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. 1998 Oct 15;17(20):6049–6060. doi: 10.1093/emboj/17.20.6049

Analysis of the functional specificity of RS domains in vivo.

B Dauwalder 1, W Mattox 1
PMCID: PMC1170931  PMID: 9774348

Abstract

A number of splicing factors contain extensive regions that are rich in arginine and serine (RS domains). These domains are thought to facilitate protein-protein interactions that are critical in the regulation of alternative splicing. Using a domain swap strategy, we have tested the ability of RS domains from several proteins to substitute in vivo for an essential RS domain in the Drosophila splicing regulator TRA-2. By several criteria, RS domains were found to vary significantly in their ability to support the splicing regulation functions of TRA-2. The RS domain of dU2AF50 functioned efficiently, while that of the dSRp55 protein did not. Moreover, we find similar differences in the ability of RS domains to direct fusion proteins to discrete subnuclear sites at which TRA-2 associates with spermatocyte chromosomes. These results indicate that RS domains are not all functionally equivalent in vivo.

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Selected References

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

  1. 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]
  2. Amrein H., Gorman M., Nöthiger R. The sex-determining gene tra-2 of Drosophila encodes a putative RNA binding protein. Cell. 1988 Dec 23;55(6):1025–1035. doi: 10.1016/0092-8674(88)90247-4. [DOI] [PubMed] [Google Scholar]
  3. Amrein H., Hedley M. L., Maniatis T. The role of specific protein-RNA and protein-protein interactions in positive and negative control of pre-mRNA splicing by Transformer 2. Cell. 1994 Feb 25;76(4):735–746. doi: 10.1016/0092-8674(94)90512-6. [DOI] [PubMed] [Google Scholar]
  4. Amrein H., Maniatis T., Nöthiger R. Alternatively spliced transcripts of the sex-determining gene tra-2 of Drosophila encode functional proteins of different size. EMBO J. 1990 Nov;9(11):3619–3629. doi: 10.1002/j.1460-2075.1990.tb07573.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Birney E., Kumar S., Krainer A. R. Analysis of the RNA-recognition motif and RS and RGG domains: conservation in metazoan pre-mRNA splicing factors. Nucleic Acids Res. 1993 Dec 25;21(25):5803–5816. doi: 10.1093/nar/21.25.5803. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bonaccorsi S., Pisano C., Puoti F., Gatti M. Y chromosome loops in Drosophila melanogaster. Genetics. 1988 Dec;120(4):1015–1034. doi: 10.1093/genetics/120.4.1015. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. 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]
  8. Cenci G., Bonaccorsi S., Pisano C., Verni F., Gatti M. Chromatin and microtubule organization during premeiotic, meiotic and early postmeiotic stages of Drosophila melanogaster spermatogenesis. J Cell Sci. 1994 Dec;107(Pt 12):3521–3534. doi: 10.1242/jcs.107.12.3521. [DOI] [PubMed] [Google Scholar]
  9. Champlin D. T., Frasch M., Saumweber H., Lis J. T. Characterization of a Drosophila protein associated with boundaries of transcriptionally active chromatin. Genes Dev. 1991 Sep;5(9):1611–1621. doi: 10.1101/gad.5.9.1611. [DOI] [PubMed] [Google Scholar]
  10. Chandler D., McGuffin M. E., Piskur J., Yao J., Baker B. S., Mattox W. Evolutionary conservation of regulatory strategies for the sex determination factor transformer-2. Mol Cell Biol. 1997 May;17(5):2908–2919. doi: 10.1128/mcb.17.5.2908. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Chandler S. D., Mayeda A., Yeakley J. M., Krainer A. R., Fu X. D. RNA splicing specificity determined by the coordinated action of RNA recognition motifs in SR proteins. Proc Natl Acad Sci U S A. 1997 Apr 15;94(8):3596–3601. doi: 10.1073/pnas.94.8.3596. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Cáceres J. F., Misteli T., Screaton G. R., Spector D. L., Krainer A. R. Role of the modular domains of SR proteins in subnuclear localization and alternative splicing specificity. J Cell Biol. 1997 Jul 28;138(2):225–238. doi: 10.1083/jcb.138.2.225. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Dauwalder B., Amaya-Manzanares F., Mattox W. A human homologue of the Drosophila sex determination factor transformer-2 has conserved splicing regulatory functions. Proc Natl Acad Sci U S A. 1996 Aug 20;93(17):9004–9009. doi: 10.1073/pnas.93.17.9004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Fu X. D., Maniatis T. Isolation of a complementary DNA that encodes the mammalian splicing factor SC35. Science. 1992 Apr 24;256(5056):535–538. doi: 10.1126/science.1373910. [DOI] [PubMed] [Google Scholar]
  15. Fu X. D. The superfamily of arginine/serine-rich splicing factors. RNA. 1995 Sep;1(7):663–680. [PMC free article] [PubMed] [Google Scholar]
  16. Goralski T. J., Edström J. E., Baker B. S. The sex determination locus transformer-2 of Drosophila encodes a polypeptide with similarity to RNA binding proteins. Cell. 1989 Mar 24;56(6):1011–1018. doi: 10.1016/0092-8674(89)90634-x. [DOI] [PubMed] [Google Scholar]
  17. Hazelrigg T., Tu C. Sex-specific processing of the Drosophila exuperantia transcript is regulated in male germ cells by the tra-2 gene. Proc Natl Acad Sci U S A. 1994 Oct 25;91(22):10752–10756. doi: 10.1073/pnas.91.22.10752. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Hedley M. L., Amrein H., Maniatis T. An amino acid sequence motif sufficient for subnuclear localization of an arginine/serine-rich splicing factor. Proc Natl Acad Sci U S A. 1995 Dec 5;92(25):11524–11528. doi: 10.1073/pnas.92.25.11524. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Heinrichs V., Baker B. S. The Drosophila SR protein RBP1 contributes to the regulation of doublesex alternative splicing by recognizing RBP1 RNA target sequences. EMBO J. 1995 Aug 15;14(16):3987–4000. doi: 10.1002/j.1460-2075.1995.tb00070.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Heinrichs V., Ryner L. C., Baker B. S. Regulation of sex-specific selection of fruitless 5' splice sites by transformer and transformer-2. Mol Cell Biol. 1998 Jan;18(1):450–458. doi: 10.1128/mcb.18.1.450. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Ito H., Fujitani K., Usui K., Shimizu-Nishikawa K., Tanaka S., Yamamoto D. Sexual orientation in Drosophila is altered by the satori mutation in the sex-determination gene fruitless that encodes a zinc finger protein with a BTB domain. Proc Natl Acad Sci U S A. 1996 Sep 3;93(18):9687–9692. doi: 10.1073/pnas.93.18.9687. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Kohtz J. D., Jamison S. F., Will C. L., Zuo P., Lührmann R., Garcia-Blanco M. A., Manley J. L. Protein-protein interactions and 5'-splice-site recognition in mammalian mRNA precursors. Nature. 1994 Mar 10;368(6467):119–124. doi: 10.1038/368119a0. [DOI] [PubMed] [Google Scholar]
  23. Kraus M. E., Lis J. T. The concentration of B52, an essential splicing factor and regulator of splice site choice in vitro, is critical for Drosophila development. Mol Cell Biol. 1994 Aug;14(8):5360–5370. doi: 10.1128/mcb.14.8.5360. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Lee C. G., Zamore P. D., Green M. R., Hurwitz J. RNA annealing activity is intrinsically associated with U2AF. J Biol Chem. 1993 Jun 25;268(18):13472–13478. [PubMed] [Google Scholar]
  25. Li H., Bingham P. M. Arginine/serine-rich domains of the su(wa) and tra RNA processing regulators target proteins to a subnuclear compartment implicated in splicing. Cell. 1991 Oct 18;67(2):335–342. doi: 10.1016/0092-8674(91)90185-2. [DOI] [PubMed] [Google Scholar]
  26. Lynch K. W., Maniatis T. Assembly of specific SR protein complexes on distinct regulatory elements of the Drosophila doublesex splicing enhancer. Genes Dev. 1996 Aug 15;10(16):2089–2101. doi: 10.1101/gad.10.16.2089. [DOI] [PubMed] [Google Scholar]
  27. Lynch K. W., Maniatis T. Synergistic interactions between two distinct elements of a regulated splicing enhancer. Genes Dev. 1995 Feb 1;9(3):284–293. doi: 10.1101/gad.9.3.284. [DOI] [PubMed] [Google Scholar]
  28. Madigan S. J., Edeen P., Esnayra J., McKeown M. att, a target for regulation by tra2 in the testes of Drosophila melanogaster, encodes alternative RNAs and alternative proteins. Mol Cell Biol. 1996 Aug;16(8):4222–4230. doi: 10.1128/mcb.16.8.4222. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Manley J. L., Tacke R. SR proteins and splicing control. Genes Dev. 1996 Jul 1;10(13):1569–1579. doi: 10.1101/gad.10.13.1569. [DOI] [PubMed] [Google Scholar]
  30. Matsuo N., Ogawa S., Imai Y., Takagi T., Tohyama M., Stern D., Wanaka A. Cloning of a novel RNA binding polypeptide (RA301) induced by hypoxia/reoxygenation. J Biol Chem. 1995 Nov 24;270(47):28216–28222. doi: 10.1074/jbc.270.47.28216. [DOI] [PubMed] [Google Scholar]
  31. Mattox W., Baker B. S. Autoregulation of the splicing of transcripts from the transformer-2 gene of Drosophila. Genes Dev. 1991 May;5(5):786–796. doi: 10.1101/gad.5.5.786. [DOI] [PubMed] [Google Scholar]
  32. Mattox W., McGuffin M. E., Baker B. S. A negative feedback mechanism revealed by functional analysis of the alternative isoforms of the Drosophila splicing regulator transformer-2. Genetics. 1996 May;143(1):303–314. doi: 10.1093/genetics/143.1.303. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Mattox W., Palmer M. J., Baker B. S. Alternative splicing of the sex determination gene transformer-2 is sex-specific in the germ line but not in the soma. Genes Dev. 1990 May;4(5):789–805. doi: 10.1101/gad.4.5.789. [DOI] [PubMed] [Google Scholar]
  34. McKeown M., Belote J. M., Baker B. S. A molecular analysis of transformer, a gene in Drosophila melanogaster that controls female sexual differentiation. Cell. 1987 Feb 13;48(3):489–499. doi: 10.1016/0092-8674(87)90199-1. [DOI] [PubMed] [Google Scholar]
  35. Mertz L. M., Rashtchian A. Nucleotide imbalance and polymerase chain reaction: effects on DNA amplification and synthesis of high specific activity radiolabeled DNA probes. Anal Biochem. 1994 Aug 15;221(1):160–165. doi: 10.1006/abio.1994.1392. [DOI] [PubMed] [Google Scholar]
  36. Pisano C., Bonaccorsi S., Gatti M. The kl-3 loop of the Y chromosome of Drosophila melanogaster binds a tektin-like protein. Genetics. 1993 Mar;133(3):569–579. doi: 10.1093/genetics/133.3.569. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. 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]
  38. Ryner L. C., Goodwin S. F., Castrillon D. H., Anand A., Villella A., Baker B. S., Hall J. C., Taylor B. J., Wasserman S. A. Control of male sexual behavior and sexual orientation in Drosophila by the fruitless gene. Cell. 1996 Dec 13;87(6):1079–1089. doi: 10.1016/s0092-8674(00)81802-4. [DOI] [PubMed] [Google Scholar]
  39. Staknis D., Reed R. SR proteins promote the first specific recognition of Pre-mRNA and are present together with the U1 small nuclear ribonucleoprotein particle in a general splicing enhancer complex. Mol Cell Biol. 1994 Nov;14(11):7670–7682. doi: 10.1128/mcb.14.11.7670. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Tian M., Maniatis T. A splicing enhancer complex controls alternative splicing of doublesex pre-mRNA. Cell. 1993 Jul 16;74(1):105–114. doi: 10.1016/0092-8674(93)90298-5. [DOI] [PubMed] [Google Scholar]
  41. 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]
  42. Vellard M., Sureau A., Soret J., Martinerie C., Perbal B. A potential splicing factor is encoded by the opposite strand of the trans-spliced c-myb exon. Proc Natl Acad Sci U S A. 1992 Apr 1;89(7):2511–2515. doi: 10.1073/pnas.89.7.2511. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Wu J. Y., Maniatis T. Specific interactions between proteins implicated in splice site selection and regulated alternative splicing. Cell. 1993 Dec 17;75(6):1061–1070. doi: 10.1016/0092-8674(93)90316-i. [DOI] [PubMed] [Google Scholar]
  44. Yannoni Y. M., White K. Association of the neuron-specific RNA binding domain-containing protein ELAV with the coiled body in Drosophila neurons. Chromosoma. 1997 Apr;105(6):332–341. doi: 10.1007/BF02529748. [DOI] [PubMed] [Google Scholar]
  45. Zahler A. M., Lane W. S., Stolk J. A., Roth M. B. SR proteins: a conserved family of pre-mRNA splicing factors. Genes Dev. 1992 May;6(5):837–847. doi: 10.1101/gad.6.5.837. [DOI] [PubMed] [Google Scholar]
  46. Zoller M. J., Smith M. Oligonucleotide-directed mutagenesis: a simple method using two oligonucleotide primers and a single-stranded DNA template. Methods Enzymol. 1987;154:329–350. doi: 10.1016/0076-6879(87)54083-6. [DOI] [PubMed] [Google Scholar]
  47. Zu K., Sikes M. L., Haynes S. R., Beyer A. L. Altered levels of the Drosophila HRB87F/hrp36 hnRNP protein have limited effects on alternative splicing in vivo. Mol Biol Cell. 1996 Jul;7(7):1059–1073. doi: 10.1091/mbc.7.7.1059. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Zuo P., Maniatis T. The splicing factor U2AF35 mediates critical protein-protein interactions in constitutive and enhancer-dependent splicing. Genes Dev. 1996 Jun 1;10(11):1356–1368. doi: 10.1101/gad.10.11.1356. [DOI] [PubMed] [Google Scholar]

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