Skip to main content
NCBI home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1996 Aug 20;93(17):8901–8906. doi: 10.1073/pnas.93.17.8901

Trypanosome U-deletional RNA editing involves guide RNA-directed endonuclease cleavage, terminal U exonuclease, and RNA ligase activities.

J Cruz-Reyes 1, B Sollner-Webb 1
PMCID: PMC38566  PMID: 8799125

Abstract

We have studied the mechanism of accurate in vitro RNA editing of Trypanosoma brucei ATPase 6 mRNA, using four mRNA-guide RNA (gRNA) pairs that specify deletion of 2, 3, or 4 U residues at editing site 1 and mitochondrial extract. This extract not only catalyzes deletion of the specified number of U residues but also exhibits a novel endonuclease activity that cleaves the input pre-mRNA in a gRNA-directed manner, precisely at the phosphodiester bond predicted in a simple enzymatic model of RNA editing. This cleavage site is inconsistent with a chimera-based editing mechanism. The U residues to be deleted, present at the 3' end of the upstream cleavage product, are then removed evidently by a 3' U-specific exonuclease and not by a reverse reaction of terminal U transferase. RNA ligase can then join the mRNA halves through their newly formed 5' P and 3' OH termini, generating mRNA faithfully edited at the first editing site. This resultant, partially edited mRNA can then undergo accurate, gRNA-directed cleavage at editing site 2, again precisely as predicted by the enzymatic editing model. All of these enzymatic activities cofractionate with the U-deletion activity and may reside in a single complex. The data imply that each round of editing is a four-step process, involving (i) gRNA-directed cleavage of the pre-mRNA at the bond immediately 5' of the region base paired to the gRNA, (ii) U deletion from or U addition to the 3' OH of the upstream mRNA half, (iii) ligation of the mRNA halves, and (iv) formation of additional base pairing between the correctly edited site and the gRNA that directs subsequent nuclease cleavage at the next editing site.

Full text

PDF
8901

Images in this article

8903Fig. 2
on p.8903
8903Fig. 3
on p.8903
8904Fig. 4
on p.8904
8904Fig. 5
on p.8904
8905Fig. 6
on p.8905

Selected References

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

  1. Adler B. K., Hajduk S. L. Mechanisms and origins of RNA editing. Curr Opin Genet Dev. 1994 Apr;4(2):316–322. doi: 10.1016/s0959-437x(05)80060-7. [DOI] [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 trypanosomes. Eur J Biochem. 1994 Apr 1;221(1):9–23. doi: 10.1111/j.1432-1033.1994.tb18710.x. [DOI] [PubMed] [Google Scholar]
  4. Bhat G. J., Koslowsky D. J., Feagin J. E., Smiley B. L., Stuart K. An extensively edited mitochondrial transcript in kinetoplastids encodes a protein homologous to ATPase subunit 6. Cell. 1990 Jun 1;61(5):885–894. doi: 10.1016/0092-8674(90)90199-o. [DOI] [PubMed] [Google Scholar]
  5. 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]
  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. 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]
  9. 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]
  10. 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]
  11. 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]
  12. 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]
  13. 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]
  14. 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]
  15. 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]
  16. 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]
  17. 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]
  18. 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]
  19. 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]
  20. Simpson L., Thiemann O. H. Sense from nonsense: RNA editing in mitochondria of kinetoplastid protozoa and slime molds. Cell. 1995 Jun 16;81(6):837–840. doi: 10.1016/0092-8674(95)90003-9. [DOI] [PubMed] [Google Scholar]
  21. 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]
  22. Sollner-Webb B., Reeder R. H. The nucleotide sequence of the initiation and termination sites for ribosomal RNA transcription in X. laevis. Cell. 1979 Oct;18(2):485–499. doi: 10.1016/0092-8674(79)90066-7. [DOI] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES