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Modulation of the microRNA-induced silencing complex loading through a specific interaction with a J-domain co-chaperone

ABSTRACT: MicroRNAs (miRNAs) are essential regulators of several biological processes. To achieve their repressive function, they are loaded onto Argonaute (AGO) proteins, forming the microRNA Induced Silencing Complex (miRISC). Thus, regulating this loading step is essential for miRNA-dependent gene regulation. In this study, we identified the co-chaperone DNJ-12 as a new interactor of ALG-1, one of the two major miRNA-specific AGOs in Caenorhabditis elegans (C. elegans). Importantly, DNJ-12 does not interact with other AGOs, making it a specific regulator of miRNA function. Furthermore, the loss of DNJ-12 leads to developmental phenotypes previously shown to be dependent on miRNA function. Using the Auxin Inducible Degron (AID) system, a powerful tool to study the spatiotemporal effects of DNJ-12 depletion on miRNA function, we show that DNJ-12 depletion hampers ALG-1 interaction with HSP70, a chaperone required for miRISC loading in vitro. Moreover, DNJ-12 depletion leads to a decrease in the levels of several miRNAs and prevents their loading onto ALG-1. This study highlights for the first time the requirement of a co-chaperone for the loading of miRNAs in vivo, provides a potential mechanism explaining AGO differential loading by small RNA, and therefore helps expand our understanding of the miRISC function in animals.

ORGANISM(S): Caenorhabditis elegans

PROVIDER: GSE241034 | GEO | 2024/04/02

REPOSITORIES: GEO

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Casein kinase 1 and 2 phosphorylate a conserved cluster of Argonaute to control microRNA functionCasein kinase 1 and 2 phosphorylate a conserved cluster of Argonaute to control microRNA function

Project description: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 C. 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

2023-09-13 | PXD044139 | Pride

Slicing-Independent RISC Activation Requires the Argonaute PAZ Domain

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2012-07-12 | E-GEOD-39146 | biostudies-arrayexpress

The Vasa homolog RDE-12 engages target mRNA and a secondary-Argonaute WAGO-1 to promote RNAi in C. elegans

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2014-06-24 | E-GEOD-54396 | biostudies-arrayexpress

High throughput sequencing of endogenous small RNAs from AGO pathway mutants

Project description:High-throughput pyrosequencing of endogenous small RNAs from >95% male enriched populations of alg-3(tm1155);alg-4(ok1041);fog-2(q71) and fog-2(q71) worms as well as purified spermatids from fem-3(q20) adult worms. Gametogenesis is thermosensitive in numerous metazoa ranging from worms to man. In C. elegans a variety of germ-line nuage- (P-granule) -associated RNA-binding proteins including the Piwi-clade Argonaute, PRG-1, have been implicated in temperature-dependent fertility. Here, we describe the role of two AGO-class paralogs, alg-3 (T22B3.2) and alg-4 (ZK757.3) in promoting male fertility at elevated temperatures. A rescuing GFP::alg-3 transgene is localized in P-granules beginning at the late pachytene stage of male gametogenesis. alg-3/4 double mutants lack a subgroup of small RNAs, named 26G-RNAs, which target and appear to down-regulate numerous spermatogenesis-expressed mRNAs. These findings add to a growing number of AGO pathways required for temperature-dependent fertility in C. elegans and support a model in which AGOs and their small RNA co-factors function to promote robustness in gene-expression networks. 3 samples examined. Small RNAs from alg-3(tm1155);alg-4(ok1041);fog-2(q71) males and fog-2(q71) males. Small RNAs from spermatids isolated from ferm-3(q20) worms.

2010-02-24 | E-GEOD-18729 | biostudies-arrayexpress

The Vasa homolog RDE-12 engages target mRNA and a secondary-Argonaute WAGO-1 to promote RNAi in C. elegans

2014-06-24 | GSE54396 | GEO

Gene expression profile of alg-3(tm1155);alg-4(ok1041) animals compared to wild type

Project description:Gametogenesis is thermosensitive in numerous metazoa ranging from worms to man. In C. elegans a variety of germ-line nuage- (P-granule) -associated RNA-binding proteins including the Piwi-clade Argonaute, PRG-1, have been implicated in temperature-dependent fertility. Here, we describe the role of two AGO-class paralogs, alg-3 (T22B3.2) and alg-4 (ZK757.3) in promoting male fertility at elevated temperatures. A rescuing GFP::alg-3 transgene is localized in P-granules beginning at the late pachytene stage of male gametogenesis. alg-3/4 double mutants lack a subgroup of small RNAs, named 26G-RNAs, which target and appear to down-regulate numerous spermatogenesis-expressed mRNAs. These findings add to a growing number of AGO pathways required for temperature-dependent fertility in C. elegans and support a model in which AGOs and their small RNA co-factors function to promote robustness in gene-expression networks.

2010-02-24 | GSE18719 | GEO

Slicing-Independent RISC Activation Requires the Argonaute PAZ Domain

2012-07-12 | GSE39146 | GEO

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

Project description: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.

2023-09-12 | GSE239453 | GEO

High throughput sequencing of endogenous small RNAs from AGO pathway mutants

2010-02-24 | GSE18729 | GEO

Cell-type-specific profiling of loaded miRNAs from Caenorhabditis elegans reveals spatial and temporal flexibility in Argonaute loading [miRNA-seq]

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2020-08-25 | GSE156716 | GEO

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