Sequestration of microRNA-mediated target repression by the Ago2-associated RNA binding protein FAM120A
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ABSTRACT:
PROVIDER: PRJNA526904 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR8723524.fastq.gz | Fastqsanger.gz | |||
| SRR8723525.fastq.gz | Fastqsanger.gz |
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Project description:Argonaute (Ago) proteins interact with various binding partners and play a pivotal role in microRNA (miRNA)-mediated silencing pathways. By utilizing immunoprecipitation followed by mass spectrometry to determine cytoplasmic Ago2 protein complexes in mouse embryonic stem cells (mESCs), we identified a putative RNA-binding protein FAM120A (also known as OSSA/C9ORF10) as an Ago2 interacting protein. Individual nucleotide resolution Cross-Linking and ImmunoPrecipitation (iCLIP) analysis revealed that FAM120A binds to homopolymeric tracts in 3´ UTRs of about 2,000 mRNAs, particularly poly(G) sequences. Comparison of FAM120A iCLIP and Ago2 iCLIP reveals that greater than one-third of mRNAs bound by Ago2 in mESCs are co-bound by FAM120A. Furthermore, such FAM120A-bound Ago2 target genes are not subject to Ago2-mediated target degradation. Reporter assays suggest that the 3´ UTRs of several FAM120A-bound miRNA target genes are less sensitive to Ago2-mediated target repression than those of FAM120A-unbound miRNA targets and FAM120A modulates them via its G-rich target sites. These findings suggest that Ago2 may exist in multiple protein complexes with varying degrees of functionality.
2019-07-09 | GSE128264 | GEO
Project description:Argonaute (Ago) proteins interact with various binding partners and play a pivotal role in microRNA (miRNA)-mediated silencing pathways. By utilizing immunoprecipitation followed by mass spectrometry to determine cytoplasmic Ago2 protein complexes in mouse embryonic stem cells (mESCs), we identified a putative RNA-binding protein FAM120A (also known as OSSA/C9ORF10) as an Ago2 interacting protein. Individual nucleotide resolution cross-linking and immunoprecipitation (iCLIP) analysis revealed that FAM120A binds to homopolymeric tracts in 3'-UTRs of about 2000 mRNAs, particularly poly(G) sequences. Comparison of FAM120A iCLIP and Ago2 iCLIP reveals that greater than one-third of mRNAs bound by Ago2 in mESCs are co-bound by FAM120A. Furthermore, such FAM120A-bound Ago2 target genes are not subject to Ago2-mediated target degradation. Reporter assays suggest that the 3'-UTRs of several FAM120A-bound miRNA target genes are less sensitive to Ago2-mediated target repression than those of FAM120A-unbound miRNA targets and FAM120A modulates them via its G-rich target sites. These findings suggest that Ago2 may exist in multiple protein complexes with varying degrees of functionality.
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Project description:Mapping of microRNA (miRNA) targets using AGO2 CLIPseq and HEAPseq has provided major insight into the function of miRNAs in various systems. HEAPseq is a powerful method because it allows for cell type-specific mapping of miRNA targets and relies on a mouse line harboring a conditional HaloTag in the endogeous Ago2 locus. However, the homozygous mice are not viable, suggesting that the insertion of the large (>1 kb) HaloTag conditional cassette in the Ago2 gene promoter region, or appending the HaloTag domain to the AGO2 protein, is deleterious to some aspects of AGO2 function. To overcome this limitation, we created tagged AGO2 mouse and mESC lines designed for minimal perturbation of miRISC function. We used a cleavable SpyTag3, which rapidly forms a covalent bond with its ligand SpyCatcher3 and is ten times smaller than HaloTag (3 vs 33 kDa)36. To minimize any perturbation on AGO2 expression and protein structure, constitutive Spy3-AGO2 mice and mESC lines were created by inserting the SpyTag3 coding sequence into exon 2 of the Ago2 gene, which is separated from the promoter region by a 38 kb intron and encodes a small unstructured region of AGO2 – the only part of the protein sequence that is not perfectly conserved between mice and humans. We then developed a method SAPseq, based on HEAPseq and benchmarked it first using mESCs.
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Project description:Mutations in human Argonaute genes, AGO1 and AGO2, are associated with neurodevelopmental disorders. Although multiple patients have been identified, the underlying molecular basis for pathogenesis remains unclear. Here, we biochemically examined five AGO2 mutations (p.L192P, p.A367P, p.T357M, p.F182del, and p.G733R) linked to different clinical severities. Except for G733R, all AGO2 mutant proteins maintained a stable fold, capable of binding and using microRNA (miRNA) guides. Kinetic studies showed that the L192P, A367P, and T357M mutants have prolonged dwell times on target RNAs, indicating impaired target release. The L192P and A367P variants also display slow target RNA association kinetics. RNA Bind-n-Seq experiments showed that in vitro, the L192P, A367P, T357M, and F182D mutants are prone to mis-targeting. In cultured murine cortical neurons, the L192P mutant altered the miRNA complement associated with AGO2, altered guide strand selectivity, and increased the accumulation of 3' isomiRs, suggesting altered miRNA loading and increased miRNA 3' end exposure. In vivo, mice carrying the p.L192P variant, but not p.G733R, demonstrated strongly reduced breeding ability, altered cortical transcriptomes, and over-repression of miRNA targets. The combined results suggest patient mutations impact AGO2 targeting dynamics in a gain-of-function manner, leading to deregulation of the neuronal transcriptome and the observed neurodevelopmental anomalies.
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Project description:MicroRNAs are small RNAs that regulate protein levels. It is commonly assumed that the expression level of a microRNA is directly correlated with its repressive activity – that is, highly expressed microRNAs will repress their target mRNAs more. Here we investigate the quantitative relationship between endogenous microRNA expression and repression for 32 mature microRNAs in Drosophila melanogaster S2 cells. In general, we find that more abundant microRNAs repress their targets to a greater degree. However, the relationship between expression and repression is nonlinear, such that a 10-fold greater microRNA concentration produces only a 10% increase in target repression. The expression/repression relationship is the same for both dominant guide microRNAs and minor mature products (so-called passenger strands/microRNA* sequences). However, we find examples of microRNAs whose cellular concentrations differ by several orders of magnitude, yet induce similar repression of target mRNAs. Likewise, microRNAs with similar expression can have very different repressive abilities. We show that the association of microRNAs with Argonaute proteins does not explain this variation in repression. The observed relationship is consistent with the limiting step in target repression being the association of the microRNA/RISC complex with the target site. These findings argue that modest changes in cellular microRNA concentration will have minor effects on repression of targets.
2014-06-12 | GSE58415 | GEO
Project description:MicroRNAs are small RNAs that regulate protein levels. It is commonly assumed that the expression level of a microRNA is directly correlated with its repressive activity M-bM-^@M-^S that is, highly expressed microRNAs will repress their target mRNAs more. Here we investigate the quantitative relationship between endogenous microRNA expression and repression for 32 mature microRNAs in Drosophila melanogaster S2 cells. In general, we find that more abundant microRNAs repress their targets to a greater degree. However, the relationship between expression and repression is nonlinear, such that a 10-fold greater microRNA concentration produces only a 10% increase in target repression. The expression/repression relationship is the same for both dominant guide microRNAs and minor mature products (so-called passenger strands/microRNA* sequences). However, we find examples of microRNAs whose cellular concentrations differ by several orders of magnitude, yet induce similar repression of target mRNAs. Likewise, microRNAs with similar expression can have very different repressive abilities. We show that the association of microRNAs with Argonaute proteins does not explain this variation in repression. The observed relationship is consistent with the limiting step in target repression being the association of the microRNA/RISC complex with the target site. These findings argue that modest changes in cellular microRNA concentration will have minor effects on repression of targets. Two replicates of S2-DRSC cells under normal conditions
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