RESUMEN
The LSM domain-containing protein LSM14/Rap55 plays a role in mRNA decapping, translational repression, and RNA granule (P-body) assembly. How LSM14 interacts with the mRNA silencing machinery, including the eIF4E-binding protein 4E-T and the DEAD-box helicase DDX6, is poorly understood. Here we report the crystal structure of the LSM domain of LSM14 bound to a highly conserved C-terminal fragment of 4E-T. The 4E-T C-terminus forms a bi-partite motif that wraps around the N-terminal LSM domain of LSM14. We also determined the crystal structure of LSM14 bound to the C-terminal RecA-like domain of DDX6. LSM14 binds DDX6 via a unique non-contiguous motif with distinct directionality as compared to other DDX6-interacting proteins. Together with mutational and proteomic studies, the LSM14-DDX6 structure reveals that LSM14 has adopted a divergent mode of binding DDX6 in order to support the formation of mRNA silencing complexes and P-body assembly.
Asunto(s)
ARN Helicasas DEAD-box/química , ARN Helicasas DEAD-box/metabolismo , Dominios y Motivos de Interacción de Proteínas , Proteínas Proto-Oncogénicas/química , Proteínas Proto-Oncogénicas/metabolismo , Interferencia de ARN/fisiología , ARN Mensajero/metabolismo , Ribonucleoproteínas/química , Ribonucleoproteínas/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Caenorhabditis elegans , Cristalografía por Rayos X , ARN Helicasas DEAD-box/genética , Drosophila melanogaster , Factor 4E Eucariótico de Iniciación/metabolismo , Células HeLa , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Mutación , Unión Proteica , Conformación Proteica , Estructura Secundaria de Proteína , Proteínas/química , Proteínas/metabolismo , Proteómica , Proteínas Proto-Oncogénicas/genética , Rec A Recombinasas/química , Proteínas Recombinantes/química , Ribonucleoproteínas/genética , Alineación de SecuenciaRESUMEN
Eukaryotic mRNA degradation often initiates with the recruitment of the CCR4-NOT deadenylase complex and decay factors to the mRNA 3' terminus. How the 3'-proximal decay machinery interacts with the 5'-terminal cap structure in order to engender mRNA decapping and 5'-3' degradation is unclear. Human 4E-T is an eIF4E-binding protein that has been reported to promote mRNA decay, albeit via an unknown mechanism. Here, we show that 4E-T is a component of the mRNA decay machinery and interacts with factors including DDX6, LSM14, and the LSM1-7-PAT1 complex. We also provide evidence that 4E-T associates with, and enhances the decay of, mRNAs targeted by the CCR4-NOT deadenylase complex, including microRNA targets. Importantly, we demonstrate that 4E-T must interact with eIF4E to engender mRNA decay. Taken together, our data support a model where 4E-T promotes mRNA turnover by physically linking the 3'-terminal mRNA decay machinery to the 5' cap via its interaction with eIF4E.