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

ABSTRACT: 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.

ORGANISM(S): Homo sapiens

PROVIDER: GSE29943 | GEO | 2011/06/30

SECONDARY ACCESSION(S): PRJNA140779

REPOSITORIES: GEO

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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. 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.

2011-06-29 | E-GEOD-29943 | biostudies-arrayexpress

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. HuR (ELAVL1) PAR-CLIP

2011-08-19 | E-GEOD-29779 | biostudies-arrayexpress

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

2011-08-19 | GSE29779 | GEO

In vivo CRISPR screening unveils RNA binding protein dependencies for leukemic stem cells and identifies ELAVL1 as a potential therapeutic target [eCLIPseq]

Project description:RNA binding protein (RBP)-directed post-transcriptional control of stem cell fate represents an underexplored mechanism driving hematopoietic stemness and transformation. We show that a unique set of RBPs are specifically enriched in leukemic stem cells (LSCs) of human primary acute myeloid leukemia (AML) but repressed in normal hematopoietic stem cells. Using an in vivo CRISPR-Cas9-mediated screening approach, we identify 33 key RBPs specifically essential for LSC-mediated MLL-AF9/NrasG12D AML. Knock-down/genetic ablation of the hit RBP Elavl1 in genetically distinct leukemias significantly reduced LSC numbers, and hampered leukemic engraftment. In human AML we show impairment of LSC-driven in vivo leukemic reconstitution and selective depletion of AML progenitors upon ELAVL1 targeting, highlighting the potential clinical importance of our findings. Profiling of the leukemic ELAVL1-mRNA interactome revealed hematopoietic differentiation, RNA splicing and mitochondrial metabolism as major pathways impacted by ELAVL1. Altogether, this work demonstrates that Elavl1 and other RBPs are critical regulators of LSC-survival and self-renewal.

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HuR (ELAVL1) RIP-chip and knockdown exon array

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.

2011-08-18 | E-GEOD-29778 | biostudies-arrayexpress

ELAVL1 iCLIP in HEK293 cells

Project description:This experiment aims to map the HuR (ELAVL1) binding to the global transcriptome in HEK293 cells.

2023-04-30 | E-MTAB-11854 | biostudies-arrayexpress

Global Target mRNA Specification and Regulation by RNA-Binding Protein ZFP36/Tristetraprolin/TTP [PAR-CLIP]

Project description:Tristetraprolin/ZFP36/TTP and ELAVL1/HuR are two disease-relevant RNA-binding proteins (RBPs) that both interact with AU-rich sequences but have antagonistic roles. While ELAVL1 binding has been profiled in several studies, the precise in vivo binding specificity of ZFP36 has not been investigated on a global scale. We determined ZFP36 binding preferences using cross-linking and immunoprecipitation in human embyonic kidney cells and examined combinatorial regulation of AU-rich elements by ZFP36 and ELAVL1. Among the targets ZFP36 binds and negatively regulates the mRNA of genes encoding proteins necessary for immune function and cancer, and other RBPs. Using partial correlation analysis, we were able to quantify the association between ZFP36 binding sites and differential target RNA abundance from ZFP36 overexpression independent of effects from confounding features, such as 3M-bM-^@M-^Y UTR length. We identified thousands of overlapping ZFP36 and ELAVL1 binding sites, in 1,313 genes. ZFP36 preferentially interacts with and regulates AU-rich sequences while ELAVL1 prefers predominantly U- and CU-rich sequences. RNA target specificity identified by global in vivo ZFP36-mRNA interactions were quantitatively similar to previously reported in vitro binding affinities. ZFP36 and ELAVL1 both bind an overlapping spectrum of RNA sequences, yet with differential relative preferences that dictate combinatorial regulatory potential. Our findings and methodology delineate an approach to untangle the in vivo combinatorial regulation by RNA-binding proteins. FLAG-HA ZFP36

2013-12-24 | E-GEOD-53184 | biostudies-arrayexpress

HuR targetome analysis in THP-1 macrophages using HuR FLASH-Seq.

Project description:FLASH, a novel method, was utilized to identify binding sites of RNA-binding proteins (RBPs) with single-nucleotide resolution (Nucleic Acids Res 2020;48:e15). In order to identify HuR target genes in THP-1 macrophages after circARCN1 regulation, we performed FLASH experiments using the anti-HuR antibody on THP-1 macrophages with circARCN1 knockdown or overexpression. The resulting FLASH products were then subjected to RNA sequencing.

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