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. 1996 Sep;16(9):5036–5047. doi: 10.1128/mcb.16.9.5036

Sex-lethal interacts with splicing factors in vitro and in vivo.

G Deshpande 1, M E Samuels 1, P D Schedl 1
PMCID: PMC231505  PMID: 8756662

Abstract

The Drosophila sex determination gene Sex-lethal controls its own expression and the expression of downstream target genes such as transformer by regulating RNA splicing. Genetic and molecular studies have established that Sxl requires the product of another gene, snf, to autoregulate the splicing of its own transcripts. snf has recently been shown to encode a Drosophila U1 and U2 small nuclear ribonucleoprotein particle protein. In the work reported here, we demonstrate that the Sxl and Snf proteins can interact directly in vitro and that these two proteins are part of an RNase-sensitive complex in vivo which can be immunoprecipitated with the Sxl antibody. Unlike bulk Snf protein, which sediments slowly in sucrose gradients, the Snf protein associated with Sxl is in a large, rapidly sedimenting complex. Detailed characterization of the Sxl-Snf complexes from cross-linked extracts indicates that these complexes contain additional small nuclear ribonucleoprotein particle proteins and the U1 and U2 small nuclear RNAs. Finally, consistent with the RNase sensitivity of the Sxl-Snf complexes, Sxl transcripts can also be immunoprecipitated by Sxl antibodies. On the basis of the physical interactions between Sxl and Snf, we present a model for Sxl splicing regulation. This model helps explain how the Sxl protein is able to promote the sex-specific splicing of Sxl transcripts, utilizing target sequences that are distant from the regulated splice sites.

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Selected References

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  1. 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]
  2. Amero S. A., Raychaudhuri G., Cass C. L., van Venrooij W. J., Habets W. J., Krainer A. R., Beyer A. L. Independent deposition of heterogeneous nuclear ribonucleoproteins and small nuclear ribonucleoprotein particles at sites of transcription. Proc Natl Acad Sci U S A. 1992 Sep 15;89(18):8409–8413. doi: 10.1073/pnas.89.18.8409. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Baker B. S. Sex in flies: the splice of life. Nature. 1989 Aug 17;340(6234):521–524. doi: 10.1038/340521a0. [DOI] [PubMed] [Google Scholar]
  4. Bell L. R., Horabin J. I., Schedl P., Cline T. W. Positive autoregulation of sex-lethal by alternative splicing maintains the female determined state in Drosophila. Cell. 1991 Apr 19;65(2):229–239. doi: 10.1016/0092-8674(91)90157-t. [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. Bernstein M., Lersch R. A., Subrahmanyan L., Cline T. W. Transposon insertions causing constitutive Sex-lethal activity in Drosophila melanogaster affect Sxl sex-specific transcript splicing. Genetics. 1995 Feb;139(2):631–648. doi: 10.1093/genetics/139.2.631. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Billings P. B., Barton J. R., Hoch S. O. A murine monoclonal antibody recognizes the 13,000 molecular weight polypeptide of the Sm small nuclear ribonucleoprotein complex. J Immunol. 1985 Jul;135(1):428–432. [PubMed] [Google Scholar]
  8. Boggs R. T., Gregor P., Idriss S., Belote J. M., McKeown M. Regulation of sexual differentiation in D. melanogaster via alternative splicing of RNA from the transformer gene. Cell. 1987 Aug 28;50(5):739–747. doi: 10.1016/0092-8674(87)90332-1. [DOI] [PubMed] [Google Scholar]
  9. Boix J., Habets W. J., van Venrooij W. J., Smith H. C. Analysis of U1snRNP-specific A protein cross-linked complexes. FEBS Lett. 1990 Oct 15;272(1-2):213–216. doi: 10.1016/0014-5793(90)80487-4. [DOI] [PubMed] [Google Scholar]
  10. Bopp D., Bell L. R., Cline T. W., Schedl P. Developmental distribution of female-specific Sex-lethal proteins in Drosophila melanogaster. Genes Dev. 1991 Mar;5(3):403–415. doi: 10.1101/gad.5.3.403. [DOI] [PubMed] [Google Scholar]
  11. Bopp D., Horabin J. I., Lersch R. A., Cline T. W., Schedl P. Expression of the Sex-lethal gene is controlled at multiple levels during Drosophila oogenesis. Development. 1993 Jul;118(3):797–812. doi: 10.1242/dev.118.3.797. [DOI] [PubMed] [Google Scholar]
  12. Burtis K. C., Baker B. S. Drosophila doublesex gene controls somatic sexual differentiation by producing alternatively spliced mRNAs encoding related sex-specific polypeptides. Cell. 1989 Mar 24;56(6):997–1010. doi: 10.1016/0092-8674(89)90633-8. [DOI] [PubMed] [Google Scholar]
  13. Butler B., Pirrotta V., Irminger-Finger I., Nöthiger R. The sex-determining gene tra of Drosophila: molecular cloning and transformation studies. EMBO J. 1986 Dec 20;5(13):3607–3613. doi: 10.1002/j.1460-2075.1986.tb04689.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Cline T. W. Autoregulatory functioning of a Drosophila gene product that establish es and maintains the sexually determined state. Genetics. 1984 Jun;107(2):231–277. doi: 10.1093/genetics/107.2.231. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Cline T. W. Evidence that sisterless-a and sisterless-b are two of several discrete "numerator elements" of the X/A sex determination signal in Drosophila that switch Sxl between two alternative stable expression states. Genetics. 1988 Aug;119(4):829–862. doi: 10.1093/genetics/119.4.829. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Deshpande G., Stukey J., Schedl P. scute (sis-b) function in Drosophila sex determination. Mol Cell Biol. 1995 Aug;15(8):4430–4440. doi: 10.1128/mcb.15.8.4430. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Estes P. A., Keyes L. N., Schedl P. Multiple response elements in the Sex-lethal early promoter ensure its female-specific expression pattern. Mol Cell Biol. 1995 Feb;15(2):904–917. doi: 10.1128/mcb.15.2.904. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Ferrandon D., Elphick L., Nüsslein-Volhard C., St Johnston D. Staufen protein associates with the 3'UTR of bicoid mRNA to form particles that move in a microtubule-dependent manner. Cell. 1994 Dec 30;79(7):1221–1232. doi: 10.1016/0092-8674(94)90013-2. [DOI] [PubMed] [Google Scholar]
  19. Flickinger T. W., Salz H. K. The Drosophila sex determination gene snf encodes a nuclear protein with sequence and functional similarity to the mammalian U1A snRNP protein. Genes Dev. 1994 Apr 15;8(8):914–925. doi: 10.1101/gad.8.8.914. [DOI] [PubMed] [Google Scholar]
  20. Gergen J. P. Dosage Compensation in Drosophila: Evidence That daughterless and Sex-lethal Control X Chromosome Activity at the Blastoderm Stage of Embryogenesis. Genetics. 1987 Nov;117(3):477–485. doi: 10.1093/genetics/117.3.477. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Govind S., Whalen A. M., Steward R. In vivo self-association of the Drosophila rel-protein dorsal. Proc Natl Acad Sci U S A. 1992 Sep 1;89(17):7861–7865. doi: 10.1073/pnas.89.17.7861. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Grabowski P. J., Seiler S. R., Sharp P. A. A multicomponent complex is involved in the splicing of messenger RNA precursors. Cell. 1985 Aug;42(1):345–353. doi: 10.1016/s0092-8674(85)80130-6. [DOI] [PubMed] [Google Scholar]
  23. Habets W. J., Hoet M. H., De Jong B. A., Van der Kemp A., Van Venrooij W. J. Mapping of B cell epitopes on small nuclear ribonucleoproteins that react with human autoantibodies as well as with experimentally-induced mouse monoclonal antibodies. J Immunol. 1989 Oct 15;143(8):2560–2566. [PubMed] [Google Scholar]
  24. Harper D. S., Fresco L. D., Keene J. D. RNA binding specificity of a Drosophila snRNP protein that shares sequence homology with mammalian U1-A and U2-B" proteins. Nucleic Acids Res. 1992 Jul 25;20(14):3645–3650. doi: 10.1093/nar/20.14.3645. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Harris S. G., Hoch S. O., Smith H. C. Chemical cross-linking of Sm and RNP antigenic proteins. Biochemistry. 1988 Jun 28;27(13):4595–4600. doi: 10.1021/bi00413a002. [DOI] [PubMed] [Google Scholar]
  26. Harris S. G., Martin T. E., Smith H. C. Reversible chemical cross-linking and ribonuclease digestion analysis of the organization of proteins in ribonucleoprotein particles. Mol Cell Biochem. 1988 Nov;84(1):17–28. doi: 10.1007/BF00235189. [DOI] [PubMed] [Google Scholar]
  27. Hodgkin J. Drosophila sex determination: a cascade of regulated splicing. Cell. 1989 Mar 24;56(6):905–906. doi: 10.1016/0092-8674(89)90619-3. [DOI] [PubMed] [Google Scholar]
  28. 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]
  29. 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]
  30. Jessen T. H., Oubridge C., Teo C. H., Pritchard C., Nagai K. Identification of molecular contacts between the U1 A small nuclear ribonucleoprotein and U1 RNA. EMBO J. 1991 Nov;10(11):3447–3456. doi: 10.1002/j.1460-2075.1991.tb04909.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Keyes L. N., Cline T. W., Schedl P. The primary sex determination signal of Drosophila acts at the level of transcription. Cell. 1992 Mar 6;68(5):933–943. doi: 10.1016/0092-8674(92)90036-c. [DOI] [PubMed] [Google Scholar]
  32. Krämer A. Purification of small nuclear ribonucleoprotein particles active in RNA processing. Methods Enzymol. 1990;181:215–232. doi: 10.1016/0076-6879(90)81124-d. [DOI] [PubMed] [Google Scholar]
  33. Lerner E. A., Lerner M. R., Janeway C. A., Jr, Steitz J. A. Monoclonal antibodies to nucleic acid-containing cellular constituents: probes for molecular biology and autoimmune disease. Proc Natl Acad Sci U S A. 1981 May;78(5):2737–2741. doi: 10.1073/pnas.78.5.2737. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Matunis E. L., Matunis M. J., Dreyfuss G. Association of individual hnRNP proteins and snRNPs with nascent transcripts. J Cell Biol. 1993 Apr;121(2):219–228. doi: 10.1083/jcb.121.2.219. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Matunis E. L., Matunis M. J., Dreyfuss G. Characterization of the major hnRNP proteins from Drosophila melanogaster. J Cell Biol. 1992 Jan;116(2):257–269. doi: 10.1083/jcb.116.2.257. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Matunis M. J., Matunis E. L., Dreyfuss G. Isolation of hnRNP complexes from Drosophila melanogaster. J Cell Biol. 1992 Jan;116(2):245–255. doi: 10.1083/jcb.116.2.245. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Oliver B., Kim Y. J., Baker B. S. Sex-lethal, master and slave: a hierarchy of germ-line sex determination in Drosophila. Development. 1993 Nov;119(3):897–908. doi: 10.1242/dev.119.3.897. [DOI] [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. Parkhurst S. M., Meneely P. M. Sex determination and dosage compensation: lessons from flies and worms. Science. 1994 May 13;264(5161):924–932. doi: 10.1126/science.8178152. [DOI] [PubMed] [Google Scholar]
  40. Paterson T., Beggs J. D., Finnegan D. J., Lührmann R. Polypeptide components of Drosophila small nuclear ribonucleoprotein particles. Nucleic Acids Res. 1991 Nov 11;19(21):5877–5882. doi: 10.1093/nar/19.21.5877. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Robberson B. L., Cote G. J., Berget S. M. Exon definition may facilitate splice site selection in RNAs with multiple exons. Mol Cell Biol. 1990 Jan;10(1):84–94. doi: 10.1128/mcb.10.1.84. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. 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]
  43. 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]
  44. Salz H. K., Maine E. M., Keyes L. N., Samuels M. E., Cline T. W., Schedl P. The Drosophila female-specific sex-determination gene, Sex-lethal, has stage-, tissue-, and sex-specific RNAs suggesting multiple modes of regulation. Genes Dev. 1989 May;3(5):708–719. doi: 10.1101/gad.3.5.708. [DOI] [PubMed] [Google Scholar]
  45. Salz H. K. The genetic analysis of snf: a Drosophila sex determination gene required for activation of Sex-lethal in both the germline and the soma. Genetics. 1992 Mar;130(3):547–554. doi: 10.1093/genetics/130.3.547. [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. Samuels M. E., Schedl P., Cline T. W. The complex set of late transcripts from the Drosophila sex determination gene sex-lethal encodes multiple related polypeptides. Mol Cell Biol. 1991 Jul;11(7):3584–3602. doi: 10.1128/mcb.11.7.3584. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. 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]
  49. Scherly D., Boelens W., van Venrooij W. J., Dathan N. A., Hamm J., Mattaj I. W. Identification of the RNA binding segment of human U1 A protein and definition of its binding site on U1 snRNA. EMBO J. 1989 Dec 20;8(13):4163–4170. doi: 10.1002/j.1460-2075.1989.tb08601.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Siebel C. W., Admon A., Rio D. C. Soma-specific expression and cloning of PSI, a negative regulator of P element pre-mRNA splicing. Genes Dev. 1995 Feb 1;9(3):269–283. doi: 10.1101/gad.9.3.269. [DOI] [PubMed] [Google Scholar]
  51. Siebel C. W., Fresco L. D., Rio D. C. The mechanism of somatic inhibition of Drosophila P-element pre-mRNA splicing: multiprotein complexes at an exon pseudo-5' splice site control U1 snRNP binding. Genes Dev. 1992 Aug;6(8):1386–1401. doi: 10.1101/gad.6.8.1386. [DOI] [PubMed] [Google Scholar]
  52. 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]
  53. 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]
  54. 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]
  55. 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]
  56. 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]
  57. 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]
  58. Wieben E. D., Pederson T. Small nuclear ribonucleoproteins of Drosophila: identification of U1 RNA-associated proteins and their behavior during heat shock. Mol Cell Biol. 1982 Aug;2(8):914–920. doi: 10.1128/mcb.2.8.914. [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. Wooley J. C., Cone R. D., Tartof D., Chung S. Y. Small nuclear ribonucleoprotein complexes of Drosophila melanogaster. Proc Natl Acad Sci U S A. 1982 Nov;79(22):6762–6766. doi: 10.1073/pnas.79.22.6762. [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. 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]
  61. Zamore P. D., Patton J. G., Green M. R. Cloning and domain structure of the mammalian splicing factor U2AF. Nature. 1992 Feb 13;355(6361):609–614. doi: 10.1038/355609a0. [DOI] [PubMed] [Google Scholar]

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