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Halo-enhanced Ago2 pulldown (HEAP) to identify miRNA targets in murine colonic tumor +/- oncogenic K-Ras-G12D

ABSTRACT: Halo-enhanced Ago2 pulldown (HEAP) to identify miRNA targets in murine colonic tumor +/- oncogenic K-Ras-G12D

PROVIDER: PRJNA784253 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR17055000.fastq.gz	Fastqsanger.gz
	SRR17055001.fastq.gz	Fastqsanger.gz
	SRR17055002.fastq.gz	Fastqsanger.gz
	SRR17055003.fastq.gz	Fastqsanger.gz
	SRR17055004.fastq.gz	Fastqsanger.gz

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Project description:The identification of miRNA targets by Ago2-CLIP methods has provided major insights into the biology of this important class of non-coding RNAs. However, these methods are technically challenging and not easily translated to an in vivo setting. To overcome these limitations and to facilitate the investigation of miRNA functions in mice, we have developed a method (HEAP) to map miRNA-mRNA binding sites. This method uses a novel mouse strain harboring a conditional Halo-Ago2 allele expressed from the Ago2 locus. By using a resin conjugated to the HaloTag ligand, Ago2-miRNA-mRNA complexes can be efficiently purified from cells and tissues expressing Halo-Ago2. We demonstrate the reproducibility and sensitivity of this method in embryonic stem cells, in embryos, in adult tissues and in autochthonous mouse models of cancers. These tools and datasets are valuable resources to the scientific community and will facilitate the characterization of miRNA functions under physiological and pathological conditions.

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Project description:K-Ras is frequently hyperactivated in human cancers through gain-of-function mutations that drive tumorigenesis. K-RasG12D, the most common oncogenic K-Ras allele, triggers massive transcriptomic and proteomic changes in the murine colon. Here, we report a comprehensive profile of physiological miRNA targets in murine colonic epithelium and tumor expressing K-RasG12D. Combining it with transcriptional, transcriptomic, and proteomic landscapes, we uncover a K-RasG12D-induced global suppression of miRNA activity that up-regulates hundreds of genes post-transcriptionally. K-RasG12D suppresses Csnk1a1 and Csnk2a1, which can decrease Ago2 phosphorylation at Ser825/829/831/835. Hypo-phosphorylated Ago2 increases binding with mRNA, reducing its regulatory activity by locking Ago2 in a small set of target transcripts. While expanding the repertoire of miRNA targets identified, it functionally decreases active Ago2, resulting in global de-repression of miRNA targets. Our findings establish a regulatory relationship among K-Ras, Csnk1a1/Csnk2a1, and Ago2 that provides a mechanistic link between oncogenic K-Ras and the up-regulation of hundreds of miRNA targets.

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