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. 1998 Jun 1;26(11):2625–2637. doi: 10.1093/nar/26.11.2625

Activities of the Sex-lethal protein in RNA binding and protein:protein interactions.

M Samuels 1, G Deshpande 1, P Schedl 1
PMCID: PMC147605  PMID: 9592147

Abstract

The Drosophila sex determination gene Sex-lethal (Sxl) controls its own expression, and the expression of downstream target genes such as transformer , by regulating pre-mRNA splicing and mRNA translation. Sxl codes an RNA-binding protein that consists of an N-terminus of approximately 100 amino acids, two 90 amino acid RRM domains, R1 and R2, and an 80 amino acid C-terminus. In the studies reported here we have examined the functional properties of the different Sxl protein domains in RNA binding and in protein:protein interactions. The two RRM domains are responsible for RNA binding. Specificity in the recognition of target RNAs requires both RRM domains, and proteins which consist of the single domains or duplicated domains have anomalous RNA recognition properties. Moreover, the length of the linker between domains can affect RNA recognition properties. Our results indicate that the two RRM domains mediate Sxl:Sxl protein interactions, and that these interactions probably occur both in cis and trans. We speculate that cis interactions between R1 and R2 play a role in RNA recognition by the Sxl protein, while trans interactions stabilize complex formation on target RNAs that contain two or more closely spaced binding sites. Finally, we show that the interaction of Sxl with the snRNP protein Snf is mediated by the R1 RRM domain.

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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. 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]
  3. Bashaw G. J., Baker B. S. The msl-2 dosage compensation gene of Drosophila encodes a putative DNA-binding protein whose expression is sex specifically regulated by Sex-lethal. Development. 1995 Oct;121(10):3245–3258. doi: 10.1242/dev.121.10.3245. [DOI] [PubMed] [Google Scholar]
  4. 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]
  5. 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]
  6. 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]
  7. 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]
  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. 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]
  10. Bopp D., Calhoun G., Horabin J. I., Samuels M., Schedl P. Sex-specific control of Sex-lethal is a conserved mechanism for sex determination in the genus Drosophila. Development. 1996 Mar;122(3):971–982. doi: 10.1242/dev.122.3.971. [DOI] [PubMed] [Google Scholar]
  11. 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]
  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. Cartegni L., Maconi M., Morandi E., Cobianchi F., Riva S., Biamonti G. hnRNP A1 selectively interacts through its Gly-rich domain with different RNA-binding proteins. J Mol Biol. 1996 Jun 14;259(3):337–348. doi: 10.1006/jmbi.1996.0324. [DOI] [PubMed] [Google Scholar]
  15. 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]
  16. 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]
  17. Deshpande G., Samuels M. E., Schedl P. D. Sex-lethal interacts with splicing factors in vitro and in vivo. Mol Cell Biol. 1996 Sep;16(9):5036–5047. doi: 10.1128/mcb.16.9.5036. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. 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]
  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. 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]
  23. 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]
  24. Hilfiker A., Amrein H., Dübendorfer A., Schneiter R., Nöthiger R. The gene virilizer is required for female-specific splicing controlled by Sxl, the master gene for sexual development in Drosophila. Development. 1995 Dec;121(12):4017–4026. doi: 10.1242/dev.121.12.4017. [DOI] [PubMed] [Google Scholar]
  25. 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]
  26. 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]
  27. 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]
  28. 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]
  29. 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]
  30. 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]
  31. 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]
  32. 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]
  33. Kühn U., Pieler T. Xenopus poly(A) binding protein: functional domains in RNA binding and protein-protein interaction. J Mol Biol. 1996 Feb 16;256(1):20–30. doi: 10.1006/jmbi.1996.0065. [DOI] [PubMed] [Google Scholar]
  34. Lee A. L., Kanaar R., Rio D. C., Wemmer D. E. Resonance assignments and solution structure of the second RNA-binding domain of sex-lethal determined by multidimensional heteronuclear magnetic resonance. Biochemistry. 1994 Nov 22;33(46):13775–13786. doi: 10.1021/bi00250a031. [DOI] [PubMed] [Google Scholar]
  35. Lutz C. S., Alwine J. C. Direct interaction of the U1 snRNP-A protein with the upstream efficiency element of the SV40 late polyadenylation signal. Genes Dev. 1994 Mar 1;8(5):576–586. doi: 10.1101/gad.8.5.576. [DOI] [PubMed] [Google Scholar]
  36. Nagai K., Oubridge C., Jessen T. H., Li J., Evans P. R. Crystal structure of the RNA-binding domain of the U1 small nuclear ribonucleoprotein A. Nature. 1990 Dec 6;348(6301):515–520. doi: 10.1038/348515a0. [DOI] [PubMed] [Google Scholar]
  37. Nietfeld W., Mentzel H., Pieler T. The Xenopus laevis poly(A) binding protein is composed of multiple functionally independent RNA binding domains. EMBO J. 1990 Nov;9(11):3699–3705. doi: 10.1002/j.1460-2075.1990.tb07582.x. [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. 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. 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]
  41. 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]
  42. Sakashita E., Sakamoto H. Protein-RNA and protein-protein interactions of the Drosophila sex-lethal mediated by its RNA-binding domains. J Biochem. 1996 Nov;120(5):1028–1033. doi: 10.1093/oxfordjournals.jbchem.a021495. [DOI] [PubMed] [Google Scholar]
  43. 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]
  44. 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]
  45. 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]
  46. 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]
  47. 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]
  48. 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]
  49. Shamoo Y., Abdul-Manan N., Williams K. R. Multiple RNA binding domains (RBDs) just don't add up. Nucleic Acids Res. 1995 Mar 11;23(5):725–728. doi: 10.1093/nar/23.5.725. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Shamoo Y., Krueger U., Rice L. M., Williams K. R., Steitz T. A. Crystal structure of the two RNA binding domains of human hnRNP A1 at 1.75 A resolution. Nat Struct Biol. 1997 Mar;4(3):215–222. doi: 10.1038/nsb0397-215. [DOI] [PubMed] [Google Scholar]
  51. 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]
  52. 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]
  53. 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]
  54. 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]
  55. 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]
  56. 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]
  57. Wang J., Dong Z., Bell L. R. Sex-lethal interactions with protein and RNA. Roles of glycine-rich and RNA binding domains. J Biol Chem. 1997 Aug 29;272(35):22227–22235. doi: 10.1074/jbc.272.35.22227. [DOI] [PubMed] [Google Scholar]
  58. Xu R. M., Jokhan L., Cheng X., Mayeda A., Krainer A. R. Crystal structure of human UP1, the domain of hnRNP A1 that contains two RNA-recognition motifs. Structure. 1997 Apr 15;5(4):559–570. doi: 10.1016/s0969-2126(97)00211-6. [DOI] [PubMed] [Google Scholar]
  59. 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]
  60. 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]
  61. 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]

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