Nuclear Argonaute:miRNA complexes recognize target sequences within chromatin and silence gene expression
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE297116 | GEO | 2025/06/29
REPOSITORIES: GEO
Similar Datasets
Project description:Mammalian RNA interference (RNAi) is often linked to regulation in the cytoplasm. While synthetic RNAs regulate transcription and splicing, endogenous functions for nuclear RNAi have been obscure. Using enhanced crosslinking immunoprecipitation (eCLIP), we mapped AGO2 binding sites within nuclear RNA. The strongest AGO binding sites mapped to miRNAs. The prevalence of individual miRNAs was similar in the cytoplasm and the nucleus. Most statistically-significant AGO2 binding was within intron boundaries. RNAseq comparing AGO1, AGO2, AGO1/2, and AGO1/2/3 knockout to wild-type cells revealed that binding was associated with both up- and down-regulation of gene expression. We observed changes of splicing at genes with significant AGO binding at exon/intron junctions or within introns. Splicing changes were confirmed by RT-PCR and could be recapitulated by synthetic duplex RNAs that target the sites of AGO binding. Inhibition of miRNA action by addition of an anti-miR reversed miRNA-mediated control of splicing. We conclude: 1) That AGO binding sites occur throughout nuclear RNA but are often within introns; 2) The distribution of miRNAs is similar in the nucleus and cytoplasm; and 3) miRNAs can control gene splicing. Nuclear RNAi has the potential to be a natural regulatory mechanism but careful study will be necessary to identify critical RNA drivers of normal physiology and disease.
2021-02-10 | GSE161559 | GEO
Project description:Growing demand in RNA-targeted therapies and promise of miRNA-based drugs creates a need for tools that can accurately identify and quantify miRNA:target interactions at scale. The experimental capture of miRNA:mRNA interactions by ligation into chimeric RNA fragments provides a direct read out of miRNA targets by enabling profiling of miRNA targets with high-throughput sequencing. However, integration of chimeric CLIP-seq into wide practical use has been limited because the inefficiency of the miRNA:mRNA ligation step (resulting in a low rate of chimeric reads in final libraries) combined with the technical complexity of the method makes it challenging to apply to miRNAs of interest at scale. Here we describe chimeric eCLIP, in which we integrate a chimeric ligation step into AGO2 eCLIP to enable chimeric read recovery, and show that removal of the cumbersome polyacrylamide gel and nitrocellulose membrane transfer step common to CLIP techniques can be omitted for chimeric AGO2 eCLIP to create a simplified high throughput version of the assay that maintains high signal-to-noise. With the increased yield of recovered miRNA:mRNA interactions in no-gel chimeric eCLIP, we show that simple enrichment steps using either PCR or on-bead probe capture can be added to chimeric eCLIP in order to target and enrich libraries for chimeric reads specific to one or more miRNAs of interest in both cell lines and tissue samples, resulting in 30- to 175-fold increases in recovery of chimeric reads for miRNAs of interest. We further show that the same probe-capture approach can be used to recover miRNA interactions for a targeted gene of interest, revealing both distinct miRNA targeting as well as co-targeted by several miRNAs from the same seed family. RNA-seq analysis of gene expression following miRNA overexpression confirmed miRNA-mediated repression of chimeric eCLIP identified targets, and indicated that chimeric eCLIP can provide additional sensitivity to detect regulated targets among genes that either contain or lack computationally predicted miRNA target sites. Thus, we believe that chimeric eCLIP will be a useful tool for quantitative profiling of miRNA targets in varied sample types at scale, and revealing a deeper picture for regulatory networks for miRNAs of biological interest.
2022-07-30 | GSE198250 | GEO
Project description:Growing demand in RNA-targeted therapies and promise of miRNA-based drugs creates a need for tools that can accurately identify and quantify miRNA:target interactions at scale. The experimental capture of miRNA:mRNA interactions by ligation into chimeric RNA fragments provides a direct read out of miRNA targets by enabling profiling of miRNA targets with high-throughput sequencing. However, integration of chimeric CLIP-seq into wide practical use has been limited because the inefficiency of the miRNA:mRNA ligation step (resulting in a low rate of chimeric reads in final libraries) combined with the technical complexity of the method makes it challenging to apply to miRNAs of interest at scale. Here we describe chimeric eCLIP, in which we integrate a chimeric ligation step into AGO2 eCLIP to enable chimeric read recovery, and show that removal of the cumbersome polyacrylamide gel and nitrocellulose membrane transfer step common to CLIP techniques can be omitted for chimeric AGO2 eCLIP to create a simplified high throughput version of the assay that maintains high signal-to-noise. With the increased yield of recovered miRNA:mRNA interactions in no-gel chimeric eCLIP, we show that simple enrichment steps using either PCR or on-bead probe capture can be added to chimeric eCLIP in order to target and enrich libraries for chimeric reads specific to one or more miRNAs of interest in both cell lines and tissue samples, resulting in 30- to 175-fold increases in recovery of chimeric reads for miRNAs of interest. We further show that the same probe-capture approach can be used to recover miRNA interactions for a targeted gene of interest, revealing both distinct miRNA targeting as well as co-targeted by several miRNAs from the same seed family. RNA-seq analysis of gene expression following miRNA overexpression confirmed miRNA-mediated repression of chimeric eCLIP identified targets, and indicated that chimeric eCLIP can provide additional sensitivity to detect regulated targets among genes that either contain or lack computationally predicted miRNA target sites. Thus, we believe that chimeric eCLIP will be a useful tool for quantitative profiling of miRNA targets in varied sample types at scale, and revealing a deeper picture for regulatory networks for miRNAs of biological interest.
2022-07-30 | GSE197483 | GEO
Project description:Growing demand in RNA-targeted therapies and promise of miRNA-based drugs creates a need for tools that can accurately identify and quantify miRNA:target interactions at scale. The experimental capture of miRNA:mRNA interactions by ligation into chimeric RNA fragments provides a direct read out of miRNA targets by enabling profiling of miRNA targets with high-throughput sequencing. However, integration of chimeric CLIP-seq into wide practical use has been limited because the inefficiency of the miRNA:mRNA ligation step (resulting in a low rate of chimeric reads in final libraries) combined with the technical complexity of the method makes it challenging to apply to miRNAs of interest at scale. Here we describe chimeric eCLIP, in which we integrate a chimeric ligation step into AGO2 eCLIP to enable chimeric read recovery, and show that removal of the cumbersome polyacrylamide gel and nitrocellulose membrane transfer step common to CLIP techniques can be omitted for chimeric AGO2 eCLIP to create a simplified high throughput version of the assay that maintains high signal-to-noise. With the increased yield of recovered miRNA:mRNA interactions in no-gel chimeric eCLIP, we show that simple enrichment steps using either PCR or on-bead probe capture can be added to chimeric eCLIP in order to target and enrich libraries for chimeric reads specific to one or more miRNAs of interest in both cell lines and tissue samples, resulting in 30- to 175-fold increases in recovery of chimeric reads for miRNAs of interest. We further show that the same probe-capture approach can be used to recover miRNA interactions for a targeted gene of interest, revealing both distinct miRNA targeting as well as co-targeted by several miRNAs from the same seed family. RNA-seq analysis of gene expression following miRNA overexpression confirmed miRNA-mediated repression of chimeric eCLIP identified targets, and indicated that chimeric eCLIP can provide additional sensitivity to detect regulated targets among genes that either contain or lack computationally predicted miRNA target sites. Thus, we believe that chimeric eCLIP will be a useful tool for quantitative profiling of miRNA targets in varied sample types at scale, and revealing a deeper picture for regulatory networks for miRNAs of biological interest.
2022-07-30 | GSE196460 | GEO
Project description:Background: MicroRNAs (miRNAs) are 22-nt small non-coding regulatory RNAs that have generally been considered to regulate gene expression at the post-transcriptional level in the cytoplasm. However, recent studies have reported that some miRNAs localize to and function in the nucleus. Methodology/Principal Findings: To determine the number of miRNAs localized to the nucleus, we systematically investigated the subcellular distribution of small RNAs (sRNAs) by independent deep sequencing the nuclear and cytoplasmic pools of 18- to 30-nucleotide sRNAs from human cells. We identified 339 nuclear and 324 cytoplasmic known miRNAs, 300 of which overlap, suggesting that the majority of miRNAs are imported into the nucleus. With the exception of a few miRNAs evidently enriched in the nuclear pool, such as the mir-29b, the ratio of miRNA abundances in the nuclear fraction versus in the cytoplasmic fraction vary to some extent. Moreover, our results revealed that a large number of tRNA 3' trailers are exported from the nucleus and accumulate in the cytoplasm. These tRNA 3' trailers accumulate in a variety of cell types, implying that the biogenesis of tRNA 3' trailers is conserved and that they have a potential functional role in vertebrate cells. Conclusion/Significance: Our results provide the first comprehensive view of the subcellular distribution of diverse sRNAs and new insights into the roles of miRNAs and tRNA 3' trailers in the cell Discovery and characterization of small RNA species through deep sequencing of nuclear and cytoplasmic small RNA fractions from 5-8F cells, a nasopharyngeal carcinoma cell line.
2010-07-14 | E-GEOD-22918 | biostudies-arrayexpress
Project description:We characterized the miRNA composition of the nucleus and the cytoplasm of uninfected cells and compared it with the one of cells infected with the endosymbiotic bacterium Wolbachia strain wMelPop-CLA. We found an overall increase of small RNAs between 18 and 28 nucleotides in both cellular compartments in Wolbachia-infected cells and identified specific miRNAs induced and/or suppressed by the Wolbachia infection. We discuss the mechanisms that the cell may use to shuttle miRNAs between the cytoplasm and the nucleus. In addition, we identified piRNAs that changed their abundance in response to Wolbachia infection. The miRNAs and piRNAs identified in this study provide promising leads for investigations into the host-endosymbiont interactions and for better understanding of how Wolbachia manipulates the host miRNA machinery in order to facilitate its persistent replication in infected cells. Examination of small RNA profile in cytoplasmic and nuclear fractions of Aag 2 and Pop cells (Aedes aegypti)
2014-02-22 | E-GEOD-55210 | biostudies-arrayexpress
Project description:Background: MicroRNAs (miRNAs) are 22-nt small non-coding regulatory RNAs that have generally been considered to regulate gene expression at the post-transcriptional level in the cytoplasm. However, recent studies have reported that some miRNAs localize to and function in the nucleus. Methodology/Principal Findings: To determine the number of miRNAs localized to the nucleus, we systematically investigated the subcellular distribution of small RNAs (sRNAs) by independent deep sequencing the nuclear and cytoplasmic pools of 18- to 30-nucleotide sRNAs from human cells. We identified 339 nuclear and 324 cytoplasmic known miRNAs, 300 of which overlap, suggesting that the majority of miRNAs are imported into the nucleus. With the exception of a few miRNAs evidently enriched in the nuclear pool, such as the mir-29b, the ratio of miRNA abundances in the nuclear fraction versus in the cytoplasmic fraction vary to some extent. Moreover, our results revealed that a large number of tRNA 3' trailers are exported from the nucleus and accumulate in the cytoplasm. These tRNA 3' trailers accumulate in a variety of cell types, implying that the biogenesis of tRNA 3' trailers is conserved and that they have a potential functional role in vertebrate cells. Conclusion/Significance: Our results provide the first comprehensive view of the subcellular distribution of diverse sRNAs and new insights into the roles of miRNAs and tRNA 3' trailers in the cell
2010-07-14 | GSE22918 | GEO
Project description:Schmitz2014 - RNA triplex formation
The model is parameterized using the
parameters for gene CCDC3 from Supplementary Table S1. The two
miRNAs which form the triplex together with CCDC3 are miR-551b and
miR-138.
This model is described in the article:
Cooperative gene regulation
by microRNA pairs and their identification using a
computational workflow.
Schmitz U, Lai X, Winter F,
Wolkenhauer O, Vera J, Gupta SK.
Nucleic Acids Res. 2014 Jul; 42(12):
7539-7552
Abstract:
MicroRNAs (miRNAs) are an integral part of gene regulation
at the post-transcriptional level. Recently, it has been shown
that pairs of miRNAs can repress the translation of a target
mRNA in a cooperative manner, which leads to an enhanced
effectiveness and specificity in target repression. However, it
remains unclear which miRNA pairs can synergize and which genes
are target of cooperative miRNA regulation. In this paper, we
present a computational workflow for the prediction and
analysis of cooperating miRNAs and their mutual target genes,
which we refer to as RNA triplexes. The workflow integrates
methods of miRNA target prediction; triplex structure analysis;
molecular dynamics simulations and mathematical modeling for a
reliable prediction of functional RNA triplexes and target
repression efficiency. In a case study we analyzed the human
genome and identified several thousand targets of cooperative
gene regulation. Our results suggest that miRNA cooperativity
is a frequent mechanism for an enhanced target repression by
pairs of miRNAs facilitating distinctive and fine-tuned target
gene expression patterns. Human RNA triplexes predicted and
characterized in this study are organized in a web resource at
www.sbi.uni-rostock.de/triplexrna/.
This model is hosted on
BioModels Database
and identified by:
BIOMD0000000530.
To cite BioModels Database, please use:
BioModels Database:
An enhanced, curated and annotated resource for published
quantitative kinetic models.
To the extent possible under law, all copyright and related or
neighbouring rights to this encoded model have been dedicated to
the public domain worldwide. Please refer to
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Public Domain Dedication for more information.
2024-09-02 | BIOMD0000000530 | BioModels
Project description:Argonaute 2 (AGO2), the catalytic engine of RNAi, is typically associated with inhibition of translation in the cytoplasm. Contrary to dogma, AGO2 has also been implicated in nuclear processes including transcription and splicing. There has been little insight into AGO2's nuclear interactions or how they might differ relative to cytoplasm. Here we investigate the interactions of cytoplasmic and nuclear AGO2 using semi-quantitative mass spectrometry. Mass spectrometry often reveals long lists of candidate proteins, complicating efforts to rigorously discriminate true interacting partners from artifacts. We prioritized candidates using orthogonal analytical strategies that compare replicate mass spectra of proteins associated with FLAG-tagged and endogenous AGO2. Interactions with TRNC6A, TRNC6B, TNRC6C, and AGO3 are conserved between nuclei and cytoplasm. TAR binding protein interacted stably with cytoplasmic AGO2 but not nuclear AGO2, consistent with strand loading in the cytoplasm. Our data suggest that the core RNAi machinery is conserved between the nucleus and cytoplasm but that accessory proteins differ. Orthogonal analysis of mass spectra is a powerful approach to streamlining identification of protein partners.
2016-02-11 | MSV000079508 | MassIVE
Project description:During microRNA-biogenesis, stem-loop structured precursors are processed in two endonucleolytic steps, giving rise to mature microRNAs (miRNAs). This process is not only regulated at the level of transcription but also posttranscriptionally by RNA-binding proteins (RBPs). Here we have used a proteomics-based pull-down approach to map and characterize the interactome of 72 pre-miRNAs in eleven cell lines. We identify over 150 RBPs that interact specifically with distinct pre-miRNAs. To demonstrate their functional relevance, we used RNAi knockdown and CRISPR/Cas-mediated knockout experiments and analyzed changes in miRNA levels. Indeed, a large number of the investigated candidates have effects on miRNA-processing under our experimental conditions, including several C3H-zinc-finger proteins. Ultimately, we show that TRIM71/Lin41 is a potent regulator of miR-29a processing and its inactivation directly affects miR-29a targets. In summary, we provide an extended database of RBPs that interact with pre-miRNAs in different cell types helping to elucidate posttranscriptional regulation of miRNA biogenesis on a global scale.
2017-04-26 | PXD004193 | Pride