ICLIP EXPERIMENTS PROTOCOLS RNA IP FREE

Other approaches to identify protein-RNA crosslink sites include mutational analysis of read-through cDNAs, such as nucleotide transitions in PAR-CLIP, or rare errors introduced by reverse transcriptase when it reads through the crosslink sites in standard HITS-CLIP methods, termed Crosslink induced mutation site (CIMS) analysis. ICLIP (individual nucleotide–resolution crosslinking and immunoprecipitation) is a variant of CLIP that enabled amplification of truncated cDNAs, which are produced when reverse transcription stops prematurely at the cross-link site. This includes the miRNA targeting AGO and TNRC6 proteins. PAR-CLIP has been employed to determine the transcriptome-wide binding sites of several known RBPs and microRNA-containing ribonucleoprotein complexes at high resolution. However, PAR-CLIP is limited mainly to cultured cells, and nucleoside cytotoxicity is a concern it has been reported that 4-SU inhibits ribosomal RNA synthesis, induces a nucleolar stress response, and reduces cell proliferation. As a result, PAR-CLIP can identify binding site locations with high accuracy. Cross-linking the 4-SU and 6-SG analogs results in thymidine to cytidine, and guanosine to adenosine transitions respectively. The isolated RNA is converted into a cDNA library and deep sequenced using high-throughput sequencing technology.

Immunoprecipitation of the RBP of interest is followed by the isolation of the cross-linked and co-immunoprecipitated RNA. Irradiation of the cells by UV light of 365 nm induces efficient cross-linking of photoreactive nucleoside-labeled cellular RNAs to interacting RBPs. The method relies on the incorporation of photoreactive ribonucleoside analogs, such as 4-thiouridine (4-SU) and 6-thioguanosine (6-SG) into nascent RNA transcripts by living cells. PAR-CLIP (photoactivatable ribonucleoside–enhanced cross-linking and immunoprecipitation) is also used for identifying the binding sites of cellular RNA-binding proteins (RBPs) and microRNA-containing ribonucleoprotein complexes (miRNPs). CLIP analysis of the RNA-binding protein Argonaute led to identification of microRNA targets by decoding microRNA-mRNA and protein-RNA interaction maps in the mouse brain and subsequently in budding yeast ( Saccharomyces cerevisiae), Caenorhabditis elegans, embryonic stem cells and tissue culture cells. In 2008, CLIP was combined with high-throughput sequencing (termed 'HITS-CLIP') to generate genome-wide protein-RNA interaction maps for Nova since then a number of other RNA-binding proteins have been studied with CLIP, including PTBP1, RbFox2 (where the was referred to as 'CLIP-seq'), SFRS1, Argonaute, hnRNP C, the Fragile-X mental retardation protein FMRP, Ptbp2 (in the mouse brain), Mbnl2, the nElavl proteins (the neuron-specific Hu proteins), and has been applied to RNA binding proteins from all kingdoms of life, including prokaryotes. Sequencing of the cDNA library identified many positions close to alternative exons, several of which were found to require Nova1/2 for their brains-specific splicing patterns. History and applications ĬLIP was originally undertaken to study interactions between the neuron-specific RNA-binding protein and splicing factors NOVA1 and NOVA2 in the mouse brain, identifying RNA binding sites that contained the expected Nova-binding motifs. cDNA is then synthesized via RT-PCR followed by high-throughput sequencing followed by mapping the reads back to the transcriptome and other computational analyses to study the interaction sites. This often leads to truncation of cDNAs at the crosslinked nucleotide, which is exploited in variants such as iCLIP to increase the resolution of the method. Proteinase K digestion is then performed in order to remove protein from the crosslinked RNA, which leaves a few amino acids at the crosslink site.

ICLIP EXPERIMENTS PROTOCOLS RNA IP FREE

In order to allow for priming of reverse transcription, RNA adapters are ligated to the 3' ends, and RNA fragments are labelled to enable the analysis of the RNA-protein complexes after they have been separated from free RNA using gel electrophoresis and membrane transfer. The cross-linked cells are then lysed, RNA is fragmented, and the protein of interest is isolated via immunoprecipitation.

Upon UV exposure, covalent bonds are formed between proteins and nucleic acids that are in close proximity (on the order of Angstroms apart). CLIP begins with the in-vivo cross-linking of RNA-protein complexes using ultraviolet light (UV).

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