RESUMEN
MicroRNA (miRNA)-mediated translational suppression of mRNAs is involved in the regulation of multiple cellular processes. A recent study showed that Nup358, a protein mutated in a neurological disorder called acute necrotizing encephalopathy 1 (ANE1), helps in the coupling of miRNA-induced silencing complex (miRISC) - consisting of miRNA, AGO and GW182/TNRC6 proteins - with the target mRNA. Here we provide a detailed characterization of the interaction between Nup358 and GW182. We identified that the N-terminal region of Nup358 directly interacts with the C-terminal silencing domain of GW182. Interestingly, ANE1-associated Nup358 mutants display reduced interaction with GW182. Consistent with this, one of the prevalent ANE1 mutations, 585th threonine (T) residue changed to methionine (M) [T585M] compromised Nup358's ability to function in the miRNA pathway. Collectively, these results suggest that the ANE1-associated mutations in Nup358 might affect the miRNA pathway and contribute to the development of ANE1.
Asunto(s)
Autoantígenos/metabolismo , Encefalopatías/genética , MicroARNs/metabolismo , Chaperonas Moleculares/metabolismo , Mutación/genética , Proteínas de Complejo Poro Nuclear/metabolismo , Proteínas de Unión al ARN/metabolismo , Animales , Autoantígenos/química , Línea Celular , Humanos , MicroARNs/genética , Unión Proteica , Dominios Proteicos , Proteínas de Unión al ARN/químicaRESUMEN
Micro-RNA mediated suppression of mRNA translation represents a major regulatory mode of post-transcriptional gene expression. Recently, the nucleoporin Nup358 was shown to interact with AGO protein, a key component of miRNA-induced silencing complex (miRISC), and facilitate the coupling of miRISC with target mRNA. Previous results suggested that SUMO-interacting motifs (SIMs) present on Nup358 mediate interaction with AGO protein. Here we show that Nup358-SIM has multiple interacting regions on AGO2, specifically within the N, PAZ and MID domains, with an affinity comparable to SIM-SUMO1 interaction. The study also unraveled specific residues involved in the interaction of AGO2 with miRNA-loading components such as Dicer and HSP90. Collectively, the results support the conclusion that multiple SIMs contribute to the association of Nup358 with AGO2.
Asunto(s)
Proteínas Argonautas/química , Proteínas Argonautas/metabolismo , Chaperonas Moleculares/química , Chaperonas Moleculares/metabolismo , Proteínas de Complejo Poro Nuclear/química , Proteínas de Complejo Poro Nuclear/metabolismo , Proteína SUMO-1/metabolismo , Secuencias de Aminoácidos , Proteínas Argonautas/genética , Sitios de Unión , ARN Helicasas DEAD-box/metabolismo , Células HEK293 , Proteínas HSP90 de Choque Térmico/metabolismo , Humanos , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Mutación Puntual , Unión Proteica , Dominios Proteicos , Ribonucleasa III/metabolismo , Eliminación de Secuencia , Resonancia por Plasmón de SuperficieRESUMEN
MicroRNA (miRNA)-guided mRNA repression, mediated by the miRNA-induced silencing complex (miRISC), is an important component of post-transcriptional gene silencing. However, how miRISC identifies the target mRNA in vivo is not well understood. Here, we show that the nucleoporin Nup358 plays an important role in this process. Nup358 localizes to the nuclear pore complex and to the cytoplasmic annulate lamellae (AL), and these structures dynamically associate with two mRNP granules: processing bodies (P bodies) and stress granules (SGs). Nup358 depletion disrupts P bodies and concomitantly impairs the miRNA pathway. Furthermore, Nup358 interacts with AGO and GW182 proteins and promotes the association of target mRNA with miRISC A well-characterized SUMO-interacting motif (SIM) in Nup358 is sufficient for Nup358 to directly bind to AGO proteins. Moreover, AGO and PIWI proteins interact with SIMs derived from other SUMO-binding proteins. Our study indicates that Nup358-AGO interaction is important for miRNA-mediated gene silencing and identifies SIM as a new interacting motif for the AGO family of proteins. The findings also support a model wherein the coupling of miRISC with the target mRNA could occur at AL, specialized domains within the ER, and at the nuclear envelope.
