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

Jae-Seong Yang (), Mireia Garriga-Canut, Nele Link, Carlo Carolis, Katrina Broadbent, Violeta Beltran-Sastre, Luis Serrano and Sebastian P. Maurer ()
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Jae-Seong Yang: The Barcelona Institute of Science and Technology (BIST)
Mireia Garriga-Canut: The Barcelona Institute of Science and Technology (BIST)
Nele Link: The Barcelona Institute of Science and Technology (BIST)
Carlo Carolis: The Barcelona Institute of Science and Technology (BIST)
Katrina Broadbent: The Barcelona Institute of Science and Technology (BIST)
Violeta Beltran-Sastre: The Barcelona Institute of Science and Technology (BIST)
Luis Serrano: The Barcelona Institute of Science and Technology (BIST)
Sebastian P. Maurer: The Barcelona Institute of Science and Technology (BIST)

Nature Communications, 2018, vol. 9, issue 1, 1-13

Abstract: 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.

Date: 2018
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DOI: 10.1038/s41467-018-06128-x

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