Small RNA was extracted from anti HuR siRNA treated and mock transfected HeLa cells to identify changes in mRNA expression. 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. As our proteomics measurements required labeling of cells in a special medium we also performed PAR-CLIP on cells grown in SILAC medium. We used, independently, 4-thiouridine (4SU) and 6-thioguanosine (6SG) to assess a possible nucleotide bias. Al 2010 but with an antibody against endogenous HuR (3A2, Santa Cruz, sc-5261) in unstressed HeLa cells. 2x100 paired end sequencing was performed according to the protocol on the Illumina HiSeq. PolyA mRNA was extracted from anti HuR siRNA treated and mock transfected HeLa cells to identify changes in mRNA expression and splicing. 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. Additionally, HuR knock down triggered strong and specific up regulation of miR-7. HuR and miRNA binding sites tended to reside nearby but generally did not overlap. Upon HuR knock down, mRNA levels and protein synthesis of thousands of target genes was down regulated, validating functionality. Surprisingly, many sites were intronic, implicating HuR in splicing. ![]() ![]() These sites were on average highly conserved, enriched for HuR binding motifs and mainly located in 3' untranslated regions. ![]() We used PAR-CLIP, a method based on RNA-protein crosslinking, to identify transcriptome wide ~26,000 HuR binding sites. The human RBP HuR/ELAVL1 is a conserved mRNA stability regulator. Post-transcriptional gene regulation relies on hundreds of RNA binding proteins (RBPs) but the function of most RBPs is unknown. Non-coding RNA profiling by high throughput sequencingĮxpression profiling by high throughput sequencing Unstressed HeLa cells and ELAVL1/HuR knock down conditions: polyA RNA-Seq, small RNA-Seq, and PAR-CLIP GEO help: Mouse over screen elements for information.
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