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The nuclear receptor ERbeta engages AGO2 in regulation of gene transcription, RNA splicing and RISC loading. Part 1

ABSTRACT: The RNA-binding protein Argonaute 2 (AGO2) is a key effector of RNA-silencing pathways It exerts a pivotal role in microRNA maturation and activity, and can modulate chromatin remodeling, transcriptional gene regulation and RNA splicing. The Estrogen Receptor beta (ERβ) is endowed with oncosuppressive activities, antagonizing hormone-induced carcinogenesis and inhibiting growth and oncogenic functions in luminal-like breast cancers (BCs), where its expression correlates with a better prognosis of the disease. Applying interaction proteomics coupled to mass spectrometry (MS) to characterize nuclear factors cooperating with ERβ in gene regulation, we identify AGO2 as a novel partner of ERβ in human BC cells. ERβ-AGO2 association was confirmed in vitro and in vivo both in the nucleus and in cytoplasm and is shown to be RNA-mediated. ChIP-Seq demonstrates AGO2 association to a large number of ERβ binding sites, and total and nascent RNA-Seq in ERβ+ vs ERβ- cells, and before and after AGO2 knock-down in ERβ+ cells, reveals a widespread involvement of this factor in ERβ-mediated regulation of gene transcription rate and RNA splicing. Moreover, isolation and sequencing by RIP-Seq of ERβ-associated long and small RNAs in the cytoplasm suggests involvement of the nuclear receptor in RISC loading, indicating that it may able to control directly also mRNA translation efficiency and stability.These results demonstrate that AGO2 can act as a pleiotropic functional partner of ERβ, indicating that both factors are endowed with multiple roles in the control of key cellular functions

INSTRUMENT(S): Illumina HiSeq 2500

ORGANISM(S): Homo sapiens

SUBMITTER: Giorgio Giurato

PROVIDER: E-MTAB-4638 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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The nuclear receptor ERbeta engages AGO2 in regulation of gene transcription, RNA splicing and RISC loading. Part 2

Project description:The RNA-binding protein Argonaute 2 (AGO2) is a key effector of RNA-silencing pathways It exerts a pivotal role in microRNA maturation and activity, and can modulate chromatin remodeling, transcriptional gene regulation and RNA splicing. The Estrogen Receptor beta (ERβ) is endowed with oncosuppressive activities, antagonizing hormone-induced carcinogenesis and inhibiting growth and oncogenic functions in luminal-like breast cancers (BCs), where its expression correlates with a better prognosis of the disease. Applying interaction proteomics coupled to mass spectrometry (MS) to characterize nuclear factors cooperating with ERβ in gene regulation, we identify AGO2 as a novel partner of ERβ in human BC cells. ERβ-AGO2 association was confirmed in vitro and in vivo both in the nucleus and in cytoplasm and is shown to be RNA-mediated. ChIP-Seq demonstrates AGO2 association to a large number of ERβ binding sites, and total and nascent RNA-Seq in ERβ+ vs ERβ- cells, and before and after AGO2 knock-down in ERβ+ cells, reveals a widespread involvement of this factor in ERβ-mediated regulation of gene transcription rate and RNA splicing. Moreover, isolation and sequencing by RIP-Seq of ERβ-associated long and small RNAs in the cytoplasm suggests involvement of the nuclear receptor in RISC loading, indicating that it may able to control directly also mRNA translation efficiency and stability.These results demonstrate that AGO2 can act as a pleiotropic functional partner of ERβ, indicating that both factors are endowed with multiple roles in the control of key cellular functions

2016-12-31 | E-MTAB-4639 | biostudies-arrayexpress

The nuclear receptor ERbeta engages AGO2 in regulation of gene transcription, RNA splicing and RISC loading. Part 5

2017-04-25 | E-MTAB-4359 | biostudies-arrayexpress

The nuclear receptor ERbeta engages AGO2 in regulation of gene transcription, RNA splicing and RISC loading. Part 4

2017-04-25 | E-MTAB-4363 | biostudies-arrayexpress

The nuclear receptor ERbeta engages AGO2 in regulation of gene transcription, RNA splicing and RISC loading. Part 3

2016-12-31 | E-MTAB-4368 | biostudies-arrayexpress

The nuclear receptor ERbeta engages AGO2 in regulation of gene transcription, RNA splicing and RISC loading

Project description:Background: The RNA-binding protein Argonaute 2 (AGO2) is a key effector of RNAsilencing pathways, exerting a pivotal role in microRNA maturation and activity, that in the cell nucleus is able to modulate chromatin remodeling, transcriptional gene regulation and RNA splicing. The Estrogen Receptor beta (ERβ), a member of the nuclear receptor superfamily of trancriptional regulators, is endowed with oncosuppressive activities, antagonizing hormone-induced carcinogenesis and inhibiting growth and oncogenic functions in luminal-like breast cancers (BCs), where its expression correlates with a better prognosis of the disease. Results: Applying interaction proteomics coupled to mass spectrometry (MS) to characterize nuclear factors cooperating with ERβ in gene regulation, we identified AGO2 as a novel partner of ERβ in human BC cells. ERβ-AGO2 association was confirmed in vitro and in vivo both in the nucleus and cytoplasm. ChIP-Seq demonstrated AGO2 association to a large number of ERβ binding sites, and total and nascent RNA-Seq in ERβ+ vs ERβ- cells, and before and after AGO2 knock-down in ERβ+ cells, revealed a widespread involvement of this factor in ERβ-mediated regulation of gene transcription rate and RNA splicing. Moreover, isolation and sequencing by RIP-Seq of ERβ-associated long and small RNAs in the cytoplasm suggested involvement of the nuclear receptor in RISC loading, indicating that it may able to control directly also mRNA translation efficiency and stability. Conclusions: These results demonstrate that AGO2 is a pleiotropic functional partner of ERβ in BC cells, indicating that both factors are endowed with multiple roles in the control of BC cell functions.

2017-09-20 | PXD006280 | Pride

MicroRNA Directly Enhances Mitochondrial Translation during Muscle Differentiation.

Project description:MicroRNAs are well known to mediate translational repression and mRNA degradation in the cytoplasm. Various microRNAs have also been detected in membrane-compartmentalized organelles, but the functional significance has remained elusive. Here we report that miR-1, a microRNA specifically induced during myogenesis, efficiently enters the mitochondria where it unexpectedly stimulates, rather than represses, the translation of specific mitochondrial genome-encoded transcripts. We show that this positive effect requires specific miR:mRNA base-pairing and Ago2, but not its functional partner GW182, which is excluded from the mitochondria. We provide evidence for the direct action of Ago2 in mitochondrial translation by Ago2 CrossLinking ImmunoPrecipitation coupled with sequencing (CLIP-seq), functional rescue with mitochondria-targeted Ago2, and selective inhibition of the microRNA machinery in the cytoplasm. These findings unveil a positive function of microRNA in mitochondrial translation and suggest a highly coordinated myogenic program via miR-1 mediated translational stimulation in the mitochondria and repression in the cytoplasm. Examination of miRNA's regulation function in mitochondria in C2C12 myoblasts cells and myotubes cells with CLIP-seq (Ago2).

2014-08-01 | E-GEOD-57596 | biostudies-arrayexpress

MicroRNA Directly Enhances Mitochondrial Translation during Muscle Differentiation.

2014-08-01 | GSE57596 | GEO

The Core RNAi Machinery is Conserved Between the Cytoplasm and Nuclei of Human Cells

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

ChIP-seq of Arabidopsis ARGONAUTE-small RNA interactions in Arabidopsis thaliana identified by high-throughput sequencing

Project description:The goal of this study was to identify small RNA-ARGONAUTE interactions in Arabidopsis. Co-immunoprecipitation assays followed by high-throughput sequencing were done to identify small RNA associated with HA-AGO2 and HA-AGO7. Experiment Overall Design: Sequencing-by-synthesis technology was used to sequence small RNA from input and co-immunoprecipitation fractions from cell lysates of inflorescence tissue from Arabidopsis plants transformed with HA epitope-tagged AGO2 or AGO7 constructs. Meaningful intepretation requires comparison between the input and co-immunoprecipitation datasets. Experiment Overall Design: Note: Raw data files requested, however, the submitter declined to provide them.

2009-08-30 | E-GEOD-12037 | biostudies-arrayexpress

Mapping ERβ genomic binding sites reveals unique genomic features and identifies EBF1 as an ERβ interactor [ChIP-Seq]

Project description:The C4-12/Flag.ERβ cell line which stably expressed Flag.ERβ is used to study ERβ genomic functions without ERα interference. Mapping ERβ binding sites in these cells reveals ERβ unique distribution and motif enrichment patterns. Accompanying our mapping results, nascent RNA profiling is performed on cells at the same treatment time. The combined results allow the identification of ERβ target genes. Gene ontology analysis reveals that ERβ targets are enriched in differentiation, development and apoptosis. Concurrently, E2 treatment suppresses proliferation in these cells. Within ERβ binding sites, while the most prevalent binding motif is the canonical ERE, motifs of known ER interactors are also enriched in ERβ binding sites. Moreover, among enriched binding motifs are those of GFI, REST and EBF1, which are unique to ERβ binding sites in these cells. Further characterization confirms the association between EBF1 and the estrogen receptors, which favors the N-terminal region of the receptor. Furthermore, EBF1 negatively regulates ERs at the protein level. In summary, by studying ERβ genomic functions in our cell model, we confirm the anti-proliferative role of ERβ and discover the novel cross talk of ERβ with EBF1 which has various implications in normal physiology. C4-12/Flag.ERβ cells were treated with 10nM E2 for 1 hour then crosslinked with 1% formaldehyde; EtOH treatment was used as control. Crosslinked samples were processed for ChIP-seq and sequenced with Illumina Genome Analyzer II and aligned to hg18. QuEST was used as the peak-calling software, using default parameters recommended to analyze transcription factor ChIP-seq data. The entire ChIP-seq process was performed once on each sample (Vehicle or E2-treated).

2013-06-21 | E-GEOD-48096 | biostudies-arrayexpress

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