RESUMO
The polypyrimidine tract binding protein (PTB) is a 58-kilodalton RNA binding protein involved in multiple aspects of messenger RNA metabolism, including the repression of alternative exons. We have determined the solution structures of the four RNA binding domains (RBDs) of PTB, each bound to a CUCUCU oligonucleotide. Each RBD binds RNA with a different binding specificity. RBD3 and RBD4 interact, resulting in an antiparallel orientation of their bound RNAs. Thus, PTB will induce RNA looping when bound to two separated pyrimidine tracts within the same RNA. This leads to structural models for how PTB functions as an alternative-splicing repressor.
Assuntos
Processamento Alternativo , Ribonucleoproteínas Nucleares Heterogêneas/química , Ribonucleoproteínas Nucleares Heterogêneas/metabolismo , Proteína de Ligação a Regiões Ricas em Polipirimidinas/química , Proteína de Ligação a Regiões Ricas em Polipirimidinas/metabolismo , RNA/química , RNA/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Sítios de Ligação , Éxons , Ribonucleoproteínas Nucleares Heterogêneas/genética , Humanos , Ligação de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Ressonância Magnética Nuclear Biomolecular , Proteínas Nucleares/metabolismo , Oligorribonucleotídeos , Proteína de Ligação a Regiões Ricas em Polipirimidinas/genética , Ligação Proteica , Conformação Proteica , Estrutura Terciária de Proteína , Ribonucleoproteínas/metabolismo , Fator de Processamento U2AFRESUMO
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.