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Homo sapiens

ABSTRACT: Transcriptome-wide identification and validation of interactions between the miRNA machinery and HuR on mRNA targets [HuR]

PROVIDER: PRJNA397429 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR5906480.fastq.gz	Fastqsanger.gz
	SRR5906481.fastq.gz	Fastqsanger.gz
	SRR5906482.fastq.gz	Fastqsanger.gz
	SRR5906483.fastq.gz	Fastqsanger.gz
	SRR5906484.fastq.gz	Fastqsanger.gz

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Transcriptome-wide identification and validation of interactions between the miRNA machinery and HuR on mRNA targets

Project description:Transcriptome-wide identification and validation of interactions between the miRNA machinery and HuR on mRNA targets

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Project description:Transcriptome-wide identification and validation of interactions between the miRNA machinery and HuR on mRNA targets [Ago2]

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Transcriptome-wide identification and validation of interactions between the miRNA machinery and HuR on mRNA targets [HuR]

Project description:The 3’ UTR of messenger RNAs serves as the regulatory region that mediates post-transcriptional control by microRNAs and RNA-binding proteins (RBPs). Aside from individual sequence-specific binding and regulation, examples of interaction between these factors at particular 3’ UTR sites have emerged in recent studies. However, the whole picture of such higher-order regulatory modules across the transcriptome is lacking. Here, we investigate the interactions between HuR, a ubiquitous RBP, and Ago2, a core effector of the miRNA pathway, at the transcriptome-wide level. Using HITS-CLIP, we map HuR and miRNA binding sites on human 3’UTRs and assess their co-occurrence. Additionally, we demonstrate global effects of HuR knockdown on Ago2 occupancy, suggesting a co-regulatory relationship. Focusing on sites of Ago2-HuR overlap, 13 candidates were screened in luciferase reporter assays, compared to miRNA site mutant controls. Eleven of the sites showed a repressive activity, which displayed significant de-repression upon subsequent testing of the reporters in Dicer-null cells, substantiating miRNA dependence. To experimentally test for HuR’s role in co-regulation, we tested the reporters in CRISPR-generated HuR KO cells. Three of the miRNA sites demonstrated altered activities, indicating that HuR has an effect on miRNA repression at those sites. Our study presents an efficient search and validation system for studying miRNA-HuR interactions, which expands our understanding of the combinatorial post-transcriptional control of gene expression at the 3’ UTR.

2018-01-19 | GSE102320 | GEO

Transcriptome-wide identification and validation of interactions between the miRNA machinery and HuR on mRNA targets [Ago2]

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Unstressed HeLa cells and ELAVL1/HuR knock down conditions: polyA RNA-Seq, small RNA-Seq, and PAR-CLIP

Project description:Post-transcriptional gene regulation relies on hundreds of RNA binding proteins (RBPs) but the function of most RBPs is unknown. The human RBP HuR/ELAVL1 is a conserved mRNA stability regulator. We used PAR-CLIP, a method based on RNA-protein crosslinking, to identify transcriptome wide ~26,000 HuR binding sites. These sites were on average highly conserved, enriched for HuR binding motifs and mainly located in 3' untranslated regions. Surprisingly, many sites were intronic, implicating HuR in splicing. Upon HuR knock down, mRNA levels and protein synthesis of thousands of target genes was down regulated, validating functionality. HuR and miRNA binding sites tended to reside nearby but generally did not overlap. Additionally, HuR knock down triggered strong and specific up regulation of miR-7. In summary, we identified thousands of direct and functional HuR targets, found a human miRNA controlled by HuR, and propose a role for HuR in splicing.

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Integrative regulatory mapping indicates that the RNA-binding protein HuR (ELAVL1) couples pre-mRNA processing and mRNA stability [sequence data]

Project description:RNA-binding proteins coordinate the fates of multiple RNAs, but the principles underlying these global interactions remain poorly understood. We elucidated regulatory mechanisms of the RNA-binding protein HuR, by integrating data from diverse high-throughput targeting technologies, specifically PAR-CLIP, RIP-chip, and whole-transcript expression profiling. The number of binding sites per transcript, degree of HuR-association, and degree of HuR-dependent RNA stabilization were positively correlated. Pre-mRNA and mature mRNA containing both intronic and 3' UTR binding sites were more highly stabilized than transcripts with only 3' UTR or only intronic binding sites, suggesting that HuR couples pre-mRNA processing with mature mRNA stability. We also observed HuR-dependent splicing changes and substantial binding of HuR in poly-pyrimidine tracts of pre-mRNAs. Comparison of the spatial patterns surrounding HuR and miRNA binding sites provided functional evidence for HuR-dependent antagonism of proximal miRNA-mediated repression. We conclude that HuR coordinates gene expression outcomes at multiple interconnected steps of RNA processing.

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Integrative regulatory mapping indicates that the RNA-binding protein HuR (ELAVL1) couples pre-mRNA processing and mRNA stability [sequence data]

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Expression data from mouse T cells after CLIP (Crosslinking immunoprecipitation) procedures (mRNA)

Project description:Molecular networks based on genome-wide mRNA and miRNA expression in activated T cells are interated by co-regulation with miRNA and protein-coding genes during immune responses, however, accurate prediction of miRs and their targets remains a challenge. Furthermore, the specific changes in the expression of miRNAs and/or mRNAs in allogeneic activated T cells during HCT would be distinct from those responding to non-specific stimulation. Recently, Ago-CLIP has been demonstrated to provide a robust platform for identification of precise miRNA-mRNA interactions by definitively distinguishing direct from indirect miRNA-target interactions. We utilized CLIP procedure followed by screening with standard microarray platforms for miRNA and mRNA transcripts to demonstrate the miR-mRNA landscape and identify novel molecular targets for modulating T cell responses.

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Imperfect centered miRNA binding sites are common and can mediate functional repression of target mRNAs [sequencing]

Project description:Whole transcriptome identification of direct targets identify a new class of miRNA::mRNA interactions.

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