Abstract
The majority of mitochondrial pre-mRNAs in kinetoplastid protozoa such as Trypanosoma, Leishmania, and Crithidia are substrates of a posttranscriptional processing reaction referred to as RNA editing. The process results in the insertion and, to a lesser extent, deletion of uridylates, thereby completing the informational content of the mRNAs. The specificity of the RNA editing reaction is provided by guide RNAs (gRNAs), which serve as templates for the editing apparatus. In addition, the process relies on mitochondrial proteins, presumably acting within a high-molecular-mass ribonucleoprotein complex. Although several enzymatic activities have been implicated in the editing process, no protein has been identified to date. Here we report the identification of a novel mitochondrial DEAD-box protein, which we termed mHel61p. Disruption of the mHEL61 alleles in insect-stage Trypanosoma brucei cells resulted in a reduced growth rate phenotype. On a molecular level, the null mutant showed significantly reduced amounts of edited mRNAs, whereas never-edited and nuclear mRNAs were unaffected. Reexpression of mHel61p in the knockout cell line restored the ability to efficiently synthesize edited mRNAs. The results suggest an involvement of mHel61p in the control of the abundance of edited mRNAs and thus reveal a novel function for DEAD-box proteins.
Full Text
The Full Text of this article is available as a PDF (923.2 KB).
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Benne R., Van den Burg J., Brakenhoff J. P., Sloof P., Van Boom J. H., Tromp M. C. Major transcript of the frameshifted coxII gene from trypanosome mitochondria contains four nucleotides that are not encoded in the DNA. Cell. 1986 Sep 12;46(6):819–826. doi: 10.1016/0092-8674(86)90063-2. [DOI] [PubMed] [Google Scholar]
- Beverley S. M., Clayton C. E. Transfection of Leishmania and Trypanosoma brucei by electroporation. Methods Mol Biol. 1993;21:333–348. doi: 10.1385/0-89603-239-6:333. [DOI] [PubMed] [Google Scholar]
- Biebinger S., Wirtz L. E., Lorenz P., Clayton C. Vectors for inducible expression of toxic gene products in bloodstream and procyclic Trypanosoma brucei. Mol Biochem Parasitol. 1997 Mar;85(1):99–112. doi: 10.1016/s0166-6851(96)02815-0. [DOI] [PubMed] [Google Scholar]
- 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]
- Brun R., Schönenberger Cultivation and in vitro cloning or procyclic culture forms of Trypanosoma brucei in a semi-defined medium. Short communication. Acta Trop. 1979 Sep;36(3):289–292. [PubMed] [Google Scholar]
- 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]
- Chomczynski P., Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987 Apr;162(1):156–159. doi: 10.1006/abio.1987.9999. [DOI] [PubMed] [Google Scholar]
- Company M., Arenas J., Abelson J. Requirement of the RNA helicase-like protein PRP22 for release of messenger RNA from spliceosomes. Nature. 1991 Feb 7;349(6309):487–493. doi: 10.1038/349487a0. [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]
- Cross G. A. Identification, purification and properties of clone-specific glycoprotein antigens constituting the surface coat of Trypanosoma brucei. Parasitology. 1975 Dec;71(3):393–417. doi: 10.1017/s003118200004717x. [DOI] [PubMed] [Google Scholar]
- 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]
- 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]
- Erickson H. P. Gene knockouts of c-src, transforming growth factor beta 1, and tenascin suggest superfluous, nonfunctional expression of proteins. J Cell Biol. 1993 Mar;120(5):1079–1081. doi: 10.1083/jcb.120.5.1079. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Feagin J. E., Jasmer D. P., Stuart K. Developmentally regulated addition of nucleotides within apocytochrome b transcripts in Trypanosoma brucei. Cell. 1987 May 8;49(3):337–345. doi: 10.1016/0092-8674(87)90286-8. [DOI] [PubMed] [Google Scholar]
- 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]
- Fuller-Pace F. V. RNA helicases: modulators of RNA structure. Trends Cell Biol. 1994 Aug;4(8):271–274. doi: 10.1016/0962-8924(94)90210-0. [DOI] [PubMed] [Google Scholar]
- Gibson T. J., Thompson J. D. Detection of dsRNA-binding domains in RNA helicase A and Drosophila maleless: implications for monomeric RNA helicases. Nucleic Acids Res. 1994 Jul 11;22(13):2552–2556. doi: 10.1093/nar/22.13.2552. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- Hartl F. U., Neupert W. Protein sorting to mitochondria: evolutionary conservations of folding and assembly. Science. 1990 Feb 23;247(4945):930–938. doi: 10.1126/science.2406905. [DOI] [PubMed] [Google Scholar]
- Hensgens L. A., Brakenhoff J., De Vries B. F., Sloof P., Tromp M. C., Van Boom J. H., Benne R. The sequence of the gene for cytochrome c oxidase subunit I, a frameshift containing gene for cytochrome c oxidase subunit II and seven unassigned reading frames in Trypanosoma brucei mitochondrial maxi-circle DNA. Nucleic Acids Res. 1984 Oct 11;12(19):7327–7344. doi: 10.1093/nar/12.19.7327. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- Hirumi H., Hirumi K. Axenic culture of African trypanosome bloodstream forms. Parasitol Today. 1994 Feb;10(2):80–84. doi: 10.1016/0169-4758(94)90402-2. [DOI] [PubMed] [Google Scholar]
- Hloch P., Schiedner G., Stahl H. Complete cDNA sequence of the human p68 protein. Nucleic Acids Res. 1990 May 25;18(10):3045–3045. doi: 10.1093/nar/18.10.3045. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hug M., Hotz H. R., Hartmann C., Clayton C. Hierarchies of RNA-processing signals in a trypanosome surface antigen mRNA precursor. Mol Cell Biol. 1994 Nov;14(11):7428–7435. doi: 10.1128/mcb.14.11.7428. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Imboden M., Blum B., DeLange T., Braun R., Seebeck T. Tubulin mRNAs of Trypanosoma brucei. J Mol Biol. 1986 Apr 5;188(3):393–402. doi: 10.1016/0022-2836(86)90163-4. [DOI] [PubMed] [Google Scholar]
- Iost I., Dreyfus M. mRNAs can be stabilized by DEAD-box proteins. Nature. 1994 Nov 10;372(6502):193–196. doi: 10.1038/372193a0. [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]
- 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]
- 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]
- Margossian S. P., Li H., Zassenhaus H. P., Butow R. A. The DExH box protein Suv3p is a component of a yeast mitochondrial 3'-to-5' exoribonuclease that suppresses group I intron toxicity. Cell. 1996 Jan 26;84(2):199–209. doi: 10.1016/s0092-8674(00)80975-7. [DOI] [PubMed] [Google Scholar]
- 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]
- 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]
- 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]
- Niemer I., Schmelzer C., Börner G. V. Overexpression of DEAD box protein pMSS116 promotes ATP-dependent splicing of a yeast group II intron in vitro. Nucleic Acids Res. 1995 Aug 11;23(15):2966–2972. [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]
- Pringle J. R., Adams A. E., Drubin D. G., Haarer B. K. Immunofluorescence methods for yeast. Methods Enzymol. 1991;194:565–602. doi: 10.1016/0076-6879(91)94043-c. [DOI] [PubMed] [Google Scholar]
- Py B., Higgins C. F., Krisch H. M., Carpousis A. J. A DEAD-box RNA helicase in the Escherichia coli RNA degradosome. Nature. 1996 May 9;381(6578):169–172. doi: 10.1038/381169a0. [DOI] [PubMed] [Google Scholar]
- 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]
- Schwer B., Guthrie C. PRP16 is an RNA-dependent ATPase that interacts transiently with the spliceosome. Nature. 1991 Feb 7;349(6309):494–499. doi: 10.1038/349494a0. [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]
- 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]
- Sowden M., Hamm J. K., Smith H. C. Overexpression of APOBEC-1 results in mooring sequence-dependent promiscuous RNA editing. J Biol Chem. 1996 Feb 9;271(6):3011–3017. doi: 10.1074/jbc.271.6.3011. [DOI] [PubMed] [Google Scholar]
- 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]
- 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]
- 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]
- Thomas J. H. Thinking about genetic redundancy. Trends Genet. 1993 Nov;9(11):395–399. doi: 10.1016/0168-9525(93)90140-d. [DOI] [PubMed] [Google Scholar]
- Wirtz E., Hartmann C., Clayton C. Gene expression mediated by bacteriophage T3 and T7 RNA polymerases in transgenic trypanosomes. Nucleic Acids Res. 1994 Sep 25;22(19):3887–3894. doi: 10.1093/nar/22.19.3887. [DOI] [PMC free article] [PubMed] [Google Scholar]