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Heterografting and RNA transport experiments have demonstrated the long-distance mobility of  StBEL5  RNA, its role in controlling tuber formation, and the function of the 503-nt 3′ untranslated region (UTR) of the RNA in mediating transport. Because the 3′ UTR of  StBEL5  is a key element in regulating several aspects of RNA metabolism, a potato leaf cDNA library was screened using the 3′ UTR of  StBEL5  as bait in the yeast three-hybrid (Y3H) system to identify putative partner RNA-binding proteins (RBPs). From this screen, 116 positive cDNA clones were isolated based on nutrient selection,  HIS3  activation, and  lacZ  induction and were sequenced and classified. Thirty-five proteins that were predicted to function in either RNA- or DNA-binding were selected from this pool. Seven were monitored for their expression profiles and further evaluated for their capacity to bind to the 3′ UTR of  StBEL5  using β-galactosidase assays in the Y3H system and RNA gel-shift assays. Among the final selections were two RBPs, a zinc finger protein, and one protein, StLSH10, from a family involved in light signaling. In this study, the Y3H system is presented as a valuable tool to screen and verify interactions between target RNAs and putative RBPs. These results can shed light on the dynamics and composition of plant RNA-protein complexes that function to regulate RNA metabolism.

Using the Yeast Three-Hybrid System to Identify Proteins that Interact with a Phloem-Mobile mRNA