Capturing microRNA targets using an RNA-induced silencing complex (RISC)-trap approach

Cambronne, Xiaolu A.; Shen, Rongkun; Auer, Paul L.; Goodman, Richard H.

doi:10.1073/pnas.1218887109

Cited by 51 publications

(50 citation statements)

References 59 publications

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“…We identified DHHC9 as a putative miR-134 target by using the RISC-trap assay that we had developed previously to capture miRNAmRNA interactions (13). This assay, performed in HEK293 cells, uses an epitope-tagged dominant negative form of the RISC component, GW182, to stabilize microRNA-mRNA complexes.…”

Section: Resultsmentioning

confidence: 99%

MicroRNA-134 activity in somatostatin interneurons regulates H-Ras localization by repressing the palmitoylation enzyme, DHHC9

Chai

Cambronne

Eichhorn

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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Significance Most brain regions comprise numerous neural cell types that are distinct in function and molecular characteristics. We examined microRNA-134 (miR-134) activity in cortical cultures using a microRNA sensor and discovered that miR-134 was induced in response to neuronal activity in somatostatin- and calretinin-expressing interneurons but not in pyramidal neurons. We identified the palmitoylation enzyme DHHC9 as a direct target of miR-134 and showed that it contributed to the regulation of H-Ras in somatostatin interneurons. H-Ras is a signaling molecule crucial for neuronal development and function. This study uncovered an activity-dependent miR-134 regulatory pathway in cortical interneurons that can potentially affect membrane localization of multiple signaling molecules.

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Section: Resultsmentioning

confidence: 99%

MicroRNA-134 activity in somatostatin interneurons regulates H-Ras localization by repressing the palmitoylation enzyme, DHHC9

Chai

Cambronne

Eichhorn

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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“…To identify what transcripts miR159 is targeting in humans, we used three independent methods, including computational prediction using software DIANA microT v3.0, RNA-Seq-based gene expression profiling of MDA-MB-231 cells transfected with synthetic miR159, and the RNA-induced silencing complex (RISC)-trap (RISCTRAP) assay [40] followed by sequencing, to identify endogenous transcripts associated with miR159 ( Figure 3A, 3B and Supplementary information, Table S5). Three genes, transcription factor 7 (TCF7), nuclear receptor coactivator 6 (NCOA6), and engrailed-2 (EN2), were identified as putative miR159 targets by all three methods.…”

Section: Mir159 Binds To and Targets Transcripts Of Human Tcf7mentioning

confidence: 99%

“…The RNA was resuspended in RNasefree H 2 O followed by miScript RT-qPCR as stated below. The RISCTRAP assay was performed as previously described [40] using biotinylated miR159 and anti-biotin beads (Sigma-Aldrich; St. Louis, MO) to pull down miR159 and associated RNAs, rather than assess total RNA. RNA-Seq was performed to identify miR159-associated RNAs which were compared with input total RNAs.…”

Section: Sodium Periodate Qpcr and Risctrap Assaymentioning

confidence: 99%

Cross-kingdom inhibition of breast cancer growth by plant miR159

Fong²,

et al. 2016

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MicroRNAs (miRNAs) are critical regulators of gene expression, and exert extensive impacts on development, physiology, and disease of eukaryotes. A high degree of parallelism is found in the molecular basis of miRNA biogenesis and action in plants and animals. Recent studies interestingly suggest a potential cross-kingdom action of plant-derived miRNAs, through dietary intake, in regulating mammalian gene expression. Although the source and scope of plant miRNAs detected in mammalian specimens remain controversial, these initial studies inspired us to determine whether plant miRNAs can be detected in Western human sera and whether these plant miRNAs are able to influence gene expression and cellular processes related to human diseases such as cancer. Here we found that Western donor sera contained the plant miRNA miR159, whose abundance in the serum was inversely correlated with breast cancer incidence and progression in patients. In human sera, miR159 was predominantly detected in the extracellular vesicles, and was resistant to sodium periodate oxidation suggesting the plant-originated 2'-O-methylation on the 3′ terminal ribose. In breast cancer cells but not non-cancerous mammary epithelial cells, a synthetic mimic of miR159 was capable of inhibiting proliferation by targeting TCF7 that encodes a Wnt signaling transcription factor, leading to a decrease in MYC protein levels. Oral administration of miR159 mimic significantly suppressed the growth of xenograft breast tumors in mice. These results demonstrate for the first time that a plant miRNA can inhibit cancer growth in mammals.

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“…The system utilizes a dominant negative mutation of the miRNA-induced silencing complex (miRISC) protein subunit GW182, which is integrated into the endogenous Argonaute (Ago)/miRISC complex and locks a microRNA/target mRNA pair in the complex and limits further processing 148 . The DYKDDDDK (FLAG epitope) tag on the dominant negative GW182 protein allows capture and isolation of the entire Ago/RISC complex containing the trapped miRNA/target mRNA pair.…”

Section: Microrna Target Identification and Quantification System (Mimentioning

confidence: 99%

Hyperlipidemia-Induced MicroRNA-155-5p Improves β-Cell Function by TargetingMafb

et al. 2017

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A high-fat diet increases bacterial lipopolysaccharide (LPS) in the circulation and thereby stimulates glucagon-like peptide 1 (GLP-1)–mediated insulin secretion by upregulating interleukin-6 (IL-6). Although microRNA-155-5p (miR-155-5p), which increases IL-6 expression, is upregulated by LPS and hyperlipidemia and patients with familial hypercholesterolemia less frequently develop diabetes, the role of miR-155-5p in the islet stress response to hyperlipidemia is unclear. In this study, we demonstrate that hyperlipidemia-associated endotoxemia upregulates miR-155-5p in murine pancreatic β-cells, which improved glucose metabolism and the adaptation of β-cells to obesity-induced insulin resistance. This effect of miR-155-5p is because of suppression of v-maf musculoaponeurotic fibrosarcoma oncogene family, protein B, which promotes β-cell function through IL-6–induced GLP-1 production in α-cells. Moreover, reduced GLP-1 levels are associated with increased obesity progression, dyslipidemia, and atherosclerosis in hyperlipidemic Mir155 knockout mice. Hence, induction of miR-155-5p expression in β-cells by hyperlipidemia-associated endotoxemia improves the adaptation of β-cells to insulin resistance and represents a protective mechanism in the islet stress response.

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Capturing microRNA targets using an RNA-induced silencing complex (RISC)-trap approach

Cited by 51 publications

References 59 publications

MicroRNA-134 activity in somatostatin interneurons regulates H-Ras localization by repressing the palmitoylation enzyme, DHHC9

MicroRNA-134 activity in somatostatin interneurons regulates H-Ras localization by repressing the palmitoylation enzyme, DHHC9

Cross-kingdom inhibition of breast cancer growth by plant miR159

Hyperlipidemia-Induced MicroRNA-155-5p Improves β-Cell Function by TargetingMafb

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