A detailed protocol for protein-protein or protein-RNA interaction screening using rec-YnH

doi:10.1038/protex.2018.112

Method Article

A detailed protocol for protein-protein or protein-RNA interaction screening using rec-YnH

https://doi.org/10.1038/protex.2018.112

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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.

Cell biology

protein-protein interactions

protein-RNA interactions

high-throughput screening

matrix-screening

Revealing which proteins, protein domains and RNAs undergo direct, physical interactions is an essential prerequisite for understanding the principles by which cellular machines operate. Assays that can detect such interactions are in high demand as they function both to generate system-level views of cellular coordination but also to generate hypothesis for subsequent mechanistic studies. Yeast two and three hybrid (Y2H, Y3H) screens, allow to detect direct protein-protein and protein-RNA interactions under physiological conditions. The Y2H assay was enhanced significantly in the past. Coupling the readout to next-generation sequencing (NGS) and multiplexed generation of genetically fused bait-prey libraries resulted in increased assay sensitivity and reproducibility while allowing screening many baits against many preys simultaneously. Improvements of Y3H, however, mostly focused on optimized RNA binding and presentation by linker RNA engineering and selection of stronger MS2-binding proteins. To date, there is no method available allowing the many-by-many direct interaction detection of full-length RBPs and RNA-fragments.

With the development of rec-YnH, we aimed at creating "one assay":http://www.nature.com/protocolexchange/system/uploads/7013/original/Detailed_Protocol_rec-YnH.pdf?1533133123 that further advances and integrates past developments in Y2H and Y3H technologies into a single, new assay pipeline allowing screening of protein libraries against protein libraries or RNA fragment libraries. At the same time, we focused on keeping the assay workflow as simple as possible to make it affordable and doable for the standard biomedical research lab. The assay pipeline combines batch-cloning and transformation with intracellular homologous recombination, liquid gel culturing, yeast two or three-hybrid based interaction detection and read out by next-generation-sequencing. This eliminates barcoding, arraying and plating steps needed for available methods. Three compatible vectors allow high-throughput interaction screening within protein libraries and, for the first time, between protein and RNA libraries. We demonstrate that the same assay pipeline can be used for rec-Y2H and rec-Y3H screening and thus create an assay for both, many-by-many protein-protein or protein-RNA interaction detection.

Step 1. Batch Gateway cloning of ORF library into pDEST vectors

pENTR-ORF clones
TE buffer (10 mM Tris, pH 8.0, 1 mM EDTA)
10 mM Tris, pH 8.0
LR clonase II enzyme mix (ThermoFisher Scientific)
NEB stable cells (New England Biolabs)
SOC media
10-cm LB-agar plates
Ampicillin
Spectinomycin
Kanomycin
1x PBS
QIAprep Spin Miniprep Kit (Qiagen)

Step 2. Competent yeast

Y2HGold (Clontech)
Loops
Plastic cuvettes
YPDA–agar plates
YPDA liquid media
50 ml tubes
250 ml flasks
500 ml flasks
Sterile H2O
TE buffer (10x)
1 M LiAc (10x)

Step 3. Linearization

I-SceI ((New England Biolabs)
I-CeuI (New England Biolabs)
Agarose gel

Step 4. Batch yeast transformation

Yeast carrier DNA (Clontech)
50% PEG (Sigma)
1 M LiAc (10x)
DMSO (Sigma)
0.9% (w/v) NaCl

Step 5. Growth in selection media

Yeast dropout agar plates

Square BioAssay dishes, 245×245 mm (Corning) for screen
10-cm petri dish for colony counting
Agar (Conda)
Minimal SD base (Clontech),
Amino Acid Dropout mixes (Clontech)
Aureobasidin A (AbA) (Clontech) when indicated

Yeast dropout liquid-gel media

5 litre flask
SeaPrep™ Agarose (Lonza) (0.5% (w/v) final concentration)
Minimal SD base (Clontech),
Amino Acid Dropout mixes (Clontech)
Aureobasidin A (Clontech) when indicated

Step 6. Colony counting and harvesting yeast

500 ml centrifuge bottles
1xPBS
Plastic cuvettes

Step 7. Yeast library DNA isolation

Zymoprep™ Yeast Plasmid Miniprep II (Zymo Research)
DNAse-free water

Step 8. Covaris shearing

microTUBE AFA Fiber Pre-Slit Snap-Cap 6x16 mm (covaris)

Step 9. Circularisation

NEBNext® End Repair Module (New England Biolabs)
MinElute PCR Purification Kit (Qiagen)
Quick Ligation™ Kit (New England Biolabs)
DNAse-free water

Step 10. R1/R2 PCR

Q5 High-Fidelity 2X Master Mix (New England Biolabs)
Primer Pr4seq_F_TS_R1 (5’- ccctacacgacgctcttccgatctcgctgcaggtcgacggatc-3’) (custom ordered from Integrated DNA Technologies)
Primer Pr4seq_R_TS_R2 (5’- ttcagacgtgtgctcttccgatctgcagctcgagctcgatggatc-3’) (custom ordered from Integrated DNA Technologies)
Agencourt AMPure XP beads (Beckman Coulter)
10 mM Tris, pH 8.0

Step 11. P5/P7 PCR

Q5 High-Fidelity 2X Master Mix (New England Biolabs)
NEBNext® Multiplex Oligos for Illumina®, Index Primers Set 1 (New England Biolabs)
DNAse-free water
Agencourt AMPure XP beads (Beckman Coulter)
10 mM Tris, pH 8.0

Step 12. Paired-end sequencing

MiSeq Reagent Kit v2 (Illumina)

o PCR machine

o Microcentrifuge

o Clinical centrifuge

o Ultracentrifuge

o Water bath at 30ºC

o Water bath at 42ºC

o Spectrophotometer

o Nanodrop

o Shaker at 30ºC

o Shaker at 37ºC

o Incubator at 30ºC

o Incubator at 37ºC

o Horizontal Electrophoresis System for DNA

o S220 Focused-ultrasonicator with AFA Technology (Covaris)

Please see attached "Detailed Protocol":http://www.nature.com/protocolexchange/system/uploads/7013/original/Detailed_Protocol_rec-YnH.pdf?1533133123 for a full protocol.

PARTS OF THE PROTOCOL

Please see Figure 1 for a "Workflow Overview":http://www.nature.com/protocolexchange/system/uploads/7059/original/Figure_1.pdf?1533558364

Part I. Bait and Prey library preparation

Step 1. Batch Gateway cloning of ORF library into pDEST vectors

Part II. Bait and Prey library fusion & Selection in liquid gel

Step 2. Competent yeast

Step 3. Linearization

Step 4. Batch yeast transformation

Step 5. Growth in liquid-gel selection media

Part III. NGS library preparation

Step 6. Harvest yeast

Step 7. Yeast library DNA isolation

Step 8. Covaris shearing

Step 9. Circularisation

Step 10. R1/R2 PCR

Step 11. P5/P7 PCR

Part IV. Paired-end sequencing

Step 12. Library quality control and paired-end MiSeq

Step 13. Analysis

PROTOCOL AT A GLANCE, DAY BY DAY

DAY 1

Step 1. Batch Gateway cloning of ORF library into pDEST vectors

Prepare pool of pENTR-ORF clones at 10 nM
Prepare pDEST for Bait and Prey at 10 nM
Set up LR reactions

Step 2. Prepare competent yeast

Streak Y2HGold on a YPDA plate

DAY 2

Step 1. Batch Gateway cloning of ORF library into pDEST vectors

Continue LR reaction
Transform LR reaction into NEB stable cells
Plate on LB-Agar with corresponding antibiotic

DAY 3

Step 1. Batch Gateway cloning of ORF library into pDEST vectors

Colony counting
Harvest colonies from LB-Agar plates
Qiaprep DNA prep of Bait and Prey pDEST-ORF pool

Step 2. Competent yeast

Grow 1 colony of Y2HGold during 8 hours
Transfer to 50 ml YPDA media
Grow overnight

Step 3. Linearization

Digest Bait and Prey pDEST-ORF pool with I-SceI/I-CeuI
Heat inactivation

DAY 4

Step 2. Competent yeast

Transfer yeast to 100 ml YPDA
Grow yeast to OD 0.5
Wash cells
Resuspend in TE/LiAc

Step 4. Batch yeast transformation

Prepare Seaprep liquid-gel selection media
Multiple transformations of yeast with linear Bait and Prey pDEST-ORF pool
Pool together all transformations
Plate dilutions for colony counting

Step 5. Growth in selection media

Add transformations to liquid-gel selection media
1 hour on ice
Grow at 30ºC for 60 hours

DAY 7

Step 6. Harvest yeast

Colony counting
Centrifuge liquid-gel selection media with yeast colonies
Wash and resuspend in PBS
OD660 to calculate concentration of cells

Step 7. Yeast library DNA isolation

For each selection media, Zymoprep™ Yeast Plasmid Miniprep of recombined Bait and Prey fragments

Step 8. Covaris shearing

Shear DNA to 1,500-bp fragments

Step 9. Circularisation

End repair
Mini Elute DNA purification
Ligation
Heat inactivation

Step 10. R1/R2 PCR

12 PCR cycles with Q5 polymerase
Purification with AMPure beads

Step 11. P5/P7 PCR

12 PCR cycles with Q5 polymerase and NEBNext® Multiplex Oligos for Illumina
Purification with AMPure beads

DAY 8 onwards

Step 12. Paired-end sequencing

Library quality control
MiSeq run

Step 13. Analysis

PARTS OF THE PROTOCOL

Part I. Bait and Prey library preparation : Day 1-3

Part II. Bait and Prey library fusion & Selection in liquid gel: Day 3-7

Part III. NGS library preparation: Day 7

Part IV. Paired-end sequencing: Day 8-9

None

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Detailed Protocol rec-YnH detailed protocol

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A detailed protocol for protein-protein or protein-RNA interaction screening using rec-YnH

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