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Analysis of High Molecular Weight RNA-induced silencing complex (HMW-RISC) in CD8+ T cells identifies miR-7 as a modulator of T cell activation

ABSTRACT: To identify the miRNAs expected to be potent in suppressing targets, we followed HMW RISC formation upon activation of CD8+ T cells. We show that while most miRNAs distribute between HMW and LMW RISC in activated T cells, several miRNAs were dominant in one complex over the other. Among these, miR-7 is enriched in HMW RISC and inhibition of miR-7 upon T cell activation leads to increased production of IL-2 and expression of IL-2-regulated proteins including the α-subunit of the IL-2 receptor, CD25; transferrin receptor, CD71; and amino acid transporter, CD98, which are direct miR-7 targets. Our data support a model where recruitment of miR-7 to HMW RISC restrains IL-2 signalling and the metabolic processes regulated by IL-2 and thus modulates T cell activation.

ORGANISM(S): Mus musculus

PROVIDER: GSE151555 | GEO | 2020/06/04

REPOSITORIES: GEO

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The distribution of individual miRNA species between high-molecular weight and low-molecular weight RISC complexes by small RNA sequencing in thyroid cancer cell lines

Project description:Thyroid cancer is the most prevalent malignancy of the endocrine system. We and others have shown that several microRNAs, which are ~21-24nt post-transcriptional gene regulators, are aberrantly expressed in anaplastic thyroid cancer (ATC) and papillary thyroid cancer (PTC) tissues and cell lines. In the cell, miRNAs are bound to Argonaute (AGO) proteins as low molecular weight RNA Induced Silencing Complexes (LMW-RISCs) that can then assemble into high molecular weight RISCs (HMW-RISCs) that also include additional proteins and mRNA. In this study, we sought to analyze the association of miRNAs across RISC complexity in ATC and PTC. For both ATC and PTC lines, miRNAs were enriched in HMW-RISC and LMW-RISC fractions compared with intermediate fractions or very low molecular weight (AGO-poor) fractions. Furthermore, 60% of all miRNAs were more abundant in LMW-RISC versus HMW- RISC fractions by ~2-4 fold. Surprisingly, miR-21-5p, one of the most abundant miRNAs in both ATC and PTC lines, was consistently one the least abundant miRNAs in HMW-RISC and the most enriched miRNA in LMW-RISC fractions. These findings suggest that miR-21 may be uniquely distributed in RISCs relative to other miRNAs. Furthermore, the methodology described here is a useful way to assess the relative magnitude of miR-21association with HMW and LMW-RISCs and may help to reveal the true roles of this miRNA in thyroid cancer development, progression, and treatment.

2020-02-28 | GSE146015 | GEO

RAVER1 interconnects lethal EMT and miR/RISC activity by the control of alternative splicing

Project description:The RAVER1 protein was proposed to serve as a co-factor in guiding the PTBP-dependent control of alternative splicing (AS). Whether RAVER1 solely acts in concert with PTBPs and how it affects cancer cell fate remained elusive. Here we provide the first comprehensive investigation of RAVER1-controlled AS in cancer cell models and reveal a pro-oncogenic role of RAVER1 in tumor growth. This unravels that RAVER1 guides AS in synergy with PTBPs but more prominently serves PTBP1-independent roles in splicing. In cancer cells, one major function of RAVER1 is the control of proliferation and apoptosis, which involves the modulation of AS events within the miR/RISC pathway. Associated with this regulatory role, RAVER1 antagonizes lethal, TGFB-driven epithelial-mesenchymal-transition (EMT) by limiting TGFB signaling. RAVER1-modulated splicing events affect the insertion of protein interaction modules in factors guiding miR/RISC-dependent gene silencing. Most prominently, in all three human TNRC6 proteins, RAVER1 controls AS of GW-enriched motifs, which are essential for AGO2-binding. Disturbance of RAVER1-guided AS events in TNRC6 proteins and other facilitators of miR/RISC activity compromise miR/RISC activity which is essential to restrict TGFB signaling and lethal EMT.

2024-01-11 | PXD043342 | Pride

Small RNA-seq from FPLC-fractionated leaf and flower crude extracts

Project description:Background: RNA interference (RNAi) is an indispensable regulatory mechanism governing developmental processes and stress responses via sequence-specific control of target RNAs mediated by the action of small, 20–24-nt-long, non-coding regulatory (s)RNAs such as micro (mi) and small interfering (si) RNAs. Biogenesis and sorting of miRNAs into ARGONAUTE (AGO) proteins are intensively investigated, however very few information is available about the presence and distribution of distinct sRNA pools in plant cells. Results: High-throughput sequencing of size-separated sRNA pools of plant crude extracts revealed that the majority of the canonical miRNAs were associated with high molecular weight RNA-induced silencing complexes co-migrating with AGO1 (HMW RISC). In contrast, the majority of 24-nt-long siRNAs were found in association with low molecular weight complexes co-migrating with AGO4 (LMW RISC). Intriguingly, we identified a large set of sRNAs in the cytoplasm, including mature miRNA sequences, in the low molecular size range corresponding to protein-unbound sRNAs. By comparing the RISC-loaded and protein-unbound pools of miRNAs, we identified miRNAs with highly different loading efficiencies. Investigation of some selected miRNAs in a transient expression system validated this finding. We also showed that the availability of RISCs is a limiting factor determining the loading efficiency of miRNAs. Conclusion: Our data reveal the existence of a regulatory checkpoint, likely controlled by information carried by the diverse miRNA precursors, determining the RISC-loading efficiencies of various miRNAs by sorting only a subset of the produced miRNAs into the biologically active RISCs.

2019-07-29 | GSE130431 | GEO

Slicing-Independent RISC Activation Requires the Argonaute PAZ Domain

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

Differential RISC association of endogenous human microRNAs predicts their inhibitory potential

Project description:It has previously been assumed that the generally high stability of microRNAs (miRNAs) reflects their tight association with Argonaute (Ago) proteins, essential components of the RNA-induced silencing complex (RISC). However, recent data have suggested that the majority of mature miRNAs are not, in fact, Ago associated. Here, we demonstrate that endogenous human miRNAs vary widely, by >100-fold, in their level of RISC association and show that the level of Ago binding is a better indicator of inhibitory potential than is the total level of miRNA expression. While miRNAs of closely similar sequence showed comparable levels of RISC association in the same cell line, these varied between different cell types. Moreover, the level of RISC association could be modulated by overexpression of complementary target mRNAs. Together, these data indicate that the level of RISC association of a given endogenous miRNA is regulated by the available RNA targetome and predicts miRNA function.

2014-05-30 | GSE58127 | GEO

Anti-inflammatory effects of differential molecular weight hyaluronic acids on UVB-induced calprotectin-mediated keratinocyte inflammation

Project description:Background: The biological functions of HA are related to its molecular weight and binding to its receptor TLR4 or CD44. Recent studies have shown that LMW-HA exhibits proinflammatory effects, while HMW-HA functions as an anti-inflammatory factor. UVB-induced epidermal inflammation is mainly mediated by endogenous molecules, such as DAMPs, that cause severe skin damage by activating TLR signaling pathways. Objective: Since both LMW- and HMW-HA have inhibitory functions on TLR-mediated macrophage inflammation, HA is assumed to suppress UVB-induced DAMP-mediated inflammation in the skin. In this study, both uLMW-HA and HMW-HA were found to inhibit UVB-induced keratinocyte inflammation. Methods: To elucidate whether HAs have anti-inflammatory effects on inflammation induced by UVB exposure, we performed whole transcriptome analysis in HaCaT cells treated with or without HAs after UVB exposure. Results: UV radiation induced upregulation or downregulation of genes (FC>1.2 or FC<0.8, q<0.05) were obtained. We analyzed network of these genes with Ingenuity Pathway analysis using information collected from databases on protein interactions. IL-6 was detected as an upstream factor of HA suppressing UV-radiation effects on HaCaT cells. Canonical pathway analysis also shows uLMW-HA downregulated NF-κB, which is located downstream of TLR4 signaling pathway, although HMW-HA did not show downregulation of NF-κB signaling pathway. Conclusions: Both uLMW-HA and HMW-HA were found to inhibit UVB-induced keratinocyte inflammation.

2022-08-05 | GSE201850 | GEO

Slicing-Independent RISC Activation Requires the Argonaute PAZ Domain

2012-07-12 | E-GEOD-39146 | biostudies-arrayexpress

Differential RISC association of endogenous human microRNAs predicts their inhibitory potential

2014-05-30 | E-GEOD-58127 | biostudies-arrayexpress

The miRNA-targeted transcriptome of porcine alveolar macrophages upon infection with Porcine Reproductive and Respiratory Syndrome Virus (RISC bound RNA)

Project description:Porcine alveolar macrophages were challenged in vitro with a low virulent strain of the Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) to identify and characterize the population of host genes subjected to miRNA post-transcriptional regulation durign early viral infection. We employed a high-throughput biochemical assay, i.e. the immunoprecipitation of RISCs followed by microarray analysis of the RISC-bound miRNA targets (RIP-Chip). qPCR analyses were carried out to validate the resolution of the microarray and to determine levels of expression of a panel of eight miRNAs (ssc-miR-142-3p, ssc-miR-142-5p, ssc-let-7f, miR-146a-5p, miR-155-5p, ssc-miR-181a, ssc-miR-21 and ssc-miR-335).

2018-04-14 | GSE98339 | GEO

Small RNA discovery in developing mouse and during human in vitro beta-cell differentiation protocol

Project description:The aim of this experiment was to identify novel, small (<600bp), capped, polyA-tailed RNA transcripts in mouse E14.5 pancreas, liver, and brain and at stages S1, S2, S3 within our human in vitro beta-cell differentiation protocol. Low-molecular-weight (LMW) and high-molecular-weight (HMW) fractions were separated.

2015-04-02 | E-MTAB-3489 | biostudies-arrayexpress

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