The Integrator complex regulates microRNA abundance through RISC loading
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ABSTRACT:
PROVIDER: PRJNA738965 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR14858700.fastq.gz | Fastqsanger.gz | |||
| SRR14858800.fastq.gz | Fastqsanger.gz | |||
| SRR14858701.fastq.gz | Fastqsanger.gz | |||
| SRR14858801.fastq.gz | Fastqsanger.gz | |||
| SRR14858702.fastq.gz | Fastqsanger.gz |
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Project description:MicroRNA (miRNA) homeostasis is crucial for the post-transcriptional regulation of their target genes and miRNA dysregulation has been linked to multiple diseases, including cancer. The mechanistic steps of miRNA biogenesis are well understood at molecular level1,2. Subsequent miRNA duplex loading into members of the Argonaute (Ago) protein family, representing the core of the RNA-induced silencing complex (RISC), is pivotal to miRNA-mediated gene silencing3-5. The Integrator complex has been previously identified as important regulator of RNA maturation, RNA polymerase II pause-release, and premature transcriptional termination, processes dependent on Integrator’s catalytical activity6-9. Here we report that absence of the Integrator complex leads to a global loss of mature miRNAs. By incorporating 4-Thiouridine (s4U) in nascent transcripts, we traced miRNA fate from biogenesis to stabilization and identified Integrator to be essential for proper miRNA assembly into RISC. Indeed, Integrator enhances miRNA-Ago2 interaction in vitro, which functionally translates to amplified miRNA target cleavage. These findings demonstrate for the first time cytoplasmic Integrator complex functions and stress an indirect role of Integrator in transcription regulation through modulation of miRNA abundance and stability.
2023-02-14 | GSE178127 | GEO
Project description:MicroRNA (miRNA) homeostasis is crucial for the posttranscriptional regulation of their target genes during development and in disease states. miRNAs are derived from primary transcripts and are processed from a hairpin precursor intermediary to a mature 22-nucleotide duplex RNA. Loading of the duplex into the Argonaute (AGO) protein family is pivotal to miRNA abundance and its posttranscriptional function. The Integrator complex plays a key role in protein coding and noncoding RNA maturation, RNA polymerase II pause-release, and premature transcriptional termination. Here, we report that loss of Integrator results in global destabilization of mature miRNAs. Enhanced ultraviolet cross-linking and immunoprecipitation of Integrator uncovered an association with duplex miRNAs before their loading onto AGOs. Tracing miRNA fate from biogenesis to stabilization by incorporating 4-thiouridine in nascent transcripts pinpointed a critical role for Integrator in miRNA assembly into AGOs.
| S-EPMC9916992 | biostudies-literature
Project description:Small RNAs regulate the genetic networks through a ribonucleoprotein complex called the RNA induced silencing complexes (RISC), which in mammals contains at its center one of four Argonaute proteins (Ago1-4). A key regulatory event in the RNAi and miRNA pathways is Ago loading, where double stranded small RNA duplexes are incorporated into RISC (pre-RISC) and then become single stranded (mature-RISC), a process that is not well understood. The Agos contain an evolutionary conserved PAZ (Piwi/Argonaute/Zwille) domain whose primary function is to bind the 3’-end of small RNAs. We created multiple Paz domain disrupted Ago mutant proteins and studied their biochemical properties and biological functionality in cells. We found that the Paz domain is dispensable for Ago loading of slicing-competent RISC. In contrast, in the absence of slicer activity or slicer substrate duplex RNAs, Paz-disrupted Agos bound duplex siRNAs but were unable to unwind/eject the passenger strand and form functional RISC complexes. We have discovered that the highly conserved Paz domain plays an important role in RISC activation, providing new mechanistic insights into how miRNAs regulate genes, as well as new insights for future design of miRNA and RNAi-based therapeutics.
2012-07-12 | GSE39146 | GEO
Project description:Small RNAs regulate the genetic networks through a ribonucleoprotein complex called the RNA induced silencing complexes (RISC), which in mammals contains at its center one of four Argonaute proteins (Ago1-4). A key regulatory event in the RNAi and miRNA pathways is Ago loading, where double stranded small RNA duplexes are incorporated into RISC (pre-RISC) and then become single stranded (mature-RISC), a process that is not well understood. The Agos contain an evolutionary conserved PAZ (Piwi/Argonaute/Zwille) domain whose primary function is to bind the 3’-end of small RNAs. We created multiple Paz domain disrupted Ago mutant proteins and studied their biochemical properties and biological functionality in cells. We found that the Paz domain is dispensable for Ago loading of slicing-competent RISC. In contrast, in the absence of slicer activity or slicer substrate duplex RNAs, Paz-disrupted Agos bound duplex siRNAs but were unable to unwind/eject the passenger strand and form functional RISC complexes. We have discovered that the highly conserved Paz domain plays an important role in RISC activation, providing new mechanistic insights into how miRNAs regulate genes, as well as new insights for future design of miRNA and RNAi-based therapeutics. Various Argonautes associated small RNA profiles were generated by deep sequencing the Agos-IP samples in HEK293 Cells transfected with corresponding Argonaute.
2012-07-12 | E-GEOD-39146 | biostudies-arrayexpress
Project description:MicroRNAs inhibits their target mRNA translation by forming RISC complex. Immunoprecipitation of RISC will pull down both microRNA and their target mRNAs. To systemetically examine the potential targets of microRNA-668 and microRNA-489, HEK293 cells were overexpressed with negative control, microRNA-668, or microRNA-489 mimics. Both total RNA samples and RNA samples from RISC complex pulled down by Ago-2 immunoprecipitation were examined by RNA-seq.
2019-07-27 | GSE117805 | GEO
Project description:Complexes containing INTS3 and either NABP1 or NABP2 were initially characterized in DNA damage responses, but their biochemical function remained unknown. Using affinity purifications and HIV Integration Targeting-Sequencing (HIT-Seq), we find that these complexes are part of the Integrator complex, which binds RNA Polymerase II and regulates specific target genes. Integrator cleaves snRNAs as part of their processing to their mature form in a mechanism that is intimately coupled with transcription termination. However, HIT-Seq reveals that Integrator also binds to the 3’ end of replication-dependent histones and promoter proximal regions of genes with polyadenylated transcripts. Depletion of Integrator subunits results in transcription termination failure, disrupting histone mRNA processing, and leading to polyadenylation of snRNAs and histone mRNAs. Furthermore, promoter proximal binding of Integrator negatively regulates expression of genes whose transcripts are normally polyadenylated. Integrator recruitment to all three gene classes is DSIF-dependent, suggesting that Integrator functions as a termination complex at DSIF-dependent RNA Polymerase II pause sites. HITseq was used to interrogate chromatin binding sites of of proteins in the complex (including INIP, INTS3, INTS9, INTS11, NABP1, NABP2, NELFA, NELFB, NELFCD, NELFE, SPT5). These proteins were fused to LEDGF-IBD and expressed in mouse LEDGF-/-MEF cells. The fusion proteins then target HIV integration to the chromatin binding sites. The HIV integration sites were identified using linker-mediated PCR and highthroughput sequencing and serve as surrogate for chromatin binding sites. Mapping was done using mouse genome mm9.
2015-01-20 | E-GEOD-65090 | biostudies-arrayexpress
Project description:Targeted gene silencing by RNAi requires the RNA-induced silencing complex (RISC), whose core component is the protein Argonaute (Ago) bound to a microRNA (miRNA) or an siRNA. In humans, Ago2 is loaded with miRNAs by the action of a specialized assembly called the RISC-loading complex (RLC), comprising the proteins Ago2, Dicer, and TRBP. Here we show that the human RLC assembles spontaneously in vitro from purified components. No cofactors or chaperones are required for the complex to form. The reconstituted RLC, containing one copy of each protein, has the dicing, slicing, guide-strand selection, and Ago2-loading activities observed for the endogenous RLC. Furthermore, once Ago2 is loaded with an miRNA, it tends to dissociate from the rest of the complex. These results lay the groundwork for future structural and functional dissection of RISC loading in humans.
| S-EPMC2206567 | biostudies-literature
Project description:Cellular homeostasis requires transcriptional outputs to be coordinated, and many events post transcription initiation can dictate the levels and functions of mature transcripts. To systematically identify regulators of inducible gene expression, we performed high-throughput RNAi screening of the Drosophila Metallothionein A (MtnA) promoter. This revealed that the Integrator complex, which has a well-established role in 3' end processing of small nuclear RNAs (snRNAs), attenuates MtnA transcription during copper stress. Integrator complex subunit 11 (IntS11) endonucleolytically cleaves MtnA transcripts, resulting in premature transcription termination and degradation of the nascent RNAs by the RNA exosome, a complex also identified in the screen. Using RNA-seq, we then identified >400 additional Drosophila protein-coding genes whose expression increases upon Integrator depletion. We focused on a subset of these genes and confirmed that Integrator is bound to their 5' ends and negatively regulates their transcription via IntS11 endonuclease activity. Many non-catalytic Integrator subunits, which are largely dispensable for snRNA processing, also have regulatory roles at these protein-coding genes, possibly by controlling Integrator recruitment or RNA polymerase II dynamics. Altogether, our results suggest that attenuation via Integrator cleavage limits production of many full-length mRNAs, allowing precise control of transcription outputs.
2019-09-12 | GSE136150 | GEO
Project description:Assembly of the RNA-induced silencing complex (RISC) requires formation of the RISC loading complex (RLC), which contains the Dicer-2 (Dcr-2)-R2D2 complex and recruits duplex siRNA to Ago2 in Drosophila melanogaster. However, the precise composition and action mechanism of Drosophila RLC remain unclear. Here we identified the missing factor of RLC as TATA-binding protein-associated factor 11 (TAF11) by genetic screen. Although it is an annotated nuclear transcription factor, we found that TAF11 also associated with Dcr-2/R2D2 and localized to cytoplasmic D2 bodies. Consistent with defective RLC assembly in taf11(-/-) ovary extract, we reconstituted the RLC in vitro using the recombinant Dcr-2-R2D2 complex, TAF11, and duplex siRNA. Furthermore, we showed that TAF11 tetramer facilitates Dcr-2-R2D2 tetramerization to enhance siRNA binding and RISC loading activities. Together, our genetic and biochemical studies define the molecular nature of the Drosophila RLC and elucidate a cytoplasmic function of TAF11 in organizing RLC assembly to enhance RNAi efficiency.
| S-EPMC4560963 | biostudies-literature
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