RESUMEN
Dicer is a member of the ribonuclease III enzyme family and processes double-stranded RNA into small functional RNAs. The variation in the domain architecture of Dicer among different species whilst preserving its biological dicing function is intriguing. Here, we describe the structure and function of a novel catalytically active RNase III protein, a non-canonical Dicer (PsDCR1), found in budding yeast Pichia stipitis. The structure of the catalytically active region (the catalytic RNase III domain and double-stranded RNA-binding domain 1 [dsRBD1]) of DCR1 showed that RNaseIII domain is structurally similar to yeast RNase III (Rnt1p) but uniquely presents dsRBD1 in a diagonal orientation, forming a catalytic core made of homodimer and large RNA-binding surface. The second dsRNA binding domain at C-terminus, which is absent in Rnt1, enhances the RNA cleavage activity. Although the cleavage pattern of PsDCR1 anchors an apical loop similar to Rnt1, the cleavage activity depended on the sequence motif at the lower stem, not the apical loop, of hairpin RNA. Through RNA sequencing and RNA mutations, we showed that RNA cleavage by PsDCR1 is determined by the stem-loop structure of the RNA substrate, suggesting the possibility that stem-loop RNA-guided gene silencing pathway exists in budding yeast.
Asunto(s)
Proteínas Fúngicas/química , Conformación de Ácido Nucleico , Multimerización de Proteína , ARN de Hongos/química , Ribonucleasa III/química , Saccharomycetales/enzimología , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Dominios Proteicos , Estructura Secundaria de Proteína , ARN de Hongos/genética , ARN de Hongos/metabolismo , Ribonucleasa III/genética , Ribonucleasa III/metabolismo , Saccharomycetales/genética , Relación Estructura-ActividadRESUMEN
microRNAs (miRNAs), a class of small and endogenous molecules that control gene expression, are broadly involved in biological processes. Although a number of cofactors that assist or antagonize let-7 miRNA biogenesis are well-established, more auxiliary factors remain to be investigated. Here, we identified SYNCRIP (Synaptotagmin Binding Cytoplasmic RNA Interacting Protein) as a new player for let-7a miRNA. SYNCRIP interacts with pri-let-7a both in vivo and in vitro. Knockdown of SYNCRIP impairs, while overexpression of SYNCRIP promotes, the expression of let-7a miRNA. A broad miRNA profiling analysis revealed that silencing of SYNCRIP regulates the expression of a set of mature miRNAs positively or negatively. In addition, SYNCRIP is associated with microprocessor complex and promotes the processing of pri-let-7a. Strikingly, the terminal loop of pri-let-7a was shown to be the main contributor for its interaction with SYNCRIP. Functional studies demonstrated that the SYNCRIP RRM2-3 domain can promote the processing of pri-let-7a. Structure-based alignment of RRM2-3 with other RNA binding proteins identified the residues likely to participate in protein-RNA interactions. Taken together, these findings suggest the promising role that SYNCRIP plays in miRNA regulation, thus providing insights into the function of SYNCRIP in eukaryotic development.