CLIP analysis of AEG1
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA433326 | ENA |
REPOSITORIES: ENA
Similar Datasets
Project description:In the present study, we performed HITS-CLIP analysis for FUS using mouse brain to extensively characterize tits RNA-binding sites and functional roles in RNA metabolisms. We identified preferential binding of FUS to stem-and-loop structures but without any discernible consensus motifs. FUS was preferentially bound to introns and 3' untranslated regions, but the exon/intron boundaries were mostly devoid of FUS-tags. Analysis of position-dependence of FUS-binding sites in regulating inclusion and skipping of exons disclosed that FUS is bound broadly around the alternatively spliced exons. Among them, however, noticeable CLIP-tags were observed in the downstream introns. We also noticed that FUS occasionally binds to the antisense strands in the promoter regions. Global analysis of CLIP-tags and expression profiles revealed that binding of FUS to the promoter antisense regions downgregulates transcription of the sense strand. HITS-CLIP (High Throughput Sequencing after Crosslinking and Immunoprecipitation) experiments targeting FUS in mouse cerebrums derived from 12-week-old C57BL/6 mice
2012-07-26 | E-GEOD-37190 | biostudies-arrayexpress
Project description:Includes HITS-CLIP and PAR-CLIP of AEG1, including negative controls
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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: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.
2011-05-15 | GSE28859 | GEO
Project description:The identification of RNAs that are recognized by RNA-binding proteins (RNA-BPs) using techniques such as Crosslinking and Immunoprecipitation (CLIP) has revolutionized the genome-wide discovery of RNA-BP RNA targets. Among the different versions of CLIP that have been developed, the use of photoactivable nucleoside analogs has resulted in high efficiency photoactivable ribonucleoside-enhanced CLIP (PAR-CLIP) in vivo. Nonetheless, PAR-CLIP has not yet been applied in prokaryotes. To determine if PAR-CLIP can be used in prokaryotes, we determined suitable conditions for the incorporation of 4-thiouridine (4SU), a photoactivable nucleoside, into E. coli RNA and for the isolation of RNA crosslinked to RNA-BPs of interest. Applying this technique to Hfq, a well-characterized regulator of small RNA (sRNA)-messenger RNA (mRNA) interactions, we showed that PAR-CLIP identified most of the known sRNA targets of Hfq, as well as functionally relevant sites of Hfq-mRNA interactions at nucleotide resolution. Based on our findings, PAR-CLIP represents an improved method to identify both the RNAs and the specific regulatory sites that are recognized by RNA-BPs in prokaryotes.
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Project description:In the present study, we performed HITS-CLIP analysis for FUS using mouse brain to extensively characterize tits RNA-binding sites and functional roles in RNA metabolisms. We identified preferential binding of FUS to stem-and-loop structures but without any discernible consensus motifs. FUS was preferentially bound to introns and 3' untranslated regions, but the exon/intron boundaries were mostly devoid of FUS-tags. Analysis of position-dependence of FUS-binding sites in regulating inclusion and skipping of exons disclosed that FUS is bound broadly around the alternatively spliced exons. Among them, however, noticeable CLIP-tags were observed in the downstream introns. We also noticed that FUS occasionally binds to the antisense strands in the promoter regions. Global analysis of CLIP-tags and expression profiles revealed that binding of FUS to the promoter antisense regions downgregulates transcription of the sense strand.
2012-07-26 | GSE37190 | GEO