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ICLIP EXPERIMENTS PROTOCOLS RNA IP CODE

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.

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Methodology and first applications to the phage T4 DNA replication system.

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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. Sanford JR, Wang X, Mort M et al (2009) Splicing factor SFRS1 recognizes a functionally diverse landscape of RNA transcripts.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.

ICLIP EXPERIMENTS PROTOCOLS RNA IP CODE

Yeo GW, Coufal NG, Liang TY et al (2009) An RNA code for the FOX2 splicing regulator revealed by mapping RNA-protein interactions in stem cells. Sugimoto Y, Konig J, Hussain S et al (2012) Analysis of CLIP and iCLIP methods for nucleotide-resolution studies of protein-RNA interactions, Genome Biol 13:R67 Langmead B, Trapnell C, Pop M et al (2009) Ultrafast and memory-efficient alignment of short DNA sequences to the human genome, Genome Biol 10:R25 Yao C, Biesinger J, Wan J et al (2012) Transcriptome-wide analyses of CstF64-RNA interactions in global regulation of mRNA alternative polyadenylation. Hafner M, Renwick N, Brown M et al (2011) RNA-ligase-dependent biases in miRNA representation in deep-sequenced small RNA cDNA libraries. Konig J, Zarnack K, Rot G et al (2010) iCLIP reveals the function of hnRNP particles in splicing at individual nucleotide resolution. Ule J, Jensen KB, Ruggiu M et al (2003) CLIP identifies Nova-regulated RNA networks in the brain. Gilbert C, Kristjuhan A, Winkler GS et al (2004) Elongator interactions with nascent mRNA revealed by RNA immunoprecipitation.

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