Abstract
The RNA editing that produces most functional mRNAs in trypanosomes is catalysed by a multiprotein complex. This complex catalyses the endoribonucleolytic cleavage, uridylate addition and removal, and RNA ligation steps of the editing process. Enzymatic and in vitro editing analyses reveal that each catalytic step contributes to the specificity of the editing and, together with the interaction between gRNA and the mRNA, results in precisely edited mRNAs. Tandem mass spectrometric analysis was used to identify the genes for several components of biochemically purified editing complexes. Their identity and presence in the editing complex were confirmed using immunochemical analyses utilizing mAbs specific to the editing complex components. The genes for two RNA ligases were identified. Genetic studies show that some, but not all, of the components of the complex are essential for editing. The TbMP52 RNA ligase is essential for editing while the TbMP48 RNA ligase is not. Editing was found to be essential in bloodstream form trypanosomes. This is surprising because mutants devoid of genes encoding RNAs that become edited survive as bloodstream forms but encouraging since editing complex components may be targets for chemotherapy.
Full Text
The Full Text of this article is available as a PDF (911.4 KB).
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- 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]
- 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]
- Estévez A. M., Simpson L. Uridine insertion/deletion RNA editing in trypanosome mitochondria--a review. Gene. 1999 Nov 29;240(2):247–260. doi: 10.1016/s0378-1119(99)00437-0. [DOI] [PubMed] [Google Scholar]
- Feagin J. E., Jasmer D. P., Stuart K. Differential mitochondrial gene expression between slender and stumpy bloodforms of Trypanosoma brucei. Mol Biochem Parasitol. 1986 Sep;20(3):207–214. doi: 10.1016/0166-6851(86)90100-3. [DOI] [PubMed] [Google Scholar]
- Gygi S. P., Rochon Y., Franza B. R., Aebersold R. Correlation between protein and mRNA abundance in yeast. Mol Cell Biol. 1999 Mar;19(3):1720–1730. doi: 10.1128/mcb.19.3.1720. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Horváth A., Berry E. A., Maslov D. A. Translation of the edited mRNA for cytochrome b in trypanosome mitochondria. Science. 2000 Mar 3;287(5458):1639–1640. doi: 10.1126/science.287.5458.1639. [DOI] [PubMed] [Google Scholar]
- Igo R. P., Jr, Palazzo S. S., Burgess M. L., Panigrahi A. K., Stuart K. Uridylate addition and RNA ligation contribute to the specificity of kinetoplastid insertion RNA editing. Mol Cell Biol. 2000 Nov;20(22):8447–8457. doi: 10.1128/mcb.20.22.8447-8457.2000. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kable M. L., Heidmann S., Stuart K. D. RNA editing: getting U into RNA. Trends Biochem Sci. 1997 May;22(5):162–166. doi: 10.1016/s0968-0004(97)01041-4. [DOI] [PubMed] [Google Scholar]
- 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]
- Köller J., Müller U. F., Schmid B., Missel A., Kruft V., Stuart K., Göringer H. U. Trypanosoma brucei gBP21. An arginine-rich mitochondrial protein that binds to guide RNA with high affinity. J Biol Chem. 1997 Feb 7;272(6):3749–3757. doi: 10.1074/jbc.272.6.3749. [DOI] [PubMed] [Google Scholar]
- Lambert L., Müller U. F., Souza A. E., Göringer H. U. The involvement of gRNA-binding protein gBP21 in RNA editing-an in vitro and in vivo analysis. Nucleic Acids Res. 1999 Mar 15;27(6):1429–1436. doi: 10.1093/nar/27.6.1429. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Lygerou Z., Christophides G., Séraphin B. A novel genetic screen for snRNP assembly factors in yeast identifies a conserved protein, Sad1p, also required for pre-mRNA splicing. Mol Cell Biol. 1999 Mar;19(3):2008–2020. doi: 10.1128/mcb.19.3.2008. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Madison-Antenucci S., Sabatini R. S., Pollard V. W., Hajduk S. L. Kinetoplastid RNA-editing-associated protein 1 (REAP-1): a novel editing complex protein with repetitive domains. EMBO J. 1998 Nov 2;17(21):6368–6376. doi: 10.1093/emboj/17.21.6368. [DOI] [PMC free article] [PubMed] [Google Scholar]
- McManus M. T., Shimamura M., Grams J., Hajduk S. L. Identification of candidate mitochondrial RNA editing ligases from Trypanosoma brucei. RNA. 2001 Feb;7(2):167–175. doi: 10.1017/s1355838201002072. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Missel A., Souza A. E., Nörskau G., Göringer H. U. Disruption of a gene encoding a novel mitochondrial DEAD-box protein in Trypanosoma brucei affects edited mRNAs. Mol Cell Biol. 1997 Sep;17(9):4895–4903. doi: 10.1128/mcb.17.9.4895. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Panigrahi A. K., Gygi S. P., Ernst N. L., Igo R. P., Jr, Palazzo S. S., Schnaufer A., Weston D. S., Carmean N., Salavati R., Aebersold R. Association of two novel proteins, TbMP52 and TbMP48, with the Trypanosoma brucei RNA editing complex. Mol Cell Biol. 2001 Jan;21(2):380–389. doi: 10.1128/MCB.21.2.380-389.2001. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- Rusché L. N., Cruz-Reyes J., Piller K. J., Sollner-Webb B. Purification of a functional enzymatic editing complex from Trypanosoma brucei mitochondria. EMBO J. 1997 Jul 1;16(13):4069–4081. doi: 10.1093/emboj/16.13.4069. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Rusché L. N., Huang C. E., Piller K. J., Hemann M., Wirtz E., Sollner-Webb B. The two RNA ligases of the Trypanosoma brucei RNA editing complex: cloning the essential band IV gene and identifying the band V gene. Mol Cell Biol. 2001 Feb;21(4):979–989. doi: 10.1128/MCB.21.4.979-989.2001. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- Schnaufer A., Panigrahi A. K., Panicucci B., Igo R. P., Jr, Wirtz E., Salavati R., Stuart K. An RNA ligase essential for RNA editing and survival of the bloodstream form of Trypanosoma brucei. Science. 2001 Feb 15;291(5511):2159–2162. doi: 10.1126/science.1058955. [DOI] [PubMed] [Google Scholar]
- 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]
- Shi H., Djikeng A., Mark T., Wirtz E., Tschudi C., Ullu E. Genetic interference in Trypanosoma brucei by heritable and inducible double-stranded RNA. RNA. 2000 Jul;6(7):1069–1076. doi: 10.1017/s1355838200000297. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Shuman S., Schwer B. RNA capping enzyme and DNA ligase: a superfamily of covalent nucleotidyl transferases. Mol Microbiol. 1995 Aug;17(3):405–410. doi: 10.1111/j.1365-2958.1995.mmi_17030405.x. [DOI] [PubMed] [Google Scholar]
- Stuart K., Allen T. E., Heidmann S., Seiwert S. D. RNA editing in kinetoplastid protozoa. Microbiol Mol Biol Rev. 1997 Mar;61(1):105–120. doi: 10.1128/mmbr.61.1.105-120.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Stuart K. RNA editing in trypanosomatid mitochondria. Annu Rev Microbiol. 1991;45:327–344. doi: 10.1146/annurev.mi.45.100191.001551. [DOI] [PubMed] [Google Scholar]
- Wirtz E., Leal S., Ochatt C., Cross G. A. A tightly regulated inducible expression system for conditional gene knock-outs and dominant-negative genetics in Trypanosoma brucei. Mol Biochem Parasitol. 1999 Mar 15;99(1):89–101. doi: 10.1016/s0166-6851(99)00002-x. [DOI] [PubMed] [Google Scholar]