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Series GSE29943 Query DataSets for GSE29943
Status Public on Jun 30, 2011
Title Unstressed HeLa cells and ELAVL1/HuR knock down conditions: polyA RNA-Seq, small RNA-Seq, and PAR-CLIP
Organism Homo sapiens
Experiment type Non-coding RNA profiling by high throughput sequencing
Expression profiling by high throughput sequencing
Summary 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.
Overall design 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.
Contributor(s) Lebedeva S, Jens M, Theil K, Schwanhaeusser B, Selbach M, Landthaler M, Rajewsky N
Citation(s) 21723171
Submission date Jun 13, 2011
Last update date May 15, 2019
Contact name Marvin Jens
Phone +493094062989
Organization name Max Delbrueck Center for Molecular Medicine
Department Systems Biology of Gene Regulatory Elements
Lab Rajewsky lab
Street address Robert Rössle Str. 10 (H. 87)
City Berlin
ZIP/Postal code 13092
Country Germany
Platforms (2)
GPL9115 Illumina Genome Analyzer II (Homo sapiens)
GPL11154 Illumina HiSeq 2000 (Homo sapiens)
Samples (9)
GSM741170 HeLa mock knockdown 5d
GSM741171 HeLa ELAVL1/HuR siRNA1 2d
GSM741172 HeLa ELAVL1/HuR siRNA1 5d
SRA SRP007498
BioProject PRJNA140779

Download family Format
SOFT formatted family file(s) SOFTHelp
MINiML formatted family file(s) MINiMLHelp
Series Matrix File(s) TXTHelp

Supplementary file Size Download File type/resource
GSE29943_RAW.tar 282.0 Mb (http)(custom) TAR (of BED, WIG)
GSE29943_consensus_premrna_sites.bed.gz 513.9 Kb (ftp)(http) BED
GSE29943_genes_fpkm_tracking.txt.gz 796.6 Kb (ftp)(http) TXT
GSE29943_sRNA_log2fc.txt.gz 17.2 Kb (ftp)(http) TXT
SRA Run SelectorHelp
Processed data provided as supplementary file
Raw data are available in SRA
Processed data are available on Series record

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