RNA and RBPs can also reversibly aggregate into granules to allow RNA storage and decay in response to stimuli. These and many other selleck chem processes are driven by large, Inhibitors,Modulators,Libraries complex networks of protein RNA Inhibitors,Modulators,Libraries interactions that provide spe cificity in gene regulation and fidelity in RNP assembly. Despite important insights regarding the necessity of RNA regulation for cellular functions, the RBP RNA interactome and its response to changing cellular condi tions have yet to be fully elucidated. Studies of RBP RNA interactions have historically relied on the identification of target transcripts bound by individual RBPs. In vitro selection of RNA sequences that bind RBPs with high affinity can identify primary sequence recognition elements.

For example, Inhibitors,Modulators,Libraries Nova proteins, which regulate mRNA splicing in neu rons, recognize the RNA consensus sequence YCAY, and Y box binding protein 1, Inhibitors,Modulators,Libraries a member of the cold shock Y box domain protein family, recognizes a CAYC RNA motif. Yet these and other primary sequence elements identified in vitro are generally short and degenerate and appear too frequently in the transcrip tome to be useful for in silico target identification. Micro array profiling of transcripts that co purify with interacting proteins has been widely used to detect transcripts stably associated with RBPs, such as mRNAs bound by translational components HuB, eIF 4E, and PABP in P19 embryonal carcinoma stem cells. Similarly, RIP Chip experiments have identified mRNAs associated with 40 yeast RBPs and uncovered a set of potential RBP recognition motifs, some of which were validated in vitro using SELEX.

Although capable of identifying mRNA targets for select RBPs, RIP Chip is prone to artifacts, including RBP RNA dissocia tion and re association after cell lysis, isolation of non specific RNAs, and indirect binding through other co purified RBPs. In addition, RIP Chip Inhibitors,Modulators,Libraries cannot detect transient interactions or resolve the exact RBP binding sites on identified transcripts. To identify transcriptome wide footprints of RBPs in vivo, UV crosslinking has been coupled with immuno purification of RBPs. CLIP takes advan tage of the photoreactivity of RNA bases, most often pyrimidines, with interacting amino acid side chains upon 254 nm UV irradiation. MDV3100 The formation of covalent linkages allows stringent purification of RBP RNA complexes and subsequent identification of cross linked RNA fragments via cDNA sequencing. Recently, a modified CLIP technique, PAR CLIP, has been introduced in which photoactivatable ribonucleoside analogs are incorporated into the transcriptome in live cells to enable efficient crosslinking using 365 nm UV irradia tion.