Casein kinase 1 and 2 phosphorylate Argonaute proteins to regulate miRNA-mediated gene silencing

MicroRNAs (miRNAs) together with Argonaute (AGO) proteins form the core of the RNA-induced silencing complex (RISC) to regulate gene expression of their target RNAs post-transcriptionally. Argonaute proteins are subjected to intensive regulation via various post-translational modifications that can affect their stability, silencing efficacy and specificity for targeted gene regulation. We report here that in Caenorhabditis elegans, two conserved serine/threonine kinases - casein kinase 1 alpha 1 (CK1A1) and casein kinase 2 (CK2) - regulate a highly conserved phosphorylation cluster of 4 Serine residues (S988:S998) on the miRNA-specific AGO protein ALG-1. We show that CK1A1 phosphorylates ALG-1 at sites S992 and S995, while CK2 phosphorylates ALG-1 at sites S988 and S998. Furthermore, we demonstrate that phospho-mimicking mutants of the entire S988:S998 cluster rescue the various developmental defects observed upon depleting CK1A1 and CK2. In humans, we show that CK1A1 also acts as a priming kinase of this cluster on AGO2. Altogether, our data suggest that phosphorylation of AGO within the cluster by CK1A1 and CK2 is required for efficient miRISC-target RNA binding and silencing. Overall design: To evaluate the effects on global miRNA levels after CK1A1 and CK2 depletion in wild-type animals by HT-sequencing The control RNAi is control treatment. And the kin-19 RNAi corresponds to CK1A1 depletion. CK1A1 is the ortholog of kin-19 in C elegans. And, kin-3 RNAi and kin-10 RNAi correspond to CK2 depletion. As CK2 is made of two subunits, kin-3 (catalytic) and kin-10 (regulatory) in C. elegans.