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Cross-linking ligation and sequencing of hybrids (qCLASH) reveals an unpredicted miRNA targetome in melanoma cells

ABSTRACT: Cross-linking ligation and sequencing of hybrids (qCLASH) reveals an unpredicted miRNA targetome in melanoma cells

PROVIDER: PRJEB35798 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	ERR3761153.fastq.gz	Fastqsanger.gz
	ERR3761154.fastq.gz	Fastqsanger.gz
	ERR3761155.fastq.gz	Fastqsanger.gz

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Cross-linking ligation and sequencing of hybrids (qCLASH) reveals an unpredicted miRNA targetome in melanoma cells

Project description:In the quick cross-linking ligation and sequencing of hybrids (qCLASH) protocol, RNA-protein (RNP) complexes of interest are UV cross-linked in living cells. The protein of interest is purified by immunoprecipitation (in this case, AGO bound to the miRNA and the target gene). The two interacting RNA molecules (e.g. miRNA-mRNA) are physically bound to each other by intermolecular RNA-RNA ligation, followed by library preparation and sequencing oh hybrids.

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Cross-Linking Ligation and Sequencing of Hybrids (qCLASH) Reveals an Unpredicted miRNA Targetome in Melanoma Cells.

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Modified cross-linking, ligation, and sequencing of hybrids (qCLASH) identifies KSHV microRNA targets in endothelial cells

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Sequencing of Argonaute-bound miRNA/mRNA hybrids reveals regulation of the unfolded protein response by microRNA-320a

Project description:MicroRNAs (miRNA) are short non-coding RNAs widely implicated in development, gene regulation, and disease progression. Most miRNAs utilize the RNase III enzymes Drosha and Dicer for biogenesis in animals. One notable exception is the RNA polymerase II transcription start sites (TSS) miRNAs whose biogenesis requires Dicer but not Drosha. The functional importance of the TSS-miRNA biogenesis pathway has remained uncertain due to their unelucidated targetomes. To better understand the function of TSS-miRNAs, we applied a modified Crosslinking, Ligation, and Sequencing of Hybrids on Argonaute (Ago-qCLASH) to identify the targets for TSS-miRNAs in HCT116 colorectal cancer cells with or without Drosha knockout (KO). We observed that miR-320a hybrids dominate in TSS-miRNA hybrids identified by Ago-qCLASH. Targets for miR-320a are enriched in the eIF2 signaling pathway, a downstream component of the unfolded protein response. Consistently, in miR-320a mimic- and inhibitor- transfected cells, differentially expressed genes are enriched in the eIF2 signaling pathway. Within the Ago-qCLASH data, we identified the endoplasmic reticulum (ER) chaperone Calnexin as a direct miR-320a target, thus connecting miR-320a to the unfolded protein response. During ER stress, but not amino acid deprivation, miR-320a up-regulates ATF4, a critical transcription factor for resolving ER stress. Our study helps to elucidate the targetome of the TSS-miRNAs in colorectal cancer cells and establishes miR-320a as a regulator of unfolded protein response.

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