ABSTRACT: Integrative regulatory mapping indicates that the RNA-binding protein HuR (ELAVL1) couples pre-mRNA processing and mRNA stability [sequence data]
Project description:Integrative regulatory mapping indicates that the RNA-binding protein HuR (ELAVL1) couples pre-mRNA processing and mRNA stability In this dataset, we employed two distinct experiments. 1) HuR RIP-chip to identify mRNA targets of HuR. 2) HuR knockdown to identify mRNAs whose expression are dependent on HuR.
Project description:Integrative regulatory mapping indicates that the RNA-binding protein HuR (ELAVL1) couples pre-mRNA processing and mRNA stability In this dataset, we employed two distinct experiments. 1) HuR RIP-chip to identify mRNA targets of HuR. 2) HuR knockdown to identify mRNAs whose expression are dependent on HuR. All 12 samples were normalized with PLIER using Affymetrix power tools. To identify RNA targets of HuR, HuR RIP samples were compared to Mock RIP samples. To identify RNA regulated by HuR, HuR knockdown samples were compared to mock knockdown samples.
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. HuR (ELAVL1) PAR-CLIP
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.
Project description:Triacylglyceride (TAG) synthesis in the small intestine determines the absorption of dietary fat, but the mechanisms underlying are largely unknown. Here, we report that the RNA-binding protein HuR (ELAVL1) promotes TAG synthesis in the small intestine. HuR associates with the 3’UTR of Dgat2 mRNA and the introns 1 of Mgat2 pre-mRNA. Association of HuR with Dgat2 3’UTR stabilizes Dgat2 mRNA, while association of HuR with intron 1 of Mgat2 pre-mRNA promotes the processing of Mgat2 pre-mRNA. Intestinal epithelium-specific HuR knockout reduces the expression of DGAT2 and MGAT2, thereby reducing the dietary fat absorption through TAG synthesis and mitigating high-fat diet (HFD)-induced non-alcoholic fatty liver disease (NAFLD) and obesity. Our findings highlight a critical role of HuR in promoting dietary fat absorption.
Project description:Triacylglyceride (TAG) synthesis in the small intestine determines the absorption of dietary fat, but the mechanisms underlying are largely unknown. Here, we report that the RNA-binding protein HuR (ELAVL1) promotes TAG synthesis in the small intestine. HuR associates with the 3’UTR of Dgat2 mRNA and the introns 1 of Mgat2 pre-mRNA. Association of HuR with Dgat2 3’UTR stabilizes Dgat2 mRNA, while association of HuR with intron 1 of Mgat2 pre-mRNA promotes the processing of Mgat2 pre-mRNA. Intestinal epithelium-specific HuR knockout reduces the expression of DGAT2 and MGAT2, thereby reducing the dietary fat absorption through TAG synthesis and mitigating high-fat diet (HFD)-induced non-alcoholic fatty liver disease (NAFLD) and obesity. Our findings highlight a critical role of HuR in promoting dietary fat absorption.
Project description:RNA binding protein, Human Antigen R (HuR/ELAVL1), regulates mRNA stability of key species involved in pancreatic ductal adenocarcinoma (PDAC) cell survival under conditions of chemotherapeutic stress, hypoxia, low glucose environment. We used RNA immunoprecipitation-microarray or RNP-IP from cytoplasmic extracts of PDAC cell lines exposed to chemotherpaeutic stress (olaparib and gemcitabine) to evaluate whole transcriptome gene expression profile associated with HuR and find novel trargtes associated with DNA repair functions of HuR.
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.
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. PolyA mRNA was extracted from anti HuR siRNA treated and mock transfected HeLa cells to identify changes in mRNA expression and splicing. 2x100 paired end sequencing was performed according to the protocol on the Illumina HiSeq. PARCLIP was performed as in Hafner et. Al 2010 but with an antibody against endogenous HuR (3A2, Santa Cruz, sc-5261) in unstressed HeLa cells. We used, independently, 4-thiouridine (4SU) and 6-thioguanosine (6SG) to assess a possible nucleotide bias. As our proteomics measurements required labeling of cells in a special medium we also performed PAR-CLIP on cells grown in SILAC medium. Altogether we performed three PAR-CLIP experiments: 4SU labeling in standard DMEM medium, 4SU labeling in SILAC medium (as a replicate) and 6SG labeling in SILAC medium. Small RNA was extracted from anti HuR siRNA treated and mock transfected HeLa cells to identify changes in mRNA expression. Sequencing was performed on Illumina GAII using the standard sRNA 36cycle protocol.