Analysis of Translation Initiation During Stress Conditions by Polysome Profiling

Summary

Here, we describe a method to analyze changes in the initiation of mRNA translation of eukaryotic cells in response to stress conditions. This method is based on the velocity separation on sucrose gradients of translating ribosomes from non-translating ribosomes.

Abstract

Precise control of mRNA translation is fundamental for eukaryotic cell homeostasis, particularly in response to physiological and pathological stress. Alterations of this program can lead to the growth of damaged cells, a hallmark of cancer development, or to premature cell death such as seen in neurodegenerative diseases. Much of what is known concerning the molecular basis for translational control has been obtained from polysome analysis using a density gradient fractionation system. This technique relies on ultracentrifugation of cytoplasmic extracts on a linear sucrose gradient. Once the spin is completed, the system allows fractionation and quantification of centrifuged zones corresponding to different translating ribosomes populations, thus resulting in a polysome profile. Changes in the polysome profile are indicative of changes or defects in translation initiation that occur in response to various types of stress. This technique also allows to assess the role of specific proteins on translation initiation, and to measure translational activity of specific mRNAs. Here we describe our protocol to perform polysome profiles in order to assess translation initiation of eukaryotic cells and tissues under either normal or stress growth conditions.

Introduction

Eukaryotic cells constantly encounter a range of harmful physiological and environmental stress conditions that require a rapid adaptive cell response. Cell stress response involves a precise balance between anti-survival and pro-survival acting factors. Disrupting this balance can have irreversible consequences leading the development of human pathologies such as cancer and neurodegenerative diseases. During the first step of the stress response, cells activate pro-survival pathways that involve the coordinated control of changes in gene expression at the level of mRNA translation.

mRNA translation in eukaryotes is a complex cellular proce....

Protocol

The protocol follows the guidelines approved by Laval's Ethical Review Board.

1. Preparation of Cell Cultures and Brain Manipulation

1. Mammalian and Drosophila Cells
   1. Grow HeLa cervical cancer cells and Schneider Drosophila embryonic cells as recommended by the American Type Culture Collection. Work with cells at a low passage.
   2. Plate cells in order to reach 80% confluence the day of the experiment. For best results, use ∼12 x 10⁶ of cells for each experimental condition. Before making extracts for polysome analysis, stimulate translation by adding f....

Results

As aforementioned, polysome profile allows the analysis of changes of translation initiation under stress conditions. Figure 1 is a simplified view of translation initiation which as described earlier is a multistep process involving an ordered assembly of translation initiation complexes. Under normal growth conditions, translation initiation complexes are converted into polyribosomes whose detection by polysome profile attest for an active translation initiation (Figure 2; Untreated). .......

Discussion

The polysome profile analysis on sucrose gradients allows measurement of translation initiation by analyzing the density of polysomes isolated from cells or tissues^9,11-14. This technique is the best (if not the unique) approach to measure translation initiation in vivo. It is used to monitor the translational status of growing cells during cell cycle¹⁵, and to assess the effects of various types of stress including viral infections, hypoxia^13,16, radiation¹⁷, and chem.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Cells
HeLa cervical cancer cells	American Type Culture Collection (Manassas, VA; ATCC)	CCL-2
Schneider Drosophila embryonic cells	American Type Culture Collection (Manassas, VA; ATCC)	CRL-1963
Culture medium and Supplements
Schneider’s Drosophila Medium	Sigma-Aldrich	SO146-500ml
DMEM	Life technologies	11995-073
FBS	Fisher Scientist Scientist	SH30396-03
Penicillin/streptomycin	Life technologies	15140122
Sucrose solutions
D-sucrose	Fisher Scientist	BP220-212
Glycerol	Sigma-Aldrich	49767
Bromophenol blue	Fisher Scientist	B3925
Lysis buffer
Tris HCl	Fisher Scientist	BP153-500
MgCl2	Sigma-Aldrich	M2670-100G
NaCl	Tekniscience	3624-05
DTT	Sigma-Aldrich	D 9779
Nonidet P40 (Igepal CA-630 )	MJS Biolynx	19628
SDS	Tekniscience	4095-02
RNase inhibitor (RnaseOUT Recombinant Ribonuclease Inhibitor)	Life technologies	10777-019
Antiproteases (complete, mini, EDTA free)	Roche	11,836,170,001
RNA Extraction
Proteinase K	Life technologies	AM2542
Phenol:chloroform	Fisher Scientist	BP1754I-400
Chloroform	Fisher Scientist	C298-500
Glycogen	Life technologies	10814-010
Isopropanol	Acros organics	327270010
Antibodies
anti-FMRP antibody	Fournier et al., Cancer Cell International, 2010