An Argonaute phosphorylation cycle promotes microRNA-mediated silencing
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE89946 | GEO | 2017/01/17
SECONDARY ACCESSION(S): PRJNA354286
REPOSITORIES: GEO
Similar Datasets
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:An Argonaute phosphorylation cycle promotes microRNA-mediated silencing
| PRJNA354286 | ENA
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:This SuperSeries is composed of the SubSeries listed below.
2018-08-31 | GSE108801 | GEO
Project description:This SuperSeries is composed of the SubSeries listed below.
2012-08-22 | GSE39749 | GEO
Project description:This SuperSeries is composed of the SubSeries listed below. Refer to individual Series
2013-05-31 | E-GEOD-41081 | biostudies-arrayexpress
Project description:Although Argonaute (AGO) proteins have been the focus of microRNA (miRNA) studies, we observed AGO-free mature miRNAs directly interacting with RNA-binding proteins, implying the sophisticated nature of fine-tuning gene regulation by miRNAs. To investigate microRNA-binding proteins (miRBPs) globally, we analyzed PAR-CLIP data sets to identify RBP quaking (QKI) as a novel miRBP for let-7b. Potential existence of AGO-free miRNAs were further verified by measuring miRNA levels in genetically engineered AGO-depleted human and mouse cells. We have shown that QKI regulates miRNA-mediated gene silencing at multiple steps, and collectively serves as an auxiliary factor empowering AGO2/let-7b-mediated gene silencing. Depletion of QKI decreases interaction of AGO2 with let-7b and target mRNA, consequently controlling target mRNA decay. This finding indicates that QKI is a complementary factor in miRNA-mediated mRNA decay. QKI, however, also suppresses the dissociation of let-7b from AGO2, and slows the assembly of AGO2/miRNA/target mRNA complexes at the single-molecule level. We also revealed that QKI overexpression suppresses cMYC expression at post-transcriptional level, and decreases proliferation and migration of HeLa cells, demonstrating that QKI is a tumour suppressor gene by in part augmenting let-7b activity. Our data show that QKI is a new type of RBP implicated in the versatile regulation of miRNA-mediated gene silencing.
| S-EPMC10878027 | biostudies-literature