A detailed protocol for protein-protein or protein-RNA interaction screening using rec-YnH
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":http://www.nature.com/protocolexchange/system/uploads/7059/original/Figure_1.pdf?1533558364 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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Detailed Protocol rec-YnH detailed protocol
Posted 25 Sep, 2018
A detailed protocol for protein-protein or protein-RNA interaction screening using rec-YnH
Posted 25 Sep, 2018
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":http://www.nature.com/protocolexchange/system/uploads/7059/original/Figure_1.pdf?1533558364 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.
Figure 1
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