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

We present a novel method for identifying RNAs which associate in vivo to p lant proteins which show RNA-binding features such as the presence of conse nsus RNA-binding motifs. The method consists of using an antibody specific for the RNA-binding protein of interest to co-immunopurify protein-associat ed RNAs from a plant extract, followed by random reverse transcription and PCR (rRT-PCR) of the isolated RNA molecules. The reverse transcription is p erformed with a fully degenerated 6-mer oligonucleotide which allows for th e generation of cDNA molecules from virtually any type of RNA. The cDNA pop ulation is then amplified by PCR using various sets of two arbitrary 10-mer oligonucleotides. The PCR products can be subsequently isolated and the or ientation of the clone determined by a strand-specific RT-PCR. As an illust ration of this approach, we show the isolation and characterization of a RN A which co-immunopurifies with the maize MA16 RNA-binding protein. (C) Else vier, Paris.