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Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1991 Aug 15;88(16):7061–7065. doi: 10.1073/pnas.88.16.7061

Limited functional equivalence of phylogenetic variation in small nuclear RNA: yeast U2 RNA with altered branchpoint complementarity inhibits splicing and produces a dominant lethal phenotype.

L Miraglia 1, S Seiwert 1, A H Igel 1, M Ares Jr 1
PMCID: PMC52233  PMID: 1871121

Abstract

U2 is a highly conserved small nuclear RNA essential for pre-mRNA splicing in mammals and yeast and for trans-splicing in trypanosomes. To test the function of variant U2 RNA structures from different organisms, we conducted phylogenetic exchanges of U2 domains. Replacing nucleotides 1-120 of yeast U2 with the corresponding region of human U2 generates a U2 RNA that is correctly folded and functions in yeast. In contrast, replacement of the branchpoint interaction region of yeast U2 with the corresponding region from trypanosome is dominant lethal. Using a GAL-U2 promoter fusion, we show that the dominant phenotype can be made conditional and that the accumulation of mutant U2 is followed rapidly by inhibition of nuclear pre-mRNA splicing. The results suggest that U2 small nuclear ribonucleoprotein particles normally participate in stable complexes with a limiting splicing factor prior to formation of U2-intron branchpoint base pairs.

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Selected References

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

  1. 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]
  2. Ares M., Jr, Mangin M., Weiner A. M. Orientation-dependent transcriptional activator upstream of a human U2 snRNA gene. Mol Cell Biol. 1985 Jul;5(7):1560–1570. doi: 10.1128/mcb.5.7.1560. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Ares M., Jr U2 RNA from yeast is unexpectedly large and contains homology to vertebrate U4, U5, and U6 small nuclear RNAs. Cell. 1986 Oct 10;47(1):49–59. doi: 10.1016/0092-8674(86)90365-x. [DOI] [PubMed] [Google Scholar]
  4. García-Blanco M. A., Jamison S. F., Sharp P. A. Identification and purification of a 62,000-dalton protein that binds specifically to the polypyrimidine tract of introns. Genes Dev. 1989 Dec;3(12A):1874–1886. doi: 10.1101/gad.3.12a.1874. [DOI] [PubMed] [Google Scholar]
  5. 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]
  6. Hannon G. J., Maroney P. A., Denker J. A., Nilsen T. W. Trans splicing of nematode pre-messenger RNA in vitro. Cell. 1990 Jun 29;61(7):1247–1255. doi: 10.1016/0092-8674(90)90689-c. [DOI] [PubMed] [Google Scholar]
  7. Hartmuth K., Barta A. Unusual branch point selection in processing of human growth hormone pre-mRNA. Mol Cell Biol. 1988 May;8(5):2011–2020. doi: 10.1128/mcb.8.5.2011. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Hausner T. P., Giglio L. M., Weiner A. M. Evidence for base-pairing between mammalian U2 and U6 small nuclear ribonucleoprotein particles. Genes Dev. 1990 Dec;4(12A):2146–2156. doi: 10.1101/gad.4.12a.2146. [DOI] [PubMed] [Google Scholar]
  9. Herskowitz I. Functional inactivation of genes by dominant negative mutations. Nature. 1987 Sep 17;329(6136):219–222. doi: 10.1038/329219a0. [DOI] [PubMed] [Google Scholar]
  10. Igel A. H., Ares M., Jr Internal sequences that distinguish yeast from metazoan U2 snRNA are unnecessary for pre-mRNA splicing. Nature. 1988 Aug 4;334(6181):450–453. doi: 10.1038/334450a0. [DOI] [PubMed] [Google Scholar]
  11. Inoue T., Cech T. R. Secondary structure of the circular form of the Tetrahymena rRNA intervening sequence: a technique for RNA structure analysis using chemical probes and reverse transcriptase. Proc Natl Acad Sci U S A. 1985 Feb;82(3):648–652. doi: 10.1073/pnas.82.3.648. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Krämer A. Presplicing complex formation requires two proteins and U2 snRNP. Genes Dev. 1988 Sep;2(9):1155–1167. doi: 10.1101/gad.2.9.1155. [DOI] [PubMed] [Google Scholar]
  13. Kunkel T. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc Natl Acad Sci U S A. 1985 Jan;82(2):488–492. doi: 10.1073/pnas.82.2.488. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. 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]
  15. Legrain P., Rosbash M. Some cis- and trans-acting mutants for splicing target pre-mRNA to the cytoplasm. Cell. 1989 May 19;57(4):573–583. doi: 10.1016/0092-8674(89)90127-x. [DOI] [PubMed] [Google Scholar]
  16. 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]
  17. McPheeters D. S., Fabrizio P., Abelson J. In vitro reconstitution of functional yeast U2 snRNPs. Genes Dev. 1989 Dec;3(12B):2124–2136. doi: 10.1101/gad.3.12b.2124. [DOI] [PubMed] [Google Scholar]
  18. Moazed D., Stern S., Noller H. F. Rapid chemical probing of conformation in 16 S ribosomal RNA and 30 S ribosomal subunits using primer extension. J Mol Biol. 1986 Feb 5;187(3):399–416. doi: 10.1016/0022-2836(86)90441-9. [DOI] [PubMed] [Google Scholar]
  19. Nelson K. K., Green M. R. Mammalian U2 snRNP has a sequence-specific RNA-binding activity. Genes Dev. 1989 Oct;3(10):1562–1571. doi: 10.1101/gad.3.10.1562. [DOI] [PubMed] [Google Scholar]
  20. 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]
  21. Noble J. C., Pan Z. Q., Prives C., Manley J. L. Splicing of SV40 early pre-mRNA to large T and small t mRNAs utilizes different patterns of lariat branch sites. Cell. 1987 Jul 17;50(2):227–236. doi: 10.1016/0092-8674(87)90218-2. [DOI] [PubMed] [Google Scholar]
  22. Parker R., Siliciano P. G., Guthrie C. Recognition of the TACTAAC box during mRNA splicing in yeast involves base pairing to the U2-like snRNA. Cell. 1987 Apr 24;49(2):229–239. doi: 10.1016/0092-8674(87)90564-2. [DOI] [PubMed] [Google Scholar]
  23. 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]
  24. Reed R., Maniatis T. The role of the mammalian branchpoint sequence in pre-mRNA splicing. Genes Dev. 1988 Oct;2(10):1268–1276. doi: 10.1101/gad.2.10.1268. [DOI] [PubMed] [Google Scholar]
  25. 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]
  26. Ruskin B., Pikielny C. W., Rosbash M., Green M. R. Alternative branch points are selected during splicing of a yeast pre-mRNA in mammalian and yeast extracts. Proc Natl Acad Sci U S A. 1986 Apr;83(7):2022–2026. doi: 10.1073/pnas.83.7.2022. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. 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]
  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. Shuster E. O., Guthrie C. Human U2 snRNA can function in pre-mRNA splicing in yeast. Nature. 1990 May 17;345(6272):270–273. doi: 10.1038/345270a0. [DOI] [PubMed] [Google Scholar]
  30. Shuster E. O., Guthrie C. Two conserved domains of yeast U2 snRNA are separated by 945 nonessential nucleotides. Cell. 1988 Oct 7;55(1):41–48. doi: 10.1016/0092-8674(88)90007-4. [DOI] [PubMed] [Google Scholar]
  31. Thomas J., Lea K., Zucker-Aprison E., Blumenthal T. The spliceosomal snRNAs of Caenorhabditis elegans. Nucleic Acids Res. 1990 May 11;18(9):2633–2642. doi: 10.1093/nar/18.9.2633. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. 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]
  33. Woolford J. L., Jr Nuclear pre-mRNA splicing in yeast. Yeast. 1989 Nov-Dec;5(6):439–457. doi: 10.1002/yea.320050604. [DOI] [PubMed] [Google Scholar]
  34. Wu J., Manley J. L. Mammalian pre-mRNA branch site selection by U2 snRNP involves base pairing. Genes Dev. 1989 Oct;3(10):1553–1561. doi: 10.1101/gad.3.10.1553. [DOI] [PubMed] [Google Scholar]
  35. Zhuang Y. A., Goldstein A. M., Weiner A. M. UACUAAC is the preferred branch site for mammalian mRNA splicing. Proc Natl Acad Sci U S A. 1989 Apr;86(8):2752–2756. doi: 10.1073/pnas.86.8.2752. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Zhuang Y., Weiner A. M. A compensatory base change in human U2 snRNA can suppress a branch site mutation. Genes Dev. 1989 Oct;3(10):1545–1552. doi: 10.1101/gad.3.10.1545. [DOI] [PubMed] [Google Scholar]

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