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

Yang, Jae-Seong; Garriga-Canut, Mireia; Link, Nele; Carolis, Carlo; Broadbent, Katrina; Beltran-Sastre, Violeta; Serrano Pubull, Luis, 1982-; Maurer, Sebastian

doi:http://dx.doi.org/10.1038/s41467-018-06128-x

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

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Yang JS, Garriga-Canut M, Link N, Carolis C, Broadbent K, Beltran-Sastre V et al. rec-YnH enables simultaneous many-by-many detection of direct protein-protein and protein-RNA interactions. Nat Commun. 2018;9(1):3747. DOI: 10.1038/s41467-018-06128-x

Abstract

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.