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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.

2020-12-15 | E-MTAB-8591 | biostudies-arrayexpress

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

Project description:MicroRNAs are key post-transcriptional gene regulators often displaying aberrant expression patterns in cancer. As microRNAs are promising disease-associated biomarkers and modulators of responsiveness to anti-cancer therapies, a solid understanding of their targetome is crucial. Despite enormous research efforts, the success rates of available tools to reliably predict microRNAs (miRNA)-target interactions remains limited. To investigate the disease-associated miRNA targetome, we have applied modified cross-linking ligation and sequencing of hybrids (qCLASH) to BRAF-mutant melanoma cells. The resulting RNA-RNA hybrid molecules provide a comprehensive and unbiased snapshot of direct miRNA-target interactions. The regulatory effects on selected miRNA target genes in predicted vs. non-predicted binding regions was validated by miRNA mimic experiments. Most miRNA-target interactions deviate from the central dogma of miRNA targeting up to 60% interactions occur via non-canonical seed pairing with a strong contribution of the 3' miRNA sequence, and over 50% display a clear bias towards the coding sequence of mRNAs. miRNAs targeting the coding sequence can directly reduce gene expression (miR-34a/CD68), while the majority of non-canonical miRNA interactions appear to have roles beyond target gene suppression (miR-100/AXL). Additionally, non-mRNA targets of miRNAs (lncRNAs) whose interactions mainly occur via non-canonical binding were identified in melanoma. This first application of CLASH sequencing to cancer cells identified over 8 K distinct miRNA-target interactions in melanoma cells. Our data highlight the importance non-canonical interactions, revealing further layers of complexity of post-transcriptional gene regulation in melanoma, thus expanding the pool of miRNA-target interactions, which have so far been omitted in the cancer field.

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

Project description:KS lesions consist of endothelial cells latently infected with KSHV which express the KSHV miRNAs. Identifying the targets of the KSHV miRNAs will help us understand their role in viral oncogenesis. Cross-Linking and Sequencing of Hybrids (CLASH) is a method for unambiguously identifying miRNA targetomes. We developed a streamlined version of CLASH, called quick CLASH (qCLASH). qCLASH requires a lower initial input of cells than its parent protocol. Additionally, a new fast-growing KSHV-negative endothelial cell line, named TIVE-EX-LTC cells, was established. qCLASH was performed on TIVE-EX-LTC cells latently infected with WT KSHV or a mutant virus lacking miR-K12-11/11*. A number of novel targets of the KSHV miRNAs were identified, including targets of miR-K12-11, the ortholog of cellular oncomiR miR-155. Many of the miRNA targets were involved in processes related to oncogenesis, such as glycolysis, angiogenesis, and cell cycle control.

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Cross-linking, ligation, and sequencing of hybrids reveals RNA-RNA interactions in yeast.

Project description:Many protein-protein and protein-nucleic acid interactions have been experimentally characterized, whereas RNA-RNA interactions have generally only been predicted computationally. Here, we describe a high-throughput method to identify intramolecular and intermolecular RNA-RNA interactions experimentally by cross-linking, ligation, and sequencing of hybrids (CLASH). As validation, we identified 39 known target sites for box C/D modification-guide small nucleolar RNAs (snoRNAs) on the yeast pre-rRNA. Novel snoRNA-rRNA hybrids were recovered between snR4-5S and U14-25S. These are supported by native electrophoresis and consistent with previously unexplained data. The U3 snoRNA was found to be associated with sequences close to the 3' side of the central pseudoknot in 18S rRNA, supporting a role in formation of this structure. Applying CLASH to the yeast U2 spliceosomal snRNA led to a revised predicted secondary structure, featuring alternative folding of the 3' domain and long-range contacts between the 3' and 5' domains. CLASH should allow transcriptome-wide analyses of RNA-RNA interactions in many organisms.

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The EBV microRNA targetome

Project description:In EBV-associated tumors, such as gastric cancer (GC) and Burkitt’s lymphoma (BL), a high proportion of microRNAs are virally encoded. To explore the targets of both viral and host microRNAs, we performed Crosslinking, Ligation, and Sequencing of Hybrids (CLASH). With this approach, we were able to quantify each Argonaute-bound microRNA-mRNA interaction in a GC (SNU719), and BL (Akata) cell line.

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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.

2021-06-23 | GSE164634 | GEO

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