doi: 10.1016/0092-8674(94)90130-9. [DOI] [PubMed] [Google Scholar]
  25. Shu H. H., Stuart K., Göringer H. U. Guide RNA molecules not engaged in RNA editing form ribonucleoprotein complexes free of mRNA. Biochim Biophys Acta. 1995 Apr 26;1261(3):349–359. doi: 10.1016/0167-4781(95)00025-c. [DOI] [PubMed] [Google Scholar]
  26. 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]
  27. 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]

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