Rapid immunopurification of ribonucleoprotein complexes of plants

Abstract

Hundreds of RNA binding proteins posttranscriptionally regulate gene expression, but relatively few have been characterized in plants. One successful approach to determine protein function has been to identify interacting molecules and the conditions of their association. The ribonucleoprotein immunopurifi cation (RIP) assay facilitates the identifi cation and quantitative comparison of RNA association to specifi c proteins under different experimental conditions. A variety of molecular techniques can be used to analyze the enriched RNAs, whether few as in the case of highly specifi c interactions, or many. Identifi cation of associated RNAs can inform hypothesis generation about the processes or pathways regulated by the target protein. Downstream analysis of associated RNA sequences can lead to the identifi cation of candidate motifs or features that mediate the protein–RNA interaction. We present a rapid method for RIP from tissues of plants that is suitable for experiments that require immediate tissue cryopreservation, such as monitoring a rapid response to an environmental stimulus.