Abstract
RNAs and RNA-binding proteins (RBPs) can interact dynamically in ribonucleoprotein (RNP) complexes that play important roles in controlling gene expression programs. One of the powerful ways to investigate changes in the association of RNAs with an RBP of interest is by immunoprecipitation (IP) analysis of native RNPs. RIP (RNP immunoprecipitation) analysis enables the rapid identification of endogenous RNAs bound to an RBP and to monitor time-dependent changes in this association, as well as changes in response to different metabolic and stress conditions. The protocol is based on the use of an antibody, typically an anti-RBP antibody, to immunoprecipitate the RNP complex. The RNA within the immunoprecipitated complex can then be isolated and further studied using different approaches such as PCR, microarray, Northern blot, and sequencing analyses. Among other advantages, RIP analysis (i) measures RNP associations in many samples relatively quickly, (ii) can be adapted easily to different endogenous RBPs, and (iii) provides extensive information at low cost. Among its limitations, RIP analysis does not inform on the specific sites of interaction of an RBP with a given target RNAs, although recent adaptations of RIP have been developed to overcome this problem. Here we provide an optimized protocol for RIP analysis that can be used to study RNA-protein interactions relevant to many areas of biology.