An Argonaute phosphorylation cycle promotes microRNA-mediated silencing
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA354286 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR5027810.fastq.gz | Fastqsanger.gz | |||
| SRR5027820.fastq.gz | Fastqsanger.gz | |||
| SRR5027830.fastq.gz | Fastqsanger.gz | |||
| SRR5027840.fastq.gz | Fastqsanger.gz | |||
| SRR5027850.fastq.gz | Fastqsanger.gz |
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Project description:MicroRNAs (miRNAs) perform critical functions in normal physiology and disease by associating with Argonaute proteins and downregulating partially complementary messenger RNAs (mRNAs). To identify new regulators of the miRNA pathway, we employed CRISPR-Cas9 genome-wide loss-of-function screening coupled with a fluorescent reporter of miRNA activity. Iterative rounds of screening revealed a novel mechanism whereby target engagement by Argonaute 2 (AGO2) triggers its hierarchical, multi-site phosphorylation by CSNK1A1 on a set of highly conserved residues (S824-S834), followed by rapid dephosphorylation by the ANKRD52-PPP6C phosphatase complex. Although genetic and biochemical studies demonstrated that AGO2 phosphorylation on these residues inhibits target mRNA binding, inactivation of this phosphorylation cycle globally impairs miRNA-mediated silencing. Analysis of the transcriptome-wide binding profile of non-phosphorylatable AGO2 revealed a dramatic expansion of the target repertoire bound at steady-state, effectively reducing the active pool of AGO2 on a per target basis. These findings support a model in which an AGO2 phosphorylation cycle stimulated by target engagement regulates miRNA:target interactions to maintain the global efficiency of miRNA-mediated silencing.
2017-01-17 | GSE89943 | GEO
Project description:MicroRNAs (miRNAs) perform critical functions in normal physiology and disease by associating with Argonaute proteins and downregulating partially complementary messenger RNAs (mRNAs). To identify new regulators of the miRNA pathway, we employed CRISPR-Cas9 genome-wide loss-of-function screening coupled with a fluorescent reporter of miRNA activity. Iterative rounds of screening revealed a novel mechanism whereby target engagement by Argonaute 2 (AGO2) triggers its hierarchical, multi-site phosphorylation by CSNK1A1 on a set of highly conserved residues (S824-S834), followed by rapid dephosphorylation by the ANKRD52-PPP6C phosphatase complex. Although genetic and biochemical studies demonstrated that AGO2 phosphorylation on these residues inhibits target mRNA binding, inactivation of this phosphorylation cycle globally impairs miRNA-mediated silencing. Analysis of the transcriptome-wide binding profile of non-phosphorylatable AGO2 revealed a dramatic expansion of the target repertoire bound at steady-state, effectively reducing the active pool of AGO2 on a per target basis. These findings support a model in which an AGO2 phosphorylation cycle stimulated by target engagement regulates miRNA:target interactions to maintain the global efficiency of miRNA-mediated silencing.
2017-01-17 | GSE89944 | GEO
Project description:MicroRNAs (miRNAs) perform critical functions in normal physiology and disease by associating with Argonaute proteins and downregulating partially complementary messenger RNAs (mRNAs). To identify new regulators of the miRNA pathway, we employed CRISPR-Cas9 genome-wide loss-of-function screening coupled with a fluorescent reporter of miRNA activity. Iterative rounds of screening revealed a novel mechanism whereby target engagement by Argonaute 2 (AGO2) triggers its hierarchical, multi-site phosphorylation by CSNK1A1 on a set of highly conserved residues (S824-S834), followed by rapid dephosphorylation by the ANKRD52-PPP6C phosphatase complex. Although genetic and biochemical studies demonstrated that AGO2 phosphorylation on these residues inhibits target mRNA binding, inactivation of this phosphorylation cycle globally impairs miRNA-mediated silencing. Analysis of the transcriptome-wide binding profile of non-phosphorylatable AGO2 revealed a dramatic expansion of the target repertoire bound at steady-state, effectively reducing the active pool of AGO2 on a per target basis. These findings support a model in which an AGO2 phosphorylation cycle stimulated by target engagement regulates miRNA:target interactions to maintain the global efficiency of miRNA-mediated silencing.
2017-01-17 | GSE89940 | GEO
Project description:MicroRNAs (miRNAs) perform critical functions in normal physiology and disease by associating with Argonaute proteins and downregulating partially complementary messenger RNAs (mRNAs). To identify new regulators of the miRNA pathway, we employed CRISPR-Cas9 genome-wide loss-of-function screening coupled with a fluorescent reporter of miRNA activity. Iterative rounds of screening revealed a novel mechanism whereby target engagement by Argonaute 2 (AGO2) triggers its hierarchical, multi-site phosphorylation by CSNK1A1 on a set of highly conserved residues (S824-S834), followed by rapid dephosphorylation by the ANKRD52-PPP6C phosphatase complex. Although genetic and biochemical studies demonstrated that AGO2 phosphorylation on these residues inhibits target mRNA binding, inactivation of this phosphorylation cycle globally impairs miRNA-mediated silencing. Analysis of the transcriptome-wide binding profile of non-phosphorylatable AGO2 revealed a dramatic expansion of the target repertoire bound at steady-state, effectively reducing the active pool of AGO2 on a per target basis. These findings support a model in which an AGO2 phosphorylation cycle stimulated by target engagement regulates miRNA:target interactions to maintain the global efficiency of miRNA-mediated silencing.
2017-01-17 | GSE89942 | GEO
Project description:MicroRNAs (miRNAs) perform critical functions in normal physiology and disease by associating with Argonaute proteins and downregulating partially complementary messenger RNAs (mRNAs). Here we use clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9) genome-wide loss-of-function screening coupled with a fluorescent reporter of miRNA activity in human cells to identify new regulators of the miRNA pathway. By using iterative rounds of screening, we reveal a novel mechanism whereby target engagement by Argonaute 2 (AGO2) triggers its hierarchical, multi-site phosphorylation by CSNK1A1 on a set of highly conserved residues (S824-S834), followed by rapid dephosphorylation by the ANKRD52-PPP6C phosphatase complex. Although genetic and biochemical studies demonstrate that AGO2 phosphorylation on these residues inhibits target mRNA binding, inactivation of this phosphorylation cycle globally impairs miRNA-mediated silencing. Analysis of the transcriptome-wide binding profile of non-phosphorylatable AGO2 reveals a pronounced expansion of the target repertoire bound at steady-state, effectively reducing the active pool of AGO2 on a per-target basis. These findings support a model in which an AGO2 phosphorylation cycle stimulated by target engagement regulates miRNA:target interactions to maintain the global efficiency of miRNA-mediated silencing.
| S-EPMC5302127 | biostudies-literature
Project description: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 new phosphorylation site, serine 642, 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 common with alteration of microRNA functions. Monitoring microRNA levels in alg-1 phosphorylation mutant animals reveal that miRNA passenger strands strongly increase but are not preferentially loaded into ALG-1, indicating that the miRNA binding defects could also lead to miRNA duplexes accumulation. Our genetic and biochemical experiments support the protein kinase A KIN-1 as the putative kinase that phosphorylates ALG-1 serine 642. Altogether, our data indicate that PKA triggers the ALG-1 phosphorylation to regulate its microRNAs association during C. elegans development.
2022-09-10 | GSE198352 | GEO
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
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
2024-10-17 | PXD043913 | Pride
Project description:This SuperSeries is composed of the SubSeries listed below.
2017-01-17 | GSE89946 | GEO
Project description:An Argonaute phosphorylation cycle promotes microRNA-mediated silencing [GeCKO screen]
| PRJNA354542 | ENA