Skip to main content
NCBI home page
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1997 Jul;17(7):3915–3923. doi: 10.1128/mcb.17.7.3915

Mitochondrial glutamate dehydrogenase from Leishmania tarentolae is a guide RNA-binding protein.

F Bringaud 1, R Stripecke 1, G C Frech 1, S Freedland 1, C Turck 1, E M Byrne 1, L Simpson 1
PMCID: PMC232244  PMID: 9199326

Abstract

To identify specific proteins interacting with guide RNAs (gRNAs) in mitochondrial ribonucleoprotein complexes from Leishmania tarentolae, fractionated and unfractionated mitochondrial extracts were subjected to UV cross-linking with added labeled gRNA and also with [alpha-32P]UTP-labeled endogenous RNA. An abundant 110-kDa protein (p110) localized in the T-V complex, which sediments in glycerol gradients at the leading edge of the 10S terminal uridylyltransferase peak, was found to interact with both types of labeled RNAs. The p110 protein was gel isolated and subjected to microsequence analysis, and the gene was cloned. The sequence revealed significant similarity with mitochondrial glutamate dehydrogenases. A polyclonal antiserum was raised against a recombinant fragment of the p110 gene and was used to demonstrate a stable and specific gRNA-binding activity by coimmunoprecipitation and competitive gel shift analyses. Complex formation was strongly inhibited by competition with poly(U) or by deletion or substitution of the gRNA 3' oligo(U) tail. Also, addition of a 3' oligo(U) tail to an unrelated transcript was sufficient for p110 binding. Both the gRNA-binding activity of the p110 protein and in vitro gRNA-independent and gRNA-dependent uridine insertion activities in the mitochondrial extract were inhibited by high concentrations of dinucleotides.

Full Text

The Full Text of this article is available as a PDF (1.8 MB).

Selected References

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

  1. 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]
  2. 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]
  3. 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]
  4. 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]
  5. 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]
  6. Braly P., Simpson L., Kretzer F. Isolation of kinetoplast-mitochondrial complexes from Leishmania tarentolae. J Protozool. 1974 Nov;21(5):782–790. doi: 10.1111/j.1550-7408.1974.tb03752.x. [DOI] [PubMed] [Google Scholar]
  7. Bringaud F., Peris M., Zen K. H., Simpson L. Characterization of two nuclear-encoded protein components of mitochondrial ribonucleoprotein complexes from Leishmania tarentolae. Mol Biochem Parasitol. 1995 Apr;71(1):65–79. doi: 10.1016/0166-6851(95)00023-t. [DOI] [PubMed] [Google Scholar]
  8. Britton K. L., Baker P. J., Rice D. W., Stillman T. J. Structural relationship between the hexameric and tetrameric family of glutamate dehydrogenases. Eur J Biochem. 1992 Nov 1;209(3):851–859. doi: 10.1111/j.1432-1033.1992.tb17357.x. [DOI] [PubMed] [Google Scholar]
  9. Byrne E., Bringaud F., Simpson L. RNA-protein interactions in the ribonucleoprotein T-complexes in a mitochondrial extract from Leishmania tarentolae. Mol Biochem Parasitol. 1995 Jun;72(1-2):65–76. doi: 10.1016/0166-6851(95)00063-7. [DOI] [PubMed] [Google Scholar]
  10. Campbell D. A. c2X75, a derivative of the cosmid vector c2XB. Nucleic Acids Res. 1989 Jan 11;17(1):458–458. doi: 10.1093/nar/17.1.458. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. 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]
  12. Chu E., Koeller D. M., Casey J. L., Drake J. C., Chabner B. A., Elwood P. C., Zinn S., Allegra C. J. Autoregulation of human thymidylate synthase messenger RNA translation by thymidylate synthase. Proc Natl Acad Sci U S A. 1991 Oct 15;88(20):8977–8981. doi: 10.1073/pnas.88.20.8977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Chu E., Takimoto C. H., Voeller D., Grem J. L., Allegra C. J. Specific binding of human dihydrofolate reductase protein to dihydrofolate reductase messenger RNA in vitro. Biochemistry. 1993 May 11;32(18):4756–4760. doi: 10.1021/bi00069a009. [DOI] [PubMed] [Google Scholar]
  14. Connell G. J., Byrne E. M., Simpson L. Guide RNA-independent and guide RNA-dependent uridine insertion into cytochrome b mRNA in a mitochondrial lysate from Leishmania tarentolae. Role of RNA secondary structure. J Biol Chem. 1997 Feb 14;272(7):4212–4218. doi: 10.1074/jbc.272.7.4212. [DOI] [PubMed] [Google Scholar]
  15. Constable A., Quick S., Gray N. K., Hentze M. W. Modulation of the RNA-binding activity of a regulatory protein by iron in vitro: switching between enzymatic and genetic function? Proc Natl Acad Sci U S A. 1992 May 15;89(10):4554–4558. doi: 10.1073/pnas.89.10.4554. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. 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]
  17. 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]
  18. Elzinga S. D., Bednarz A. L., van Oosterum K., Dekker P. J., Grivell L. A. Yeast mitochondrial NAD(+)-dependent isocitrate dehydrogenase is an RNA-binding protein. Nucleic Acids Res. 1993 Nov 25;21(23):5328–5331. doi: 10.1093/nar/21.23.5328. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. 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]
  20. Frech G. C., Simpson L. Uridine insertion into preedited mRNA by a mitochondrial extract from Leishmania tarentolae: stereochemical evidence for the enzyme cascade model. Mol Cell Biol. 1996 Aug;16(8):4584–4589. doi: 10.1128/mcb.16.8.4584. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Gottlieb E., Steitz J. A. Function of the mammalian La protein: evidence for its action in transcription termination by RNA polymerase III. EMBO J. 1989 Mar;8(3):851–861. doi: 10.1002/j.1460-2075.1989.tb03446.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. 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]
  23. 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]
  24. 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]
  25. Hentze M. W. Enzymes as RNA-binding proteins: a role for (di)nucleotide-binding domains? Trends Biochem Sci. 1994 Mar;19(3):101–103. doi: 10.1016/0968-0004(94)90198-8. [DOI] [PubMed] [Google Scholar]
  26. 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]
  27. Kapoor M., Vijayaraghavan Y., Kadonaga R., LaRue K. E. NAD(+)-specific glutamate dehydrogenase of Neurospora crassa: cloning, complete nucleotide sequence, and gene mapping. Biochem Cell Biol. 1993 Mar-Apr;71(3-4):205–219. doi: 10.1139/o93-032. [DOI] [PubMed] [Google Scholar]
  28. Kyrpides N. C., Ouzounis C. A. Nucleic acid-binding metabolic enzymes: living fossils of stereochemical interactions? J Mol Evol. 1995 Jun;40(6):564–569. doi: 10.1007/BF00160502. [DOI] [PubMed] [Google Scholar]
  29. 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]
  30. 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]
  31. Lima B. D., Simpson L. Sequence-dependent in vivo importation of tRNAs into the mitochondrion of Leishmania tarentolae. RNA. 1996 May;2(5):429–440. [PMC free article] [PubMed] [Google Scholar]
  32. Miller R. L., Wang A. L., Wang C. C. Identification of Giardia lamblia isolates susceptible and resistant to infection by the double-stranded RNA virus. Exp Parasitol. 1988 Jun;66(1):118–123. doi: 10.1016/0014-4894(88)90056-2. [DOI] [PubMed] [Google Scholar]
  33. Milligan J. F., Groebe D. R., Witherell G. W., Uhlenbeck O. C. Oligoribonucleotide synthesis using T7 RNA polymerase and synthetic DNA templates. Nucleic Acids Res. 1987 Nov 11;15(21):8783–8798. doi: 10.1093/nar/15.21.8783. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. 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]
  35. Missel A., Nörskau G., Shu H. H., Göringer H. U. A putative RNA helicase of the DEAD box family from Trypanosoma brucei. Mol Biochem Parasitol. 1995 Dec;75(1):123–126. doi: 10.1016/0166-6851(95)02511-1. [DOI] [PubMed] [Google Scholar]
  36. 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]
  37. Peris M., Simpson A. M., Grunstein J., Liliental J. E., Frech G. C., Simpson L. Native gel analysis of ribonucleoprotein complexes from a Leishmania tarentolae mitochondrial extract. Mol Biochem Parasitol. 1997 Mar;85(1):9–24. doi: 10.1016/s0166-6851(96)02795-8. [DOI] [PubMed] [Google Scholar]
  38. 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]
  39. 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]
  40. 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]
  41. Preiss T., Hall A. G., Lightowlers R. N. Identification of bovine glutamate dehydrogenase as an RNA-binding protein. J Biol Chem. 1993 Nov 25;268(33):24523–24526. [PubMed] [Google Scholar]
  42. 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]
  43. Saiki R. K., Gelfand D. H., Stoffel S., Scharf S. J., Higuchi R., Horn G. T., Mullis K. B., Erlich H. A. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science. 1988 Jan 29;239(4839):487–491. doi: 10.1126/science.2448875. [DOI] [PubMed] [Google Scholar]
  44. 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]
  45. 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]
  46. 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]
  47. Simpson L., Braly P. Synchronization of Leishmania tarentolae by hydroxyurea. J Protozool. 1970 Nov;17(4):511–517. doi: 10.1111/j.1550-7408.1970.tb04719.x. [DOI] [PubMed] [Google Scholar]
  48. Singh R., Green M. R. Sequence-specific binding of transfer RNA by glyceraldehyde-3-phosphate dehydrogenase. Science. 1993 Jan 15;259(5093):365–368. doi: 10.1126/science.8420004. [DOI] [PubMed] [Google Scholar]
  49. Smith H. O., Tabiti K., Schaffner G., Soldati D., Albrecht U., Birnstiel M. L. Two-step affinity purification of U7 small nuclear ribonucleoprotein particles using complementary biotinylated 2'-O-methyl oligoribonucleotides. Proc Natl Acad Sci U S A. 1991 Nov 1;88(21):9784–9788. doi: 10.1073/pnas.88.21.9784. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Stripecke R., Hentze M. W. Bacteriophage and spliceosomal proteins function as position-dependent cis/trans repressors of mRNA translation in vitro. Nucleic Acids Res. 1992 Nov 11;20(21):5555–5564. doi: 10.1093/nar/20.21.5555. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Taussig R., Gilman A. G. Mammalian membrane-bound adenylyl cyclases. J Biol Chem. 1995 Jan 6;270(1):1–4. doi: 10.1074/jbc.270.1.1. [DOI] [PubMed] [Google Scholar]
  52. Theriot J. A. Worm sperm and advances in cell locomotion. Cell. 1996 Jan 12;84(1):1–4. doi: 10.1016/s0092-8674(00)80068-9. [DOI] [PubMed] [Google Scholar]
  53. Xu C., Ray D. S. Isolation of proteins associated with kinetoplast DNA networks in vivo. Proc Natl Acad Sci U S A. 1993 Mar 1;90(5):1786–1789. doi: 10.1073/pnas.90.5.1786. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Yoo C. J., Wolin S. L. La proteins from Drosophila melanogaster and Saccharomyces cerevisiae: a yeast homolog of the La autoantigen is dispensable for growth. Mol Cell Biol. 1994 Aug;14(8):5412–5424. doi: 10.1128/mcb.14.8.5412. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Molecular and Cellular Biology are provided here courtesy of Taylor & Francis

RESOURCES