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.
Highlights
Knowing which proteins and RNAs directly interact is essential for understanding cellular mechanisms
We discover new putative interactions between specific microtubule end-binding proteins (EBs) and plus-end-tracking proteins (+TIPs)10, and show that quantitative measures of protein abundance obtained via mass spectrometry (MS)11 depend on the number of nodes between baits and preys
For rec-Y3H screening, RNA fragment subcloning by Gibson assembly (Supplementary Fig. 3) into pMS22H creates hybrids of RNA fragments fused to MS2-binding stem-loop sequences
Summary
Knowing which proteins and RNAs directly interact is essential for understanding cellular mechanisms. 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. We combined batch transfer of DNA into screening vectors with batch library transformation, highly efficient DNA assembly by homologous recombination in yeast, liquid–gel culturing, and NGS-based readout of interactions using ORF sequences as identifiers to avoid any arraying, plating, or barcoding steps. We demonstrate recYnH by focussing on problems typically faced by biomedical research laboratories: (i) understanding the interactions between hundreds of proteins in a given pathway, (ii) mapping the interactions of multiple protein domains, (iii) predicting the putative architecture of a coprecipitated complex, and (iv) detecting the RNA targets of a number of RBPs at once. This is the first time that high-throughput screening is adapted and used for simultaneous multi-domain interaction mapping and multi-protein–RNA interaction mapping
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