Name: Genome-wide Quantification of Translation in Budding Yeast by Ribosome Profiling (Video) | JoVE Journal
Uploaded: 2017-12-21T14:00:00.000+00:00
Duration: 12 min 57 s
Description: Learn ribosome profiling for genome-wide translation analysis in Saccharomyces cerevisiae. This sensitive method enables quantitative measurement of protein synthesis and translational regulation mechanisms.

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This work was supported by the National Institutes of Health grants AG040191 and AG054566&#160;to VML. This research was conducted while VML was an AFAR Research Grant recipient from the American Federation for Aging Research.

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The authors declare that they have no competing financial interests.

The Ribo-Seq approach has emerged as a powerful technology for the analysis of mRNA translation in vivo at the genome-wide level 3. Studies using this approach, which allows monitoring translation with single-codon resolution, has contributed to our understanding of translational regulation. Despite its advantages, Ribo-Seq has several limitations. Ribosomal RNA (rRNA) fragments are always co-purified during isolation of ribosome-protected footprints decreasing the yield of useful sequenc...

mRNA translation is one of the fundamental processes in the cell, which plays an important role in the regulation of protein expression. Therefore, mRNA translation is tightly controlled in response to different internal and external physiological stimuli 1,2. However, the mechanisms of translational regulation remain understudied. Here, we describe the protocol for the genome-wide quantification of translation in budding yeast by ribosome profiling. The overall goal of the ribosome profiling technique is to study and quantify the translation of specific mRNAs under different cellular conditions. This technique uses next-generation sequencing to quantitatively analyze ribosome occupancy throughout the genome and allows monitoring the rate of protein synthesis in vivo at the single codon resolution 3,4. Currently, this method provides the most advanced means of measuring the levels of protein translation, and has proven to be a useful discovery tool providing information that cannot be revealed by other currently available techniques, e.g. microarrays or translation state array analysis (TSAA) 5. As ribosome profiling reports on the combined changes in transcript levels and translational output, it also provides much greater sensitivity compared to other methods.
This approach is based on deep sequencing of ribosome-protected mRNA fragments 3. During protein translation, ribosomes protect ~ 28 nt portions of the mRNA (called footprints) 6. By determining the sequence of the ribosome-protected fragments, Ribo-Seq can map the position of ribosomes on the translated mRNA and identify which regions of mRNA are likely to be actively translated into protein 3,7. In addition, we can quantitatively measure the translation of mRNA by counting the number of footprints that align to a given mRNA transcript.
In order to isolate the ribosome-protected fragments, cell lysates are initially treated with a translation inhibitor to stall the ribosomes followed by ribonuclease digestion. Whereas free mRNA and portions of translated mRNAs not protected by ribosomes are degraded by ribonuclease, the ribosome-protected mRNA fragments can be recovered by purifying intact ribosome-footprint complexes. These mRNA footprints are then converted into cDNA library and analyzed by deep sequencing (Figure 1). In parallel to ribosome profiling, intact mRNA is extracted from the same sample and sequenced. By comparing the level of translation identified by Ribo-Seq with mRNA abundance measurements, we can identify genes that are specifically up- or down-regulated at the level of translation and calculate translation efficiency of mRNA at the genome-wide level. While the protocol described in this article is specific for yeast, it should be also useful for&#160;researchers who will try to establish the Ribo-Seq protocol in other systems.

<table><tbody><tr><td>0.45 &amp;mu;M membrane filters</td><td>Millipore</td><td>HVLP04700</td><td></td></tr><tr><td>0.5 M EDTA</td><td>Invitrogen</td><td>AM9261</td><td></td></tr><tr><td>0.5 mL centrifugal filters (100 kDa MWCO)</td><td>Millipore</td><td>UFC510024</td><td></td></tr><tr><td>1 M Tris-HCl, pH 7.0</td><td>Invitrogen</td><td>AM9850G</td><td></td></tr><tr><td>1 M Tris-HCl, pH 7.5 (pH 8.0 at 4&amp;deg;C)</td><td>Invitrogen</td><td>15567-027</td><td></td></tr><tr><td>10X TBE buffer</td><td>Invitrogen</td><td>AM9863</td><td></td></tr><tr><td>10% TBE-urea gel</td><td>Invitrogen</td><td>EC6875BOX</td><td></td></tr><tr><td>15% TBE-urea gel</td><td>Invitrogen</td><td>EC6885BOX</td><td></td></tr><tr><td>2 M MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>RPI</td><td>M24500-10.0</td><td></td></tr><tr><td>2X TBE-urea sample buffer</td><td>Invitrogen</td><td>LC6876</td><td></td></tr><tr><td>3M NaOAc, pH 5.5</td><td>Invitrogen</td><td>AM9740</td><td></td></tr><tr><td>5&#039; Deadenylase (10 U/&amp;mu;L)</td><td>Epicentre</td><td>DA11101K</td><td></td></tr><tr><td>5X Nucleic acid sample loading buffer</td><td>Bio-Rad</td><td>161-0767</td><td></td></tr><tr><td>8% TBE gel</td><td>Invitrogen</td><td>EC6215BOX</td><td></td></tr><tr><td>Acid-Phenol:Chloroform, pH 4.5 (with IAA, 125:24:1)</td><td>Invitrogen</td><td>AM9722</td><td></td></tr><tr><td>Blue light transilluminator</td><td>Clare Chemical Research</td><td>DR-46B</td><td></td></tr><tr><td>Chrome-steel beads, 3.2 mm</td><td>BioSpec Products</td><td>11079132c</td><td></td></tr><tr><td>Cryogrinder</td><td>Biospec product</td><td>3110BX</td><td></td></tr><tr><td>Cycloheximide</td><td>RPI</td><td>C81040-5.0</td><td></td></tr><tr><td>Data Acquisition System</td><td>DATAQ Instruments</td><td>DI-245</td><td></td></tr><tr><td>Deoxynucleotide (dNTP) solution mix (10 mM)</td><td>NEB</td><td>N0447L</td><td></td></tr><tr><td>Glycogen</td><td>Invitrogen</td><td>AM9510</td><td></td></tr><tr><td>Gradient fractionation system</td><td>Brandel</td><td>BR-184X</td><td></td></tr><tr><td>High-fidelity DNA polymerase (2,000 U/mL)</td><td>NEB</td><td>M0530S</td><td>Supplied with 5X Phusion HF Buffer</td></tr><tr><td>Next-generation sequencing library quantification kit</td><td>Kapa Biosystems</td><td>KK4824</td><td></td></tr><tr><td>Nucleic acid gel stain</td><td>Invitrogen</td><td>S11494</td><td></td></tr><tr><td>Optima XE-90 ultracentrifuge</td><td>Beckman Coulter</td><td>A94471</td><td></td></tr><tr><td>Poly(A) mRNA isolation kit</td><td>Invitrogen</td><td>61011</td><td></td></tr><tr><td>Rec J exonuclease (10 U/&amp;mu;L)</td><td>Epicentre</td><td>RJ411250</td><td></td></tr><tr><td>Reverse transcriptase (200 U/&amp;mu;L)</td><td>Invitrogen</td><td>18080093</td><td>Supplied with 5X first-strand buffer and 0.1 M DTT</td></tr><tr><td>RNA fragmentation buffer</td><td>NEB</td><td>E6186A</td><td></td></tr><tr><td>RNase I (100 U/&amp;mu;L)</td><td>Invitrogen</td><td>AM2295</td><td></td></tr><tr><td>RNase inhibitor (20 U/&amp;mu;L)</td><td>Invitrogen</td><td>AM2696</td><td></td></tr><tr><td>Silicone rubber caps</td><td>BioSpec Products</td><td>2008</td><td></td></tr><tr><td>ssDNA ligase (100 U/&amp;mu;L)</td><td>Epicentre</td><td>CL9021K</td><td>Supplied with 10X CircLigase II buffer and 50 mM MnCl&lt;sub&gt;2&lt;/sub&gt;</td></tr><tr><td>Stainless steel microvials, 1.8 mL</td><td>BioSpec Products</td><td>2007</td><td></td></tr><tr><td>Sucrose</td><td>RPI</td><td>S24060-5000.0</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>New Era Pump Systems</td><td>NE-300</td><td></td></tr><tr><td>T4 polynucleotide kinase (10,000 U/mL)</td><td>NEB</td><td>M0201S</td><td>Supplied with 10X T4 polynucleotide kinase buffer</td></tr><tr><td>T4 RNA ligase 2 truncated KQ (200,000 U/mL)</td><td>NEB</td><td>M0373S</td><td>Supplied with 10X T4 RNA ligase buffer and 50% PEG8000</td></tr><tr><td>Thermal cycler</td><td>Bio-Rad</td><td>1851148</td><td></td></tr><tr><td>Thinwall polyallomer tubes, 13.2 mL</td><td>Beckman Coulter</td><td>331372</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma Aldrich</td><td>X100-100ML</td><td></td></tr><tr><td>UV monitor</td><td>Bio-Rad</td><td>7318160</td><td></td></tr><tr><td>Saccharomyces cerevisiae strain BY4741</td><td>Open Biosystems</td><td>YSC1048</td><td></td></tr></tbody></table>

genome-wide quantification of translation in budding yeast by ribosome profiling

Genome-wide Quantification of Translation in Budding Yeast by Ribosome Profiling

Translation of mRNA into proteins is a complex process involving several layers of regulation. It is often assumed that changes in mRNA transcription reflect changes in protein synthesis, but many exceptions have been observed. Recently, a technique called ribosome profiling (or Ribo-Seq) has emerged as a powerful method that allows identification, with high accuracy, which regions of mRNA are translated into proteins and quantification of translation at the genome-wide level. Here, we present a generalized protocol for genome-wide quantification of translation using Ribo-Seq in budding yeast. In addition, combining Ribo-Seq data with mRNA abundance measurements allows us to simultaneously quantify translation efficiency of thousands of mRNA transcripts in the same sample and compare changes in these parameters in response to experimental manipulations or in different physiological states. We describe a detailed protocol for generation of ribosome footprints using nuclease digestion, isolation of intact ribosome-footprint complexes via sucrose gradient fractionation, and preparation of DNA libraries for deep sequencing along with appropriate quality controls necessary to ensure accurate analysis of in vivo translation.

Detailed pipelines for bioinformatic analysis of ribosome profiling data have been described previously 8,9. In addition, several research groups have developed bioinformatics tools for differential gene expression analysis and processing of sequencing data, which are specific for ribosome profiling method 10,11,12,<sup class="...

Translational regulation plays an important role in the control of protein abundance. Here, we describe a high-throughput method for quantitative analysis of translation in the budding yeast Saccharomyces cerevisiae.

Translation of mRNA into proteins is a complex process involving several layers of regulation. It is often assumed that changes in mRNA transcription ...

genome-wide-quantification-translation-budding-yeast-ribosome

Research

JoVE Journal

Biochemistry

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.

Analysis of Translation Initiation During Stress Conditions by Polysome Profiling

Ribosomes play a central role in protein synthesis. Polyribosome (polysome) fractionation by sucrose density gradient centrifugation allows direct determination of translation efficiencies of individual mRNAs on a genome-wide scale. In addition, this method can be used for biochemical analysis of ribosome- and polysome-associated factors such as chaperones and signaling molecules.

Polysome Fractionation and Analysis of Mammalian Translatomes on a Genome-wide Scale

The ability of cells to adapt to stress is crucial for their survival. Regulation of mRNA translation is one such adaptation strategy, providing for rapid regulation of the proteome. Here, we provide a standardized polysome profiling protocol to identify specific mRNAs that are selectively translated under stress conditions.

Assessment of Selective mRNA Translation in Mammalian Cells by Polysome Profiling

This manuscript describes a method to visualize and quantify localized translation events in subcellular compartments. The approach proposed in this manuscript requires a basic confocal imaging system and reagents and is rapid and cost-effective.

Quantitative Immunofluorescence to Measure Global Localized Translation

The protocol described here is based on the genome-wide quantification of newly synthesized mRNA purified from yeast cells labeled with 4-thiouracil. This method allows to measure mRNA synthesis uncoupled from mRNA decay and, thus, provides an accurate measurement of RNA polymerase II transcription.

Saccharomyces cerevisiae Metabolic Labeling with 4-thiouracil and the Quantification of Newly Synthesized mRNA As a Proxy for RNA Polymerase II Activity

Here we describe the stages of sample collection and preparation for RIBO-seq in bacteria. Sequencing of the libraries prepared according to these guidelines results in sufficient data for comprehensive bioinformatic analysis. The protocol we present is simple, uses standard laboratory equipment and takes seven days from lysis to obtaining libraries.

RIBO-seq in Bacteria: a Sample Collection and Library Preparation Protocol for NGS Sequencing

We present a technique to rapidly stabilize translational (protein biosynthesis) complexes with formaldehyde crosslinking in live yeast and mammalian cells. The approach enables dissecting transient intermediates and dynamic RNA:protein interactions. The crosslinked complexes can be used in multiple downstream applications such as in deep sequencing-based profiling methods, microscopy, and mass-spectrometry.

Rapid In Vivo Fixation and Isolation of Translational Complexes from Eukaryotic Cells

This protocol describes how to generate a polysome profile without using automated gradient makers or gradient fractionation systems.

Polysome Profiling without Gradient Makers or Fractionation Systems

Co-translational interactions play a crucial role in nascent-chain modifications, targeting, folding, and assembly pathways. Here, we describe Selective Ribosome Profiling, a method for in vivo, direct analysis of these interactions in the model eukaryote Saccharomyces cerevisiae.

Global Identification of Co-Translational Interaction Networks by Selective Ribosome Profiling

Translating ribosomes decode three nucleotides per codon into peptides. Their movement along mRNA, captured by ribosome profiling, produces the footprints exhibiting characteristic triplet periodicity. This protocol describes how to use RiboCode to decipher this prominent feature from ribosome profiling data to identify actively translated open reading frames at the whole-transcriptome level.

De novo Identification of Actively Translated Open Reading Frames with Ribosome Profiling Data

Learn ribosome profiling for genome-wide translation analysis in Saccharomyces cerevisiae. This sensitive method enables quantitative measurement of protein synthesis and translational regulation mechanisms.

Genome-wide Quantification of Translation in Budding Yeast by Ribosome Profiling

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