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
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA636360 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR11901800_1.fastq.gz | Fastqsanger.gz | |||
| SRR11901800_2.fastq.gz | Fastqsanger.gz | |||
| SRR11901801_1.fastq.gz | Fastqsanger.gz | |||
| SRR11901801_2.fastq.gz | Fastqsanger.gz | |||
| SRR11901789_1.fastq.gz | Fastqsanger.gz |
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Project description: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.
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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
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.
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Project description:Introgression of a high molecular weight glutenin subunit (HMW-GS) gene, 1Ay21*, into commercial wheat cultivars increased overall grain protein content and bread-making quality by unknown mechanisms. As well as increased abundance of 1Ay HMW-GS, 115 differentially expressed proteins (DEPs) were discovered between three cultivars and corresponding introgressed near-isogenic lines (NILs). Functional category analysis showed that the DEPs were predominantly other storage proteins, and proteins involved in protein synthesis, protein folding, protein degradation, stress response and grain development. Nearly half the genes encoding the DEPs showed strong co-expression patterns during grain development. Promoters of these genes are enriched in elements associated with transcription initiation and light response, indicating a potential connection between these cis-elements and grain protein accumulation. A model of how this HMW-GS enhances the abundance of machinery for protein synthesis and maturation during grain filling is proposed. This analysis not only provides insights into how introgression of the 1Ay21* improves grain protein content, but also directs selection of protein candidates for future wheat quality breeding programmes.
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Project description:Zymo HMW Nanopore R10.4.1 Early access
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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.
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Project description:ARGONAUTE1 (AGO1) binds directly to small regulatory RNA and is a key effector protein of post-transcriptional gene silencing mediated by microRNA (miRNA) and small interfering RNA (siRNA). The formation of an RNA induced silencing complex (RISC) of AGO1 and small RNA requires the function of the Heat Shock Protein 70/90 chaperone system. Some functions of AGO1 occur in association with endomembranes, in particular the rough endoplasmic reticulum (rER), but proteins interacting with AGO1 in membrane fractions remain unidentified. In this study, we show that the farnesylated Heat Shock Protein 40 homologs, J2 and J3, associate with AGO1 in membrane fractions in a manner that involves protein farnesylation. We also show that three changes in AGO1 function are detectable in mutants in protein farnesylation and J2/J3. First, perturbations of the HSP40/70/90 pathway by mutation of j3, hsp90 and farnesyl transferase affect the amounts of AGO1 associated with membranes. Second, miRNA association with membrane-bound AGO1, and with membrane-bound polysomes is increased in farnesyl transferase and farnesylationdeficient J2/J3 mutants. Third, silencing by non-cell autonomously acting short interfering RNAs (siRNAs) is impaired. These observations highlight the involvement of farnesylated J2/J3 in small RNA-mediated gene regulation, and suggest that the importance of chaperone-AGO1 interaction is not limited to the RISC assembly process.
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