A three-hybrid screen identifies mRNAs controlled by a regulatory protein

Abstract

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.

Mutations in the Saccharomyces cerevisiae Kinase Cbk1p Lead to a Fertility Defect That Can Be Suppressed by the Absence of Brr1p or Mpt5p (Puf5p), Proteins Involved in RNA Metabolism

“…Systematic studies have suggested that Mpt5p can bind to .200 mRNAs, indicating that Mpt5p could regulate many cellular processes (Gerber et al 2004;Seay et al 2006). Several Mpt5p mRNA targets are well characterized such as HO, involved in mating type switching, and STE7 and TEC1, two components of the (f )-MAPK pathway.…”

A 5′ cytosine binding pocket in Puf3p specifies regulation of mitochondrial mRNAs

“…RNA was isolated using the MasterPure Yeast RNA Purification Kit (Epicentre Biotechnologies). Northern blot analysis was performed as described previously (42). Decay data were analyzed using GraphPad Prism software.…”

Selections that optimize RNA display in the yeast three-hybrid system

“…In 2006, Seay et al (2006) reported a Y3H selection to identify 39-UTR sequences recognized by the PUF protein Mpt5. The investigators ligated z50-150-bp fragments of yeast chromosomal DNA into pIIIA-MS2-2 for expression of test transcripts.…”

“…A protein (''prey'') is expressed as a fusion with the Gal4 transcription activation domain. The Y3H has been successfully used to confirm natural or artificial RNAprotein interactions and to identify novel RNA partners for target proteins by selection from RNA or protein libraries (SenGupta et al 1996;Zhang et al 1999Zhang et al , 2000Kraemer et al 2000;Maher 2001, 2003;van Zon et al 2001;Bernstein et al 2002Bernstein et al , 2005Sokolowski et al 2003;Spiridonov et al 2003;Klimek-Tomczak et al 2004;Hook et al 2005;Riley et al 2006;Seay et al 2006;Konig et al 2007;Stumpf et al 2008;Koh et al 2009;Wurster et al 2009). …”