A PCR-based method to identify plant protein-associated RNAs

We present a novel method for identifying RNAs which associate in vivo to plant proteins which show RNA-binding features such as the presence of consensus RNA-binding motifs. The method consists of using an antibody specific for the RNA-binding protein of interest to co-immunopurify protein-associated RNAs from a plant extract, followed by random reverse transcription and PCR (rRT-PCR) of the isolated RNA molecules. The reverse transcription is performed with a fully degenerated 6-mer oligonucleotide which allows for the generation of cDNA molecules from virtually any type of RNA. The cDNA population is then amplified by PCR using various sets of two arbitrary 10-mer oligonucleotides. The PCR products can be subsequently isolated and the orientation of the clone determined by a strand-specific RT-PCR. As an illustration of this approach, we show the isolation and characterization of a RNA which co-immunopurifies with the maize MA16 RNA-binding protein.