RESUMEN
Congenital hydrocephalus (CH) is a common neurological disorder affecting many newborns. Imbalanced neurogenesis is a major cause of CH. Multiple CH-associated mutations are within the RNA-binding domain of Trim71, a conserved, stem cell-specific RNA-binding protein. How these mutations alter stem cell fate is unclear. Here, we show that the CH-associated mutations R595H and R783H in Trim71 accelerate differentiation and enhance neural lineage commitment in mouse embryonic stem cells (mESCs), and reduce binding to mRNAs targeted by wild-type Trim71, consistent with previous reports. Unexpectedly, however, each mutant binds an ectopic and distinct repertoire of target mRNAs. R595H-Trim71, but not R783H-Trim71 nor wild-type Trim71, binds the mRNA encoding ß-catenin and represses its translation. Increasing ß-catenin by overexpression or treatment with a Wnt agonist specifically restores differentiation of R595H-Trim71 mESCs. These results suggest that Trim71 mutations give rise to unique gain-of-function pathological mechanisms in CH. Further, our studies suggest that disruption of the Wnt/ß-catenin signaling pathway can be used to stratify disease etiology and develop precision medicine approaches for CH.
Asunto(s)
Hidrocefalia , beta Catenina , Animales , Ratones , beta Catenina/genética , Mutación con Ganancia de Función , Diferenciación Celular/genética , Mutación/genética , Hidrocefalia/genética , Vía de Señalización Wnt/genéticaRESUMEN
Congenital hydrocephalus (CH) is a major cause of childhood morbidity. Mono-allelic mutations in Trim71, a conserved stem-cell-specific RNA-binding protein, cause CH; however, the molecular basis for pathogenesis mediated by these mutations remains unknown. Here, using mouse embryonic stem cells as a model, we reveal that the mouse R783H mutation (R796H in human) alters Trim71's mRNA substrate specificity and leads to accelerated stem-cell differentiation and neural lineage commitment. Mutant Trim71, but not wild-type Trim71, binds Lsd1 (Kdm1a) mRNA and represses its translation. Specific inhibition of this repression or a slight increase of Lsd1 in the mutant cells alleviates the defects in stem cell differentiation and neural lineage commitment. These results determine a functionally relevant target of the CH-causing Trim71 mutant that can potentially be a therapeutic target and provide molecular mechanistic insights into the pathogenesis of this disease.
Asunto(s)
Hidrocefalia , Proteínas de Motivos Tripartitos , Animales , Humanos , Ratones , Histona Demetilasas/genética , Histona Demetilasas/metabolismo , Hidrocefalia/genética , Células Madre Embrionarias de Ratones/metabolismo , Mutación , Biosíntesis de Proteínas , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas de Motivos Tripartitos/genética , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
Argonaute (AGO) proteins are evolutionarily conserved RNA-binding proteins that control gene expression through the small RNAs they interact with. Whether AGOs have regulatory roles independent of RNAs, however, is unknown. Here, we show that AGO1 controls cell fate decisions through facilitating protein folding. We found that in mouse embryonic stem cells (mESCs), while AGO2 facilitates differentiation via the microRNA (miRNA) pathway, AGO1 controls stemness independently of its binding to small RNAs. We determined that AGO1 specifically interacts with HOP, a co-chaperone for the HSP70 and HSP90 chaperones, and enhances the folding of a set of HOP client proteins with intrinsically disordered regions. This AGO1-mediated facilitation of protein folding is important for maintaining stemness in mESCs. Our results demonstrate divergent functions between AGO1 and AGO2 in controlling cellular states and identify an RNA-independent function of AGO1 in controlling gene expression and cell fate decisions.
Asunto(s)
Proteínas Argonautas , Diferenciación Celular , Células Madre Embrionarias de Ratones , Pliegue de Proteína , Animales , Proteínas Argonautas/metabolismo , Proteínas Argonautas/genética , Ratones , Células Madre Embrionarias de Ratones/metabolismo , Células Madre Embrionarias de Ratones/citología , MicroARNs/genética , MicroARNs/metabolismo , Factores Eucarióticos de Iniciación/metabolismo , Factores Eucarióticos de Iniciación/genética , Proteínas HSP90 de Choque Térmico/metabolismo , Proteínas HSP90 de Choque Térmico/genética , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/genética , Linaje de la CélulaRESUMEN
microRNAs associate with Argonaute proteins, forming the microRNA-induced silencing complex (miRISC), to repress target gene expression post-transcriptionally. Although microRNAs are critical regulators in mammalian cell differentiation, our understanding of how microRNA machinery, such as the miRISC, are regulated during development is still limited. We previously showed that repressing the production of one Argonaute protein, Ago2, by Trim71 is important for mouse embryonic stem cells (mESCs) self-renewal (Liu et al., 2021). Here, we show that among the four Argonaute proteins in mammals, Ago2 is the major developmentally regulated Argonaute protein in mESCs. Moreover, in pluripotency, besides the Trim71-mediated regulation of Ago2 (Liu et al., 2021), Mir182/Mir183 also repress Ago2. Specific inhibition of this microRNA-mediated repression results in stemness defects and accelerated differentiation through the let-7 microRNA pathway. These results reveal a microRNA-mediated regulatory circuit on microRNA machinery that is critical to maintaining pluripotency.