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Advancing Functional Utility of PAR-CLIP by Quantifying Background Binding to mRNAs and lncRNAs

ABSTRACT: We developed a method for measuring non-specific background in PAR-CLIP data demonstrating that covalently crosslinked background binding is common, reproducible and apparently universal. Furthermore, we show that quantitative determination of background is essential for identifying targets of weakly binding RNA-binding proteins and can substantially improve motif analysis.

ORGANISM(S): Homo sapiens

PROVIDER: GSE50989 | GEO | 2013/11/07

SECONDARY ACCESSION(S): PRJNA219483

REPOSITORIES: GEO

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Advancing Functional Utility of PAR-CLIP by Quantifying Background Binding to mRNAs and lncRNAs

Project description:We developed a method for measuring non-specific background in PAR-CLIP data demonstrating that covalently crosslinked background binding is common, reproducible and apparently universal. Furthermore, we show that quantitative determination of background is essential for identifying targets of weakly binding RNA-binding proteins and can substantially improve motif analysis. To define background binding events in PAR-CLIP data we performed the standard PAR-CLIP protocol (Hafner et al., Cell 2010.) on lysates expressing a commonly used non-RBP control, FLAG-GFP. After FLAG-tag immunopurification of UV 365nm irradiated lysates prepared from cells supplemented with 4-thiouridine (4SU), RNA was partially digested with RNase T1, radiolabeled and separated by SDS-PAGE. Reads were sequenced by Illumina HiSeq. PAR-CLIP was also performed for HuR. Included as well is a total from lysates treated like PAR-CLIP, but without immunoprecipitation (see sample description for more detail).

2013-11-07 | E-GEOD-50989 | biostudies-arrayexpress

Advancing the functional utility of PAR-CLIP by quantifying background binding to mRNAs and lncRNAs.

Project description:BackgroundSequence specific RNA binding proteins are important regulators of gene expression. Several related crosslinking-based, high-throughput sequencing methods, including PAR-CLIP, have recently been developed to determine direct binding sites of global protein-RNA interactions. However, no studies have quantitatively addressed the contribution of background binding to datasets produced by these methods.ResultsWe measured non-specific RNA background in PAR-CLIP data, demonstrating that covalently crosslinked background binding is common, reproducible and apparently universal among laboratories. We show that quantitative determination of background is essential for identifying targets of most RNA-binding proteins and can substantially improve motif analysis. We also demonstrate that by applying background correction to an RNA binding protein of unknown binding specificity, Caprin1, we can identify a previously unrecognized RNA recognition element not otherwise apparent in a PAR-CLIP study.ConclusionsEmpirical background measurements of global RNA-protein crosslinking are a necessary addendum to other experimental controls, such as performing replicates, because covalently crosslinked background signals are reproducible and otherwise unavoidable. Recognizing and quantifying the contribution of background extends the utility of PAR-CLIP and can improve mechanistic understanding of protein-RNA specificity, protein-RNA affinity and protein-RNA association dynamics.

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Transcriptome-wide identification of RNA-binding protein and microRNA target sites by PAR-CLIP: sequencing data

Project description:We applied 4-thiouridine to cultured cells expressing the FLAG/HA-tagged RNA-binding proteins (RBPs) followed by UV 365 nm irradiation. The crosslinked RNA-protein complexes were isolated by immunoprecipitation, and the covalently bound RNA was partially digested with RNase T1 and radiolabeled. The radiolabeled RNPs were subsequenctly separated by SDS-PAGE, the crosslinked RNA segments recovered and converted into a cDNA library and sequenced.

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Transcriptome-wide identification of RNA-binding protein and microRNA target sites by PAR-CLIP: sequencing data

2010-05-21 | E-GEOD-21918 | biostudies-arrayexpress

Chip-chip from E. coli MG1655 cells with different methods to detect SeqA and σ32 binding and causes of high background

Project description:Reinvestigation of the chromatin immunoprecipitation procedure led us to discover four causes of high background: non-unique sequences, incomplete reversion of crosslinks, washing with spin-columns and insufficient RNase treatment. We used a publishd method giving a high background signal and a modified chromatin immunoprecipitation method which could greatly reduce the false positive rate and apply it to analyze genome wide binding of SeqA and σ32 binding in E. coli. RNA polymerase binding in wt E. coli MG1655 with old method (two biological relpicates); comparison of SeqA-binding to wt with old and modified method (two biological replicates each); comparison of SeqA-binding with old and modified method to E. coli ΔseqA (one array each); comparison of crosslinked/reversed to non crosslinked E. coli chrom. DNA; σ32 binding at 30°C and 43°C (two biological replicates each); σ32 binding at 30°C and 43°C with shortened RNase digestion during ChIP (one array each).

2010-07-12 | E-GEOD-19053 | biostudies-arrayexpress

RNAs bound by the glucocorticoid receptor during dexamethasone treatment

Project description:The RNA binding ability of the glucocorticoid receptor (GR) remains an understudied area of GR regulation. Through in vitro binding assays, we identified hairpin RNAs as GR's preferred binding motif. To study how GR-bound RNAs change with dexamethasone treatment, we first generated stable U2OS cells expressing wild-type GR-HaloTag. We then treated the cells with dexamethsone every hour over three hours, UV crosslinked, harvested, and pulled down GR using a specific and covalently-bound HaloTag ligand. GR-bound RNAs were then isolated, library prepped, and sequenced.

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Transcriptome-wide identification of RNA-binding protein and microRNA target sites by PAR-CLIP: QKI data

Project description:To assess whether the transcripts identified by PAR-CLIP are regulated by the RNA-binding protein (RBP) Quaking (QKI), we analyzed the mRNA levels of mock-transfected and QKI-specific siRNA-transfected cells with microarrays. Transcripts crosslinked to QKI were significantly upregulated upon siRNA transfection, indicating that QKI negatively regulates bound mRNAs (Figure 3H of PMID 20371350), consistent with previous reports of QKI being a repressor.

2010-04-29 | GSE21574 | GEO

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eIF3 PAR-CLIP in 293T cells

Project description:Regulation of protein synthesis is fundamental for all aspects of eukaryotic biology by controlling development, homeostasis, and stress responses. The 13-subunit, 800-kDa eukaryotic initiation factor 3 (eIF3) organizes initiation factor and ribosome interactions required for productive translation. However, current understanding of eIF3 function does not explain genetic evidence correlating eIF3 deregulation with tissue-specific cancers and developmental defects. Here we report the genome-wide discovery of human transcripts that interact with eIF3 using photo-activatable crosslinking and immunoprecipitation (PAR-CLIP). eIF3 binds to a highly specific programme of messenger RNAs (mRNAs) involved in cell growth control processes, including cell cycling, differentiation, and apoptosis, via the mRNA 5' untranslated region (5' UTR). Surprisingly, functional analysis of the interaction between eIF3 and two mRNAs encoding cell proliferation regulators, c-Jun and BTG1, reveals that eIF3 employs different modes of RNA stem loop binding to exert either translational activation or repression. Our findings illuminate a new role for eIF3 in governing a specialized repertoire of gene expression and suggest that binding of eIF3 to specific mRNAs could be targeted to control carcinogenesis. 293T cells were treated with 4-thiouridine and protein-RNA complexes were crosslinked, and eIF3-RNA complexes were immunoprecipitated.

2015-04-06 | E-GEOD-65004 | biostudies-arrayexpress

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