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. 1989 Apr;86(8):2752–2756. doi: 10.1073/pnas.86.8.2752

UACUAAC is the preferred branch site for mammalian mRNA splicing.

Y A Zhuang 1, A M Goldstein 1, A M Weiner 1
PMCID: PMC286996  PMID: 2704744

Abstract

The conserved branch-site sequence UAC-UAAC is known to form base pairs with the complementary sequence GUAGUA in U2 small nuclear RNA (snRNA) during mRNA splicing in the yeast Saccharomyces cerevisiae. Although the GUAGUA element is conserved in mammalian U2 snRNA, mammalian branch sites conform only weakly to a YURAC consensus and can even be deleted without obvious effects on the efficiency of splicing in vivo. To understand why the GUAGUA element of U2 is conserved in evolution but the branch site is not, we have devised two different competitive assays for branch-site selection using the first intron of the human beta-globin gene. We find that a sequence resembling UACUAAC is the most efficient branch site for mammalian mRNA splicing both in vivo and in vitro. Our results suggest that in mammals U2 snRNA can form base pairs with the branch site and the interaction between U2 and the branch site can be augmented or replaced by an interaction between the spliceosome and some other element of the intron or exons, perhaps the conserved polypyrimidine tract located immediately upstream from the 3' splice site.

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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 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]
  2. 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]
  3. Brow D. A., Guthrie C. Spliceosomal RNA U6 is remarkably conserved from yeast to mammals. Nature. 1988 Jul 21;334(6179):213–218. doi: 10.1038/334213a0. [DOI] [PubMed] [Google Scholar]
  4. Cech T. R. The generality of self-splicing RNA: relationship to nuclear mRNA splicing. Cell. 1986 Jan 31;44(2):207–210. doi: 10.1016/0092-8674(86)90751-8. [DOI] [PubMed] [Google Scholar]
  5. Chabot B., Black D. L., LeMaster D. M., Steitz J. A. The 3' splice site of pre-messenger RNA is recognized by a small nuclear ribonucleoprotein. Science. 1985 Dec 20;230(4732):1344–1349. doi: 10.1126/science.2933810. [DOI] [PubMed] [Google Scholar]
  6. Chabot B., Steitz J. A. Multiple interactions between the splicing substrate and small nuclear ribonucleoproteins in spliceosomes. Mol Cell Biol. 1987 Jan;7(1):281–293. doi: 10.1128/mcb.7.1.281. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. 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]
  8. Couto J. R., Tamm J., Parker R., Guthrie C. A trans-acting suppressor restores splicing of a yeast intron with a branch point mutation. Genes Dev. 1987 Jul;1(5):445–455. doi: 10.1101/gad.1.5.445. [DOI] [PubMed] [Google Scholar]
  9. 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]
  10. Frendewey D., Krämer A., Keller W. Different small nuclear ribonucleoprotein particles are involved in different steps of splicing complex formation. Cold Spring Harb Symp Quant Biol. 1987;52:287–298. doi: 10.1101/sqb.1987.052.01.034. [DOI] [PubMed] [Google Scholar]
  11. Fu X. Y., Ge H., Manley J. L. The role of the polypyrimidine stretch at the SV40 early pre-mRNA 3' splice site in alternative splicing. EMBO J. 1988 Mar;7(3):809–817. doi: 10.1002/j.1460-2075.1988.tb02879.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Gerke V., Steitz J. A. A protein associated with small nuclear ribonucleoprotein particles recognizes the 3' splice site of premessenger RNA. Cell. 1986 Dec 26;47(6):973–984. doi: 10.1016/0092-8674(86)90812-3. [DOI] [PubMed] [Google Scholar]
  13. 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]
  14. 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]
  15. Hernandez N. Formation of the 3' end of U1 snRNA is directed by a conserved sequence located downstream of the coding region. EMBO J. 1985 Jul;4(7):1827–1837. doi: 10.1002/j.1460-2075.1985.tb03857.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Hornig H., Aebi M., Weissmann C. Effect of mutations at the lariat branch acceptor site on beta-globin pre-mRNA splicing in vitro. Nature. 1986 Dec 11;324(6097):589–591. doi: 10.1038/324589a0. [DOI] [PubMed] [Google Scholar]
  17. 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]
  18. Keller E. B., Noon W. A. Intron splicing: a conserved internal signal in introns of animal pre-mRNAs. Proc Natl Acad Sci U S A. 1984 Dec;81(23):7417–7420. doi: 10.1073/pnas.81.23.7417. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. 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]
  20. 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]
  21. Krainer A. R., Maniatis T., Ruskin B., Green M. R. Normal and mutant human beta-globin pre-mRNAs are faithfully and efficiently spliced in vitro. Cell. 1984 Apr;36(4):993–1005. doi: 10.1016/0092-8674(84)90049-7. [DOI] [PubMed] [Google Scholar]
  22. 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]
  23. Lamond A. I., Konarska M. M., Grabowski P. J., Sharp P. A. Spliceosome assembly involves the binding and release of U4 small nuclear ribonucleoprotein. Proc Natl Acad Sci U S A. 1988 Jan;85(2):411–415. doi: 10.1073/pnas.85.2.411. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Langford C. J., Gallwitz D. Evidence for an intron-contained sequence required for the splicing of yeast RNA polymerase II transcripts. Cell. 1983 Jun;33(2):519–527. doi: 10.1016/0092-8674(83)90433-6. [DOI] [PubMed] [Google Scholar]
  25. 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]
  26. Nelson K. K., Green M. R. Splice site selection and ribonucleoprotein complex assembly during in vitro pre-mRNA splicing. Genes Dev. 1988 Mar;2(3):319–329. doi: 10.1101/gad.2.3.319. [DOI] [PubMed] [Google Scholar]
  27. 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]
  28. Pikielny C. W., Rymond B. C., Rosbash M. Electrophoresis of ribonucleoproteins reveals an ordered assembly pathway of yeast splicing complexes. 1986 Nov 27-Dec 3Nature. 324(6095):341–345. doi: 10.1038/324341a0. [DOI] [PubMed] [Google Scholar]
  29. Rautmann G., Breathnach R. A role for branchpoints in splicing in vivo. 1985 May 30-Jun 5Nature. 315(6018):430–432. doi: 10.1038/315430a0. [DOI] [PubMed] [Google Scholar]
  30. Reed R., Maniatis T. A role for exon sequences and splice-site proximity in splice-site selection. Cell. 1986 Aug 29;46(5):681–690. doi: 10.1016/0092-8674(86)90343-0. [DOI] [PubMed] [Google Scholar]
  31. 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]
  32. 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]
  33. Ruskin B., Green M. R. An RNA processing activity that debranches RNA lariats. Science. 1985 Jul 12;229(4709):135–140. doi: 10.1126/science.2990042. [DOI] [PubMed] [Google Scholar]
  34. Ruskin B., Green M. R. Specific and stable intron-factor interactions are established early during in vitro pre-mRNA splicing. Cell. 1985 Nov;43(1):131–142. doi: 10.1016/0092-8674(85)90018-2. [DOI] [PubMed] [Google Scholar]
  35. Ruskin B., Greene J. M., Green M. R. Cryptic branch point activation allows accurate in vitro splicing of human beta-globin intron mutants. Cell. 1985 Jul;41(3):833–844. doi: 10.1016/s0092-8674(85)80064-7. [DOI] [PubMed] [Google Scholar]
  36. 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]
  37. Sharp P. A. On the origin of RNA splicing and introns. Cell. 1985 Sep;42(2):397–400. doi: 10.1016/0092-8674(85)90092-3. [DOI] [PubMed] [Google Scholar]
  38. 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]
  39. Swanson M. S., Dreyfuss G. RNA binding specificity of hnRNP proteins: a subset bind to the 3' end of introns. EMBO J. 1988 Nov;7(11):3519–3529. doi: 10.1002/j.1460-2075.1988.tb03228.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Tazi J., Alibert C., Temsamani J., Reveillaud I., Cathala G., Brunel C., Jeanteur P. A protein that specifically recognizes the 3' splice site of mammalian pre-mRNA introns is associated with a small nuclear ribonucleoprotein. Cell. 1986 Dec 5;47(5):755–766. doi: 10.1016/0092-8674(86)90518-0. [DOI] [PubMed] [Google Scholar]
  41. Zhuang Y., Leung H., Weiner A. M. The natural 5' splice site of simian virus 40 large T antigen can be improved by increasing the base complementarity to U1 RNA. Mol Cell Biol. 1987 Aug;7(8):3018–3020. doi: 10.1128/mcb.7.8.3018. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Zhuang Y., Weiner A. M. A compensatory base change in U1 snRNA suppresses a 5' splice site mutation. Cell. 1986 Sep 12;46(6):827–835. doi: 10.1016/0092-8674(86)90064-4. [DOI] [PubMed] [Google Scholar]
  43. van der Veen R., Kwakman J. H., Grivell L. A. Mutations at the lariat acceptor site allow self-splicing of a group II intron without lariat formation. EMBO J. 1987 Dec 1;6(12):3827–3831. doi: 10.1002/j.1460-2075.1987.tb02719.x. [DOI] [PMC free article] [PubMed] [Google Scholar]

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