Skip to main content
PMC home page
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1991 Jun;11(6):3180–3190. doi: 10.1128/mcb.11.6.3180

Maturation of polycistronic pre-mRNA in Trypanosoma brucei: analysis of trans splicing and poly(A) addition at nascent RNA transcripts from the hsp70 locus.

J Huang 1, L H van der Ploeg 1
PMCID: PMC360171  PMID: 1710026

Abstract

Numerous protein-coding genes of the protozoan Trypanosoma brucei are arranged in tandem arrays that are transcribed polycistronically. The pre-mRNA transcripts are processed by trans splicing, leading to the addition of a capped 39-nucleotide (nt) miniexon and by poly(A) addition. We wished to determine the order of the RNA processing events at the hsp70 locus and address the potential occurrence of cotranscriptional RNA processing. We determined the rate of transcriptional elongation at the hsp70 locus in isolated nuclei, which measured between 20 and 40 nt/min. This low rate of RNA chain elongation allowed us to label the 3' end of hsp70 nascent RNA with a short (about 180-nt) 32P tail. The structure of the labeled nascent hsp70 RNA could then be analyzed by RNase T1 and RNase T1/RNase A mapping. We show that the trans splicing of hsp70 pre-mRNA did not occur immediately after the synthesis of the 3' splice acceptor site, and nascent RNA molecules that contained about 550 nt of RNA beyond the 3' splice acceptor site still had not acquired a miniexon. In contrast, nascent RNA with a 5' end that mapped to the polyadenylation site of the hsp70 genes could be detected, indicating that maturation of the pre-mRNA in trypanosomes involves a rapid cleavage of the nascent hsp70 RNA (within seconds after synthesis of the site) for poly(A) addition. Our data suggest that polycistronic pre-mRNA is unlikely to be synthesized in toto and rather appears to be processed cotranscriptionally by cleavage for poly(A) addition.

Full text

PDF
3180

Images in this article

3183Image
on p.3183
3184Image
on p.3184
3185Image
on p.3185
3187Image
on p.3187

Selected References

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

  1. Beyer A. L., Osheim Y. N. Splice site selection, rate of splicing, and alternative splicing on nascent transcripts. Genes Dev. 1988 Jun;2(6):754–765. doi: 10.1101/gad.2.6.754. [DOI] [PubMed] [Google Scholar]
  2. Birnstiel M. L., Busslinger M., Strub K. Transcription termination and 3' processing: the end is in site! Cell. 1985 Jun;41(2):349–359. doi: 10.1016/s0092-8674(85)80007-6. [DOI] [PubMed] [Google Scholar]
  3. 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]
  4. 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]
  5. Brody E., Abelson J. The "spliceosome": yeast pre-messenger RNA associates with a 40S complex in a splicing-dependent reaction. Science. 1985 May 24;228(4702):963–967. doi: 10.1126/science.3890181. [DOI] [PubMed] [Google Scholar]
  6. Bruzik J. P., Steitz J. A. Spliced leader RNA sequences can substitute for the essential 5' end of U1 RNA during splicing in a mammalian in vitro system. Cell. 1990 Sep 7;62(5):889–899. doi: 10.1016/0092-8674(90)90264-f. [DOI] [PubMed] [Google Scholar]
  7. Bruzik J. P., Van Doren K., Hirsh D., Steitz J. A. Trans splicing involves a novel form of small nuclear ribonucleoprotein particles. Nature. 1988 Oct 6;335(6190):559–562. doi: 10.1038/335559a0. [DOI] [PubMed] [Google Scholar]
  8. 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]
  9. Cornelissen A. W., Verspieren M. P., Toulmé J. J., Swinkels B. W., Borst P. The common 5' terminal sequence on trypanosome mRNAs: a target for anti-messenger oligodeoxynucleotides. Nucleic Acids Res. 1986 Jul 25;14(14):5605–5614. doi: 10.1093/nar/14.14.5605. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Cox R. F. Quantitation of elongating form A and B RNA polymerases in chick oviduct nuclei and effects of estradiol. Cell. 1976 Mar;7(3):455–465. doi: 10.1016/0092-8674(76)90176-8. [DOI] [PubMed] [Google Scholar]
  11. Darnell J. E., Jr Variety in the level of gene control in eukaryotic cells. Nature. 1982 Jun 3;297(5865):365–371. doi: 10.1038/297365a0. [DOI] [PubMed] [Google Scholar]
  12. Frendewey D., Keller W. Stepwise assembly of a pre-mRNA splicing complex requires U-snRNPs and specific intron sequences. Cell. 1985 Aug;42(1):355–367. doi: 10.1016/s0092-8674(85)80131-8. [DOI] [PubMed] [Google Scholar]
  13. 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]
  14. 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]
  15. Gottesdiener K., Chung H. M., Brown S. D., Lee M. G., Van der Ploeg L. H. Characterization of VSG gene expression site promoters and promoter-associated DNA rearrangement events. Mol Cell Biol. 1991 May;11(5):2467–2480. doi: 10.1128/mcb.11.5.2467. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Grabowski P. J., Padgett R. A., Sharp P. A. Messenger RNA splicing in vitro: an excised intervening sequence and a potential intermediate. Cell. 1984 Jun;37(2):415–427. doi: 10.1016/0092-8674(84)90372-6. [DOI] [PubMed] [Google Scholar]
  17. Grabowski P. J., Seiler S. R., Sharp P. A. A multicomponent complex is involved in the splicing of messenger RNA precursors. Cell. 1985 Aug;42(1):345–353. doi: 10.1016/s0092-8674(85)80130-6. [DOI] [PubMed] [Google Scholar]
  18. 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]
  19. Hofer E., Darnell J. E., Jr The primary transcription unit of the mouse beta-major globin gene. Cell. 1981 Feb;23(2):585–593. doi: 10.1016/0092-8674(81)90154-9. [DOI] [PubMed] [Google Scholar]
  20. Johnson P. J., Kooter J. M., Borst P. Inactivation of transcription by UV irradiation of T. brucei provides evidence for a multicistronic transcription unit including a VSG gene. Cell. 1987 Oct 23;51(2):273–281. doi: 10.1016/0092-8674(87)90154-1. [DOI] [PubMed] [Google Scholar]
  21. 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]
  22. Kooter J. M., van der Spek H. J., Wagter R., d'Oliveira C. E., van der Hoeven F., Johnson P. J., Borst P. The anatomy and transcription of a telomeric expression site for variant-specific surface antigens in T. brucei. Cell. 1987 Oct 23;51(2):261–272. doi: 10.1016/0092-8674(87)90153-x. [DOI] [PubMed] [Google Scholar]
  23. 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]
  24. Lai C. J., Dhar R., Khoury G. Mapping the spliced and unspliced late lytic SV40 RNAs. Cell. 1978 Aug;14(4):971–982. doi: 10.1016/0092-8674(78)90351-3. [DOI] [PubMed] [Google Scholar]
  25. 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]
  26. Laird P. W., ten Asbroek A. L., Borst P. Controlled turnover and 3' trimming of the trans splicing precursor of Trypanosoma brucei. Nucleic Acids Res. 1987 Dec 23;15(24):10087–10103. doi: 10.1093/nar/15.24.10087. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Lee M. G., Atkinson B. L., Giannini S. H., Van der Ploeg L. H. Structure and expression of the hsp 70 gene family of Leishmania major. Nucleic Acids Res. 1988 Oct 25;16(20):9567–9585. doi: 10.1093/nar/16.20.9567. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Lee M. G., Polvere R. I., Van der Ploeg L. H. Evidence for segmental gene conversion between a cognate hsp 70 gene and the temperature-sensitively transcribed hsp70 genes of Trypanosoma brucei. Mol Biochem Parasitol. 1990 Jun;41(2):213–220. doi: 10.1016/0166-6851(90)90184-n. [DOI] [PubMed] [Google Scholar]
  29. Lee M. G., Van der Ploeg L. H. Frequent independent duplicative transpositions activate a single VSG gene. Mol Cell Biol. 1987 Jan;7(1):357–364. doi: 10.1128/mcb.7.1.357. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Lee M. G., Van der Ploeg L. H. Homologous recombination and stable transfection in the parasitic protozoan Trypanosoma brucei. Science. 1990 Dec 14;250(4987):1583–1587. doi: 10.1126/science.2177225. [DOI] [PubMed] [Google Scholar]
  31. 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]
  32. Lindell T. J., Weinberg F., Morris P. W., Roeder R. G., Rutter W. J. Specific inhibition of nuclear RNA polymerase II by alpha-amanitin. Science. 1970 Oct 23;170(3956):447–449. doi: 10.1126/science.170.3956.447. [DOI] [PubMed] [Google Scholar]
  33. Manley J. L., Sharp P. A., Gefter M. L. Rna synthesis in isolated nuclei processing of adenovirus serotype 2 late messenger rna precursors. J Mol Biol. 1982 Aug 25;159(4):581–599. doi: 10.1016/0022-2836(82)90102-4. [DOI] [PubMed] [Google Scholar]
  34. Marzluff W. F., Jr, Pan C. J., Cooper D. L. RNA synthesis in myeloma cells synchronized by isoleucine starvation. Nucleic Acids Res. 1978 Nov;5(11):4177–4193. doi: 10.1093/nar/5.11.4177. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Melton D. A., Krieg P. A., Rebagliati M. R., Maniatis T., Zinn K., Green M. R. Efficient in vitro synthesis of biologically active RNA and RNA hybridization probes from plasmids containing a bacteriophage SP6 promoter. Nucleic Acids Res. 1984 Sep 25;12(18):7035–7056. doi: 10.1093/nar/12.18.7035. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Mory Y. Y., Gefter M. L. Synthesis of messenger RNA-like molecules in isolated myeloma nuclei. Nucleic Acids Res. 1977 Jun;4(6):1739–1757. doi: 10.1093/nar/4.6.1739. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Mottram J., Perry K. L., Lizardi P. M., Lührmann R., Agabian N., Nelson R. G. Isolation and sequence of four small nuclear U RNA genes of Trypanosoma brucei subsp. brucei: identification of the U2, U4, and U6 RNA analogs. Mol Cell Biol. 1989 Mar;9(3):1212–1223. doi: 10.1128/mcb.9.3.1212. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Muhich M. L., Boothroyd J. C. Polycistronic transcripts in trypanosomes and their accumulation during heat shock: evidence for a precursor role in mRNA synthesis. Mol Cell Biol. 1988 Sep;8(9):3837–3846. doi: 10.1128/mcb.8.9.3837. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Muhich M. L., Boothroyd J. C. Synthesis of trypanosome hsp70 mRNA is resistant to disruption of trans-splicing by heat shock. J Biol Chem. 1989 May 5;264(13):7107–7110. [PubMed] [Google Scholar]
  40. 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]
  41. Nevins J. R., Darnell J. E., Jr Steps in the processing of Ad2 mRNA: poly(A)+ nuclear sequences are conserved and poly(A) addition precedes splicing. Cell. 1978 Dec;15(4):1477–1493. doi: 10.1016/0092-8674(78)90071-5. [DOI] [PubMed] [Google Scholar]
  42. Osheim Y. N., Miller O. L., Jr, Beyer A. L. RNP particles at splice junction sequences on Drosophila chorion transcripts. Cell. 1985 Nov;43(1):143–151. doi: 10.1016/0092-8674(85)90019-4. [DOI] [PubMed] [Google Scholar]
  43. 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]
  44. Ralph D., Huang J., Van der Ploeg L. H. Physical identification of branched intron side-products of splicing in Trypanosoma brucei. EMBO J. 1988 Aug;7(8):2539–2545. doi: 10.1002/j.1460-2075.1988.tb03102.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Roop D. R., Nordstrom J. L., Tsai S. Y., Tsai M. J., O'Malley B. W. Transcription of structural and intervening sequences in the ovalbumin gene and identification of potential ovalbumin mRNA precursors. Cell. 1978 Oct;15(2):671–685. doi: 10.1016/0092-8674(78)90035-1. [DOI] [PubMed] [Google Scholar]
  46. 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]
  47. Sharp P. A. Splicing of messenger RNA precursors. Science. 1987 Feb 13;235(4790):766–771. doi: 10.1126/science.3544217. [DOI] [PubMed] [Google Scholar]
  48. Shea C., Lee M. G., Van der Ploeg L. H. VSG gene 118 is transcribed from a cotransposed pol I-like promoter. Cell. 1987 Aug 14;50(4):603–612. doi: 10.1016/0092-8674(87)90033-x. [DOI] [PubMed] [Google Scholar]
  49. Smith M. M., Reeve A. E., Huang R. C. Analysis of RNA initiated in isolated mouse myeloma nuclei using purine nucleoside 5'[gamma-S]triphosphates as affinity probes. Cell. 1978 Oct;15(2):615–626. doi: 10.1016/0092-8674(78)90030-2. [DOI] [PubMed] [Google Scholar]
  50. 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]
  51. Tilghman S. M., Curtis P. J., Tiemeier D. C., Leder P., Weissmann C. The intervening sequence of a mouse beta-globin gene is transcribed within the 15S beta-globin mRNA precursor. Proc Natl Acad Sci U S A. 1978 Mar;75(3):1309–1313. doi: 10.1073/pnas.75.3.1309. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Tschudi C., Richards F. F., Ullu E. The U2 RNA analogue of Trypanosoma brucei gambiense: implications for a splicing mechanism in trypanosomes. Nucleic Acids Res. 1986 Nov 25;14(22):8893–8903. doi: 10.1093/nar/14.22.8893. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Tschudi C., Ullu E. Destruction of U2, U4, or U6 small nuclear RNA blocks trans splicing in trypanosome cells. Cell. 1990 May 4;61(3):459–466. doi: 10.1016/0092-8674(90)90527-l. [DOI] [PubMed] [Google Scholar]
  54. Tschudi C., Ullu E. Polygene transcripts are precursors to calmodulin mRNAs in trypanosomes. EMBO J. 1988 Feb;7(2):455–463. doi: 10.1002/j.1460-2075.1988.tb02833.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Ullu E., Tschudi C. Permeable trypanosome cells as a model system for transcription and trans-splicing. Nucleic Acids Res. 1990 Jun 11;18(11):3319–3326. doi: 10.1093/nar/18.11.3319. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Van Doren K., Hirsh D. Trans-spliced leader RNA exists as small nuclear ribonucleoprotein particles in Caenorhabditis elegans. Nature. 1988 Oct 6;335(6190):556–559. doi: 10.1038/335556a0. [DOI] [PubMed] [Google Scholar]
  57. Van der Ploeg L. H. Discontinuous transcription and splicing in trypanosomes. Cell. 1986 Nov 21;47(4):479–480. doi: 10.1016/0092-8674(86)90608-2. [DOI] [PubMed] [Google Scholar]
  58. Walder J. A., Eder P. S., Engman D. M., Brentano S. T., Walder R. Y., Knutzon D. S., Dorfman D. M., Donelson J. E. The 35-nucleotide spliced leader sequence is common to all trypanosome messenger RNA's. Science. 1986 Aug 1;233(4763):569–571. doi: 10.1126/science.3523758. [DOI] [PubMed] [Google Scholar]
  59. Weber J., Jelinek W., Darnell J. E., Jr The definition of a large viral transcription unit late in Ad2 infection of HeLa cells: mapping of nascent RNA molecules labeled in isolated nuclei. Cell. 1977 Apr;10(4):611–616. doi: 10.1016/0092-8674(77)90093-9. [DOI] [PubMed] [Google Scholar]
  60. White T. C., Borst P. RNA end-labeling and RNA ligase activities can produce a circular rRNA in whole cell extracts from trypanosomes. Nucleic Acids Res. 1987 Apr 24;15(8):3275–3290. doi: 10.1093/nar/15.8.3275. [DOI] [PMC free article] [PubMed] [Google Scholar]
  61. Zomerdijk J. C., Ouellette M., ten Asbroek A. L., Kieft R., Bommer A. M., Clayton C. E., Borst P. The promoter for a variant surface glycoprotein gene expression site in Trypanosoma brucei. EMBO J. 1990 Sep;9(9):2791–2801. doi: 10.1002/j.1460-2075.1990.tb07467.x. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES