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. 1988 May;7(5):1431–1437. doi: 10.1002/j.1460-2075.1988.tb02960.x

Trypanosome trans-splicing utilizes 2'-5' branches and a corresponding debranching activity.

R E Sutton 1, J C Boothroyd 1
PMCID: PMC458393  PMID: 3409870

Abstract

The 5' ends of trypanosome mRNAs consist of an identical sequence of 39 nucleotides which is derived from a discrete transcript of approximately 140 nucleotides (medRNA). It has been proposed that generation of chimeric mRNAs in trypanosomes occurs by the process of trans-splicing involving medRNA and an acceptor RNA. Part of the basis for this suggestion comes from the ability of HeLa cell extracts (known to contain debranching activity) to catalyze the release of the intron portion of medRNA (minRNA) implying a Y-branched intermediate in the splicing process. Here we provide direct chemical analysis that miniRNA is attached to higher mol. wt RNA molecules by a 2'-5' phosphodiester bond (i.e. as a branched structure). We also demonstrate that trypanosomes have substantial amounts of debranching activity which is similar in nature to that of HeLa cells. These results provide further evidence for trans-splicing in trypanosomes and highlights its similarity to cis-splicing in other eukaryotes.

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Selected References

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

  1. Arenas J., Hurwitz J. Purification of a RNA debranching activity from HeLa cells. J Biol Chem. 1987 Mar 25;262(9):4274–4279. [PubMed] [Google Scholar]
  2. Boothroyd J. C. Antigenic variation in African trypanosomes. Annu Rev Microbiol. 1985;39:475–502. doi: 10.1146/annurev.mi.39.100185.002355. [DOI] [PubMed] [Google Scholar]
  3. Borst P. Discontinuous transcription and antigenic variation in trypanosomes. Annu Rev Biochem. 1986;55:701–732. doi: 10.1146/annurev.bi.55.070186.003413. [DOI] [PubMed] [Google Scholar]
  4. 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]
  5. Campbell D. A., Thornton D. A., Boothroyd J. C. Apparent discontinuous transcription of Trypanosoma brucei variant surface antigen genes. 1984 Sep 27-Oct 3Nature. 311(5984):350–355. doi: 10.1038/311350a0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. De Lange T., Berkvens T. M., Veerman H. J., Frasch A. C., Barry J. D., Borst P. Comparison of the genes coding for the common 5' terminal sequence of messenger RNAs in three trypanosome species. Nucleic Acids Res. 1984 Jun 11;12(11):4431–4443. doi: 10.1093/nar/12.11.4431. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. De Lange T., Liu A. Y., Van der Ploeg L. H., Borst P., Tromp M. C., Van Boom J. H. Tandem repetition of the 5' mini-exon of variant surface glycoprotein genes: a multiple promoter for VSG gene transcription? Cell. 1983 Oct;34(3):891–900. doi: 10.1016/0092-8674(83)90546-9. [DOI] [PubMed] [Google Scholar]
  8. De Lange T., Michels P. A., Veerman H. J., Cornelissen A. W., Borst P. Many trypanosome messenger RNAs share a common 5' terminal sequence. Nucleic Acids Res. 1984 May 11;12(9):3777–3790. doi: 10.1093/nar/12.9.3777. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Dignam J. D., Lebovitz R. M., Roeder R. G. Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucleic Acids Res. 1983 Mar 11;11(5):1475–1489. doi: 10.1093/nar/11.5.1475. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Dorfman D. M., Donelson J. E. Characterization of the 1.35 kilobase DNA repeat unit containing the conserved 35 nucleotides at the 5'-termini of variable surface glycoprotein mRNAs in Trypanosoma brucei. Nucleic Acids Res. 1984 Jun 25;12(12):4907–4920. doi: 10.1093/nar/12.12.4907. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Freistadt M. S., Cross G. A., Branch A. D., Robertson H. D. Direct analysis of the mini-exon donor RNA of Trypanosoma brucei: detection of a novel cap structure also present in messenger RNA. Nucleic Acids Res. 1987 Dec 10;15(23):9861–9879. doi: 10.1093/nar/15.23.9861. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Glass D. J., Polvere R. I., Van der Ploeg L. H. Conserved sequences and transcription of the hsp70 gene family in Trypanosoma brucei. Mol Cell Biol. 1986 Dec;6(12):4657–4666. doi: 10.1128/mcb.6.12.4657. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Green M. R. Pre-mRNA splicing. Annu Rev Genet. 1986;20:671–708. doi: 10.1146/annurev.ge.20.120186.003323. [DOI] [PubMed] [Google Scholar]
  14. Hornig H., Aebi M., Weissmann C. Effect of mutations at the lariat branch acceptor site on beta-globin pre-mRNA splicing in vitro. Nature. 1986 Dec 11;324(6097):589–591. doi: 10.1038/324589a0. [DOI] [PubMed] [Google Scholar]
  15. Jacquier A., Rosbash M. RNA splicing and intron turnover are greatly diminished by a mutant yeast branch point. Proc Natl Acad Sci U S A. 1986 Aug;83(16):5835–5839. doi: 10.1073/pnas.83.16.5835. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Kierzek R., Kopp D. W., Edmonds M., Caruthers M. H. Chemical synthesis of branched RNA. Nucleic Acids Res. 1986 Jun 25;14(12):4751–4764. doi: 10.1093/nar/14.12.4751. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Konarska M. M., Grabowski P. J., Padgett R. A., Sharp P. A. Characterization of the branch site in lariat RNAs produced by splicing of mRNA precursors. Nature. 1985 Feb 14;313(6003):552–557. doi: 10.1038/313552a0. [DOI] [PubMed] [Google Scholar]
  18. Kooter J. M., De Lange T., Borst P. Discontinuous synthesis of mRNA in trypanosomes. EMBO J. 1984 Oct;3(10):2387–2392. doi: 10.1002/j.1460-2075.1984.tb02144.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Krause M., Hirsh D. A trans-spliced leader sequence on actin mRNA in C. elegans. Cell. 1987 Jun 19;49(6):753–761. doi: 10.1016/0092-8674(87)90613-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Laird P. W., Zomerdijk J. C., de Korte D., Borst P. In vivo labelling of intermediates in the discontinuous synthesis of mRNAs in Trypanosoma brucei. EMBO J. 1987 Apr;6(4):1055–1062. doi: 10.1002/j.1460-2075.1987.tb04858.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Langford C. J., Klinz F. J., Donath C., Gallwitz D. Point mutations identify the conserved, intron-contained TACTAAC box as an essential splicing signal sequence in yeast. Cell. 1984 Mar;36(3):645–653. doi: 10.1016/0092-8674(84)90344-1. [DOI] [PubMed] [Google Scholar]
  22. Lanham S. M., Godfrey D. G. Isolation of salivarian trypanosomes from man and other mammals using DEAE-cellulose. Exp Parasitol. 1970 Dec;28(3):521–534. doi: 10.1016/0014-4894(70)90120-7. [DOI] [PubMed] [Google Scholar]
  23. Michiels F., Matthyssens G., Kronenberger P., Pays E., Dero B., Van Assel S., Darville M., Carvador A., Steinert M., Hamers R. Gene activation and re-expression of a Trypanosoma brucei variant surface glycoprotein. EMBO J. 1983;2(7):1185–1192. doi: 10.1002/j.1460-2075.1983.tb01565.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Milhausen M., Nelson R. G., Sather S., Selkirk M., Agabian N. Identification of a small RNA containing the trypanosome spliced leader: a donor of shared 5' sequences of trypanosomatid mRNAs? Cell. 1984 Oct;38(3):721–729. doi: 10.1016/0092-8674(84)90267-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Muhich M. L., Hughes D. E., Simpson A. M., Simpson L. The monogenetic kinetoplastid protozoan, Crithidia fasciculata, contains a transcriptionally active, multicopy mini-exon sequence. Nucleic Acids Res. 1987 Apr 10;15(7):3141–3153. doi: 10.1093/nar/15.7.3141. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Murphy W. J., Watkins K. P., Agabian N. Identification of a novel Y branch structure as an intermediate in trypanosome mRNA processing: evidence for trans splicing. Cell. 1986 Nov 21;47(4):517–525. doi: 10.1016/0092-8674(86)90616-1. [DOI] [PubMed] [Google Scholar]
  27. Myers R. M., Larin Z., Maniatis T. Detection of single base substitutions by ribonuclease cleavage at mismatches in RNA:DNA duplexes. Science. 1985 Dec 13;230(4731):1242–1246. doi: 10.1126/science.4071043. [DOI] [PubMed] [Google Scholar]
  28. Nakazato H., Edmonds M. Purification of messenger RNA and heterogeneous nuclear RNA containing poly(a) sequences. Methods Enzymol. 1974;29:431–443. doi: 10.1016/0076-6879(74)29035-9. [DOI] [PubMed] [Google Scholar]
  29. Nelson R. G., Parsons M., Barr P. J., Stuart K., Selkirk M., Agabian N. Sequences homologous to the variant antigen mRNA spliced leader are located in tandem repeats and variable orphons in trypanosoma brucei. Cell. 1983 Oct;34(3):901–909. doi: 10.1016/0092-8674(83)90547-0. [DOI] [PubMed] [Google Scholar]
  30. Nelson R. G., Parsons M., Selkirk M., Newport G., Barr P. J., Agabian N. Sequences homologous to variant antigen mRNA spliced leader in Trypanosomatidae which do not undergo antigenic variation. Nature. 1984 Apr 12;308(5960):665–667. doi: 10.1038/308665a0. [DOI] [PubMed] [Google Scholar]
  31. Newman A. J., Lin R. J., Cheng S. C., Abelson J. Molecular consequences of specific intron mutations on yeast mRNA splicing in vivo and in vitro. Cell. 1985 Aug;42(1):335–344. doi: 10.1016/s0092-8674(85)80129-x. [DOI] [PubMed] [Google Scholar]
  32. Padgett R. A., Grabowski P. J., Konarska M. M., Seiler S., Sharp P. A. Splicing of messenger RNA precursors. Annu Rev Biochem. 1986;55:1119–1150. doi: 10.1146/annurev.bi.55.070186.005351. [DOI] [PubMed] [Google Scholar]
  33. Parsons M., Nelson R. G., Watkins K. P., Agabian N. Trypanosome mRNAs share a common 5' spliced leader sequence. Cell. 1984 Aug;38(1):309–316. doi: 10.1016/0092-8674(84)90552-X. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Peebles C. L., Perlman P. S., Mecklenburg K. L., Petrillo M. L., Tabor J. H., Jarrell K. A., Cheng H. L. A self-splicing RNA excises an intron lariat. Cell. 1986 Jan 31;44(2):213–223. doi: 10.1016/0092-8674(86)90755-5. [DOI] [PubMed] [Google Scholar]
  35. Perry K. L., Watkins K. P., Agabian N. Trypanosome mRNAs have unusual "cap 4" structures acquired by addition of a spliced leader. Proc Natl Acad Sci U S A. 1987 Dec;84(23):8190–8194. doi: 10.1073/pnas.84.23.8190. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Ruskin B., Green M. R. An RNA processing activity that debranches RNA lariats. Science. 1985 Jul 12;229(4709):135–140. doi: 10.1126/science.2990042. [DOI] [PubMed] [Google Scholar]
  37. Silberklang M., Gillum A. M., RajBhandary U. L. Use of in vitro 32P labeling in the sequence analysis of nonradioactive tRNAs. Methods Enzymol. 1979;59:58–109. doi: 10.1016/0076-6879(79)59072-7. [DOI] [PubMed] [Google Scholar]
  38. Sninsky J. J., Last J. A., Gilham P. T. The use of terminal blocking groups for the specific joining of oligonucleotides in RNA ligase reactions containing equimolar concentrations of acceptor and donor molecules. Nucleic Acids Res. 1976 Nov;3(11):3157–3166. doi: 10.1093/nar/3.11.3157. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Sogin M. L., Elwood H. J., Gunderson J. H. Evolutionary diversity of eukaryotic small-subunit rRNA genes. Proc Natl Acad Sci U S A. 1986 Mar;83(5):1383–1387. doi: 10.1073/pnas.83.5.1383. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Sutton R. E., Boothroyd J. C. Evidence for trans splicing in trypanosomes. Cell. 1986 Nov 21;47(4):527–535. doi: 10.1016/0092-8674(86)90617-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Sutton R. E., Boothroyd J. C. The cap of both miniexon-derived RNA and mRNA of trypanosomes is 7-methylguanosine. Mol Cell Biol. 1988 Jan;8(1):494–496. doi: 10.1128/mcb.8.1.494. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Van der Ploeg L. H., Cornelissen A. W., Michels P. A., Borst P. Chromosome rearrangements in Trypanosoma brucei. Cell. 1984 Nov;39(1):213–221. doi: 10.1016/0092-8674(84)90207-1. [DOI] [PubMed] [Google Scholar]
  43. Van der Ploeg L. H., Liu A. Y., Michels P. A., De Lange T., Borst P., Majumder H. K., Weber H., Veeneman G. H., Van Boom J. RNA splicing is required to make the messenger RNA for a variant surface antigen in trypanosomes. Nucleic Acids Res. 1982 Jun 25;10(12):3591–3604. doi: 10.1093/nar/10.12.3591. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Wallace J. C., Edmonds M. Polyadenylylated nuclear RNA contains branches. Proc Natl Acad Sci U S A. 1983 Feb;80(4):950–954. doi: 10.1073/pnas.80.4.950. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Yost H. J., Lindquist S. RNA splicing is interrupted by heat shock and is rescued by heat shock protein synthesis. Cell. 1986 Apr 25;45(2):185–193. doi: 10.1016/0092-8674(86)90382-x. [DOI] [PubMed] [Google Scholar]
  46. van der Veen R., Arnberg A. C., van der Horst G., Bonen L., Tabak H. F., Grivell L. A. Excised group II introns in yeast mitochondria are lariats and can be formed by self-splicing in vitro. Cell. 1986 Jan 31;44(2):225–234. doi: 10.1016/0092-8674(86)90756-7. [DOI] [PubMed] [Google Scholar]

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