Analysis of Translation in the Developing Mouse Brain using Polysome Profiling

Summary

The development of the mammalian brain requires proper control of gene expression at the level of translation. Here, we describe a polysome profiling system with an easy-to-assemble sucrose gradient-making and fractionation platform to assess the translational status of mRNAs in the developing brain.

Abstract

The proper development of the mammalian brain relies on a fine balance of neural stem cell proliferation and differentiation into different neural cell types. This balance is tightly controlled by gene expression that is fine-tuned at multiple levels, including transcription, post-transcription and translation. In this regard, a growing body of evidence highlights a critical role of translational regulation in coordinating neural stem cell fate decisions. Polysome fractionation is a powerful tool for the assessment of mRNA translational status at both global and individual gene levels. Here, we present an in-house polysome profiling pipeline to assess translational efficiency in cells from the developing mouse cerebral cortex. We describe the protocols for sucrose gradient preparation, tissue lysis, ultracentrifugation and fractionation-based analysis of mRNA translational status.

Introduction

During the development of the mammalian brain, neural stem cells proliferate and differentiate to generate neurons and glia^1,2 . The perturbation of this process can lead to alterations in brain structure and function, as seen in many neurodevelopmental disorders^3,4. The proper behavior of neural stem cells requires the orchestrated expression of specific genes⁵. While the epigenetic and transcriptional control of these genes has been intensively studied, recent findings suggest that gene regulation at other levels also contribu....

Protocol

All animal use was supervised by the Animal Care Committee at the University of Calgary. CD1 mice used for the experiment were purchased from commercial vendor.

1. Preparation of solutions

NOTE To prevent RNA degradation, spray workbench and all equipment with RNase decontamination solution. RNase-free tips are used for the experiment. All solutions are prepared in RNase-free water.

1. Prepare cycloheximide stock solution (100 mg/mL) in DMSO and store at -20 °C.
2. Prepare 2.2 M sucrose stock solution by adding 75.3 g of sucrose to RNase-free water and topping up the volume to 100 mL (for ~16....

Results

As a demonstration, the cortical lysate containing 75 µg RNA (pooled from 8 embryos) was separated by the sucrose gradient into 12 fractions. Peaks of UV absorbance at 254 nm identified fractions containing the 40S subunit, 60S subunit, 80S monosome and polysomes (Figure 4A). Analysis of fractions by western blot for the large ribosomal subunit, Rpl10 showed its presence in the 60S subunit (fraction 3), monosome (fraction 4) and polysomes (fractions 5-12) (Figure 4B.......

Discussion

Polysome profiling is a commonly used and powerful technique to assess the translational status at both single gene and genome-wide levels¹⁴ . In this report, we present a protocol of polysome profiling using a home-assembled platform and its application to analyze the developing mouse cortex. This cost-effective platform is easy to assemble and generate robust, reproducible sucrose gradients and polysome profiling with high sensitivity.

It is worthy to note that the pr.......

Materials

Name	Company	Catalog Number	Comments
1.5 mL RNA free microtubes	Axygen	MCT-150-C
10 cm dish	Greiner-Bio	664160
1M MgCl2	Invitrogen	AM9530G
21-23G needle	BD	305193
2M KCl	Invitrogen	AM8640G
30 mL syringe	BD	302832
Blunt end needle	VWR	20068-781
Breadboard	Thorlabs	MB2530/M
Bromophenol blue	Sigma	115-39-9
CD1 mouse		Charles River Laboratory
Curved tip forceps	Sigma	#Z168785
Cycloheximide		Sigma	66-81-9
Data acquisition software TracerDAQ	Measurement Computing
Digital converter		Measurement Computing	USB-1208LS
Direct-zol RNA miniprep kit	Zymo	R2070
Dithiothreitol (DTT)	Bio-basic	12-03-3483
DMSO	Bioshop	67-68-5
Dumont No.5 forceps	Sigma	#F6521
Fraction collector	Bio-Rad	Model 2110
HBSS	Wisent	311-513-CL
Linear stage actuator	Rattmmotor	CBX1605-100A
Luciferase control RNA	Promega	L4561
Maxima first strand cDNA synthesis kit	Themo Fisher	M1681
Miniature V-clamp	Thorlabs	VH1/M
Mini-series breadboard	Thorlabs	MSB7515/M
Mini-series optical post	Thorlabs	MS2R/M
Mini-series pedestal post holder base	Thorlabs	MBA1
NaCl	Bio-basic	7647-14-5
Neurobasal media	Gibco	21103-049
Ø12.7 mm aluminum post	Thorlabs	TRA150/M
Parafilm	Bemis	PM992
PerfeCTa SYBR green fastmix	Quanta Bio	CA101414-274
Phosphate buffered saline (PBS)	Wisent	311-010-CL
Puromycin	Bioshop	58-58-2
Right-angle clamp	Thorlabs	RA90/M
Right-angle Ø1/2" to Ø6 mm post clamp	Thorlabs	RA90TR/M
Rnase AWAY	Molecular BioProducts	7002
RNase free tips	Frogga Bio	FT10, FT200, FT1000
RNase free water	Wisent	809-115-CL
RNasin	Promega	N2111
Slim right-angle bracket	Thorlabs	AB90B/M
Small V-clamp	Thorlabs	VC1/M
Sodium deoxycholate	Sigma	302-95-4
Stepper motor driver	SongHe	TB6600
Sucrose	Bioshop	57501
SW 41 Ti rotor	Beckman Coulter	331362
Syringe pump	Harvard Apparatus	70-4500
Syringe pump	Harvard Apparatus	70-4500
Triton-X-100	Bio-basic	9002-93-1
Trizol	Thermofisher Scientific	15596018
Tube piercer	Brandel	BR-184
Ultracentrifuge		Beckman Coulter	L8-70M
Ultracentrifuge tubes	Beckman Coulter	331372
UltraPure 1M Tris-HCl pH 7.5	Invitrogen	15567-027
UNO project super starter kit	Elegoo	EL-KIT-003
UV monitor	Bio-Rad	EM-1 Econo
Vertical bracket	Thorlabs	VB01A/M