Skip to main content
NCBI home page
The EMBO Journal logoLink to The EMBO Journal
. 1993 Mar;12(3):1239–1248. doi: 10.1002/j.1460-2075.1993.tb05765.x

The trans-spliceosomal U2 snRNP protein 40K of Trypanosoma brucei: cloning and analysis of functional domains reveals homology to a mammalian snRNP protein.

M Cross 1, B Wieland 1, Z Palfi 1, A Günzl 1, U Röthlisberger 1, H W Lahm 1, A Bindereif 1
PMCID: PMC413327  PMID: 8458336

Abstract

Through immunoscreening we have isolated a cDNA encoding the trans-spliceosomal U2 snRNP-specific 40 kDa protein of Trypanosoma brucei. The protein has a predicted molecular weight of 36.6 kDa and shows 31% amino acid identity with the human U2 snRNP A' protein of 28.4 kDa. The homology between the trypanosome and human protein sequences is restricted to the N-terminal half where they share a series of six leucine repeat motifs. Sequence alignment revealed three 40K-specific regions: a C-terminal extension and two insertions, one of which makes up a seventh leucine repeat. Bacterially expressed 40K protein efficiently bound RNA by itself in a nonspecific manner; this general RNA binding activity was located to a region in the C-terminal half overlapping with the leucine repeat domain. U2 RNA-specific interaction required the presence of other trypanosome proteins and depended upon the loop IV sequence of U2 RNA. Deletion analysis of the 40K protein demonstrated the leucine repeats, including the 40K-specific, seventh repeat, to be essential for specific U2 RNP assembly, most likely through their role as an interface for protein-protein interaction.

Full text

PDF
1239

Images in this article

1241Image
on p.1241
1242Image
on p.1242
1243Image
on p.1243
1244Image
on p.1244
1245Image
on p.1245
1246Image
on p.1246

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. Bentley R. C., Keene J. D. Recognition of U1 and U2 small nuclear RNAs can be altered by a 5-amino-acid segment in the U2 small nuclear ribonucleoprotein particle (snRNP) B" protein and through interactions with U2 snRNP-A' protein. Mol Cell Biol. 1991 Apr;11(4):1829–1839. doi: 10.1128/mcb.11.4.1829. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Boelens W., Scherly D., Beijer R. P., Jansen E. J., Dathan N. A., Mattaj I. W., van Venrooij W. J. A weak interaction between the U2A' protein and U2 snRNA helps to stabilize their complex with the U2B" protein. Nucleic Acids Res. 1991 Feb 11;19(3):455–460. doi: 10.1093/nar/19.3.455. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. 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]
  5. Calnan B. J., Biancalana S., Hudson D., Frankel A. D. Analysis of arginine-rich peptides from the HIV Tat protein reveals unusual features of RNA-protein recognition. Genes Dev. 1991 Feb;5(2):201–210. doi: 10.1101/gad.5.2.201. [DOI] [PubMed] [Google Scholar]
  6. Cross M., Günzl A., Palfi Z., Bindereif A. Analysis of small nuclear ribonucleoproteins (RNPs) in Trypanosoma brucei: structural organization and protein components of the spliced leader RNP. Mol Cell Biol. 1991 Nov;11(11):5516–5526. doi: 10.1128/mcb.11.11.5516. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Devereux J., Haeberli P., Smithies O. A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 1984 Jan 11;12(1 Pt 1):387–395. doi: 10.1093/nar/12.1part1.387. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Fresco L. D., Harper D. S., Keene J. D. Leucine periodicity of U2 small nuclear ribonucleoprotein particle (snRNP) A' protein is implicated in snRNP assembly via protein-protein interactions. Mol Cell Biol. 1991 Mar;11(3):1578–1589. doi: 10.1128/mcb.11.3.1578. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Guthrie C., Patterson B. Spliceosomal snRNAs. Annu Rev Genet. 1988;22:387–419. doi: 10.1146/annurev.ge.22.120188.002131. [DOI] [PubMed] [Google Scholar]
  10. Günzl A., Cross M., Bindereif A. Domain structure of U2 and U4/U6 small nuclear ribonucleoprotein particles from Trypanosoma brucei: identification of trans-spliceosomal specific RNA-protein interactions. Mol Cell Biol. 1992 Feb;12(2):468–479. doi: 10.1128/mcb.12.2.468. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Hartshorne T., Agabian N. A new U2 RNA secondary structure provided by phylogenetic analysis of trypanosomatid U2 RNAs. Genes Dev. 1990 Dec;4(12A):2121–2131. doi: 10.1101/gad.4.12a.2121. [DOI] [PubMed] [Google Scholar]
  12. Hewick R. M., Hunkapiller M. W., Hood L. E., Dreyer W. J. A gas-liquid solid phase peptide and protein sequenator. J Biol Chem. 1981 Aug 10;256(15):7990–7997. [PubMed] [Google Scholar]
  13. Laird P. W. Trans splicing in trypanosomes--archaism or adaptation? Trends Genet. 1989 Jul;5(7):204–208. doi: 10.1016/0168-9525(89)90082-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Lazinski D., Grzadzielska E., Das A. Sequence-specific recognition of RNA hairpins by bacteriophage antiterminators requires a conserved arginine-rich motif. Cell. 1989 Oct 6;59(1):207–218. doi: 10.1016/0092-8674(89)90882-9. [DOI] [PubMed] [Google Scholar]
  15. Lin K. H., Cheng S. Y. An efficient method to purify active eukaryotic proteins from the inclusion bodies in Escherichia coli. Biotechniques. 1991 Dec;11(6):748, 750, 752-3. [PubMed] [Google Scholar]
  16. 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]
  17. Palfi Z., Bindereif A. Immunological characterization and intracellular localization of trans-spliceosomal small nuclear ribonucleoproteins in Trypanosoma brucei. J Biol Chem. 1992 Oct 5;267(28):20159–20163. [PubMed] [Google Scholar]
  18. Palfi Z., Günzl A., Cross M., Bindereif A. Affinity purification of Trypanosoma brucei small nuclear ribonucleoproteins reveals common and specific protein components. Proc Natl Acad Sci U S A. 1991 Oct 15;88(20):9097–9101. doi: 10.1073/pnas.88.20.9097. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Scherly D., Boelens W., Dathan N. A., van Venrooij W. J., Mattaj I. W. Major determinants of the specificity of interaction between small nuclear ribonucleoproteins U1A and U2B'' and their cognate RNAs. Nature. 1990 Jun 7;345(6275):502–506. doi: 10.1038/345502a0. [DOI] [PubMed] [Google Scholar]
  20. Scherly D., Dathan N. A., Boelens W., van Venrooij W. J., Mattaj I. W. The U2B'' RNP motif as a site of protein-protein interaction. EMBO J. 1990 Nov;9(11):3675–3681. doi: 10.1002/j.1460-2075.1990.tb07579.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Sillekens P. T., Beijer R. P., Habets W. J., van Verooij W. J. Molecular cloning of the cDNA for the human U2 snRNA-specific A' protein. Nucleic Acids Res. 1989 Mar 11;17(5):1893–1906. doi: 10.1093/nar/17.5.1893. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Smiley B. L., Stadnyk A. W., Myler P. J., Stuart K. The trypanosome leucine repeat gene in the variant surface glycoprotein expression site encodes a putative metal-binding domain and a region resembling protein-binding domains of yeast, Drosophila, and mammalian proteins. Mol Cell Biol. 1990 Dec;10(12):6436–6444. doi: 10.1128/mcb.10.12.6436. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Smith D. B., Johnson K. S. Single-step purification of polypeptides expressed in Escherichia coli as fusions with glutathione S-transferase. Gene. 1988 Jul 15;67(1):31–40. doi: 10.1016/0378-1119(88)90005-4. [DOI] [PubMed] [Google Scholar]
  24. Sommerville J. RNA-binding proteins: masking proteins revealed. Bioessays. 1992 May;14(5):337–339. doi: 10.1002/bies.950140509. [DOI] [PubMed] [Google Scholar]
  25. Studier F. W., Rosenberg A. H., Dunn J. J., Dubendorff J. W. Use of T7 RNA polymerase to direct expression of cloned genes. Methods Enzymol. 1990;185:60–89. doi: 10.1016/0076-6879(90)85008-c. [DOI] [PubMed] [Google Scholar]
  26. 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]
  27. 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]
  28. Tschudi C., Williams S. P., Ullu E. Conserved sequences in the U2 snRNA-encoding genes of Kinetoplastida do not include the putative branchpoint recognition region. Gene. 1990 Jul 2;91(1):71–77. doi: 10.1016/0378-1119(90)90164-m. [DOI] [PubMed] [Google Scholar]
  29. Zamore P. D., Zapp M. L., Green M. R. Gene expression. RNA binding: beta s and basics. Nature. 1990 Dec 6;348(6301):485–486. doi: 10.1038/348485a0. [DOI] [PubMed] [Google Scholar]

Articles from The EMBO Journal are provided here courtesy of Nature Publishing Group

RESOURCES