Name: analysis of translation in the developing mouse brain using polysome profiling
Uploaded: 2021-05-22T14:00:00
Description: Watch this Scientific Journal Video about Analysis of Translation in the Developing Mouse Brain using Polysome Profiling at JoVE.com

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.

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.
<ol>
	<li>Prepare cycloheximide stock solution (100 mg/mL) in DMSO and store at -2...

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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...

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 contributes to the coordination of neural stem cell proliferation and differentiation6,7,8,9,10. Thus, addressing the translational control programs will greatly advance our understanding of the mechanisms underlying neural stem cell fate decision and brain development.
Three main techniques with different strengths have been widely applied to assess the translational status of mRNA, including ribosome profiling, translating ribosome affinity purification (TRAP) and polysome profiling. Ribosome profiling uses RNA sequencing to determine ribosome-protected mRNA fragments, allowing the global analysis of the number and location of translating ribosomes on each transcript to indirectly infer the translation rate by comparing it to transcript abundance11. TRAP takes advantage of epitope-tagged ribosomal proteins to capture ribosome-bound mRNAs12. Given that the tagged ribosomal proteins can be expressed in specific cell types using genetic approaches, TRAP allows the analysis of translation in a cell type-specific manner. In comparison, polysome profiling, which uses sucrose density gradient fractionation to separate free and poorly-translated portion (lighter monosomes) from those being actively translated by ribosomes (heavier polysomes), provides a direct measurement of ribosome density on mRNA13. One advantage this technique offers is its versatility to study the translation of specific mRNA of interest as well as genome-wide translatome analysis14.
In this paper, we describe a detailed protocol of polysome profiling to analyze the developing mouse cerebral cortex. We use a home-assembled system to prepare sucrose density gradients and collect fractions for downstream applications. The protocol presented here can be adapted easily to analyze other types of tissues and organisms.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1.5 mL RNA free microtubes</td>
			<td>Axygen</td>
			<td>MCT-150-C</td>
			<td></td>
		</tr>
		<tr>
			<td>10 cm dish</td>
			<td>Greiner-Bio</td>
			<td>664160</td>
			<td></td>
		</tr>
		<tr>
			<td>1M MgCl2</td>
			<td>Invitrogen</td>
			<td>AM9530G</td>
			<td></td>
		</tr>
		<tr>
			<td>21-23G needle</td>
			<td>BD</td>
			<td>305193</td>
			<td></td>
		</tr>
		<tr>
			<td>2M KCl</td>
			<td>Invitrogen</td>
			<td>AM8640G</td>
			<td></td>
		</tr>
		<tr>
			<td>30 mL syringe</td>
			<td>BD</td>
			<td>302832</td>
			<td></td>
		</tr>
		<tr>
			<td>Blunt end needle</td>
			<td>VWR</td>
			<td>20068-781</td>
			<td></td>
		</tr>
		<tr>
			<td>Breadboard</td>
			<td>Thorlabs</td>
			<td>MB2530/M</td>
			<td></td>
		</tr>
		<tr>
			<td>Bromophenol blue</td>
			<td>Sigma</td>
			<td>115-39-9</td>
			<td></td>
		</tr>
		<tr>
			<td>CD1 mouse</td>
			<td></td>
			<td>Charles River Laboratory</td>
			<td></td>
		</tr>
		<tr>
			<td>Curved tip forceps</td>
			<td>Sigma</td>
			<td>#Z168785</td>
			<td></td>
		</tr>
		<tr>
			<td>Cycloheximide</td>
			<td></td>
			<td>Sigma</td>
			<td>66-81-9</td>
		</tr>
		<tr>
			<td>Data acquisition software TracerDAQ</td>
			<td>Measurement Computing</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Digital converter</td>
			<td></td>
			<td>Measurement Computing</td>
			<td>USB-1208LS</td>
		</tr>
		<tr>
			<td>Direct-zol RNA miniprep kit</td>
			<td>Zymo</td>
			<td>R2070</td>
			<td></td>
		</tr>
		<tr>
			<td>Dithiothreitol (DTT)</td>
			<td>Bio-basic</td>
			<td>12-03-3483</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Bioshop</td>
			<td>67-68-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont No.5 forceps</td>
			<td>Sigma</td>
			<td>#F6521</td>
			<td></td>
		</tr>
		<tr>
			<td>Fraction collector</td>
			<td>Bio-Rad</td>
			<td>Model 2110</td>
			<td></td>
		</tr>
		<tr>
			<td>HBSS</td>
			<td>Wisent</td>
			<td>311-513-CL</td>
			<td></td>
		</tr>
		<tr>
			<td>Linear stage actuator</td>
			<td>Rattmmotor</td>
			<td>CBX1605-100A</td>
			<td></td>
		</tr>
		<tr>
			<td>Luciferase control RNA</td>
			<td>Promega</td>
			<td>L4561</td>
			<td></td>
		</tr>
		<tr>
			<td>Maxima first strand cDNA synthesis kit</td>
			<td>Themo Fisher</td>
			<td>M1681</td>
			<td></td>
		</tr>
		<tr>
			<td>Miniature V-clamp</td>
			<td>Thorlabs</td>
			<td>VH1/M</td>
			<td></td>
		</tr>
		<tr>
			<td>Mini-series breadboard</td>
			<td>Thorlabs</td>
			<td>MSB7515/M</td>
			<td></td>
		</tr>
		<tr>
			<td>Mini-series optical post</td>
			<td>Thorlabs</td>
			<td>MS2R/M</td>
			<td></td>
		</tr>
		<tr>
			<td>Mini-series pedestal post holder base</td>
			<td>Thorlabs</td>
			<td>MBA1</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Bio-basic</td>
			<td>7647-14-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Neurobasal media</td>
			<td>Gibco</td>
			<td>21103-049</td>
			<td></td>
		</tr>
		<tr>
			<td>&#216;12.7 mm aluminum post</td>
			<td>Thorlabs</td>
			<td>TRA150/M</td>
			<td></td>
		</tr>
		<tr>
			<td>Parafilm</td>
			<td>Bemis</td>
			<td>PM992</td>
			<td></td>
		</tr>
		<tr>
			<td>PerfeCTa SYBR green fastmix</td>
			<td>Quanta Bio</td>
			<td>CA101414-274</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline (PBS)</td>
			<td>Wisent</td>
			<td>311-010-CL</td>
			<td></td>
		</tr>
		<tr>
			<td>Puromycin</td>
			<td>Bioshop</td>
			<td>58-58-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Right-angle clamp</td>
			<td>Thorlabs</td>
			<td>RA90/M</td>
			<td></td>
		</tr>
		<tr>
			<td>Right-angle &#216;1/2&#34; to &#216;6 mm post clamp</td>
			<td>Thorlabs</td>
			<td>RA90TR/M</td>
			<td></td>
		</tr>
		<tr>
			<td>Rnase AWAY</td>
			<td>Molecular BioProducts</td>
			<td>7002</td>
			<td></td>
		</tr>
		<tr>
			<td>RNase free tips</td>
			<td>Frogga Bio</td>
			<td>FT10, FT200, FT1000</td>
			<td></td>
		</tr>
		<tr>
			<td>RNase free water</td>
			<td>Wisent</td>
			<td>809-115-CL</td>
			<td></td>
		</tr>
		<tr>
			<td>RNasin</td>
			<td>Promega</td>
			<td>N2111</td>
			<td></td>
		</tr>
		<tr>
			<td>Slim right-angle bracket</td>
			<td>Thorlabs</td>
			<td>AB90B/M</td>
			<td></td>
		</tr>
		<tr>
			<td>Small V-clamp</td>
			<td>Thorlabs</td>
			<td>VC1/M</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium deoxycholate</td>
			<td>Sigma</td>
			<td>302-95-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Stepper motor driver</td>
			<td>SongHe</td>
			<td>TB6600</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Bioshop</td>
			<td>57501</td>
			<td></td>
		</tr>
		<tr>
			<td>SW 41 Ti rotor</td>
			<td>Beckman Coulter</td>
			<td>331362</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe pump</td>
			<td>Harvard Apparatus</td>
			<td>70-4500</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe pump</td>
			<td>Harvard Apparatus</td>
			<td>70-4500</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton-X-100</td>
			<td>Bio-basic</td>
			<td>9002-93-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Trizol</td>
			<td>Thermofisher Scientific</td>
			<td>15596018</td>
			<td></td>
		</tr>
		<tr>
			<td>Tube piercer</td>
			<td>Brandel</td>
			<td>BR-184</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultracentrifuge</td>
			<td></td>
			<td>Beckman Coulter</td>
			<td>L8-70M</td>
		</tr>
		<tr>
			<td>Ultracentrifuge tubes</td>
			<td>Beckman Coulter</td>
			<td>331372</td>
			<td></td>
		</tr>
		<tr>
			<td>UltraPure 1M Tris-HCl pH 7.5</td>
			<td>Invitrogen</td>
			<td>15567-027</td>
			<td></td>
		</tr>
		<tr>
			<td>UNO project super starter kit</td>
			<td>Elegoo</td>
			<td>EL-KIT-003</td>
			<td></td>
		</tr>
		<tr>
			<td>UV monitor</td>
			<td>Bio-Rad</td>
			<td>EM-1 Econo</td>
			<td></td>
		</tr>
		<tr>
			<td>Vertical bracket</td>
			<td>Thorlabs</td>
			<td>VB01A/M</td>
			<td></td>
		</tr>
	</tbody>
</table>

analysis of translation in the developing mouse brain using polysome profiling

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.

As a demonstration, the cortical lysate containing 75 &#181;g 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...

Watch this Scientific Journal Video about Analysis of Translation in the Developing Mouse Brain using Polysome Profiling at JoVE.com

Analysis of Translation in the Developing Mouse Brain using Polysome Profiling

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 ...

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