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Nat Commun. 2018 Sep 14;9(1):3747. doi: 10.1038/s41467-018-06128-x.

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

Author information

1
Centre for Genomic Regulation (CRG), The Barcelona Institute of Science and Technology (BIST), Doctor Aiguader 88, 08003, Barcelona, Spain.
2
Universitat Pompeu Fabra (UPF), 08002, Barcelona, Spain.
3
Institució Catalana de Recerca i Estudis Avançats (ICREA), Pg. Lluis Companys 23, 08010, Barcelona, Spain.
4
Centre for Genomic Regulation (CRG), The Barcelona Institute of Science and Technology (BIST), Doctor Aiguader 88, 08003, Barcelona, Spain. jae-seong.yang@crg.es.
5
Universitat Pompeu Fabra (UPF), 08002, Barcelona, Spain. jae-seong.yang@crg.es.

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.

PMID:
30217970
PMCID:
PMC6138660
DOI:
10.1038/s41467-018-06128-x
[Indexed for MEDLINE]
Free PMC Article

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