RESUMO
The sequence-specific RNA-binding proteins SRp20 and 9G8 are the smallest members of the serine- and arginine-rich (SR) protein family, well known for their role in splicing. They also play a role in mRNA export, in particular of histone mRNAs. We present the solution structures of the free 9G8 and SRp20 RNA recognition motifs (RRMs) and of SRp20 RRM in complex with the RNA sequence 5'CAUC3'. The SRp20-RNA structure reveals that although all 4 nt are contacted by the RRM, only the 5' cytosine is primarily recognized in a specific way. This might explain the numerous consensus sequences found by SELEX (systematic evolution of ligands by exponential enrichment) for the RRM of 9G8 and SRp20. Furthermore, we identify a short arginine-rich peptide adjacent to the SRp20 and 9G8 RRMs, which does not contact RNA but is necessary and sufficient for interaction with the export factor Tip-associated protein (TAP). Together, these results provide a molecular description for mRNA and TAP recognition by SRp20 and 9G8.
Assuntos
Núcleo Celular/metabolismo , Modelos Moleculares , Proteínas de Transporte Nucleocitoplasmático/metabolismo , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Transporte Ativo do Núcleo Celular/fisiologia , Motivos de Aminoácidos , Humanos , Ressonância Magnética Nuclear Biomolecular , Proteínas Nucleares , Proteínas de Transporte Nucleocitoplasmático/química , Proteínas de Transporte Nucleocitoplasmático/genética , Peptídeos/química , Peptídeos/genética , Peptídeos/metabolismo , Ligação Proteica/genética , Estrutura Terciária de Proteína , RNA Mensageiro/química , RNA Mensageiro/genética , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Fatores de Processamento de Serina-ArgininaRESUMO
The polypyrimidine tract binding protein (PTB) is a 58 kDa protein involved in many aspects of RNA metabolism. In this study, we focused our attention on the structure of the two C-terminal RNA recognition motifs (RRM3 and RRM4) of PTB. In a previous study, it was found that the two RRMs are independent in the free state. We recently determined the structure of the same fragment in complex with RNA and found that the two RRMs interact extensively. This difference made us re-evaluate in detail the free protein structure and in particular the interdomain interface. We used a combination of NMR spectroscopy and segmental isotopic labeling to unambiguously study and characterize the interdomain interactions. An improved segmental isotopic labeling protocol was used, enabling us to unambiguously identify 130 interdomain NOEs between the two RRMs and to calculate a very precise structure. The structure reveals a large interdomain interface, resulting in a very unusual positioning of the two RRM domains relative to one another.
Assuntos
Ribonucleoproteínas Nucleares Heterogêneas/química , Proteína de Ligação a Regiões Ricas em Polipirimidinas/química , Motivos de Aminoácidos/genética , Isótopos de Carbono , Ribonucleoproteínas Nucleares Heterogêneas/genética , Ribonucleoproteínas Nucleares Heterogêneas/metabolismo , Humanos , Marcação por Isótopo , Espectroscopia de Ressonância Magnética , Mutagênese , Isótopos de Nitrogênio , Proteína de Ligação a Regiões Ricas em Polipirimidinas/genética , Proteína de Ligação a Regiões Ricas em Polipirimidinas/metabolismo , Estrutura Terciária de Proteína/genética , RNA Mensageiro/metabolismoRESUMO
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.