rec-YnH enables simultaneous many-by-many detection of direct protein-protein and protein-RNA interactions

Nat Commun. 2018 Sep 14;9(1):3747. doi: 10.1038/s41467-018-06128-x.


Knowing which proteins and RNAs directly interact is essential for understanding cellular mechanisms. Unfortunately, discovering such interactions is costly and often unreliable. To overcome these limitations, we developed rec-YnH, a new yeast two and three-hybrid-based screening pipeline capable of detecting interactions within protein libraries or between protein libraries and RNA fragment pools. rec-YnH combines batch cloning and transformation with intracellular homologous recombination to generate bait-prey fusion libraries. By developing interaction selection in liquid-gels and using an ORF sequence-based readout of interactions via next-generation sequencing, we eliminate laborious plating and barcoding steps required by existing methods. We use rec-Y2H to simultaneously map interactions of protein domains and reveal novel putative interactors of PAR proteins. We further employ rec-Y2H to predict the architecture of published coprecipitated complexes. Finally, we use rec-Y3H to map interactions between multiple RNA-binding proteins and RNAs-the first time interactions between protein and RNA pools are simultaneously detected.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cloning, Molecular
  • High-Throughput Nucleotide Sequencing
  • High-Throughput Screening Assays
  • Protein Interaction Maps*
  • RNA / metabolism*
  • RNA-Binding Proteins / metabolism*
  • Saccharomyces cerevisiae / metabolism*
  • Saccharomyces cerevisiae Proteins / metabolism*
  • Two-Hybrid System Techniques*


  • RNA-Binding Proteins
  • Saccharomyces cerevisiae Proteins
  • RNA