ABSTRACT
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.
Subject(s)
Caenorhabditis elegans Proteins , MicroRNAs , Animals , Humans , Argonaute Proteins/genetics , Argonaute Proteins/metabolism , Caenorhabditis elegans Proteins/genetics , Caenorhabditis elegans Proteins/metabolism , Casein Kinase I/genetics , Casein Kinase I/metabolism , MicroRNAs/genetics , MicroRNAs/metabolism , RNA-Induced Silencing Complex/genetics , RNA-Induced Silencing Complex/metabolism , Caenorhabditis elegans/genetics , Caenorhabditis elegans/metabolism , Gene Silencing , Serine/metabolism , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolismABSTRACT
Argonaute proteins are at the core of the microRNA-mediated gene silencing pathway essential for animals. In C. elegans, the microRNA-specific Argonautes ALG-1 and ALG-2 regulate multiple processes required for proper animal developmental timing and viability. Here we identified a phosphorylation site on ALG-1 that modulates microRNA association. Mutating ALG-1 serine 642 into a phospho-mimicking residue impairs microRNA binding and causes embryonic lethality and post-embryonic phenotypes that are consistent with alteration of microRNA functions. Monitoring microRNA levels in alg-1 phosphorylation mutant animals shows that microRNA passenger strands increase in abundance but are not preferentially loaded into ALG-1, indicating that the miRNA binding defects could lead to microRNA duplex accumulation. Our genetic and biochemical experiments support protein kinase A (PKA) KIN-1 as the putative kinase that phosphorylates ALG-1 serine 642. Our data indicate that PKA triggers ALG-1 phosphorylation to regulate its microRNA association during C. elegans development.
Subject(s)
Caenorhabditis elegans Proteins , MicroRNAs , Animals , Caenorhabditis elegans/metabolism , Argonaute Proteins/genetics , Argonaute Proteins/metabolism , Caenorhabditis elegans Proteins/metabolism , MicroRNAs/genetics , MicroRNAs/metabolism , Phosphorylation , RNA-Binding Proteins/metabolism , Serine/metabolismABSTRACT
Once loaded onto Argonaute proteins, microRNAs form a silencing complex called miRISC that targets mostly the 3'UTR of mRNAs to silence their translation. How microRNAs are transported to and from their target mRNA remains poorly characterized. While some reports linked intracellular trafficking to microRNA activity, it is still unclear how these pathways coordinate for proper microRNA-mediated gene silencing and turnover. Through a forward genetic screen using Caenorhabditis elegans, we identified the RabGAP tbc-11 as an important factor for the microRNA pathway. We show that TBC-11 acts mainly through the small GTPase RAB-6 and that its regulation is required for microRNA function. The absence of functional TBC-11 increases the pool of microRNA-unloaded Argonaute ALG-1 that is likely associated to endomembranes. Furthermore, in this condition, this pool of Argonaute accumulates in a perinuclear region and forms a high molecular weight complex. Altogether, our data suggest that the alteration of TBC-11 generates a fraction of ALG-1 that cannot bind to target mRNAs, leading to defective gene repression. Our results establish the importance of intracellular trafficking for microRNA function and demonstrate the involvement of a small GTPase and its GAP in proper Argonaute localization in vivo.
Subject(s)
Caenorhabditis elegans Proteins/genetics , Protein Biosynthesis , RNA-Binding Proteins/genetics , rab GTP-Binding Proteins/genetics , 3' Untranslated Regions/genetics , Animals , Caenorhabditis elegans/genetics , Gene Expression Regulation, Developmental/genetics , Gene Silencing , MicroRNAs/genetics , RNA, Messenger/geneticsABSTRACT
Three recent studies (Dodson and Kennedy, 2019; Lev et al., 2019; Ouyang et al., 2019) reveal that germ granule formation is necessary to protect germline-expressed genes from improper small RNA-mediated silencing. Loss of this protection leads to accumulation of small RNAs, impacting gene expression in multiple subsequent generations.
