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.

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Journal Article | Research Support, Non-U.S. Gov't

Authors

Yang JS, Garriga-Canut M, Link N, Carolis C, Broadbent K, Beltran-Sastre V, Serrano L, Maurer SP

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