A recent study analyzing previously published PAR-CLIP datasets showed that if no control dataset is used for correction, up to 45 % of the called binding sites overlap with background binding sites. īesides background noise, such as the signal from sticky RNA fragments or non-specific crosslink events within CL motifs, the binding of background proteins is a major challenge in the analysis of CLIP-seq data. showed that certain polypyrimidine-rich k-mers, which they call crosslink-associated (CL) motifs, are enriched at read start sites in both input and target eCLIP data compared to upstream regions. The crosslinking sequence bias can also be observed within the eCLIP input data, since it “represent RNAs crosslinked to many different RBPs and should reflect the sequence preferences at crosslink sites that are common to a mixture of RBPs”. To infer target-specific RBP binding regions from iCLIP/eCLIP data, it is crucial to account for different sources of biases, such as transcript abundances, crosslinking sequence preferences, and mappability. This input control is sampled prior to the immunoprecipitation and thus, contains the signal of a non-specific background. While previous CLIP-seq experiments often had matched IgG control experiments, which suffer from sparsity and high amplification rates, the eCLIP-seq protocol is designed to generate a size-matched input control. To date, eCLIP datasets for more than 120 different proteins have been published by the ENCODE consortium. Like iCLIP, it provides single-nucleotide resolution by capturing truncated cDNAs but, due to the optimization of several steps, it improves the specificity of called binding sites. Another protocol called eCLIP was published in 2016. Recently, various improvements to the protocol were proposed to alleviate previous limitations. As a consequence, valuable information about the exact crosslink site can be retained from truncated cDNAs, or more precisely from the read start sites they cause. ICLIP-seq uses a cleavable adapter in combination with an additional circularization step, which allows all cDNA fragments to be amplified and sequenced. The fraction of truncated and thus, lost fragments is typically over 80 %. However, due to the ligation of an adapter at the 5 ′ end of the RNA fragments, the HITS-CLIP and PAR-CLIP methods capture only cDNAs that are entirely read by the reverse transcriptase, i.e., not truncated. Such diagnostic events can be used to localize the crosslink position. These crosslinks subsequently increase the probability for base transitions, deletions, and truncations during the reverse transcription. All methods use UV light, which causes the formation of crosslinks at protein–RNA interaction sites. The most commonly used protocols in this field are HITS-CLIP, photoactivatable ribonucleoside-enhanced CLIP (PAR-CLIP) and since 2010, individual-nucleotide CLIP (iCLIP). State-of-the-art technologies using crosslinking and immunoprecipitation combined with high-throughput sequencing (CLIP-seq) allow genome-wide binding site detection with high resolution. To understand fully the regulatory processes mediated by RBPs, it is crucial to determine accurately the full landscape of interactions for a protein of interest. RBPs bind on several sites of both coding and non-coding RNAs with a more or less strong binding affinity for both RNA sequence and structure. The interactions between RNAs and RNA binding proteins (RBPs) play essential roles in both transcriptional and post-transcriptional gene regulation.
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