However, HITS-CLIP protocol is still required for some optimization due to experimental complication, low efficiency and time-consuming, whose library has to be generated from very small amounts of RNAs. Crosslinking immunoprecipitation (CLIP), coupled with high-throughput sequencing (HITS-CLIP), is a powerful technique for investigating the molecular mechanisms underlying disease pathogenesis by comprehensive identification of RBP target sequences at the transcriptome level. Sei E, Conrad NK (2014) UV cross-linking of interacting RNA and protein in cultured cells.RNA-binding proteins (RBPs) control many types of post-transcriptional regulation, including mRNA splicing, mRNA stability, and translational efficiency, by directly binding to their target RNAs and their mutation and dysfunction are often associated with several human neurological diseases and tumorigenesis.This approach can provide key mechanistic insight into the function of these newly identified ncRNAs. This technique utilizes UV light to cross-link the cells, which takes advantage of the fact that UV light will only cross-link proteins and nucleic acids that are directly interacting. This protocol describes one technique that can be used to study this, ultra-violet light cross-linking RNA immunoprecipitation (UV-RIP), which uses an antibody to pull down a specific protein of interest and then detects RNA that is bound to it. As we delve deeper into studying their mechanisms of action, it becomes important to understand how they play these roles, in particular by understanding what proteins these ncRNAs interact with. These nonprotein-coding RNAs (ncRNAs) come in many different forms, and they have been shown to have a variety of functions within the cell, influencing processes such as gene expression, mRNA splicing, and transport, just as a few examples. With the many advances in genome-wide sequencing, it has been discovered that much more of the genome is transcribed into RNA than previously appreciated. Sei E, Conrad NK (2014) UV cross-linking of interacting RNA and protein in cultured cells.Huppertz I, Attig J, D'Ambrogio A et al (2014) iCLIP: protein-RNA interactions at nucleotide resolution.Licatalosi DD, Mele A, Fak JJ et al (2008) HITS-CLIP yields genome-wide insights into brain alternative RNA processing.Ule J, Jensen K, Mele A, Darnell RB (2005) CLIP: a method for identifying protein-RNA interaction sites in living cells.Lai F, Orom UA, Cesaroni M et al (2013) Activating RNAs associate with mediator to enhance chromatin architecture and transcription.Schaukowitch K, Joo JY, Liu X et al (2014) Enhancer RNA facilitates NELF release from immediate early genes.Kim TK, Hemberg M, Gray JM et al (2010) Widespread transcription at neuronal activity-regulated enhancers.Darnell RB (2010) HITS-CLIP: panoramic views of protein-RNA regulation in living cells.Brimacombe R, Stiege W, Kyriatsoulis A et al (1988) Intra-RNA and RNA-protein cross-linking techniques in Escherichia coli ribosomes.Greenberg JR (1979) Ultraviolet light-induced crosslinking of mRNA to proteins.Methodology and first applications to the phage T4 DNA replication system. Hockensmith JW, Kubasek WL, Vorachek WR et al (1986) Laser cross-linking of nucleic acids to proteins.
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