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Method Article
A detailed protocol for protein-protein or protein-RNA interaction screening using rec-YnH
Jae-Seong Yang
Centre for Genomic Regulation (CRG), The Barcelona Institute of Science and Technology (BIST), Doctor Aiguader 88, Barcelona 08003, Spain
Corresponding Author
Mireia Garriga-Canut
Centre for Genomic Regulation (CRG), The Barcelona Institute of Science and Technology (BIST), Doctor Aiguader 88, Barcelona 08003, Spain
Corresponding Author
Sebastian P. Maurer
Centre for Genomic Regulation (CRG), The Barcelona Institute of Science and Technology (BIST), Doctor Aiguader 88, Barcelona 08003, Spain
Corresponding Author
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This work is licensed under a CC BY 4.0 License. Read Full License
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.
Keywords
protein-protein interactions, protein-RNA interactions, high-throughput screening, matrix-screening
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Detailed Protocol rec-YnH detailed protocol
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Associated Publications
rec-YnH enables simultaneous many-by-many detection of direct protein–protein and protein–RNA interactions
by Jae-Seong Yang, Mireia Garriga-Canut, Nele Link, +5
Nature Communications (14 September, 2018)
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This is a preprint. It has not completed peer review.
Method Article
A detailed protocol for protein-protein or protein-RNA interaction screening using rec-YnH
Jae-Seong Yang
Centre for Genomic Regulation (CRG), The Barcelona Institute of Science and Technology (BIST), Doctor Aiguader 88, Barcelona 08003, Spain
Corresponding Author
Mireia Garriga-Canut
Centre for Genomic Regulation (CRG), The Barcelona Institute of Science and Technology (BIST), Doctor Aiguader 88, Barcelona 08003, Spain
Corresponding Author
Sebastian P. Maurer
Centre for Genomic Regulation (CRG), The Barcelona Institute of Science and Technology (BIST), Doctor Aiguader 88, Barcelona 08003, Spain
Corresponding Author
Badges
Prescreen
This manuscript passed our Prescreen assessment
Complete author information
Appropriate declaration statements
Potential risks to human health
Prescreen
History
CURRENT STATUS: Posted
Version 1
Posted 25 Sep, 2018
No community comments so far
Metrics
Comments: 0
HTML Views: ...
Subject Areas
Cell biology
License:
This work is licensed under a CC BY 4.0 License. Read Full License
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
2 comments flagged for moderation
If you posted a comment and think it was mistakenly removed, please contact customer support at [email protected].
Associated Publications
rec-YnH enables simultaneous many-by-many detection of direct protein–protein and protein–RNA interactions
by Jae-Seong Yang, Mireia Garriga-Canut, Nele Link, +5
Nature Communications (14 September, 2018)