Global Identification of Co-Translational Interaction Networks by Selective Ribosome Profiling

Summary

Co-translational interactions play a crucial role in nascent-chain modifications, targeting, folding, and assembly pathways. Here, we describe Selective Ribosome Profiling, a method for in vivo, direct analysis of these interactions in the model eukaryote Saccharomyces cerevisiae.

Abstract

In recent years, it has become evident that ribosomes not only decode our mRNA but also guide the emergence of the polypeptide chain into the crowded cellular environment. Ribosomes provide the platform for spatially and kinetically controlled binding of membrane-targeting factors, modifying enzymes, and folding chaperones. Even the assembly into high-order oligomeric complexes, as well as protein-protein network formation steps, were recently discovered to be coordinated with synthesis.

Here, we describe Selective Ribosome Profiling, a method developed to capture co-translational interactions in vivo. We will detail the various affinity purification steps required for capturing ribosome-nascent-chain complexes together with co-translational interactors, as well as the mRNA extraction, size exclusion, reverse transcription, deep-sequencing, and big-data analysis steps, required to decipher co-translational interactions in near-codon resolution.

Introduction

Selective Ribosome Profiling (SeRP) is the only method, to date, that captures and characterizes co-translational interactions, in vivo, in a direct manner^1,2,3,4,5,6. SeRP enables global profiling of interactions of any factor with translating ribosomes in near codon resolution^2,7.

The method relies on flash freezing of growing cells and preserving active t....

Protocol

1. Generating strains for Selective Ribosome Profiling

NOTE: Selective Ribosome Profiling (SeRP) is a method that relies on affinity purification of factors of interest, to assess their mode of interaction with ribosomes-nascent chain complexes. Homologous recombination⁹, as well as CRISPR/Cas9¹⁰ based methods are utilized to fuse various factors of interest with tags for affinity purifications. Such tags are GFP, for GFP-trap affinity purifications, TAP-tag for IgG-Sepharose beads purifications as well as AVI-Tag purified by avidin or streptavidin, to list a few successful ....

Results

As illustrated in the flow chart of this protocol (Figure 1), cells were grown to log phase, and then collected swiftly by filtration and lysed by cryogenic grinding. The lysate was then divided into two: one for total ribosome-protected mRNA footprints and the other for selected ribosome-protected mRNA footprints, on which we performed affinity purification to pull-down the tagged protein-ribosome-nascent chains complexes. We ensured tagged protein expression and the success of the pull-dow.......

Discussion

Here, the protocol details the Selective Ribosome Profiling approach for capturing co-translational interactions in near codon resolution. As the ribosome rises as a hub for coordinating the nascent-chain emergence into the crowded cytoplasm, this is a crucial method to identify and characterize the various co-translational interactions required to ensure a functional proteome, as well as for studying various diseases. To date, SeRP is the only method that can capture and characterize these interactions, in a direct.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
3'-Phosphorylated 28 nt RNA control oligonucleotide	IDT	custom order	RNase free HPLC purification; 5'-AUGUAGUCGGAGUCGAGGCGC GACGCGA/3Phos/-3'
Absolute ethanol	VWR	20821
Acid phenol–chloroform	Ambion	AM9722
Antibody: mouse monclonal anti-HA	Merck	11583816001	12CA5
Aprotinin	Roth	A162.3
ATP*	NEB	P0756S	10 mM
Bacto agar	BD	214030
Bacto peptone	BD	211820
Bacto tryptone	BD	211699
Bacto yeast extract	BD	212720
Bestatin hydrochloride	Roth	2937.2
Chloroform	Merck	102445
CircLigase II ssDNA Ligase*	Epicentre	CL9025K	100 U/μL
Colloidal Coomassie staining solution	Roth	4829
cOmplete, EDTA-free protease inhibitor cocktail tablets	Roche Diagnostics	29384100
Cycloheximide	Biological Industries	A0879
DEPC treated and sterile filtered water*	Sigma	95284
D-Glucose anhydrous	Merck	G5767-500G
Diethylpyrocarbonate	Roth	K028
Dimethylsulfoxide*	Sigma-Aldrich	276855
DNA ladder, 10 bp O'RangeRuler*	Thermo Fisher Scientific	SM1313
DNA loading dye*	Thermo Fisher Scientific	R0631	6×
DNase I, recombinant	Roche	4716728001	RNAse free
dNTP solution set*	NEB	N0446S
EDTA*	Roth	8043
Glycerol	VWR	24388.260.
Glycine solution	Sigma-Aldrich	67419-1ML-F	1 M
GlycoBlue	Ambion	AM9516	15 mg/mL
HEPES	Roth	HN78.3
HF Phusion polymerase*	NEB	M0530L
HK from S. cerevisiae	Sigma-Aldrich	H6380-1.5KU
Hydrochloric acid	AppliChem	A1305
Isopropanol	Sigma-Aldrich	33539
Isopropyl β-D-1-thiogalactopyranoside	Roth	CN08
Kanamycin	Roth	T832.4
KCl	Roth	6781.1
KH2PO4	Roth	3904.1
Leupeptin	Roth	CN33.4
Linker L(rt)	IDT	custom order
Liquid nitrogen
MgCl2	Roth	KK36.3
Na2HPO4	Roth	P030.2
Na2HPO4·2H2O	Roth	T879.3
NaCl*	Invitrogen	AM97606	5 M
NaH2PO4·H2O	Roth	K300.2
NHS-activated Sepharose 4 fast-flow beads	GE Life Sciences	17090601
Nonidet P 40 substitute	Sigma	74385
Pepstatin A	Roth	2936.2
Phenylmethyl sulfonyl fluoride	Roth	6367
Precast gels	Bio-Rad	5671034	10% and 12%
RNase I	Ambion	AM2294
SDS, 20%	Ambion	AM9820	RNase free
Sodium acetate*	Ambion	AM9740	3 M, pH 5.5
Sodium azide	Merck	S8032-100G
Sodium chloride	Roth	9265
Sodium hydroxide*	Sigma	S2770	1 N
Sucrose	Sigma-Aldrich	16104
SUPERase-In RNase Inhibitor	Ambion	AM2694
Superscript III Reverse Transciptase*	Invitrogen	18080-044
SYBR Gold*	Invitrogen	S11494
T4 polynucleotide kinase*	NEB	M0201L
T4 RNA ligase 2*	NEB	M0242L
TBE polyacrylamide gel*	Novex	EC6215BOX	8%
TBE–urea polyacrylamide gel*	Novex	EC68752BOX	10%
TBE–urea polyacrylamide gel*	Novex	EC6885BOX	15%
TBE–urea sample buffer*	Novex	LC6876	2×
Tris	Roth	4855
Tris*	Ambion	AM9851	1 M, pH 7.0
Tris*	Ambion	AM9856	1 M, pH 8.0
UltraPure 10× TBE buffer*	Invitrogen	15581-044
* - for library preparation
gasket and spring clamp , 90 mm,	Millipore	XX1009020
ground joint flask 1 L ,	Millipore	XX1504705