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

Sung Ki Cho,Tyrell Carr,David J Hannapel,Il-Ho Kang

doi:10.3389/fpls.2012.00189

Abstract

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.

Highlights

INTRODUCTIONPhloem plays important roles in transporting nutrients, and as a conduit for moving signal RNAs and proteins
For plant development, phloem plays important roles in transporting nutrients, and as a conduit for moving signal RNAs and proteins
These results clearly demonstrate the utility of the Y3H system in identifying candidate RNA-binding proteins (RBPs)

Summary

INTRODUCTION

Phloem plays important roles in transporting nutrients, and as a conduit for moving signal RNAs and proteins. RNA-binding proteins for StBEL5 RNA long-distance transport, controlling translation, and regulating stability (Banerjee et al, 2006, 2009), suggesting the presence of cis-elements in this UTR that are recognized by RNA-binding partners. There are several useful biochemical approaches to analyze RNA-protein interactions, the yeast three-hybrid (Y3H) system (Sengupta et al, 1996; Hook et al, 2005) represents a simple but powerful tool for searching a large collection of cDNAs to identify proteins that bind a specific RNA of interest (Cassiday and Maher III, 2003; Gonsalvez et al, 2003; Maniataki et al, 2003; Moore et al, 2003; Campalans et al, 2004; Hwang et al, 2005). Seven proteins were selected based on their putative RNA-binding properties for further analyses and RNA gel-shift assays These results clearly demonstrate the utility of the Y3H system in identifying candidate RBPs

MATERIALS AND METHODS

Findings

CONCLUSION