title: RGG-boxes of the EWS oncoprotein repress a range of transcriptional activation domains.
abstract: The Ewings Sarcoma Oncoprotein (EWS) interacts with several components of the mammalian transcriptional and pre-mRNA splicing machinery and is also found in the cytoplasm and even on the cell surface. The apparently diverse cellular functions of EWS are, however, not well characterized. EWS harbours a potent N-terminal transcriptional activation domain (the EAD) that is revealed in the context of oncogenic EWS-fusion proteins (EFPs) and a C-terminal RNA-binding domain (RBD) that recruits pre-mRNA splicing factors and may couple transcription and splicing. In contrast to EFPs, the presumed transcriptional role of normal EWS remains enigmatic. Here, we report that multiple RGG-boxes within the RBD are necessary and sufficient for cis-repression of the EAD and that RGG-boxes can also repress in-trans, within dimeric partners. Lys can functionally substitute for Arg, indicating that the basic nature of the Arg side chain is the critical determinant of RGG-box-mediated repression. In addition to the EAD, RGG-boxes can repress a broad range of activation domains (including those of VP16, E1a and CREB), but repression can be alleviated by the simultaneous presence of more than one activation domain. We therefore propose that a key function of RGG boxes within native EWS is to restrict promiscuous activation by the EAD while still allowing EWS to enter functional transcription complexes and participate in other transactions involving pre-mRNAs.
authors.affiliation: Department of Biology, Hong Kong University of Science & Technology Clear Water Bay, Kowloon Hong Kong, SAR, China.
authors.forename: Deepa
authors.initials: D
authors.lastname: Alex
authors.forename: Kevin A W
authors.initials: KA
authors.lastname: Lee
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D002460:Cell Line; D004252:DNA Mutational Analysis; D019281:Dimerization; D006801:Humans; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D034802:RNA-Binding Protein EWS / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012097:Repressor Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015534:Trans-Activators / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015533:Transcriptional Activation
chemical_list: D034802:RNA-Binding Protein EWS; D012097:Repressor Proteins; D015534:Trans-Activators
references.citation: Nucleic Acids Res. 1999 Feb 1;27(3):817-21
references.pmid: 9889278
references.citation: Cell. 1999 Apr 2;97(1):5-8
references.pmid: 10199396
references.citation: Cell. 1999 Apr 2;97(1):17-27
references.pmid: 10199399
references.citation: Mol Cancer Res. 2002 Nov;1(1):48-55
references.pmid: 12496368
references.citation: J Biol Chem. 2003 Feb 14;278(7):5427-32
references.pmid: 12459554
references.citation: Chromosoma. 2003 Apr;111(7):461-9
references.pmid: 12707784
references.citation: Curr Biol. 1999 May 6;9(9):485-8
references.pmid: 10322114
references.citation: Curr Opin Oncol. 1999 Jul;11(4):275-84
references.pmid: 10416880
references.citation: Mol Cell Biol. 1999 Oct;19(10):6833-44
references.pmid: 10490622
references.citation: Genes Dev. 2003 Aug 1;17(15):1909-20
references.pmid: 12897056
references.citation: Brain Res Mol Brain Res. 2003 Nov 6;119(1):37-49
references.pmid: 14597228
references.citation: Genomics. 2004 Jan;83(1):130-9
references.pmid: 14667816
references.citation: FEBS Lett. 2004 Apr 23;564(1-2):188-98
references.pmid: 15094065
references.citation: J Virol. 1984 Nov;52(2):483-91
references.pmid: 6208378
references.citation: Proc Natl Acad Sci U S A. 1986 Sep;83(18):6682-6
references.pmid: 2875459
references.citation: Oncogene. 1999 Dec 23;18(56):8000-10
references.pmid: 10637511
references.citation: J Biol Chem. 2000 Aug 11;275(32):24865-71
references.pmid: 10827180
references.citation: J Biol Chem. 2000 Jul 28;275(30):23053-8
references.pmid: 10767297
references.citation: Physiol Genomics. 1999 Nov 11;1(3):127-38
references.pmid: 11015571
references.citation: J Biol Chem. 2000 Dec 1;275(48):37612-8
references.pmid: 10982800
references.citation: J Biol Chem. 2001 Mar 2;276(9):6807-16
references.pmid: 11098054
references.citation: Cancer Res. 2001 Mar 15;61(6):2690-5
references.pmid: 11289149
references.citation: J Biol Chem. 2001 Apr 20;276(16):13395-401
references.pmid: 11278855
references.citation: Oncogene. 2001 Mar 22;20(12):1519-24
references.pmid: 11313895
references.citation: Oncogene. 2001 Mar 29;20(14):1756-64
references.pmid: 11313922
references.citation: Cancer Res. 2001 May 1;61(9):3586-90
references.pmid: 11325824
references.citation: J Biol Chem. 2001 Jun 1;276(22):18681-7
references.pmid: 11278906
references.citation: J Biol Chem. 2001 Jun 22;276(25):22317-22
references.pmid: 11301318
references.citation: Oncogene. 2001 Jul 12;20(31):4161-8
references.pmid: 11464282
references.citation: Oncogene. 2001 Sep 10;20(40):5747-54
references.pmid: 11607824
references.citation: Oncogene. 2001 Oct 11;20(46):6653-9
references.pmid: 11709699
references.citation: J Biol Chem. 2002 Jan 4;277(1):535-43
references.pmid: 11673470
references.citation: Mol Cell Biol. 2002 May;22(9):2918-27
references.pmid: 11940650
references.citation: Proc Natl Acad Sci U S A. 1991 Jan 1;88(1):224-8
references.pmid: 1898773
references.citation: Proc Natl Acad Sci U S A. 1992 Apr 1;89(7):2723-6
references.pmid: 1557378
references.citation: EMBO J. 1992 Jul;11(7):2655-64
references.pmid: 1628625
references.citation: J Virol. 1992 Aug;66(8):4803-13
references.pmid: 1321270
references.citation: New Biol. 1992 May;4(5):421-9
references.pmid: 1381215
references.citation: EMBO J. 1992 Dec;11(13):5005-12
references.pmid: 1464322
references.citation: Genes Dev. 1993 Feb;7(2):173-9
references.pmid: 8436289
references.citation: Annu Rev Biochem. 1993;62:289-321
references.pmid: 8352591
references.citation: Biochim Biophys Acta. 1993 Sep 23;1174(3):221-33
references.pmid: 8373801
references.citation: Nat Genet. 1993 Aug;4(4):341-5
references.pmid: 8401579
references.citation: Proc Natl Acad Sci U S A. 1993 Oct 1;90(19):9046-50
references.pmid: 8105470
references.citation: Science. 1994 Jul 29;265(5172):615-21
references.pmid: 8036511
references.citation: Oncogene. 1994 Oct;9(10):3087-97
references.pmid: 8084618
references.citation: Mol Cell Biol. 1994 Nov;14(11):7204-10
references.pmid: 7935435
references.citation: Oncogene. 1994 Dec;9(12):3717-29
references.pmid: 7970732
references.citation: J Biol Chem. 1994 Dec 9;269(49):31124-8
references.pmid: 7983053
references.citation: Oncogene. 1995 May 4;10(9):1749-56
references.pmid: 7753552
references.citation: Mol Cell Biol. 1995 Aug;15(8):4562-71
references.pmid: 7623847
references.citation: Nucleic Acids Res. 1995 Jul 11;23(13):2389-95
references.pmid: 7630716
references.citation: Oncogene. 1996 Jan 4;12(1):159-67
references.pmid: 8552387
references.citation: J Biol Chem. 1996 Feb 16;271(7):3420-7
references.pmid: 8631943
references.citation: EMBO J. 1996 Sep 16;15(18):5022-31
references.pmid: 8890175
references.citation: Biochemistry. 1997 Apr 29;36(17):5185-92
references.pmid: 9136880
references.citation: J Cell Sci. 1997 Aug;110 ( Pt 15):1741-50
references.pmid: 9264461
references.citation: J Biol Chem. 1997 Oct 24;272(43):27369-77
references.pmid: 9341188
references.citation: Mol Endocrinol. 1998 Jan;12(1):4-18
references.pmid: 9440806
references.citation: Mol Cell Biol. 1998 Mar;18(3):1489-97
references.pmid: 9488465
references.citation: J Biol Chem. 1998 Feb 27;273(9):4838-42
references.pmid: 9478924
references.citation: Oncogene. 1998 Mar 26;16(12):1625-31
references.pmid: 9569031
references.citation: Proc Natl Acad Sci U S A. 1998 Jun 9;95(12):6699-704
references.pmid: 9618475
references.citation: J Biol Chem. 1998 Jun 19;273(25):15521-7
references.pmid: 9624140
references.citation: Oncogene. 1998 Aug 6;17(5):603-10
references.pmid: 9704926
references.citation: J Biol Chem. 1998 Oct 23;273(43):27761-4
references.pmid: 9774382
references.citation: J Biol Chem. 1998 Oct 30;273(44):29224-9
references.pmid: 9786934