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. 1994 Oct 11;22(20):4050–4056. doi: 10.1093/nar/22.20.4050

Trypanosoma brucei mitochondria contain RNA helicase activity.

A Missel 1, H U Göringer 1
PMCID: PMC331889  PMID: 7524033

Abstract

Mitochondrial gene expression in kinetoplastid organisms such as Trypanosoma, Leishmania and Crithidia requires a posttranscriptional RNA processing event known as kRNA editing. During editing, uridine nucleotides get inserted and deleted into pre-mRNAs directed by small, metabolically stable RNAs, termed guide RNAs. Although the precise mechanism of the reaction is not understood, the accepted working model describes the formation of extended anti-parallel RNA helices between gRNA molecules with pre- and partially edited mRNAs as intermediates. These duplex structures must be separated to ensure the sequential action of multiple gRNAs in a 3' to 5' polarity on the mRNA molecule. In spite of this fact, no unwinding activity has heretofore been identified in kinetoplastid mitochondria. We report the characterisation of a RNA helicase activity within Trypanosoma brucei mitochondrial extracts. The activity unwinds 25- and 48 bp, tailed RNA duplex structures but fails to separate DNA strands. It can be destroyed by heat denaturation as well as by proteinase K treatment. The activity requires magnesium cations and acts in a NTP/dNTP dependent manner. Hydrolysis of a nucleoside triphosphate is required rather than mere NTP binding as deduced from a comparison of unwinding in the presence of ATP and AMP-PCP. RNA duplexes mimicking presumed kRNA editing intermediates are substrates of the unwinding activity and therefore, we address the possible involvement of a RNA helicase activity during kRNA editing.

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Selected References

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

  1. Agabian N. Trans splicing of nuclear pre-mRNAs. Cell. 1990 Jun 29;61(7):1157–1160. doi: 10.1016/0092-8674(90)90674-4. [DOI] [PubMed] [Google Scholar]
  2. Bass B. L., Weintraub H. A developmentally regulated activity that unwinds RNA duplexes. Cell. 1987 Feb 27;48(4):607–613. doi: 10.1016/0092-8674(87)90239-x. [DOI] [PubMed] [Google Scholar]
  3. Bass B. L., Weintraub H. An unwinding activity that covalently modifies its double-stranded RNA substrate. Cell. 1988 Dec 23;55(6):1089–1098. doi: 10.1016/0092-8674(88)90253-x. [DOI] [PubMed] [Google Scholar]
  4. Benne R. RNA-editing in trypanosome mitochondria. Biochim Biophys Acta. 1989 Mar 1;1007(2):131–139. doi: 10.1016/0167-4781(89)90031-6. [DOI] [PubMed] [Google Scholar]
  5. 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]
  6. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  7. Claude A., Arenas J., Hurwitz J. The isolation and characterization of an RNA helicase from nuclear extracts of HeLa cells. J Biol Chem. 1991 Jun 5;266(16):10358–10367. [PubMed] [Google Scholar]
  8. Cross M., Günzl A., Palfi Z., Bindereif A. Analysis of small nuclear ribonucleoproteins (RNPs) in Trypanosoma brucei: structural organization and protein components of the spliced leader RNP. Mol Cell Biol. 1991 Nov;11(11):5516–5526. doi: 10.1128/mcb.11.11.5516. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Devereux J., Haeberli P., Smithies O. A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 1984 Jan 11;12(1 Pt 1):387–395. doi: 10.1093/nar/12.1part1.387. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. 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]
  11. 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]
  12. 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]
  13. Hemmingsen S. M., Woolford C., van der Vies S. M., Tilly K., Dennis D. T., Georgopoulos C. P., Hendrix R. W., Ellis R. J. Homologous plant and bacterial proteins chaperone oligomeric protein assembly. Nature. 1988 May 26;333(6171):330–334. doi: 10.1038/333330a0. [DOI] [PubMed] [Google Scholar]
  14. Hirling H., Scheffner M., Restle T., Stahl H. RNA helicase activity associated with the human p68 protein. Nature. 1989 Jun 15;339(6225):562–564. doi: 10.1038/339562a0. [DOI] [PubMed] [Google Scholar]
  15. Jaramillo M., Dever T. E., Merrick W. C., Sonenberg N. RNA unwinding in translation: assembly of helicase complex intermediates comprising eukaryotic initiation factors eIF-4F and eIF-4B. Mol Cell Biol. 1991 Dec;11(12):5992–5997. doi: 10.1128/mcb.11.12.5992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Koll H., Guiard B., Rassow J., Ostermann J., Horwich A. L., Neupert W., Hartl F. U. Antifolding activity of hsp60 couples protein import into the mitochondrial matrix with export to the intermembrane space. Cell. 1992 Mar 20;68(6):1163–1175. doi: 10.1016/0092-8674(92)90086-r. [DOI] [PubMed] [Google Scholar]
  17. Koslowsky D. J., Bhat G. J., Read L. K., Stuart K. Cycles of progressive realignment of gRNA with mRNA in RNA editing. Cell. 1991 Nov 1;67(3):537–546. doi: 10.1016/0092-8674(91)90528-7. [DOI] [PubMed] [Google Scholar]
  18. 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]
  19. Koslowsky D. J., Riley G. R., Feagin J. E., Stuart K. Guide RNAs for transcripts with developmentally regulated RNA editing are present in both life cycle stages of Trypanosoma brucei. Mol Cell Biol. 1992 May;12(5):2043–2049. doi: 10.1128/mcb.12.5.2043. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Lee C. G., Hurwitz J. A new RNA helicase isolated from HeLa cells that catalytically translocates in the 3' to 5' direction. J Biol Chem. 1992 Mar 5;267(7):4398–4407. [PubMed] [Google Scholar]
  21. Maslov D. A., Simpson L. The polarity of editing within a multiple gRNA-mediated domain is due to formation of anchors for upstream gRNAs by downstream editing. Cell. 1992 Aug 7;70(3):459–467. doi: 10.1016/0092-8674(92)90170-h. [DOI] [PubMed] [Google Scholar]
  22. Nishi K., Morel-Deville F., Hershey J. W., Leighton T., Schnier J. An eIF-4A-like protein is a suppressor of an Escherichia coli mutant defective in 50S ribosomal subunit assembly. Nature. 1988 Dec 1;336(6198):496–498. doi: 10.1038/336496a0. [DOI] [PubMed] [Google Scholar]
  23. Ntambi J. M., Shapiro T. A., Ryan K. A., Englund P. T. Ribonucleotides associated with a gap in newly replicated kinetoplast DNA minicircles from Trypanosoma equiperdum. J Biol Chem. 1986 Sep 5;261(25):11890–11895. [PubMed] [Google Scholar]
  24. PENNINGTON R. J. Biochemistry of dystrophic muscle. Mitochondrial succinate-tetrazolium reductase and adenosine triphosphatase. Biochem J. 1961 Sep;80:649–654. doi: 10.1042/bj0800649. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. 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]
  26. 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]
  27. Read L. K., Göringer H. U., Stuart K. Assembly of mitochondrial ribonucleoprotein complexes involves specific guide RNA (gRNA)-binding proteins and gRNA domains but does not require preedited mRNA. Mol Cell Biol. 1994 Apr;14(4):2629–2639. doi: 10.1128/mcb.14.4.2629. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Rohrer S. P., Michelotti E. F., Torri A. F., Hajduk S. L. Transcription of kinetoplast DNA minicircles. Cell. 1987 Jun 5;49(5):625–632. doi: 10.1016/0092-8674(87)90538-1. [DOI] [PubMed] [Google Scholar]
  29. Ruby S. W., Abelson J. Pre-mRNA splicing in yeast. Trends Genet. 1991 Mar;7(3):79–85. doi: 10.1016/0168-9525(91)90276-V. [DOI] [PubMed] [Google Scholar]
  30. Schimmang T., Tollervey D., Kern H., Frank R., Hurt E. C. A yeast nucleolar protein related to mammalian fibrillarin is associated with small nucleolar RNA and is essential for viability. EMBO J. 1989 Dec 20;8(13):4015–4024. doi: 10.1002/j.1460-2075.1989.tb08584.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Schmid S. R., Linder P. D-E-A-D protein family of putative RNA helicases. Mol Microbiol. 1992 Feb;6(3):283–291. doi: 10.1111/j.1365-2958.1992.tb01470.x. [DOI] [PubMed] [Google Scholar]
  32. Shuman S. Vaccinia virus RNA helicase: an essential enzyme related to the DE-H family of RNA-dependent NTPases. Proc Natl Acad Sci U S A. 1992 Nov 15;89(22):10935–10939. doi: 10.1073/pnas.89.22.10935. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Simpson L. RNA editing--a novel genetic phenomenon? Science. 1990 Oct 26;250(4980):512–513. doi: 10.1126/science.1700474. [DOI] [PubMed] [Google Scholar]
  34. Simpson L., Shaw J. RNA editing and the mitochondrial cryptogenes of kinetoplastid protozoa. Cell. 1989 May 5;57(3):355–366. doi: 10.1016/0092-8674(89)90911-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Spithill T. W., Shimer S. P., Hill G. C. Inhibitory effects of chloramphenicol isomers and other antibiotics on protein synthesis and respiration in procyclic Trypanosoma brucei brucei. Mol Biochem Parasitol. 1981 Feb;2(3-4):235–255. doi: 10.1016/0166-6851(81)90103-1. [DOI] [PubMed] [Google Scholar]
  36. Stepien P. P., Margossian S. P., Landsman D., Butow R. A. The yeast nuclear gene suv3 affecting mitochondrial post-transcriptional processes encodes a putative ATP-dependent RNA helicase. Proc Natl Acad Sci U S A. 1992 Aug 1;89(15):6813–6817. doi: 10.1073/pnas.89.15.6813. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Stuart K., Gobright E., Jenni L., Milhausen M., Thomashow L., Agabian N. The IsTaR 1 serodeme of Trypanosoma brucei: development of a new serodeme. J Parasitol. 1984 Oct;70(5):747–754. [PubMed] [Google Scholar]
  38. Séraphin B., Simon M., Boulet A., Faye G. Mitochondrial splicing requires a protein from a novel helicase family. Nature. 1989 Jan 5;337(6202):84–87. doi: 10.1038/337084a0. [DOI] [PubMed] [Google Scholar]
  39. Woods A., Sherwin T., Sasse R., MacRae T. H., Baines A. J., Gull K. Definition of individual components within the cytoskeleton of Trypanosoma brucei by a library of monoclonal antibodies. J Cell Sci. 1989 Jul;93(Pt 3):491–500. doi: 10.1242/jcs.93.3.491. [DOI] [PubMed] [Google Scholar]
  40. Zuker M., Jaeger J. A., Turner D. H. A comparison of optimal and suboptimal RNA secondary structures predicted by free energy minimization with structures determined by phylogenetic comparison. Nucleic Acids Res. 1991 May 25;19(10):2707–2714. doi: 10.1093/nar/19.10.2707. [DOI] [PMC free article] [PubMed] [Google Scholar]

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