JoVE Logo
Faculty Resource Center

Sign In





Representative Results






Analysis of Translation in the Developing Mouse Brain using Polysome Profiling

Published: May 22nd, 2021



1Department of Biochemistry and Molecular Biology; Cumming School of Medicine, University of Calgary, 2Department of Medical Genetics, Cumming School of Medicine, University of Calgary, 3Alberta Children's Hospital Research Institute, University of Calgary

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.

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.

During the development of the mammalian brain, neural stem cells proliferate and differentiate to generate neurons and glia1,2 . The perturbation of this process can lead to alterations in brain structure and function, as seen in many neurodevelopmental disorders3,4. 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....

Log in or to access full content. Learn more about your institution’s access to JoVE content here

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 -2.......

Log in or to access full content. Learn more about your institution’s access to JoVE content here

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.......

Log in or to access full content. Learn more about your institution’s access to JoVE content here

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.......

Log in or to access full content. Learn more about your institution’s access to JoVE content here

This work was funded by a NSERC Discovery Grant (RGPIN/04246-2018 to G.Y.). G.Y. is a Canada Research Chair. S.K. was funded by Mitacs Globalink Graduate Fellowship and ACHRI Graduate Student Scholarship.


Log in or to access full content. Learn more about your institution’s access to JoVE content here

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

  1. Götz, M., Huttner, W. B. The cell biology of neurogenesis. Nature Reviews Molecular Cell Biology. 6, 777-788 (2005).
  2. Guillemot, F. Spatial and temporal specification of neural fates by transcription factor codes. Development. 134, 3771-3780 (2007).
  3. Fiddes, I. T., et al. Human-Specific NOTCH2NL Genes Affect Notch Signaling and Cortical Neurogenesis. Cell. 173, 1356-1369 (2018).
  4. Lennox, A. L., et al. Pathogenic DDX3X mutations impair RNA metabolism and neurogenesis during fetal cortical development. Neuron. 106, 404-420 (2020).
  5. Martynoga, B., Drechsel, D., Guillemot, F. Molecular control of neurogenesis: A view from the mammalian cerebral cortex. Cold Spring Harbor Perspective Biology. 4 (10), 008359 (2012).
  6. Amadei, G., et al. A Smaug2-based translational repression complex determines the balance between precursor maintenance versus differentiation during mammalian neurogenesis. Journal of Neurosci. 35, 15666-15681 (2015).
  7. Yang, G., Smibert, C. A., Kaplan, D. R., Miller, F. D. An eIF4E1/4E-T complex determines the genesis of neurons from precursors by translationally repressing a proneurogenic transcription program. Neuron. 84, 723-739 (2014).
  8. Yang, G., et al. A Glo1-Methylglyoxal pathway that is perturbed in maternal diabetes regulates embryonic and adult neural stem cell pools in murine offspring. Cell Reports. 17, 1022-1036 (2016).
  9. Kraushar, M. L., et al. Temporally defined neocortical translation and polysome assembly are determined by the RNA-binding protein Hu antigen R. Proceedings of the National Academy of Science U. S. A. 111, 3815-3824 (2014).
  10. Rodrigues, D. C., et al. Methylglyoxal couples metabolic and translational control of Notch signalling in mammalian neural stem cells. Nature Communications. 11, 2018 (2020).
  11. Iwasaki, S., Ingolia, N. T. The growing toolbox for protein synthesis studies. Trends in Biochemical Sciences. 42, 612-624 (2017).
  12. Chekulaeva, M., Landthaler, M. Eyes on translation. Molecular Cell. 63, 918-925 (2016).
  13. Faye, M. D., Graber, T. E., Holcik, M. Assessment of Selective mRNA Translation in Mammalian Cells by Polysome Profiling. Journal of Visualized Experiments. (92), e52295 (2014).
  14. Chassé, H., Boulben, S., Costache, V., Cormier, P., Morales, J. Analysis of translation using polysome profiling. Nucleic Acids Research. 45, 15 (2017).
  15. Schneider-Poetsch, T., et al. Inhibition of eukaryotic translation elongation by cycloheximide and lactimidomycin. Nature Chemical Biology. 6, 209-217 (2010).
  16. Kraushar, M. L., et al. Thalamic WNT3 secretion spatiotemporally regulates the neocortical ribosome signature and mRNA translation to specify neocortical cell subtypes. Journal of Neuroscience: Official Journal of Society of Neuroscience. 35, 10911-10926 (2015).
  17. Gandin, V., et al. Polysome fractionation and analysis of mammalian translatomes on a genome-wide scale. Journal of Visualized Experiments. (7), e51455 (2014).

This article has been published

Video Coming Soon

JoVE Logo


Terms of Use





Copyright © 2024 MyJoVE Corporation. All rights reserved