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. 1989 Jun 26;17(12):4647–4660. doi: 10.1093/nar/17.12.4647

In vitro 3' end processing and poly(A) tailing of RNA in Trypanosoma cruzi.

T A Zwierzynski 1, G Widmer 1, G A Buck 1
PMCID: PMC318022  PMID: 2473439

Abstract

Pre-mRNA in kinetoplastids is processed to maturity following unique pathways requiring a transplicing event that links a common 39 nucleotide leader to the 5' termini of the mature mRNAs. The mechanisms of this reaction and other steps of mRNA processing; i.e., 5' capping and 3' cleavage and polyadenylation, have not been resolved. Herein, we describe a 3' polyadenylation activity in cell-free extracts prepared from nuclei isolated from Trypanosoma cruzi, the kinetoplastid agent of Chagas' Disease. Synthetic RNA transcripts incubated in these extracts in the presence of ATP are 3' polyadenylated. This polyadenylation activity is sensitive to heat or pre-treatment of the extract with Micrococcal nuclease, suggesting that an RNA-protein complex is required. As these are characteristics of polyadenylation activities in other eukaryotes, we believe that this activity may participate in the in vivo trypanosome mRNA polyadenylation system. Several other modification activities specific for RNA 3' termini, including terminal nucleotide transferases, a tRNA CCA maturation activity, and a 3' exonuclease were also identified in these T. cruzi nuclear extracts.

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Selected References

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

  1. 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]
  2. 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]
  3. 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]
  4. England T. E., Bruce A. G., Uhlenbeck O. C. Specific labeling of 3' termini of RNA with T4 RNA ligase. Methods Enzymol. 1980;65(1):65–74. doi: 10.1016/s0076-6879(80)65011-3. [DOI] [PubMed] [Google Scholar]
  5. Feagin J. E., Abraham J. M., Stuart K. Extensive editing of the cytochrome c oxidase III transcript in Trypanosoma brucei. Cell. 1988 May 6;53(3):413–422. doi: 10.1016/0092-8674(88)90161-4. [DOI] [PubMed] [Google Scholar]
  6. Feagin J. E., Shaw J. M., Simpson L., Stuart K. Creation of AUG initiation codons by addition of uridines within cytochrome b transcripts of kinetoplastids. Proc Natl Acad Sci U S A. 1988 Jan;85(2):539–543. doi: 10.1073/pnas.85.2.539. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Gilmartin G. M., McDevitt M. A., Nevins J. R. Multiple factors are required for specific RNA cleavage at a poly(A) addition site. Genes Dev. 1988 May;2(5):578–587. doi: 10.1101/gad.2.5.578. [DOI] [PubMed] [Google Scholar]
  8. Hashimoto C., Steitz J. A. A small nuclear ribonucleoprotein associates with the AAUAAA polyadenylation signal in vitro. Cell. 1986 May 23;45(4):581–591. doi: 10.1016/0092-8674(86)90290-4. [DOI] [PubMed] [Google Scholar]
  9. Hernandez N., Keller W. Splicing of in vitro synthesized messenger RNA precursors in HeLa cell extracts. Cell. 1983 Nov;35(1):89–99. doi: 10.1016/0092-8674(83)90211-8. [DOI] [PubMed] [Google Scholar]
  10. Humphrey T., Christofori G., Lucijanic V., Keller W. Cleavage and polyadenylation of messenger RNA precursors in vitro occurs within large and specific 3' processing complexes. EMBO J. 1987 Dec 20;6(13):4159–4168. doi: 10.1002/j.1460-2075.1987.tb02762.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Konarska M. M., Padgett R. A., Sharp P. A. Recognition of cap structure in splicing in vitro of mRNA precursors. Cell. 1984 Oct;38(3):731–736. doi: 10.1016/0092-8674(84)90268-x. [DOI] [PubMed] [Google Scholar]
  12. Krainer A. R., Maniatis T. Multiple factors including the small nuclear ribonucleoproteins U1 and U2 are necessary for pre-mRNA splicing in vitro. Cell. 1985 Oct;42(3):725–736. doi: 10.1016/0092-8674(85)90269-7. [DOI] [PubMed] [Google Scholar]
  13. McDevitt M. A., Gilmartin G. M., Reeves W. H., Nevins J. R. Multiple factors are required for poly(A) addition to a mRNA 3' end. Genes Dev. 1988 May;2(5):588–597. doi: 10.1101/gad.2.5.588. [DOI] [PubMed] [Google Scholar]
  14. 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]
  15. Moore C. L., Skolnik-David H., Sharp P. A. Sedimentation analysis of polyadenylation-specific complexes. Mol Cell Biol. 1988 Jan;8(1):226–233. doi: 10.1128/mcb.8.1.226. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Morel C., Chiari E., Camargo E. P., Mattei D. M., Romanha A. J., Simpson L. Strains and clones of Trypanosoma cruzi can be characterized by pattern of restriction endonuclease products of kinetoplast DNA minicircles. Proc Natl Acad Sci U S A. 1980 Nov;77(11):6810–6814. doi: 10.1073/pnas.77.11.6810. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Shaw J. M., Feagin J. E., Stuart K., Simpson L. Editing of kinetoplastid mitochondrial mRNAs by uridine addition and deletion generates conserved amino acid sequences and AUG initiation codons. Cell. 1988 May 6;53(3):401–411. doi: 10.1016/0092-8674(88)90160-2. [DOI] [PubMed] [Google Scholar]
  18. 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]
  19. Skolnik-David H., Moore C. L., Sharp P. A. Electrophoretic separation of polyadenylation-specific complexes. Genes Dev. 1987 Sep;1(7):672–682. doi: 10.1101/gad.1.7.672. [DOI] [PubMed] [Google Scholar]
  20. Takagaki Y., Ryner L. C., Manley J. L. Separation and characterization of a poly(A) polymerase and a cleavage/specificity factor required for pre-mRNA polyadenylation. Cell. 1988 Mar 11;52(5):731–742. doi: 10.1016/0092-8674(88)90411-4. [DOI] [PubMed] [Google Scholar]
  21. Walseth T. F., Johnson R. A. The enzymatic preparation of [alpha-(32)P]nucleoside triphosphates, cyclic [32P] AMP, and cyclic [32P] GMP. Biochim Biophys Acta. 1979 Mar 28;562(1):11–31. doi: 10.1016/0005-2787(79)90122-9. [DOI] [PubMed] [Google Scholar]
  22. 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]
  23. Wilusz J., Shenk T. A 64 kd nuclear protein binds to RNA segments that include the AAUAAA polyadenylation motif. Cell. 1988 Jan 29;52(2):221–228. doi: 10.1016/0092-8674(88)90510-7. [DOI] [PubMed] [Google Scholar]

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