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RNA-binding proteins (RBPs) mediate important co- and post-transcriptional gene regulation by binding pre-mRNA in a sequence- and/or structure-specific manner. For a comprehensive understanding of RBP function, transcriptome-wide mapping of the RNA-binding sites is essential, and CLIP-seq methods have been developed to elucidate protein/RNA interactions at high resolution. CLIP-seq combines protein/RNA UV-crosslinking with immunoprecipitation (CLIP) followed by high-throughput sequencing of crosslinked RNA fragments. To overcome the limitations of low RNA-protein crosslinking efficiency in standard CLIP-seq, photoactivatable-ribonucleoside-enhanced CLIP (PAR-CLIP) has been developed. Here, living cells or whole organisms are fed photo-activatable nucleoside analogs that are incorporated into nascent RNA transcripts before UV treatment. This allows greater crosslinking efficiency at comparable radiation doses for enhanced RNA recovery and separation of crosslinked target RNA fragments from background RNA degradation products. Moreover, it facilitates the generation of specific UV-induced mutations that mark the crosslinking nucleotide and allow transcriptome-wide identification of RBP binding sites at single-nucleotide resolution. © by 2017 John Wiley & Sons, Inc.

Every eukaryotic RNA transcript undergoes extensive post-transcriptional processing from the moment of transcription up through degradation. This regulation is performed by a distinct cohort of RNA-binding proteins which recognize their target transcript by both its primary sequence and secondary structure. Here, we describe protein interaction profile sequencing (PIP-seq), a technique that uses ribonuclease-based footprinting followed by high-throughput sequencing to globally assess both protein-bound RNA sequences and RNA secondary structure. PIP-seq utilizes single- and double-stranded RNA-specific nucleases in the absence of proteins to infer RNA secondary structure. These libraries are also compared to samples that undergo nuclease digestion in the presence of proteins in order to find enriched protein-bound sequences. Combined, these four libraries provide a comprehensive, transcriptome-wide view of RNA secondary structure and RNA protein interaction sites from a single experimental technique. © 2016 by John Wiley & Sons, Inc.

This unit describes the combination of chromatin immunoprecipitation (ChIP) with microarray hybridization to determine the genome-wide occupancy profile of a DNA-associated protein. After conventional ChIP, the immunoprecipitated material is amplified by a two-step process involving primer extension followed by PCR in the presence of a modified nucleotide. The amplified DNA is fluorescently labeled in a reaction that couples dye to the modified nucleotide, and the labeled sample is hybridized to a microarray representing a complete genome. This method allows the study of a protein's pattern of DNA association across an entire genome with no need for prior knowledge of potential DNA targets. Keywords: Chromatin immunoprecipitation; ChIP; microarray; amplification; PCR; dye coupling; protein-DNA interactions; ChIP-chip; ChIP-on-chip; genome-wide location; hybridization; whole-genome analysis

Similar to chromatin immunoprecipitation (ChIP), RNA immunoprecipitation (RIP) can be used to detect the association of individual proteins with specific nucleic acid regions, in this case on RNA. Live cells are treated with formaldehyde to generate protein-RNA cross-links between molecules that are in close proximity in vivo. RNA sequences that cross-link with a given protein are isolated by immunoprecipitation of the protein, and reversal of the formaldehyde cross-linking permits recovery and quantitative analysis of the immunoprecipitated RNA by reverse transcription PCR. The basics of RIP are very similar to those of ChIP, but with some important caveats. This unit describes the RIP procedure for Saccharomyces cerevisiae. Although the corresponding steps for metazoan cells have not yet been worked out, it is likely that the yeast procedure can easily be adapted for use in other organisms.

Sequential Chromatin Immunoprecipitation (SeqChIP) is a powerful technique for analyzing the simultaneous association of two different proteins with genomic DNA sequences in vivo. Cellular Protein-DNA complexes are cross-linked with formaldehyde (unit Unavailable ), and are purified via two successive immunoprecipitations, with each immunoprecipitation targeting a different protein. Protein-DNA cross-links are then reversed and DNA sequences of interest are analyzed by quantitative PCR. At each genomic region, calculated SeqChIP co-occupancy values are compared to occupancy values of singly immunoprecipitated samples. The extent of enrichment brought about by the second immunoprecipitation relative to the singly immunoprecipitated sample is directly correlated with the degree of co-occupancy between the two proteins at the genomic location assayed. In principle, the technique is not limited to Saccharomyces cerevisiae. Cells from a wide variety of organisms can be used. Keywords: chromatin; immunoprecipitation; protein-DNA interactions; in vivo crosslinking

Coprecipitation of proteins from whole-cell extracts is a valuable approach to testing for physical interactions between proteins of interest. When a precipitating antibody is used, this method is referred to as co-immunoprecipitation. Coprecipitation can be used to study interactions between known proteins and as a means of identifying components of a complex. This unit describes basic approaches to immunoprecipitating tagged proteins from whole-cell extracts. The extract is prepared under nondenaturing conditions, the protein of interest is precipitated, and the precipitate is tested for a second specifically associated protein. The approach can be used for native or epitope-tagged proteins for which antibodies are available, or for recombinant proteins that bind with high affinity to a molecule that can be coupled to a solid-phase matrix. An associated protein is detected by separating the precipitated proteins by SDS-PAGE and immunoblotting with a second antibody that recognizes the putative associated protein. Keywords: co-immunoprecipitation; immunoprecipitation; protein interactions; protein tags; antibody; immunoblot