In vivo and transcriptome-wide identification of RNA-binding protein target sites [PAR-CLIP]
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ABSTRACT:
ORGANISM(S): Caenorhabditis elegans
PROVIDER: GSE33569 | GEO | 2011/12/09
SECONDARY ACCESSION(S): PRJNA154339
REPOSITORIES: GEO
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Project description:Animal mRNAs are regulated by hundreds of RNA binding proteins (RBPs). The identification of RBP targets is crucial for understanding their function. A recent method, PAR-CLIP, uses photoreactive nucleosides to crosslink RBPs to target RNAs in cells prior to immunoprecipitation. Here, we establish iPAR-CLIP (in vivo PAR-CLIP) to determine, at nucleotide resolution, transcriptome-wide target sites of GLD-1, a conserved, germline-specific translational repressor in C. elegans. We identified 439 reproducible targets and demonstrate an excellent dynamic range of target detection by iPAR-CLIP. Upon GLD-1 knock-down, protein but not mRNA expression of the 439 targets was specifically and highly significantly upregulated, demonstrating functionality. Finally, we discovered strongly conserved GLD-1 binding sites nearby the start codon of target genes. We propose that GLD-1 interacts with the translation machinery nearby the start codon, a so far unknown mode of gene regulation in eukaryotes.
2011-12-09 | GSE33573 | GEO
Project description:Animal mRNAs are regulated by hundreds of RNA binding proteins (RBPs). The identification of RBP targets is crucial for understanding their function. A recent method, PAR-CLIP, uses photoreactive nucleosides to crosslink RBPs to target RNAs in cells prior to immunoprecipitation. Here, we establish iPAR-CLIP (in vivo PAR-CLIP) to determine, at nucleotide resolution, transcriptome-wide target sites of GLD-1, a conserved, germline-specific translational repressor in C. elegans. We identified 439 reproducible targets and demonstrate an excellent dynamic range of target detection by iPAR-CLIP. Upon GLD-1 knock-down, protein but not mRNA expression of the 439 targets was specifically and highly significantly upregulated, demonstrating functionality. Finally, we discovered strongly conserved GLD-1 binding sites nearby the start codon of target genes. We propose that GLD-1 interacts with the translation machinery nearby the start codon, a so far unknown mode of gene regulation in eukaryotes. PolyA mRNA was extracted from young adult wildtype (N2) worms and young adult germline less worms (glp-4(bn2) TS) to identify and quantify genes expressed in the young adult germline by sequencing. 2x100 paired end sequencing was performed according to the protocol on the Illumina HiSeq 2000.
2011-12-09 | E-GEOD-33573 | biostudies-arrayexpress
Project description:Animal mRNAs are regulated by hundreds of RNA binding proteins (RBPs). The identification of RBP targets is crucial for understanding their function. A recent method, PAR-CLIP, uses photoreactive nucleosides to crosslink RBPs to target RNAs in cells prior to immunoprecipitation. Here, we establish iPAR-CLIP (in vivo PAR-CLIP) to determine, at nucleotide resolution, transcriptome-wide target sites of GLD-1, a conserved, germline-specific translational repressor in C. elegans. We identified 439 reproducible targets and demonstrate an excellent dynamic range of target detection by iPAR-CLIP. Upon GLD-1 knock-down, protein but not mRNA expression of the 439 targets was specifically and highly significantly upregulated, demonstrating functionality. Finally, we discovered strongly conserved GLD-1 binding sites nearby the start codon of target genes. We propose that GLD-1 interacts with the translation machinery nearby the start codon, a so far unknown mode of gene regulation in eukaryotes. Arrested L1 worms were grown in liquid culture supplemented with 2mM 4SU or 6SG. 250,000 worms were sufficient for one iPAR-CLIP experiment. Living adult worms were transferred to NGM plates and crosslinked on ice using a Stratalinker (Stratagene) with customized 365nm UV-lamps (energy setting: 2J/cm2). Worms were lysed on ice by douncing in NP40 lysis buffer (50 mM HEPES-K pH 7.5, 150 mM KCl, 2 mM EDTA, 0.5% (v/v) NP-40, 0.5 mM DTT, protease inhibitor cocktail (Roche)). Cleared lysates were treated with RNase T1 (Fermentas) (final concentration 1U/?l) for 15 min at 22ºC. GLD-1::GFP::FLAG fusion proteins were immunoprecipitated for 1h at 4ºC using anti-FLAG antibody (Sigma, F3165) coupled to Protein G magnetic beads (Invitrogen). For one iPAR-CLIP experiment (1ml cleared lysate obtained from 250,000 worms), 300µl beads and 150µg antibody were used. Immunoprecipitates were treated with RNase T1 (100U/?l) for exactly 12 min at 22 ºC. Subsequently, PAR-CLIP was carried out as described previously (Hafner et al, 2010). cDNA libraries were sequenced on a Genome Analyzer II (Illumina).
2011-12-09 | E-GEOD-33569 | biostudies-arrayexpress
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
Project description:RNA-binding proteins (RBPs) are critical regulators of gene expression and elucidating the interactions of RBPs with their RNA targets is necessary to understand how combinations of RBPs control transcriptome expression. The Quaking-related (QR) sub-family of STAR domain RBPs includes developmental regulators and tumor suppressors such as C. elegans GLD-1, which functions as a master regulator of germ line development. To understand how GLD-1 interacts with the transcriptome, we identified GLD-1 associated mRNAs by a ribonomic approach. The scale of GLD-1 mRNA interactions allowed us to determine rules governing GLD-1 target selection and to derive a predictive model where GLD-1 association with mRNA is based on the number and strength of 7-mer GLD-1 binding elements (GBEs) within UTRs. GLD-1/mRNA interactions were quantified, and predictions were verified both in vitro and in live animals, including by M-bM-^@M-^XtransplantationM-bM-^@M-^Y experiments where M-bM-^@M-^XweakM-bM-^@M-^Y and M-bM-^@M-^XstrongM-bM-^@M-^Y GBEs imposed translational repression of increasing strength on a non-target mRNA.Importantly, this study provides a unique quantitative picture of how an RBP interacts with its mRNA targets. As combinatorial regulation by multiple RBPs is thought to regulate gene expression, quantification of RBP/mRNA interactions should be a way to predict and potentially modify biological outcomes of complex posttranscriptional regulatory networks, and our analysis suggests that such an approach is possible. Wild-type (N2) or gld-1(q485); ozIs2 [gld-1::gfp::flag] worms were synchronized and harvested as young adults. To identify GLD-1 associated transcripts, two independent IP strategies were performed: 1. Comparison of anti-FLAG IP to anti-MYC IP on the gld-1(q485); ozIs2 [gld-1::gfp::flag] worm extract, 2. Comparison of anti-FLAG IP on the gld-1(q485); ozIs2 [gld-1::gfp::flag] worm extract to anti-FLAG IP on wild type extract. All IPs were performed in triplicate (12 IPs).
2011-02-04 | E-GEOD-21591 | biostudies-arrayexpress
Project description:Crosslinking and immunoprecipitation (CLIP) is increasingly used to map transcriptome-wide binding sites of RNA-binding proteins (RBPs). We developed a method for CLIP data analysis and applied it to compare 254 nm CLIP with PAR-CLIP, which involves crosslinking of photoreactive nucleotides with 365 nm UV light. We found small differences in the accuracy of these methods in identifying binding sites of HuR, a protein that binds low-complexity sequences and Argonaute 2, which has a complex binding specificity. We show that crosslink-induced mutations lead to single-nucleotide resolution for both PAR-CLIP and CLIP. Our results confirm the expectation from original CLIP publications that RNA-binding proteins do not protect sufficiently their sites under the denaturing conditions used during the CLIP procedure, and we show that extensive digestion with sequence-specific ribonucleases strongly biases the set of recovered binding sites. We finally show that this bias can be substantially reduced by milder nuclease digestion conditions. We performed duplicate experiments for each variant of the CLIP protocol (CLIP, PAR-CLIP), each protein (HuR, Ago2), and enzymatic digestion (complete T1 digestion, mild MNase digestion). In addition, we performed a single PAR-CLIP experiment with mild T1 digestion.
2011-05-15 | E-GEOD-28859 | biostudies-arrayexpress
Project description:RNA-binding proteins (RBPs) are critical regulators of gene expression and elucidating the interactions of RBPs with their RNA targets is necessary to understand how combinations of RBPs control transcriptome expression. The Quaking-related (QR) sub-family of STAR domain RBPs includes developmental regulators and tumor suppressors such as C. elegans GLD-1, which functions as a master regulator of germ line development. To understand how GLD-1 interacts with the transcriptome, we identified GLD-1 associated mRNAs by a ribonomic approach. The scale of GLD-1 mRNA interactions allowed us to determine rules governing GLD-1 target selection and to derive a predictive model where GLD-1 association with mRNA is based on the number and strength of 7-mer GLD-1 binding elements (GBEs) within UTRs. GLD-1/mRNA interactions were quantified, and predictions were verified both in vitro and in live animals, including by ‘transplantation’ experiments where ‘weak’ and ‘strong’ GBEs imposed translational repression of increasing strength on a non-target mRNA.Importantly, this study provides a unique quantitative picture of how an RBP interacts with its mRNA targets. As combinatorial regulation by multiple RBPs is thought to regulate gene expression, quantification of RBP/mRNA interactions should be a way to predict and potentially modify biological outcomes of complex posttranscriptional regulatory networks, and our analysis suggests that such an approach is possible.
2011-02-04 | GSE21591 | GEO
Project description:Human LIN28A and B are RNA-binding proteins (RBPs) conserved in animals with important roles during development and stem cell reprogramming. We used Photoactivatable-Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation (PAR-CLIP) in HEK293 cells and identified a largely overlapping set of ~3,000 mRNAs at ~9,500 sites located in the 3M-bM-^@M-^YUTR and CDS. In vitro and in vivo, LIN28 preferentially bound single-stranded RNA containing a uridine-rich element and one or more flanking guanosines, and appeared to be able to disrupt base-pairing to access these elements when embedded in predicted secondary structure. In HEK293 cells, LIN28 protein binding mildly stabilized target mRNAs and increased protein abundance. The top targets were its own mRNAs and those of other RBPs and cell-cycle regulators. Alteration of LIN28 protein levels also negatively regulated the abundance of some, but not all let-7 miRNA family members, indicating sequence-specific binding of let-7 precursors to LIN28 proteins and competition with cytoplasmic miRNA biogenesis factors. LIN28 protein PAR-CLIP
2013-02-26 | E-GEOD-44615 | biostudies-arrayexpress
Project description:We employed PAR-CLIP, a recently developed method based on RNA-protein crosslinking, to identify Cirbp and Rbm3 binding sites at transcriptome-level. Genome-wide RNA-seq analysis indicated that cold temperature leads to extensive 3’UTR lengthening whereas the loss of Cirbp or Rbm3 resulted in 3’UTR shortening. Combining with PAR-CLIP results, we found that these two RBPs repress the polyadenylation adjacent to their binding sites.
2013-05-01 | GSE40468 | GEO
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.
2011-06-30 | GSE29943 | GEO