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 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.
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.
2. Preparation of sucrose gradient
NOTE: Accuracy in preparation of sucrose gradients is critical in obtaining consistent and reproducible results.
Sucrose solution | 10% | 20% | 30% | 40% | 50% |
2.2 M sucrose | 2 mL | 4 mL | 6 mL | 8 mL | 10 mL |
10X salt solution | 1.5 mL | 1.5 mL | 1.5 mL | 1.5 mL | 1.5 mL |
Cycloheximide | 15 µL | 15 µL | 15 µL | 15 µL | 15 µL |
Water | 11.5 mL | 9.5 mL | 7.5 mL | 5.5 mL | 3.5 mL |
Total volume | 15 mL | 15 mL | 15 mL | 15 mL | 15 mL |
Table 1: Sucrose dilutions for preparations of sucrose gradients.
NOTE: Always prepare sucrose gradients in multiples of two to balance weight during ultracentrifugation.
3. Tissue dissection
NOTE: Pregnant mice were euthanized by cervical dislocation preceded by anesthesia with 5% isoflurane.
4. Cell lysis
Solution | Final concentration | Volume |
Tris-HCl (pH 7.5) | 20 mM | 100 µL |
KCl | 100 mM | 250 µL |
MgCl2 | 5 mM | 25 µL |
Triton X-100 | 1% (v/v) | 500 µL |
Sodium deoxycholate | 0.5% (w/v) | 500 µL |
Dithiothreitol (DTT) | 1 mM | 5 µL |
Cycloheximide | 100 µg/mL | 5 µL |
RNase free water | Top up to 5 mL | |
Total | 5 mL | 5 mL |
Table 2: Preparation of polysome lysis buffer.
NOTE: Supplement lysis buffer with protease and phosphatase inhibitors.
5. Sample loading and ultracentrifugation
6. Fractionation and sample collection
NOTE: A home-assembled fractionating, recording and collecting system is used for the analysis and collecting samples from the gradients (Figure 3, see Device components).
7. Extraction of RNA
8. Reverse transcription and real-time PCR
9. Sucrose gradient making system assembly
NOTE: Follow the steps to assemble each component (as described in Table 3) of the sucrose gradient maker (Figure 1).
Component | Item |
B1 | Linear stage actuator |
E1 | Stepper motor driver |
E2 | UNO project super starter kit |
A1 | Breadboard |
A2 | Vertical bracket |
B2 | Slim right-angle bracket |
C1 | Mini-series breadboard |
C5 | Small V-clamp |
D4 | Miniature V-clamp |
C2 | Ø12.7 mm aluminum post |
C4, D3 | Mini-series optical post |
D1 | Ø12.7 mm aluminum post |
C3, D2 | Right-angle Ø1/2" to Ø6 mm post clamp |
C6 | Mini-series pedestal post holder base |
D5 | Blunt end needle |
F | Syringe pump |
Table 3: Gradient making system components.
10. Fractionating and detecting system assembly (Figure 2).
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). In contrast, cytoplasmic proteins Gapdh and Csde1 were not associated with ribosomes but were enriched in fractions containing free RNA (fraction 1) (Figure 4B). Consistent with the separation of proteins in different fractions, we found that gapdh and sox2 mRNAs were highly enriched in the fractions containing heavy polysomes (more than three ribosomes in fractions 7-12), suggesting that gapdh and sox2 mRNAs are efficiently translated in the developing cortex (Figure 4C,D). In contrast, rpl7 and rpl34 mRNAs were enriched in the fraction containing monosomes, suggesting repressed translation (Figure 4E,F)16. These results validated our polysome fractionation protocol.
Figure 1: Setup for sucrose gradient preparation. (A) Home-assembled sucrose gradient maker consisting of a linear stage actuator, a tube holder on the motorized stage, a stage controller set, a metal blunt-end needle mounted on a needle holder, and an automated syringe pump with a syringe connected to the needle (see Table 3). (B) To prepare the sucrose gradient, the ultracentrifuge tube is placed in the tube holder. Sucrose solutions are dispensed through the blunt-end needle using a syringe pump. Please click here to view a larger version of this figure.
Figure 2: Dissection of the cortical tissues from the development mouse embryo. (A) Image showing an E12.5 CD1 embryo fixed to a Ø 6 cm plate using 21G-23G needles. (B) Image showing the dissected cortex after removal of the skin, skull and meninges (marked with white dashed lines). Please click here to view a larger version of this figure.
Figure 3: Setup for fractionating, recording and sample collecting. Image depicting the home-assembled fractionation, recording and sample collection system comprising of an automated syringe pump, a tube piercer, a fraction collector and a UV monitor with a digital convertor connected to a laptop for the continuous monitoring of UV absorbance. Data acquisition is handled by the commercial data acquisition software. Please click here to view a larger version of this figure.
Figure 4: Polysome profiling analysis of the developing mouse cortex. (A) UV absorbance showing fractions containing free RNA (fraction 1), 40S subunit (fraction 2), 60S subunit (fraction 3), 80S monosomes (fraction 4) and polysomes (fraction 5-12). 75 µg total RNA pooled from 8 embryos was used. (B) Western blots showing the distributions of Gapdh, Rpl10 and Csde1 proteins in fractions. qPCR analysis showing the distribution of gapdh (C), sox2 (D), rpl7 (E) and rpl34 (F) mRNAs in fractions. Please click here to view a larger version of this figure.
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 preparation of consistent and good quality sucrose gradients is critical to obtain reproducible polysome profiling results17. Changes in the volume of 10-50% sucrose solutions during gradient preparation could cause the shift of the polysome peaks and inconsistency of results in downstream analysis. Moreover, disturbing the gradient during the insertion and removal of the needle could contribute to the inconsistency of gradient preparation. Compared to other approaches and commercial devices, our home-made gradient making system used a motorized stage to reduce disturbance of gradients during preparation and a syringe pump to accurately dispense sucrose solutions, which offers a cheap and simple solution for gradient preparation.
Applying the polysome profiling platform and protocol present here to the developing mouse cortex, we found that the ribosomal protein Rpl10 and cytoplasmic proteins Gapdh and Csde1 were distributed in the correct fractions. Moreover, the highly translated gapdh and sox2 mRNAs were enriched in polysomal fractions, while translationally repressed rpl34 and rpl7 mRNAs showed less enrichement in polysomes, which validate our platform and protocol. With a similar approach, the translational status of other genes in the developing cortex can be determined individually by real-time PCR. Of note, polysome profiling has been used to analyse translational status in the developing brain at the genome-wide level. In this regard, fractions containing polysomes can be combined and extracted RNA can be analysed using deep sequencing. The platform and protocol present here can be adapted and further optimized to suit translatomic studies using other cell types and tissues. Considering the availability of cortical tissues limited by experimental conditions (e.g., <50 µg RNA), additional optimization of the protocol may provide further increases in the efficiency and reproducibility of the experiments.
While polysome profiling provides insights into the translational status of mRNAs in the developing cortex, it has limitations to analyze specific cell types at later developmental stages, when different neural cell types are present. To address this question, polysome profiling can be integrated with TRAP by pull-down of epitope tagged ribosomal protein expressed under a tissue specific promoter11.
In conclusion, the platform and protocol that we report here for sucrose gradient making and fractionation provide an economical solution to polysome profiling experiments in the context of brain development as well as other biological contexts.
The authors have nothing to disclose.
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.
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 |