A three-hybrid screen identifies mRNAs controlled by a regulatory protein.
RNA-protein interactions are important in many biological contexts. Identification of the networks that connect regulatory proteins to one another and to the mRNAs they control is a critical need. Here, we use a yeast three-hybrid screening approach to identify RNAs that bind a known RNA regulatory protein, the Saccharomyces cerevisiae PUF protein, Mpt5p. The assay selects RNAs that bind in vivo using simple phenotypes and reporter genes. It enables rapid analyses of the affinity and specificity of the interaction. We show that the method identifies mRNAs that are genuinely regulated by the protein in vivo, and that it complements biochemical strategies, yielding a set of mRNAs that overlap with, but are distinct from, those obtained by biochemical means. The approach we describe facilitates construction of protein-RNA linkage maps.
Seay
Daniel
D
Department of Biochemistry, University of Wisconsin-Madison, WI 53706, USA.
Hook
Brad
B
Evans
Katie
K
Wickens
Marvin
M
D001681:Biological Assay; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism*; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D017930:Genes, Reporter; D009154:Mutation; D014176:Protein Biosynthesis; D012331:RNA, Fungal / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D016601:RNA-Binding Proteins; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D012680:Sensitivity and Specificity; D020798:Two-Hybrid System Techniques
D018797:Cell Cycle Proteins; D005656:Fungal Proteins; C449502:MPT5 protein, S cerevisiae; D012331:RNA, Fungal; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins