Mapping microRNA-target interactions in abundant cell types with Spy3-AGO2 Pull-down [CLIP-seq]
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE289638 | GEO | 2025/04/02
REPOSITORIES: GEO
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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:To elucidate miRNA-mediated temporal crosstalk during productive infection, we identified genome-wide miRNA target sites using Argonaute-crosslinking and immunoprecipitation followed by high-throughput sequencing (AGO-CLIPseq) in human cytomegalovirus (HCMV)-infected cells and evaluated the targeting efficacy by applying our new AGO-CLIPseq enrichment (ACE)-scoring algorithm.
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Project description:Argonaute 2 (AGO2) is a cytoplasmic component of the miRNA pathway with essential roles in development and disease. Yet, little is known about its regulation in vivo. Here, we show that in mouse quiescent cells, AGO2 localizes almost exclusively to the nucleus. In quiescent splenocytes, AGO2’s subcellular localization is modulated by the Pi3K-AKT-mTOR pathway, a well-established regulator of quiescence. Signaling through this pathway in proliferating cells promotes AGO2 cytoplasmic accumulation at least in part by stimulating the expression of TNRC6, an AGO2 binding partner in the miRNA pathway. In contrast, low signaling in quiescent cells enables AGO2 to accumulate in the nucleus where it binds to young mobile transposons co-transcriptionally. Loss of AGO2 in quiescent cells results in upregulation of transposon transcripts suggesting it performs functions analogous to those of PIWI in the germline. Our results point to an essential but previously unrecognized nuclear role for AGO2 during mammalian quiescence as part of a genome-defense system against transposable elements.
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Project description:Argonaute 2 (AGO2) is a cytoplasmic component of the miRNA pathway with essential roles in development and disease. Yet, little is known about its regulation in vivo. Here, we show that in mouse quiescent cells, AGO2 localizes almost exclusively to the nucleus. In quiescent splenocytes, AGO2’s subcellular localization is modulated by the Pi3K-AKT-mTOR pathway, a well-established regulator of quiescence. Signaling through this pathway in proliferating cells promotes AGO2 cytoplasmic accumulation at least in part by stimulating the expression of TNRC6, an AGO2 binding partner in the miRNA pathway. In contrast, low signaling in quiescent cells enables AGO2 to accumulate in the nucleus where it binds to young mobile transposons co-transcriptionally. Loss of AGO2 in quiescent cells results in upregulation of transposon transcripts suggesting it performs functions analogous to those of PIWI in the germline. Our results point to an essential but previously unrecognized nuclear role for AGO2 during mammalian quiescence as part of a genome-defense system against transposable elements.
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Project description:Argonaute 2 (AGO2) is a cytoplasmic component of the miRNA pathway with essential roles in development and disease. Yet, little is known about its regulation in vivo. Here, we show that in mouse quiescent cells, AGO2 localizes almost exclusively to the nucleus. In quiescent splenocytes, AGO2’s subcellular localization is modulated by the Pi3K-AKT-mTOR pathway, a well-established regulator of quiescence. Signaling through this pathway in proliferating cells promotes AGO2 cytoplasmic accumulation at least in part by stimulating the expression of TNRC6, an AGO2 binding partner in the miRNA pathway. In contrast, low signaling in quiescent cells enables AGO2 to accumulate in the nucleus where it binds to young mobile transposons co-transcriptionally. Loss of AGO2 in quiescent cells results in upregulation of transposon transcripts suggesting it performs functions analogous to those of PIWI in the germline. Our results point to an essential but previously unrecognized nuclear role for AGO2 during mammalian quiescence as part of a genome-defense system against transposable elements.
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