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Analysis of CLIP and iCLIP methods for nucleotide-resolution studies of protein-RNA interactions.

ABSTRACT: UV cross-linking and immunoprecipitation (CLIP) and individual-nucleotide resolution CLIP (iCLIP) are methods to study protein-RNA interactions in untreated cells and tissues. Here, we analyzed six published and two novel data sets to confirm that both methods identify protein-RNA cross-link sites, and to identify a slight uridine preference of UV-C-induced cross-linking. Comparing Nova CLIP and iCLIP data revealed that cDNA deletions have a preference for TTT motifs, whereas iCLIP cDNA truncations are more likely to identify clusters of YCAY motifs as the primary Nova binding sites. In conclusion, we demonstrate how each method impacts the analysis of protein-RNA binding specificity.

SUBMITTER: Sugimoto Y

PROVIDER: S-EPMC4053741 | biostudies-literature | 2012 Aug

REPOSITORIES: biostudies-literature

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Analysis of CLIP and iCLIP methods for nucleotide-resolution studies of protein-RNA interactions.

Sugimoto Yoichiro Y König Julian J Hussain Shobbir S Zupan Blaž B Curk Tomaž T Frye Michaela M Ule Jernej J

Genome biology 20120803 8

UV cross-linking and immunoprecipitation (CLIP) and individual-nucleotide resolution CLIP (iCLIP) are methods to study protein-RNA interactions in untreated cells and tissues. Here, we analyzed six published and two novel data sets to confirm that both methods identify protein-RNA cross-link sites, and to identify a slight uridine preference of UV-C-induced cross-linking. Comparing Nova CLIP and iCLIP data revealed that cDNA deletions have a preference for TTT motifs, whereas iCLIP cDNA truncati ...[more]

PMID: 22863408

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Analysis of CLIP and iCLIP methods for nucleotide-resolution studies of protein-RNA interactions

Project description:UV cross-linking and immunoprecipitation (CLIP) and individual-nucleotide resolution CLIP (iCLIP) are the most frequently used methods to study protein-RNA interactions in the intact cells and tissues, but their relative advantages or inherent biases have not been evaluated. To benchmark CLIP and iCLIP method, we performed iCLIP with Nova protein, which is the most extensively studied protein by CLIP. Further, we assessed UV-C-induced cross-linking preferences, by exploiting the UV-independent formation of covalent RNA cross-links of the mutant RNA methylase NSUN2.

2012-07-20 | E-MTAB-1008 | biostudies-arrayexpress

Analysis of CLIP and iCLIP methods for nucleotide-resolution studies of protein-RNA interactions

Project description:Analysis of CLIP and iCLIP methods for nucleotide-resolution studies of protein-RNA interactions

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Mapping in vivo protein-RNA interactions at single-nucleotide resolution from HITS-CLIP data.

Project description:Mammalian RNA complexity is regulated through interactions of RNA-binding proteins (RBPs) with their target transcripts. High-throughput sequencing together with UV-crosslinking and immunoprecipitation (HITS-CLIP) is able to globally map RBP-binding footprint regions at a resolution of ~30-60 nucleotides. Here we describe a systematic way to analyze HITS-CLIP data to identify exact crosslink sites, and thereby determine protein-RNA interactions at single-nucleotide resolution. We found that reverse transcriptase used in CLIP frequently skips the crosslinked amino-acid-RNA adduct, resulting in a nucleotide deletion. Genome-wide analysis of these crosslinking-induced mutation sites (CIMS) in HITS-CLIP data for Nova and Argonaute (Ago) proteins in mouse brain tissue revealed deletions in ~8-20% of mRNA tags, which mapped to Nova and Ago binding sites on mRNA or miRNA. CIMS analysis provides a general and more precise means of mapping protein-RNA interactions than currently available methods and insight into the biochemical properties of such interactions in living tissues.

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Global protein-RNA interaction mapping at single nucleotide resolution by iCLIP-seq.

Project description:Eukaryotic genomes encode a large number of RNA-binding proteins, which play critical roles in many aspects of gene regulation. To functionally characterize these proteins, a key step is to map their interactions with target RNAs. UV crosslinking and immunoprecipitation coupled with high-throughput sequencing has become the standard method for this purpose. Here we describe the detailed procedure that we have used to characterize the protein-RNA interactions of the mRNA 3' processing factors.

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Improved binding site assignment by high-resolution mapping of RNA-protein interactions using iCLIP.

Project description:Individual-nucleotide resolution crosslinking and immunoprecipitation (iCLIP) allows the determination of crosslinking sites of RNA-binding proteins (RBPs) on RNAs. iCLIP is based on ultraviolet light crosslinking of RBPs to RNA, reverse transcription and high-throughput sequencing of fragments terminating at the site of crosslinking. As a result, start sites of iCLIP fragments are expected to cluster with a narrow distribution, typically representing the site of direct interaction between the RBP and the RNA. Here we show that for several RBPs (eIF4A3, PTB, SRSF3, SRSF4 and hnRNP L), the start sites of iCLIP fragments show a fragment length-dependent broader distribution that can be shifted to positions upstream of the known RNA-binding site. We developed an analysis tool that identifies these shifts and can improve the positioning of RBP binding sites.

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Temporal-iCLIP captures co-transcriptional RNA-protein interactions.

Project description:Dynamic RNA-protein interactions govern the co-transcriptional packaging of RNA polymerase II (RNAPII)-derived transcripts. Yet, our current understanding of this process in vivo primarily stems from steady state analysis. To remedy this, we here conduct temporal-iCLIP (tiCLIP), combining RNAPII transcriptional synchronisation with UV cross-linking of RNA-protein complexes at serial timepoints. We apply tiCLIP to the RNA export adaptor, ALYREF; a component of the Nuclear Exosome Targeting (NEXT) complex, RBM7; and the nuclear cap binding complex (CBC). Regardless of function, all tested factors interact with nascent RNA as it exits RNAPII. Moreover, we demonstrate that the two transesterification steps of pre-mRNA splicing temporally separate ALYREF and RBM7 binding to splicing intermediates, and that exon-exon junction density drives RNA 5'end binding of ALYREF. Finally, we identify underappreciated steps in snoRNA 3'end processing performed by RBM7. Altogether, our data provide a temporal view of RNA-protein interactions during the early phases of transcription.

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Revised iCLIP-seq Protocol for Profiling RNA-protein Interaction Sites at Individual Nucleotide Resolution in Living Cells.

Project description:Individual nucleotide resolution UV cross-linking and immunoprecipitation followed by high-throughput sequencing (iCLIP-seq) is a powerful technique that is used to identify RNA-binding proteins' (RBP) binding sites on target RNAs and to characterize the molecular basis of posttranscriptional regulatory pathways. Several variants of CLIP have been developed to improve its efficiency and simplify the protocol [e.g., iCLIP2 and enhanced CLIP (eCLIP)]. We have recently reported that transcription factor SP1 functions in the regulation of alternative cleavage and polyadenylation through direct RNA binding. We utilized a modified iCLIP method to identify RNA-binding sites for SP1 and several of the cleavage and polyadenylation complex subunits, including CFIm25, CPSF7, CPSF100, CPSF2, and Fip1. Our revised protocol takes advantage of several features of the eCLIP procedure and also improves on certain steps of the original iCLIP method, including optimization of circularization of cDNA. Herein, we describe a step-by-step procedure for our revised iCLIP-seq protocol, that we designate as iCLIP-1.5, and provide alternative approaches for certain difficult-to-CLIP proteins. Key features Identification of RNA-binding sites of RNA-binding proteins (RBPs) at nucleotide resolution. iCLIP-seq provides precise positional and quantitative information on the RNA-binding sites of RBPs in living cells. iCLIP facilitates the identification of sequence motifs recognized by RBPs. Allows quantitative analysis of genome-wide changes in protein-RNA interactions. Revised iCLIP-1.5 protocol is more efficient and highly robust; it provides higher coverage even for low-input samples. Graphical overview.

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Implementation of PAR-CLIP to characterize RNA-protein interactions in prokaryotes at nucleotide resolution

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.

2022-10-18 | MSV000090547 | MassIVE

iCLIP reveals the function of hnRNP particles in splicing at individual nucleotide resolution.

Project description:In the nucleus of eukaryotic cells, nascent transcripts are associated with heterogeneous nuclear ribonucleoprotein (hnRNP) particles that are nucleated by hnRNP C. Despite their abundance, however, it remained unclear whether these particles control pre-mRNA processing. Here, we developed individual-nucleotide resolution UV cross-linking and immunoprecipitation (iCLIP) to study the role of hnRNP C in splicing regulation. iCLIP data show that hnRNP C recognizes uridine tracts with a defined long-range spacing consistent with hnRNP particle organization. hnRNP particles assemble on both introns and exons but remain generally excluded from splice sites. Integration of transcriptome-wide iCLIP data and alternative splicing profiles into an 'RNA map' indicates how the positioning of hnRNP particles determines their effect on the inclusion of alternative exons. The ability of high-resolution iCLIP data to provide insights into the mechanism of this regulation holds promise for studies of other higher-order ribonucleoprotein complexes.

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Individual-nucleotide resolution CLIP and high-throughput sequencing to map hnRNP L-RNA interactions in HeLa cells

Project description:Individual-nucleotide resolution UV-crosslinking and immunoprecipitation (iCLIP) combined with high-throughput sequencing was used to generate a transcriptome-wide binding map of hnRNP L. Supplementary file GSE37560_hnRNPL_crosslink_site.bed includes filtered crosslink sites of hnRNPL: combining data from all 3 experiments. 3 biological replicates of hnRNP L-specific and control (Flag) co-immunoprecipitated RNA after UV-crosslinking in HeLa cells

2014-02-13 | E-GEOD-37560 | biostudies-arrayexpress

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