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. 1993 Jul 15;90(14):6771–6775. doi: 10.1073/pnas.90.14.6771

The Saccharomyces cerevisiae PRP21 gene product is an integral component of the prespliceosome.

J E Arenas 1, J N Abelson 1
PMCID: PMC47014  PMID: 8341697

Abstract

In Saccharomyces cerevisiae, the prp21 mutation causes accumulation of unspliced pre-mRNA at the nonpermissive temperature. We have cloned the PRP21 gene by complementation of its temperature-sensitive phenotype and found it to be the same as SPP91, an extragenic suppressor of the prp9 mutation previously studied in vivo by Chapon and Legrain [Chapon, C. & Legrain, P. (1992) EMBO J. 11, 3279-3288]. We have analyzed the effects of the prp21 mutation on splicing in vitro and have found that PRP21 is a splicing factor required for prespliceosome assembly. We also have analyzed the interaction of PRP21 with splicing complexes using anti-PRP21 antibodies and found that the RNA components of the prespliceosome--U1 and U2 small nuclear RNA (snRNA) particles and pre-mRNA--are specifically coimmunoprecipitated under splicing conditions in the presence of 0.2 M KCl. At higher KCl concentrations, U1 snRNP dissociates from splicing complexes; nevertheless, U2 snRNA and pre-mRNA are still efficiently immunoprecipitated. Immunoprecipitation of both U1 and U2 snRNA as well as pre-mRNA is ATP-dependent and requires a pre-mRNA capable of supporting prespliceosome assembly. Analysis of the unbound complexes in native gels confirmed that prespliceosomes are specifically immunoprecipitated by anti-PRP21 antibodies. These results demonstrate that PRP21 is an integral component of the prespliceosome and establishes a stable interaction with U2 snRNP and/or pre-mRNA in that complex.

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Selected References

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

  1. Abovich N., Legrain P., Rosbash M. The yeast PRP6 gene encodes a U4/U6 small nuclear ribonucleoprotein particle (snRNP) protein, and the PRP9 gene encodes a protein required for U2 snRNP binding. Mol Cell Biol. 1990 Dec;10(12):6417–6425. doi: 10.1128/mcb.10.12.6417. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Anderson G. J., Bach M., Lührmann R., Beggs J. D. Conservation between yeast and man of a protein associated with U5 small nuclear ribonucleoprotein. Nature. 1989 Dec 14;342(6251):819–821. doi: 10.1038/342819a0. [DOI] [PubMed] [Google Scholar]
  3. Ares M., Jr, Igel A. H. Lethal and temperature-sensitive mutations and their suppressors identify an essential structural element in U2 small nuclear RNA. Genes Dev. 1990 Dec;4(12A):2132–2145. doi: 10.1101/gad.4.12a.2132. [DOI] [PubMed] [Google Scholar]
  4. Banroques J., Abelson J. N. PRP4: a protein of the yeast U4/U6 small nuclear ribonucleoprotein particle. Mol Cell Biol. 1989 Sep;9(9):3710–3719. doi: 10.1128/mcb.9.9.3710. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bindereif A., Green M. R. An ordered pathway of snRNP binding during mammalian pre-mRNA splicing complex assembly. EMBO J. 1987 Aug;6(8):2415–2424. doi: 10.1002/j.1460-2075.1987.tb02520.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bjørn S. P., Soltyk A., Beggs J. D., Friesen J. D. PRP4 (RNA4) from Saccharomyces cerevisiae: its gene product is associated with the U4/U6 small nuclear ribonucleoprotein particle. Mol Cell Biol. 1989 Sep;9(9):3698–3709. doi: 10.1128/mcb.9.9.3698. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Brown J. D., Plumpton M., Beggs J. D. The genetics of nuclear pre-mRNA splicing: a complex story. Antonie Van Leeuwenhoek. 1992 Aug;62(1-2):35–46. doi: 10.1007/BF00584461. [DOI] [PubMed] [Google Scholar]
  8. Cameron J. R., Loh E. Y., Davis R. W. Evidence for transposition of dispersed repetitive DNA families in yeast. Cell. 1979 Apr;16(4):739–751. doi: 10.1016/0092-8674(79)90090-4. [DOI] [PubMed] [Google Scholar]
  9. Chapon C., Legrain P. A novel gene, spp91-1, suppresses the splicing defect and the pre-mRNA nuclear export in the prp9-1 mutant. EMBO J. 1992 Sep;11(9):3279–3288. doi: 10.1002/j.1460-2075.1992.tb05406.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Cheng S. C., Abelson J. Fractionation and characterization of a yeast mRNA splicing extract. Proc Natl Acad Sci U S A. 1986 Apr;83(8):2387–2391. doi: 10.1073/pnas.83.8.2387. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Cheng S. C., Abelson J. Spliceosome assembly in yeast. Genes Dev. 1987 Nov;1(9):1014–1027. doi: 10.1101/gad.1.9.1014. [DOI] [PubMed] [Google Scholar]
  12. Dalbadie-McFarland G., Abelson J. PRP5: a helicase-like protein required for mRNA splicing in yeast. Proc Natl Acad Sci U S A. 1990 Jun;87(11):4236–4240. doi: 10.1073/pnas.87.11.4236. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. 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]
  14. Guthrie C. Messenger RNA splicing in yeast: clues to why the spliceosome is a ribonucleoprotein. Science. 1991 Jul 12;253(5016):157–163. doi: 10.1126/science.1853200. [DOI] [PubMed] [Google Scholar]
  15. Herman P. K., Emr S. D. Characterization of VPS34, a gene required for vacuolar protein sorting and vacuole segregation in Saccharomyces cerevisiae. Mol Cell Biol. 1990 Dec;10(12):6742–6754. doi: 10.1128/mcb.10.12.6742. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Ito H., Fukuda Y., Murata K., Kimura A. Transformation of intact yeast cells treated with alkali cations. J Bacteriol. 1983 Jan;153(1):163–168. doi: 10.1128/jb.153.1.163-168.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Konarska M. M., Sharp P. A. Electrophoretic separation of complexes involved in the splicing of precursors to mRNAs. Cell. 1986 Sep 12;46(6):845–855. doi: 10.1016/0092-8674(86)90066-8. [DOI] [PubMed] [Google Scholar]
  18. Konarska M. M., Sharp P. A. Interactions between small nuclear ribonucleoprotein particles in formation of spliceosomes. Cell. 1987 Jun 19;49(6):763–774. doi: 10.1016/0092-8674(87)90614-3. [DOI] [PubMed] [Google Scholar]
  19. Legrain P., Seraphin B., Rosbash M. Early commitment of yeast pre-mRNA to the spliceosome pathway. Mol Cell Biol. 1988 Sep;8(9):3755–3760. doi: 10.1128/mcb.8.9.3755. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Lin R. J., Newman A. J., Cheng S. C., Abelson J. Yeast mRNA splicing in vitro. J Biol Chem. 1985 Nov 25;260(27):14780–14792. [PubMed] [Google Scholar]
  21. Lossky M., Anderson G. J., Jackson S. P., Beggs J. Identification of a yeast snRNP protein and detection of snRNP-snRNP interactions. Cell. 1987 Dec 24;51(6):1019–1026. doi: 10.1016/0092-8674(87)90588-5. [DOI] [PubMed] [Google Scholar]
  22. Lustig A. J., Lin R. J., Abelson J. The yeast RNA gene products are essential for mRNA splicing in vitro. Cell. 1986 Dec 26;47(6):953–963. doi: 10.1016/0092-8674(86)90810-x. [DOI] [PubMed] [Google Scholar]
  23. Nagai K., Thøgersen H. C. Synthesis and sequence-specific proteolysis of hybrid proteins produced in Escherichia coli. Methods Enzymol. 1987;153:461–481. doi: 10.1016/0076-6879(87)53072-5. [DOI] [PubMed] [Google Scholar]
  24. Rose M. D., Novick P., Thomas J. H., Botstein D., Fink G. R. A Saccharomyces cerevisiae genomic plasmid bank based on a centromere-containing shuttle vector. Gene. 1987;60(2-3):237–243. doi: 10.1016/0378-1119(87)90232-0. [DOI] [PubMed] [Google Scholar]
  25. Rosenberg A. H., Lade B. N., Chui D. S., Lin S. W., Dunn J. J., Studier F. W. Vectors for selective expression of cloned DNAs by T7 RNA polymerase. Gene. 1987;56(1):125–135. doi: 10.1016/0378-1119(87)90165-x. [DOI] [PubMed] [Google Scholar]
  26. Ruby S. W., Abelson J. An early hierarchic role of U1 small nuclear ribonucleoprotein in spliceosome assembly. Science. 1988 Nov 18;242(4881):1028–1035. doi: 10.1126/science.2973660. [DOI] [PubMed] [Google Scholar]
  27. Ruby S. W., Abelson J. Pre-mRNA splicing in yeast. Trends Genet. 1991 Mar;7(3):79–85. doi: 10.1016/0168-9525(91)90276-V. [DOI] [PubMed] [Google Scholar]
  28. Seraphin B., Rosbash M. Identification of functional U1 snRNA-pre-mRNA complexes committed to spliceosome assembly and splicing. Cell. 1989 Oct 20;59(2):349–358. doi: 10.1016/0092-8674(89)90296-1. [DOI] [PubMed] [Google Scholar]
  29. Smith D. E., Fisher P. A. Identification, developmental regulation, and response to heat shock of two antigenically related forms of a major nuclear envelope protein in Drosophila embryos: application of an improved method for affinity purification of antibodies using polypeptides immobilized on nitrocellulose blots. J Cell Biol. 1984 Jul;99(1 Pt 1):20–28. doi: 10.1083/jcb.99.1.20. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Vijayraghavan U., Company M., Abelson J. Isolation and characterization of pre-mRNA splicing mutants of Saccharomyces cerevisiae. Genes Dev. 1989 Aug;3(8):1206–1216. doi: 10.1101/gad.3.8.1206. [DOI] [PubMed] [Google Scholar]
  31. Vijayraghavan U., Parker R., Tamm J., Iimura Y., Rossi J., Abelson J., Guthrie C. Mutations in conserved intron sequences affect multiple steps in the yeast splicing pathway, particularly assembly of the spliceosome. EMBO J. 1986 Jul;5(7):1683–1695. doi: 10.1002/j.1460-2075.1986.tb04412.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Zavanelli M. I., Ares M., Jr Efficient association of U2 snRNPs with pre-mRNA requires an essential U2 RNA structural element. Genes Dev. 1991 Dec;5(12B):2521–2533. doi: 10.1101/gad.5.12b.2521. [DOI] [PubMed] [Google Scholar]

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