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. 2004 Apr 14;47(4):527–535. doi: 10.1016/0092-8674(86)90617-3

Evidence for Trans splicing in trypanosomes

Richard E Sutton 1, John C Boothroyd 1
PMCID: PMC7133315  PMID: 3022935

Abstract

The 5′ ends of trypanosome mRNAs consist of an identical sequence of 35 nucleotides. This “mini-exon” sequence is derived from the 5′ end of a 137 nucleotide RNA (medRNA). The remainder of each mRNA is derived from a protein-coding exon that is not linked to the mini-exon. We propose that medRNA is spliced in trans to de-novo-initiated transcripts of protein-coding genes. This trans splicing model predicts that the downstream portion of medRNA will be part of a branched structure and then be released as a free product (minRNA). We demonstrate that significant levels of minRNA exist in trypanosome RNA. Furthermore, minRNA can be released from high molecular weight RNA by a HeLa cell S100 “debranching” extract. We conclude that trnas splicing is the physiological process by which mature mRNA molecules are synthesized in trypanosomes.

References

  1. Berk A.J., Sharp P.A. Sizing and mapping of early adenovirus mRNAs by gel electrophoresis of S1 endonuclease-digested hybrids. Cell. 1977;12:721–732. doi: 10.1016/0092-8674(77)90272-0. [DOI] [PubMed] [Google Scholar]
  2. Bernards A., De Lange T., Michels P.A.M., Liu A.Y.C., Huisman M.J., Borst P. Two modes of activation of a single surface antigen gene of Trypanosoma brucei. Cell. 1984;36:163–170. doi: 10.1016/0092-8674(84)90085-0. [DOI] [PubMed] [Google Scholar]
  3. Boothroyd J.C. Antigenic variation in African trypanosomes. Ann. Rev. Microbiol. 1985;39:475–502. doi: 10.1146/annurev.mi.39.100185.002355. [DOI] [PubMed] [Google Scholar]
  4. Boothroyd J.C., Cross G.A.M. Transcripts coding for different variant surface glycoproteins in Trypanosoma brucei have a short identical exon at their 5′-end. Gene. 1982;20:279–287. doi: 10.1016/0378-1119(82)90046-4. [DOI] [PubMed] [Google Scholar]
  5. Boothroyd J.C., Campbell D.A., Sutton R.E. Expression of surface antigen genes in Trypanosoma brucei involves a novel system of discontinuous transcription. In: Lerner R.A., Channock R.M., Brown F., editors. Modern Approaches to Vaccines. Cold Spring Harbor Laboratory; Cold Spring Harbor, New York: 1985. pp. 61–66. [Google Scholar]
  6. Borst P. Discontinuous transcription and antigenic variation in trypanosomes. Ann. Rev. Biochem. 1986;55:701–732. doi: 10.1146/annurev.bi.55.070186.003413. [DOI] [PubMed] [Google Scholar]
  7. Campbell D.A., Thornton D.A., Boothroyd J.C. Apparent discontinuous transcription of Trypanosoma brucei surface antigen genes. Nature. 1984;311:350–355. doi: 10.1038/311350a0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Cech T.R., Bass B.L. Biological catalysis by RNA. Ann. Rev. Biochem. 1986;55:599–629. doi: 10.1146/annurev.bi.55.070186.003123. [DOI] [PubMed] [Google Scholar]
  9. Clayton C.E. Structure and regulated expression of genes encoding fructose biphosphate aldolase in Trypanosoma brucei. EMBO J. 1985;4:2997–3003. doi: 10.1002/j.1460-2075.1985.tb04035.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Cross G.A.M. Identification, purification and properties of clone-specific glycoprotein antigens constituting the surface coat of Trypanosoma brucei. Parasitology. 1975;71:393–417. doi: 10.1017/s003118200004717x. [DOI] [PubMed] [Google Scholar]
  11. De Lange T., Liu A.Y.C., Van der Ploeg L.H.T., Borst P., Tromp M.C., Van Boom J.H. Tandem repetition of the 5′ miniexon of variant surface glycoprotein genes: a multiple promoter for VSG gene transcription? Cell. 1983;34:891–900. doi: 10.1016/0092-8674(83)90546-9. [DOI] [PubMed] [Google Scholar]
  12. De Lange T., Michels P.A.M., Veerman H.J.G., Cornelissen A.W.C.A., Borst P. Many trypanosome messenger RNAs share a common 5′ terminal sequence. Nucl. Acids Res. 1984;12:3777–3789. doi: 10.1093/nar/12.9.3777. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. De Lange T., Berkvens T.M., Veerman H.J.G., Carlos A., Frasch C., Barry J.D., Borst P. Comparison of the genes coding for the common 5′ terminal sequence of messenger RNAs in three trypanosome species. Nucl. Acids Res. 1984;12:4431–4443. doi: 10.1093/nar/12.11.4431. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Dorfman D., Donelson J. Characterization of the 1.35 kb DNA repeat unit containing the conserved 35 nucleotides at the 5′-termini of VSG mRNAs in Trypanosoma brucei. Nucl. Acids Res. 1984;12:4907–4920. doi: 10.1093/nar/12.12.4907. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Gilbert W., Marchionni M., McKnight G. On the antiquity of introns. Cell. 1986;46:151–154. doi: 10.1016/0092-8674(86)90730-0. [DOI] [PubMed] [Google Scholar]
  16. Gonzalez A., Lerner T.J., Huccas M., Sosa-Pineda B., Nogueira N., Lizardi P.M. Apparent generation of a segmented mRNA from two separate tandem gene families in Trypanosoma cruzi. Nucl. Acids Res. 1985;13:5789–5804. doi: 10.1093/nar/13.16.5789. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Guyaux M., Cornelissen A.W.C.A., Pays E., Steinert M., Borst P. Trypanosoma brucei: a surface antigen mRNA is discontinuously transcribed from two distinct chromosomes. EMBO J. 1985;4:995–998. doi: 10.1002/j.1460-2075.1985.tb03729.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Kimmel B.E., Samson S., Hirschberg R., Yarbrough L.R. Tubulin genes of the African trypanosome Trypanosoma brucei rhodesiense: nucleotide sequence of a 3.7-kb fragment containing genes for alpha and beta tubulins. Gene. 1985;35:237–248. doi: 10.1016/0378-1119(85)90002-2. [DOI] [PubMed] [Google Scholar]
  19. Konarska M.M., Padgett R.A., Sharp P.A. Trans splicing of mRNA precursors in vitro. Cell. 1985;42:165–171. doi: 10.1016/s0092-8674(85)80112-4. [DOI] [PubMed] [Google Scholar]
  20. Kooter J., De Lange T., Borst P. Discontinuous synthesis of mRNA in trypanosomes. EMBO J. 1984;3:2387–2392. doi: 10.1002/j.1460-2075.1984.tb02144.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Lanham S.M., Godfrey D.G. Isolation of salivarian trypanosomes from man and other mammals using DEAE-cellulose. Exp. Parasitol. 1970;28:521–534. doi: 10.1016/0014-4894(70)90120-7. [DOI] [PubMed] [Google Scholar]
  22. Liu A.Y.C., Van der Ploeg L.H.T., Rijsewijk F.A.M., Borst P. The transposition unit of VSG gene 118 of Trypanosoma brucei: presence of repeated elements at its border and absence of promoter-associated sequences. J. Mol. Biol. 1983;167:57–75. doi: 10.1016/s0022-2836(83)80034-5. [DOI] [PubMed] [Google Scholar]
  23. Makino S., Stohlman S.A., Lai M.M.C. Vol. 83. 1986. Leader sequences of murine coronavirus mRNAs can be freely reassorted: evidence for the role of free leader RNA in transcription; pp. 4204–4208. (Proc. Natl. Acad. Sci. USA). [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Maniatis T., Fritsch E.F., Sambrook J. Cold Spring Harbor Laboratory; Cold Spring Harbor, New York: 1982. (Molecular Cloning: A Laboratory Manual). [Google Scholar]
  25. Maxam A.M., Gilbert W. Sequencing end-labeled DNA with base-specific chemical cleavages. Methods Enzymol. 1980;65:499–560. doi: 10.1016/s0076-6879(80)65059-9. [DOI] [PubMed] [Google Scholar]
  26. Michels P.A.M., Poliszczak A., Osinga K.A., Misset O., van Beeumen J., Werenga R.K., Borst P., Opperdoes F.R. Two tandemly linked identical genes code for the glycosomal glyceraldehyde-phosphate dehydrogenase in Trypanosoma brucei. EMBO J. 1986;5:1049–1056. doi: 10.1002/j.1460-2075.1986.tb04321.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Michiels F., Matthyssens G., Kronenberger P., Pays E., Dero B., Van Assel S., Darville M., Cravador A., Steinert M., Hamers R. Gene activation and re-expression of a Trypanosoma brucei variant surface glycoprotein. EMBO J. 1983;2:1185–1192. doi: 10.1002/j.1460-2075.1983.tb01565.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Milhausen M., Nelson R.G., Sather S., Selkirk M., Agabian N. Identification of a small RNA containing the trypanosome spliced leader: a donor of the shared 5′ sequences of trypanosomatid mRNAs? Cell. 1984;38:721–729. doi: 10.1016/0092-8674(84)90267-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. 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]
  30. 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;34:901–909. doi: 10.1016/0092-8674(83)90547-0. [DOI] [PubMed] [Google Scholar]
  31. Nelson R.G., Parsons M., Selkirk M., Newport G., Barr P.J., Agabian N. Sequences homologous to the variant antigen mRNA spliced leader are present in Trypanosomatidae which do not undergo antigenic variation. Nature. 1984;308:665–667. doi: 10.1038/308665a0. [DOI] [PubMed] [Google Scholar]
  32. Padgett R.A., Konarska M.M., Grabowski P.J., Hardy S.F., Sharp P.A. Lariat RNAs as intermediates and products in the splicing of messenger RNA precursors. Science. 1984;225:898–903. doi: 10.1126/science.6206566. [DOI] [PubMed] [Google Scholar]
  33. Padgett R.A., Grabowski P.J., Konarska M.M., Seiler S., Sharp P.A. Splicing of messenger RNA precursors. Ann. Rev. Biochem. 1986;55:1119–1150. doi: 10.1146/annurev.bi.55.070186.005351. [DOI] [PubMed] [Google Scholar]
  34. Parsons M., Nelson R.G., Watkins K.P., Agabian N. Trypanosome mRNAs share a common 5′ spliced leader sequence. Cell. 1984;38:309–316. doi: 10.1016/0092-8674(84)90552-X. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Pays E., Lheureux M., Steinert M. Structure and expression of a Trypanosoma brucei gambiense variant specific antigen gene. Nucl. Acids Res. 1982;10:3149–3163. doi: 10.1093/nar/10.10.3149. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Ruskin B., Green M.R. An RNA processing activity that debranches RNA lariats. Science. 1985;229:135–140. doi: 10.1126/science.2990042. [DOI] [PubMed] [Google Scholar]
  37. Ruskin B., Krainer A.R., Maniatis T., Green M.R. Excision of an intact intron as a novel lariat structure during pre-mRNA splicing in vitro. Cell. 1984;38:317–331. doi: 10.1016/0092-8674(84)90553-1. [DOI] [PubMed] [Google Scholar]
  38. Sanger F., Coulson A.R. The use of thin acrylamide gels for DNA sequencing. FEBS Lett. 1978;87:107–110. doi: 10.1016/0014-5793(78)80145-8. [DOI] [PubMed] [Google Scholar]
  39. Sather S., Agabian N. Vol. 82. 1985. A 5′ spliced leader is added in trans to both alpha- and beta-tubulin transcripts in Trypanosoma brucei; pp. 5695–5699. (Proc. Natl. Acad. Sci. USA). [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Sharp P.A. On the origin of RNA splicing and introns. Cell. 1985;42:397–400. doi: 10.1016/0092-8674(85)90092-3. [DOI] [PubMed] [Google Scholar]
  41. Sharp P.A., Konarska M.M. Does trans splicing in vitro require base pairing between RNAs? Response to Solnick. Cell. 1986;44:211. doi: 10.1016/0092-8674(86)90752-x. [DOI] [PubMed] [Google Scholar]
  42. Sogin M.L., Elwood H.J., Gunderson J.H. Vol. 83. 1986. Evolutionary diversity of eukaryotic small-subunit rRNA genes; pp. 1383–1387. (Proc. Natl. Acad. Sci. USA). [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Solnick D. Trans splicing of mRNA precursors. Cell. 1985;42:157–164. doi: 10.1016/s0092-8674(85)80111-2. [DOI] [PubMed] [Google Scholar]
  44. Solnick D. Does trans splicing in vitro require base pairing between RNAs? Cell. 1986;44:211. doi: 10.1016/0092-8674(86)90752-x. [DOI] [PubMed] [Google Scholar]
  45. Swinkels B.W., Gibson W.C., Osinga K.A., Kramer R., Veeneman G.H., van Boom J.H., Borst P. Characterization of the gene for the microbody (glycosomal) triosephosphate isomerase of Trypanosoma brucei. EMBO J. 1986;5:1291–1298. doi: 10.1002/j.1460-2075.1986.tb04358.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Tschudi C., Young A.S., Ruben L., Patton C.L., Richards F.F. Vol. 82. 1985. Calmodulin genes in trypanosomes are tandemly repeated and produce multiple mRNAs with a common 5′ leader sequence; pp. 3998–4002. (Proc. Natl. Acad. Sci. USA). [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Van der Ploeg L.H.T., Liu A.Y.C., Michels P.A.M., De Lange T., Borst P., Majumder K., Weber H., Veeneman G.H. RNA splicing is required to make the messenger RNA for a variant surface antigen in trypanosomes. Nucl. Acids Res. 1982;10:3591–3604. doi: 10.1093/nar/10.12.3591. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Van der Ploeg L.H.T., Cornelissen A.W.C.A., Michels P.A.M., Borst P. Chromosome rearrangement in Trypanosoma brucei. Cell. 1984;39:213–221. doi: 10.1016/0092-8674(84)90207-1. [DOI] [PubMed] [Google Scholar]

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