Exon repression by polypyrimidine tract binding protein

RNA - Tập 11 Số 5 - Trang 699-716 - 2005
Batoul Amir-Ahmady1, Paul L. Boutz1, Vadim Markovtsov1, Martin Phillips1, Douglas L. Black1
1Department of Microbiology, Immunology & Molecular Genetics, 2Department of Chemistry and Biochemistry, and 3Howard Hughes Medical Institute, University of California, Los Angeles, California 90095-1662, USA

Tóm tắt

Polypyrimidine tract binding protein (PTB) is known to silence the splicing of many alternative exons. However, exons repressed by PTB are affected by other RNA regulatory elements and proteins. This makes it difficult to dissect the structure of the pre-mRNP complexes that silence splicing, and to understand the role of PTB in this process. We determined the minimal requirements for PTB-mediated splicing repression. We find that the minimal sequence for high affinity binding by PTB is relatively large, containing multiple polypyrimidine elements. Analytical ultracentrifugation and proteolysis mapping of RNA cross-links on the PTB protein indicate that most PTB exists as a monomer, and that a polypyrimidine element extends across multiple PTB domains. The high affinity site is bound initially by a PTB monomer and at higher concentrations by additional PTB molecules. Significantly, this site is not sufficient for splicing repression when placed in the 3′ splice site of a strong test exon. Efficient repression requires a second binding site within the exon itself or downstream from it. This second site enhances formation of a multimeric PTB complex, even if it does not bind well to PTB on its own. These experiments show that PTB can be sufficient to repress splicing of an otherwise constitutive exon, without binding sites for additional regulatory proteins and without competing with U2AF binding. The minimal complex mediating splicing repression by PTB requires two binding sites bound by an oligomeric PTB complex.

Từ khóa


Tài liệu tham khảo

10.1261/rna.2131603

10.1021/bi980113j

10.1146/annurev.biochem.72.121801.161720

10.1007/978-3-662-09728-1_7

1997, RNA, 3, 405

10.1093/emboj/18.7.1939

10.1128/MCB.20.19.7388-7400.2000

1995, Mol. Cell. Biol., 15, 6377, 10.1128/MCB.15.11.6377

1997, Mol. Cell. Biol., 17, 4667, 10.1128/MCB.17.8.4667

10.1016/S1097-2765(02)00479-3

1999, Mol. Cell. Biol., 19, 69, 10.1128/MCB.19.1.69

10.1016/S1097-2765(00)80260-9

Cohn, E.J. and Edsall, J.T. 1943. Density and apparent specific volume of proteins. In Proteins, amino acids and peptides as ions and dipolar ions, pp. 370–381. Reinold, New York.

10.1093/emboj/19.12.3132

10.1016/S0092-8674(01)00566-9

1999, Mol. Cell. Biol., 19, 251, 10.1128/MCB.19.1.251

1991, Mol. Cell. Biol., 11, 6075

10.1016/S0076-6879(80)65011-3

10.1002/jcb.10687

1994, EMBO J., 13, 3861, 10.1002/j.1460-2075.1994.tb06697.x

1998, RNA, 4, 85

1987, Eur. J. Biochem., 163, 113, 10.1111/j.1432-1033.1987.tb10743.x

1989, Eur. J. Biochem., 180, 577, 10.1111/j.1432-1033.1989.tb14684.x

10.1261/rna.2191903

10.1091/mbc.E03-09-0692

10.1074/jbc.M203633200

10.1021/bi00200a028

2003, Cancer Res., 63, 6154

10.1038/ng1207

10.1016/S0959-440X(00)00250-5

Laue, T.M., Shah, B.D., Ridgeway, T.M., and Pelletier, S.L. 1992. Computer-aided interpretation of analytical ultracentrifugation of analytical sedimentation data for proteins. In Analytical ultracentrifugation in biochemistry and polymer science, pp. 90–125. The Royal Society of Chemistry, Redwood Press limited, Melksham, Wilt-shire, England.

1995, RNA, 1, 234

10.1017/S1355838202015029

10.1128/MCB.20.20.7463-7479.2000

10.3109/10409239709108550

10.1093/nar/15.21.8783

1997, Mol. Cell. Biol., 17, 6537, 10.1128/MCB.17.11.6537

1992, J. Biol. Chem., 267, 25480, 10.1016/S0021-9258(19)74066-6

10.1093/nar/22.19.3854

1998, Biochem. J., 331, 169, 10.1042/bj3310169

1997, RNA, 3, 764

10.1021/bi9711745

10.1073/pnas.110128397

10.1261/rna.5229704

2004, Structure (Camb.), 12, 1631, 10.1016/j.str.2004.07.008

10.1126/science.7761834

10.1016/S0968-0004(00)01604-2

1999, Mol. Cell. Biol., 19, 2699, 10.1128/MCB.19.4.2699

10.1093/emboj/20.20.5748

10.1016/S0960-9822(06)00361-7

10.1146/annurev.biophys.27.1.407

10.1128/MCB.21.10.3281-3288.2001

10.1016/S1097-2765(02)00645-7

10.1017/S1355838201010214

10.1093/nar/30.2.456

10.1074/jbc.M312743200

10.1016/S1097-2765(01)00409-9

Thông tin
Thông tin xuất bản

RNA

Tập 11 Số 5

699-716

Thông tin tác giả