Skip to main content
PMC home page
RNA logoLink to RNA
. 2000 Jun;6(6):901–911. doi: 10.1017/s1355838200000376

Differential recognition of the polypyrimidine-tract by the general splicing factor U2AF65 and the splicing repressor sex-lethal.

R Singh 1, H Banerjee 1, M R Green 1
PMCID: PMC1369966  PMID: 10864047

Abstract

The polypyrimidine-tract (Py-tract) adjacent to 3' splice sites is an essential splicing signal and is recognized by several proteins, including the general splicing factor U2AF65 and the highly specific splicing repressor Sex-lethal (SXL). They both contain ribonucleoprotein-consensus RNA-binding motifs. However, U2AF65 recognizes a wide variety of Py-tracts, whereas SXL recognizes specific Py-tracts such as the nonsex-specific Py-tract of the transformer pre-mRNA. It is not understood how these seemingly similar proteins differentially recognize the Py-tract. To define these interactions, we used chemical interference and protection assays, saturation mutagenesis, and RNAs containing modified nucleotides. We find that these proteins recognize distinct features of the RNA. First, although uracils within the Py-tract are protected from chemical modification by both of these proteins, modification of any one of seven uracils by hydrazine, or any of eight phosphates by ethylnitrosourea strongly interfered with the binding of SXL only. Second, the 2' hydroxyl groups or backbone conformation appeared important for the binding of SXL, but not U2AF65. Third, although any of the bases (cytosine >> adenine > guanine) could substitute for uracils for U2AF65 binding, only guanine partially substituted for certain uracils for SXL binding. The different dependence on individual contacts and nucleotide preference may provide a basis for the different RNA-binding specificities and thus functions of U2AF65 and SXL in 3' splice site choice.

Full Text

The Full Text of this article is available as a PDF (287.2 KB).

Selected References

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

  1. Adams M. D., Rudner D. Z., Rio D. C. Biochemistry and regulation of pre-mRNA splicing. Curr Opin Cell Biol. 1996 Jun;8(3):331–339. doi: 10.1016/s0955-0674(96)80006-8. [DOI] [PubMed] [Google Scholar]
  2. Albrecht E. B., Salz H. K. The Drosophila sex determination gene snf is utilized for the establishment of the female-specific splicing pattern of Sex-lethal. Genetics. 1993 Jul;134(3):801–807. doi: 10.1093/genetics/134.3.801. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bashaw G. J., Baker B. S. The regulation of the Drosophila msl-2 gene reveals a function for Sex-lethal in translational control. Cell. 1997 May 30;89(5):789–798. doi: 10.1016/s0092-8674(00)80262-7. [DOI] [PubMed] [Google Scholar]
  4. Been M. D., Cech T. R. Selection of circularization sites in a group I IVS RNA requires multiple alignments of an internal template-like sequence. Cell. 1987 Sep 11;50(6):951–961. doi: 10.1016/0092-8674(87)90522-8. [DOI] [PubMed] [Google Scholar]
  5. Bell L. R., Maine E. M., Schedl P., Cline T. W. Sex-lethal, a Drosophila sex determination switch gene, exhibits sex-specific RNA splicing and sequence similarity to RNA binding proteins. Cell. 1988 Dec 23;55(6):1037–1046. doi: 10.1016/0092-8674(88)90248-6. [DOI] [PubMed] [Google Scholar]
  6. Burd C. G., Dreyfuss G. Conserved structures and diversity of functions of RNA-binding proteins. Science. 1994 Jul 29;265(5172):615–621. doi: 10.1126/science.8036511. [DOI] [PubMed] [Google Scholar]
  7. Chang K. A., Kuroda M. I. Modulation of MSL1 abundance in female Drosophila contributes to the sex specificity of dosage compensation. Genetics. 1998 Oct;150(2):699–709. doi: 10.1093/genetics/150.2.699. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Cline T. W., Meyer B. J. Vive la différence: males vs females in flies vs worms. Annu Rev Genet. 1996;30:637–702. doi: 10.1146/annurev.genet.30.1.637. [DOI] [PubMed] [Google Scholar]
  9. Conway L., Wickens M. Modification interference analysis of reactions using RNA substrates. Methods Enzymol. 1989;180:369–379. doi: 10.1016/0076-6879(89)80112-0. [DOI] [PubMed] [Google Scholar]
  10. Deo R. C., Bonanno J. B., Sonenberg N., Burley S. K. Recognition of polyadenylate RNA by the poly(A)-binding protein. Cell. 1999 Sep 17;98(6):835–845. doi: 10.1016/s0092-8674(00)81517-2. [DOI] [PubMed] [Google Scholar]
  11. Ding J., Hayashi M. K., Zhang Y., Manche L., Krainer A. R., Xu R. M. Crystal structure of the two-RRM domain of hnRNP A1 (UP1) complexed with single-stranded telomeric DNA. Genes Dev. 1999 May 1;13(9):1102–1115. doi: 10.1101/gad.13.9.1102. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Ehresmann C., Baudin F., Mougel M., Romby P., Ebel J. P., Ehresmann B. Probing the structure of RNAs in solution. Nucleic Acids Res. 1987 Nov 25;15(22):9109–9128. doi: 10.1093/nar/15.22.9109. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Ellington A. D., Szostak J. W. In vitro selection of RNA molecules that bind specific ligands. Nature. 1990 Aug 30;346(6287):818–822. doi: 10.1038/346818a0. [DOI] [PubMed] [Google Scholar]
  14. Fleckner J., Zhang M., Valcárcel J., Green M. R. U2AF65 recruits a novel human DEAD box protein required for the U2 snRNP-branchpoint interaction. Genes Dev. 1997 Jul 15;11(14):1864–1872. doi: 10.1101/gad.11.14.1864. [DOI] [PubMed] [Google Scholar]
  15. Gebauer F., Merendino L., Hentze M. W., Valcárcel J. The Drosophila splicing regulator sex-lethal directly inhibits translation of male-specific-lethal 2 mRNA. RNA. 1998 Feb;4(2):142–150. [PMC free article] [PubMed] [Google Scholar]
  16. Gish G., Eckstein F. DNA and RNA sequence determination based on phosphorothioate chemistry. Science. 1988 Jun 10;240(4858):1520–1522. doi: 10.1126/science.2453926. [DOI] [PubMed] [Google Scholar]
  17. Gozani O., Potashkin J., Reed R. A potential role for U2AF-SAP 155 interactions in recruiting U2 snRNP to the branch site. Mol Cell Biol. 1998 Aug;18(8):4752–4760. doi: 10.1128/mcb.18.8.4752. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Granadino B., Campuzano S., Sánchez L. The Drosophila melanogaster fl(2)d gene is needed for the female-specific splicing of Sex-lethal RNA. EMBO J. 1990 Aug;9(8):2597–2602. doi: 10.1002/j.1460-2075.1990.tb07441.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Granadino B., Penalva L. O., Green M. R., Valcárcel J., Sánchez L. Distinct mechanisms of splicing regulation in vivo by the Drosophila protein Sex-lethal. Proc Natl Acad Sci U S A. 1997 Jul 8;94(14):7343–7348. doi: 10.1073/pnas.94.14.7343. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Granadino B., San Juán A., Santamaria P., Sánchez L. Evidence of a dual function in fl(2)d, a gene needed for Sex-lethal expression in Drosophila melanogaster. Genetics. 1992 Mar;130(3):597–612. doi: 10.1093/genetics/130.3.597. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. 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]
  22. Handa N., Nureki O., Kurimoto K., Kim I., Sakamoto H., Shimura Y., Muto Y., Yokoyama S. Structural basis for recognition of the tra mRNA precursor by the Sex-lethal protein. Nature. 1999 Apr 15;398(6728):579–585. doi: 10.1038/19242. [DOI] [PubMed] [Google Scholar]
  23. Horabin J. I., Schedl P. Sex-lethal autoregulation requires multiple cis-acting elements upstream and downstream of the male exon and appears to depend largely on controlling the use of the male exon 5' splice site. Mol Cell Biol. 1993 Dec;13(12):7734–7746. doi: 10.1128/mcb.13.12.7734. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Inoue K., Hoshijima K., Sakamoto H., Shimura Y. Binding of the Drosophila sex-lethal gene product to the alternative splice site of transformer primary transcript. Nature. 1990 Mar 29;344(6265):461–463. doi: 10.1038/344461a0. [DOI] [PubMed] [Google Scholar]
  25. Ishikawa F., Matunis M. J., Dreyfuss G., Cech T. R. Nuclear proteins that bind the pre-mRNA 3' splice site sequence r(UUAG/G) and the human telomeric DNA sequence d(TTAGGG)n. Mol Cell Biol. 1993 Jul;13(7):4301–4310. doi: 10.1128/mcb.13.7.4301. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Kanaar R., Lee A. L., Rudner D. Z., Wemmer D. E., Rio D. C. Interaction of the sex-lethal RNA binding domains with RNA. EMBO J. 1995 Sep 15;14(18):4530–4539. doi: 10.1002/j.1460-2075.1995.tb00132.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Kanaar R., Roche S. E., Beall E. L., Green M. R., Rio D. C. The conserved pre-mRNA splicing factor U2AF from Drosophila: requirement for viability. Science. 1993 Oct 22;262(5133):569–573. doi: 10.1126/science.7692602. [DOI] [PubMed] [Google Scholar]
  28. Kelley R. L., Solovyeva I., Lyman L. M., Richman R., Solovyev V., Kuroda M. I. Expression of msl-2 causes assembly of dosage compensation regulators on the X chromosomes and female lethality in Drosophila. Cell. 1995 Jun 16;81(6):867–877. doi: 10.1016/0092-8674(95)90007-1. [DOI] [PubMed] [Google Scholar]
  29. Kelley R. L., Wang J., Bell L., Kuroda M. I. Sex lethal controls dosage compensation in Drosophila by a non-splicing mechanism. Nature. 1997 May 8;387(6629):195–199. doi: 10.1038/387195a0. [DOI] [PubMed] [Google Scholar]
  30. Lee A. L., Volkman B. F., Robertson S. A., Rudner D. Z., Barbash D. A., Cline T. W., Kanaar R., Rio D. C., Wemmer D. E. Chemical shift mapping of the RNA-binding interface of the multiple-RBD protein sex-lethal. Biochemistry. 1997 Nov 25;36(47):14306–14317. doi: 10.1021/bi970830y. [DOI] [PubMed] [Google Scholar]
  31. McKeown M. Alternative mRNA splicing. Annu Rev Cell Biol. 1992;8:133–155. doi: 10.1146/annurev.cb.08.110192.001025. [DOI] [PubMed] [Google Scholar]
  32. Milligan J. F., Groebe D. R., Witherell G. W., Uhlenbeck O. C. Oligoribonucleotide synthesis using T7 RNA polymerase and synthetic DNA templates. Nucleic Acids Res. 1987 Nov 11;15(21):8783–8798. doi: 10.1093/nar/15.21.8783. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Moazed D., Noller H. F. Transfer RNA shields specific nucleotides in 16S ribosomal RNA from attack by chemical probes. Cell. 1986 Dec 26;47(6):985–994. doi: 10.1016/0092-8674(86)90813-5. [DOI] [PubMed] [Google Scholar]
  34. Nadal-Ginard B., Smith C. W., Patton J. G., Breitbart R. E. Alternative splicing is an efficient mechanism for the generation of protein diversity: contractile protein genes as a model system. Adv Enzyme Regul. 1991;31:261–286. doi: 10.1016/0065-2571(91)90017-g. [DOI] [PubMed] [Google Scholar]
  35. Nagai K. RNA-protein complexes. Curr Opin Struct Biol. 1996 Feb;6(1):53–61. doi: 10.1016/s0959-440x(96)80095-9. [DOI] [PubMed] [Google Scholar]
  36. O'Neil M. T., Belote J. M. Interspecific comparison of the transformer gene of Drosophila reveals an unusually high degree of evolutionary divergence. Genetics. 1992 May;131(1):113–128. doi: 10.1093/genetics/131.1.113. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Oliver B., Perrimon N., Mahowald A. P. Genetic evidence that the sans fille locus is involved in Drosophila sex determination. Genetics. 1988 Sep;120(1):159–171. doi: 10.1093/genetics/120.1.159. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Oubridge C., Ito N., Evans P. R., Teo C. H., Nagai K. Crystal structure at 1.92 A resolution of the RNA-binding domain of the U1A spliceosomal protein complexed with an RNA hairpin. Nature. 1994 Dec 1;372(6505):432–438. doi: 10.1038/372432a0. [DOI] [PubMed] [Google Scholar]
  39. Potashkin J., Naik K., Wentz-Hunter K. U2AF homolog required for splicing in vivo. Science. 1993 Oct 22;262(5133):573–575. doi: 10.1126/science.8211184. [DOI] [PubMed] [Google Scholar]
  40. Price S. R., Evans P. R., Nagai K. Crystal structure of the spliceosomal U2B"-U2A' protein complex bound to a fragment of U2 small nuclear RNA. Nature. 1998 Aug 13;394(6694):645–650. doi: 10.1038/29234. [DOI] [PubMed] [Google Scholar]
  41. Robertson D. L., Joyce G. F. Selection in vitro of an RNA enzyme that specifically cleaves single-stranded DNA. Nature. 1990 Mar 29;344(6265):467–468. doi: 10.1038/344467a0. [DOI] [PubMed] [Google Scholar]
  42. 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]
  43. Rymond B. C., Rosbash M. A chemical modification/interference study of yeast pre-mRNA spliceosome assembly and splicing. Genes Dev. 1988 Apr;2(4):428–439. doi: 10.1101/gad.2.4.428. [DOI] [PubMed] [Google Scholar]
  44. Sakamoto H., Inoue K., Higuchi I., Ono Y., Shimura Y. Control of Drosophila Sex-lethal pre-mRNA splicing by its own female-specific product. Nucleic Acids Res. 1992 Nov 11;20(21):5533–5540. doi: 10.1093/nar/20.21.5533. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Sakashita E., Sakamoto H. Characterization of RNA binding specificity of the Drosophila sex-lethal protein by in vitro ligand selection. Nucleic Acids Res. 1994 Oct 11;22(20):4082–4086. doi: 10.1093/nar/22.20.4082. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Samuels M. E., Bopp D., Colvin R. A., Roscigno R. F., Garcia-Blanco M. A., Schedl P. RNA binding by Sxl proteins in vitro and in vivo. Mol Cell Biol. 1994 Jul;14(7):4975–4990. doi: 10.1128/mcb.14.7.4975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Singh R., Valcárcel J., Green M. R. Distinct binding specificities and functions of higher eukaryotic polypyrimidine tract-binding proteins. Science. 1995 May 26;268(5214):1173–1176. doi: 10.1126/science.7761834. [DOI] [PubMed] [Google Scholar]
  48. Sosnowski B. A., Belote J. M., McKeown M. Sex-specific alternative splicing of RNA from the transformer gene results from sequence-dependent splice site blockage. Cell. 1989 Aug 11;58(3):449–459. doi: 10.1016/0092-8674(89)90426-1. [DOI] [PubMed] [Google Scholar]
  49. Sosnowski B. A., Davis D. D., Boggs R. T., Madigan S. J., McKeown M. Multiple portions of a small region of the Drosophila transformer gene are required for efficient in vivo sex-specific regulated RNA splicing and in vitro sex-lethal binding. Dev Biol. 1994 Jan;161(1):302–312. doi: 10.1006/dbio.1994.1030. [DOI] [PubMed] [Google Scholar]
  50. Steinmann-Zwicky M. Sex determination in Drosophila: the X-chromosomal gene liz is required for Sxl activity. EMBO J. 1988 Dec 1;7(12):3889–3898. doi: 10.1002/j.1460-2075.1988.tb03275.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Tuerk C., Gold L. Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase. Science. 1990 Aug 3;249(4968):505–510. doi: 10.1126/science.2200121. [DOI] [PubMed] [Google Scholar]
  52. Valcárcel J., Gaur R. K., Singh R., Green M. R. Interaction of U2AF65 RS region with pre-mRNA branch point and promotion of base pairing with U2 snRNA [corrected]. Science. 1996 Sep 20;273(5282):1706–1709. doi: 10.1126/science.273.5282.1706. [DOI] [PubMed] [Google Scholar]
  53. Valcárcel J., Singh R., Zamore P. D., Green M. R. The protein Sex-lethal antagonizes the splicing factor U2AF to regulate alternative splicing of transformer pre-mRNA. Nature. 1993 Mar 11;362(6416):171–175. doi: 10.1038/362171a0. [DOI] [PubMed] [Google Scholar]
  54. Vlassov V. V., Giege R., Ebel J. P. The tertiary structure of yeast tRNAPhe in solution studied by phosphodiester bond modification with ethylnitrosourea. FEBS Lett. 1980 Oct 20;120(1):12–16. doi: 10.1016/0014-5793(80)81034-9. [DOI] [PubMed] [Google Scholar]
  55. Wang J., Bell L. R. The Sex-lethal amino terminus mediates cooperative interactions in RNA binding and is essential for splicing regulation. Genes Dev. 1994 Sep 1;8(17):2072–2085. doi: 10.1101/gad.8.17.2072. [DOI] [PubMed] [Google Scholar]
  56. Yanowitz J. L., Deshpande G., Calhoun G., Schedl P. D. An N-terminal truncation uncouples the sex-transforming and dosage compensation functions of sex-lethal. Mol Cell Biol. 1999 Apr;19(4):3018–3028. doi: 10.1128/mcb.19.4.3018. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Zamore P. D., Green M. R. Identification, purification, and biochemical characterization of U2 small nuclear ribonucleoprotein auxiliary factor. Proc Natl Acad Sci U S A. 1989 Dec;86(23):9243–9247. doi: 10.1073/pnas.86.23.9243. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Zhou S., Yang Y., Scott M. J., Pannuti A., Fehr K. C., Eisen A., Koonin E. V., Fouts D. L., Wrightsman R., Manning J. E. Male-specific lethal 2, a dosage compensation gene of Drosophila, undergoes sex-specific regulation and encodes a protein with a RING finger and a metallothionein-like cysteine cluster. EMBO J. 1995 Jun 15;14(12):2884–2895. doi: 10.1002/j.1460-2075.1995.tb07288.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. Zorio D. A., Blumenthal T. U2AF35 is encoded by an essential gene clustered in an operon with RRM/cyclophilin in Caenorhabditis elegans. RNA. 1999 Apr;5(4):487–494. doi: 10.1017/s1355838299982225. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from RNA are provided here courtesy of The RNA Society

RESOURCES