Skip to main content
NCBI home page
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1993 Aug 25;21(17):4067–4072. doi: 10.1093/nar/21.17.4067

Differential response to RNA trans-splicing signals within the phosphoglycerate kinase gene cluster in Trypanosoma brucei.

N Kapotas 1, V Bellofatto 1
PMCID: PMC310004  PMID: 8371981

Abstract

In trypanosomatids, nuclear pre-mRNA splicing is exclusively a trans-splicing reaction in which a capped, 39 nt exon, the mini-exon, is positioned 5' to an open reading frame. Differential RNA splicing might reflect specific mini-exon and 3' splice site interactions. To test this hypothesis, we compared the efficiency of mini-exon addition to three natural 3' splice acceptor sites (SASs) located within a single pre-mRNA transcript. In Trypanosoma brucei, the phosphoglycerate kinase A, B and C genes (PGK A, B and C) are co-expressed as three consecutive sequences on a polycistronic pre-mRNA. This pre-mRNA gives rise to unequal amounts of PGK A, B and C mRNAs. When the SAS from each gene was placed upstream of the luciferase open reading frame and the resultant constructs transiently transfected into T. brucei procyclic cells, luciferase activity levels indicated differential SAS utilization. Enzyme activity was low when the SAS from the A gene was present. Levels were indistinguishable when the B and C SASs were compared. After replacing luciferase with chloramphenicol acetyl transferase in the test constructs, enzyme activities were shown to directly correlate with mRNA amounts. Thus, poor splicing efficiency accounts for the differential expression of the PGK A mRNA during PGK pre-mRNA maturation. This reaction appears to reflect the polypyrimidine pattern within the 3' splice acceptor site.

Full text

PDF
4067

Images in this article

4070Image
on p.4070

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. Agami R., Shapira M. Nucleotide sequence of the spliced leader RNA gene from Leishmania mexicana amazonensis. Nucleic Acids Res. 1992 Apr 11;20(7):1804–1804. doi: 10.1093/nar/20.7.1804. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Alexander K., Parsons M. A phosphoglycerate kinase-like molecule localized to glycosomal microbodies: evidence that the topogenic signal is not at the C-terminus. Mol Biochem Parasitol. 1991 May;46(1):1–10. doi: 10.1016/0166-6851(91)90193-a. [DOI] [PubMed] [Google Scholar]
  4. Bellofatto V., Cooper R., Cross G. A. Discontinuous transcription in Leptomonas seymouri: presence of intact and interrupted mini-exon gene families. Nucleic Acids Res. 1988 Aug 11;16(15):7437–7456. doi: 10.1093/nar/16.15.7437. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bellofatto V., Cross G. A. Expression of a bacterial gene in a trypanosomatid protozoan. Science. 1989 Jun 9;244(4909):1167–1169. doi: 10.1126/science.2499047. [DOI] [PubMed] [Google Scholar]
  6. Brown S. D., Huang J., Van der Ploeg L. H. The promoter for the procyclic acidic repetitive protein (PARP) genes of Trypanosoma brucei shares features with RNA polymerase I promoters. Mol Cell Biol. 1992 Jun;12(6):2644–2652. doi: 10.1128/mcb.12.6.2644. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. 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]
  8. Clayton C. E., Fueri J. P., Itzhaki J. E., Bellofatto V., Sherman D. R., Wisdom G. S., Vijayasarathy S., Mowatt M. R. Transcription of the procyclic acidic repetitive protein genes of Trypanosoma brucei. Mol Cell Biol. 1990 Jun;10(6):3036–3047. doi: 10.1128/mcb.10.6.3036. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Clayton C. Developmental regulation of nuclear gene expression in Trypanosoma brucei. Prog Nucleic Acid Res Mol Biol. 1992;43:37–66. doi: 10.1016/s0079-6603(08)61043-0. [DOI] [PubMed] [Google Scholar]
  10. Curotto de Lafaille M. A., Laban A., Wirth D. F. Gene expression in Leishmania: analysis of essential 5' DNA sequences. Proc Natl Acad Sci U S A. 1992 Apr 1;89(7):2703–2707. doi: 10.1073/pnas.89.7.2703. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Gibson W. C., Swinkels B. W., Borst P. Post-transcriptional control of the differential expression of phosphoglycerate kinase genes in Trypanosoma brucei. J Mol Biol. 1988 May 20;201(2):315–325. doi: 10.1016/0022-2836(88)90140-4. [DOI] [PubMed] [Google Scholar]
  12. Green M. R. Biochemical mechanisms of constitutive and regulated pre-mRNA splicing. Annu Rev Cell Biol. 1991;7:559–599. doi: 10.1146/annurev.cb.07.110191.003015. [DOI] [PubMed] [Google Scholar]
  13. Huang J., Van der Ploeg L. H. Requirement of a polypyrimidine tract for trans-splicing in trypanosomes: discriminating the PARP promoter from the immediately adjacent 3' splice acceptor site. EMBO J. 1991 Dec;10(12):3877–3885. doi: 10.1002/j.1460-2075.1991.tb04957.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Kapler G. M., Zhang K., Beverley S. M. Sequence and S1 nuclease mapping of the 5' region of the dihydrofolate reductase-thymidylate synthase gene of Leishmania major. Nucleic Acids Res. 1987 Apr 24;15(8):3369–3383. doi: 10.1093/nar/15.8.3369. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Layden R. E., Eisen H. Alternate trans splicing in Trypanosoma equiperdum: implications for splice site selection. Mol Cell Biol. 1988 Mar;8(3):1352–1360. doi: 10.1128/mcb.8.3.1352. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Le Blancq S. M., Swinkels B. W., Gibson W. C., Borst P. Evidence for gene conversion between the phosphoglycerate kinase genes of Trypanosoma brucei. J Mol Biol. 1988 Apr 5;200(3):439–447. doi: 10.1016/0022-2836(88)90534-7. [DOI] [PubMed] [Google Scholar]
  17. Lee M. G., Van der Ploeg L. H. Transcription of the heat shock 70 locus in Trypanosoma brucei. Mol Biochem Parasitol. 1990 Jun;41(2):221–231. doi: 10.1016/0166-6851(90)90185-o. [DOI] [PubMed] [Google Scholar]
  18. Mowatt M. R., Clayton C. E. Developmental regulation of a novel repetitive protein of Trypanosoma brucei. Mol Cell Biol. 1987 Aug;7(8):2838–2844. doi: 10.1128/mcb.7.8.2838. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Nilsen T. W. Trans-splicing in nematodes. Exp Parasitol. 1989 Nov;69(4):413–416. doi: 10.1016/0014-4894(89)90191-4. [DOI] [PubMed] [Google Scholar]
  20. Osinga K. A., Swinkels B. W., Gibson W. C., Borst P., Veeneman G. H., Van Boom J. H., Michels P. A., Opperdoes F. R. Topogenesis of microbody enzymes: a sequence comparison of the genes for the glycosomal (microbody) and cytosolic phosphoglycerate kinases of Trypanosoma brucei. EMBO J. 1985 Dec 30;4(13B):3811–3817. doi: 10.1002/j.1460-2075.1985.tb04152.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Parsons M., Hill T. Elevated phosphoglycerate kinase mRNA but not protein in monomorphic Trypanosoma brucei: implications for stage-regulation and post-transcriptional control. Mol Biochem Parasitol. 1989 Mar 15;33(3):215–227. doi: 10.1016/0166-6851(89)90083-2. [DOI] [PubMed] [Google Scholar]
  22. Patzelt E., Perry K. L., Agabian N. Mapping of branch sites in trans-spliced pre-mRNAs of Trypanosoma brucei. Mol Cell Biol. 1989 Oct;9(10):4291–4297. doi: 10.1128/mcb.9.10.4291. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Pays E., Coquelet H., Tebabi P., Pays A., Jefferies D., Steinert M., Koenig E., Williams R. O., Roditi I. Trypanosoma brucei: constitutive activity of the VSG and procyclin gene promoters. EMBO J. 1990 Oct;9(10):3145–3151. doi: 10.1002/j.1460-2075.1990.tb07512.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Rajkovic A., Davis R. E., Simonsen J. N., Rottman F. M. A spliced leader is present on a subset of mRNAs from the human parasite Schistosoma mansoni. Proc Natl Acad Sci U S A. 1990 Nov;87(22):8879–8883. doi: 10.1073/pnas.87.22.8879. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Rudenko G., Le Blancq S., Smith J., Lee M. G., Rattray A., Van der Ploeg L. H. Procyclic acidic repetitive protein (PARP) genes located in an unusually small alpha-amanitin-resistant transcription unit: PARP promoter activity assayed by transient DNA transfection of Trypanosoma brucei. Mol Cell Biol. 1990 Jul;10(7):3492–3504. doi: 10.1128/mcb.10.7.3492. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Ruskin B., Zamore P. D., Green M. R. A factor, U2AF, is required for U2 snRNP binding and splicing complex assembly. Cell. 1988 Jan 29;52(2):207–219. doi: 10.1016/0092-8674(88)90509-0. [DOI] [PubMed] [Google Scholar]
  27. Sather S., Agabian N. A 5' spliced leader is added in trans to both alpha- and beta-tubulin transcripts in Trypanosoma brucei. Proc Natl Acad Sci U S A. 1985 Sep;82(17):5695–5699. doi: 10.1073/pnas.82.17.5695. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Sherman D. R., Janz L., Hug M., Clayton C. Anatomy of the parp gene promoter of Trypanosoma brucei. EMBO J. 1991 Nov;10(11):3379–3386. doi: 10.1002/j.1460-2075.1991.tb04902.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Swinkels B. W., Evers R., Borst P. The topogenic signal of the glycosomal (microbody) phosphoglycerate kinase of Crithidia fasciculata resides in a carboxy-terminal extension. EMBO J. 1988 Apr;7(4):1159–1165. doi: 10.1002/j.1460-2075.1988.tb02926.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Tessier L. H., Keller M., Chan R. L., Fournier R., Weil J. H., Imbault P. Short leader sequences may be transferred from small RNAs to pre-mature mRNAs by trans-splicing in Euglena. EMBO J. 1991 Sep;10(9):2621–2625. doi: 10.1002/j.1460-2075.1991.tb07804.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Tschudi C., Young A. S., Ruben L., Patton C. L., Richards F. F. Calmodulin genes in trypanosomes are tandemly repeated and produce multiple mRNAs with a common 5' leader sequence. Proc Natl Acad Sci U S A. 1985 Jun;82(12):3998–4002. doi: 10.1073/pnas.82.12.3998. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Ullu E., Matthews K. R., Tschudi C. Temporal order of RNA-processing reactions in trypanosomes: rapid trans splicing precedes polyadenylation of newly synthesized tubulin transcripts. Mol Cell Biol. 1993 Jan;13(1):720–725. doi: 10.1128/mcb.13.1.720. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Vijayasarathy S., Ernest I., Itzhaki J. E., Sherman D., Mowatt M. R., Michels P. A., Clayton C. E. The genes encoding fructose bisphosphate aldolase in Trypanosoma brucei are interspersed with unrelated genes. Nucleic Acids Res. 1990 May 25;18(10):2967–2975. doi: 10.1093/nar/18.10.2967. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Zamore P. D., Patton J. G., Green M. R. Cloning and domain structure of the mammalian splicing factor U2AF. Nature. 1992 Feb 13;355(6361):609–614. doi: 10.1038/355609a0. [DOI] [PubMed] [Google Scholar]

Articles from Nucleic Acids Research are provided here courtesy of Oxford University Press

RESOURCES