RESUMEN
RNA-binding proteins recognizing unique sequences within large transcriptomes serve as a powerful tool to control RNA metabolism. Pumilio and fem-3 mRNA-binding factor (PUF) proteins are considered good candidates for such tools, because they are typically composed of eight highly homologous repeat segments and can be designed to recognize arbitrary 8â nt RNA sequences. However, a specific 8â nt RNA sequence is found at multiple sites in various RNAs in the transcriptome, making it difficult to specifically target a single RNA. Designer PUF proteins recognizing longer RNA sequences should achieve more selective binding. Here, we propose an approach for creating 16-repeat PUFs capable of targeting a single, unique mRNA in the transcriptome. Our design is simple and involves either the tandem alignment of two PUF segments or the nesting of one PUF segment within another. Designed 16-repeat PUFs bound to the target RNA sequence without partial recognition derived from the original 8-repeat PUF. Furthermore, based on our strategy, expression of an endogenous mRNA was selectively and effectively modulated, demonstrating the applicability of 16-repeat PUF proteins for regulating endogenous RNA metabolism.
Asunto(s)
Regulación de la Expresión Génica/genética , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/metabolismo , Animales , Ensayo de Cambio de Movilidad Electroforética , Células HEK293 , Humanos , Ratones , Células 3T3 NIH , ARN Mensajero/genética , Proteínas de Unión al ARN/genéticaRESUMEN
We established a method for converting TALE-DNA binding to luminescence, by combining a TALE and a split luciferase system. Furthermore, using a methylation-sensitive TALE, sequence-specific 5mC detection of genomic DNA was achieved in live cells. This study provides a new strategy for exploring the biological functions of 5mC.
Asunto(s)
Citosina/análisis , Metilación de ADN , Efectores Tipo Activadores de la Transcripción/química , ADN , Células HCT116 , Humanos , Elementos de Nucleótido Esparcido Largo , LuciferasasRESUMEN
New methods to control local RNA methylation are needed to elucidate the function of individual m6A. Here, fusion proteins between the programmable RNA binding protein PUF and the m6A demethylase FTO or METTL14 methyltransferase were designed. FTO-PUFs and METTL14-PUFs showed sequence-specific RNA demethylation and methylation activities, respectively.