Name: genome-wide quantification of translation in budding yeast by ribosome profiling
Uploaded: 2017-12-21T14:00:00.000+00:00
Duration: 12 min 57 s
Description: Watch this Scientific Journal Video about Genome-wide Quantification of Translation in Budding Yeast by Ribosome Profiling at JoVE.com

この仕事は健康補助金 AG040191 と VML に AG054566 研究機構によって支えられました。VML が加齢医学研究のためのアメリカ連合から遠く研究助成受信者間に調査しました。

<ol>
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著者は、彼らは競合する金銭的な利益があることを宣言します。

リボ Seq アプローチは、mRNA 翻訳体内でゲノムのレベル3の解析のための強力な技術として浮上しています。シングル コドン解像度変換を監視でき、この手法を使用して研究は翻訳制御の私達の理解に貢献しています。その利点にもかかわらずリボ Seq いくつか制限があります。リボソーム RNA (rRNA) フラグメントは、リボソーム保護足跡リボ Seq 実験9<s...

mRNA の翻訳は、タンパク質発現の調節に重要な役割を果たしている細胞の基本的なプロセスの 1 つです。したがって、mRNA の翻訳は、さまざまな内部および外部生理的刺激1,2への応答では厳重します。ただし、翻訳制御機構のまま流。ここでは、プロファイリング ・ リボソームで翻訳出芽酵母におけるゲノム定量化のプロトコルについて述べる.リボソーム プロファイリング技術の全体的な目標は、研究し、細胞の異なる条件の下で特定の Mrna の翻訳を定量化することです。このテクニックは、ゲノム全体のリボソーム占有率を定量的に解析する次世代シーケンスを使用して、タンパク質合成生体内単一コドン解像度3,4の率を監視できます。現在、このメソッド タンパク質翻訳のレベルを測定する最も先進的な手段を提供して他の現在利用可能な技術、マイクロ アレイなどによって明らかにすることができない情報を提供する便利な検出ツールをあると証明したか翻訳の状態配列解析 (TSAA) 5。リボソームのプロファイリングと並進出力のトラン スクリプト レベルでそれらの変更内容をレポートとしてそれはまた他の方法と比べて多くの高い感度を提供します。
このアプローチは、リボソームで保護された mRNA の断片3のディープ シーケンスに基づいています。リボゾームの保護タンパク質翻訳中 〜 (足跡と呼ばれる) mRNA の6の 28 の nt 部分。リボソームで保護されたフラグメントのシーケンスを決定すると、リボ Seq は翻訳 mRNA にリボソームの位置をマップし、積極的にタンパク質3,7と訳されるが mRNA のどの領域を確認できます。さらに、特定の mRNA のコピーに揃える足跡の数をカウントすることによって mRNA の翻訳定量的測定できます。
リボソーム保護断片を分離するためにセル lysates は当初リボヌクレアーゼ消化続いてリボソームを失速する翻訳阻害剤と扱われます。無料の mRNA およびリボゾームによって保護されていない翻訳 Mrna の部分はリボヌクレアーゼによって低下するのに対し、フット プリントとはそのままリボソーム複合体を浄化することによって mRNA のリボソーム保護断片を回復できます。これらの mRNA の足跡は cDNA ライブラリに変換し、ディープ シーケンス (図 1) によって分析します。リボソームのプロファイリングに並行して、そのまま mRNA を同じサンプルから抽出してシーケンスします。リボ Seq mRNA 豊富な測定と識別される翻訳のレベルを比較すると、特にアップ- または調整される翻訳のレベルでは、ある遺伝子を識別し、ゲノム全体のレベルでの mRNA の翻訳の効率を計算できます。この資料で説明されているプロトコルはイースト菌の特定が、他のシステムでリボ Seq プロトコルを確立しようとして、研究者の役に立つまたする必要があります。

<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

MRNA のタンパク質への翻訳は、規制のいくつかの層を含む複雑なプロセスです。MRNA 転写の変化蛋白質合成に変更を反映するが、多くの例外を観察されているとしばしば見なされます。最近では、リボソーム プロファイル (またはリボ Seq) と呼ばれる手法は、mRNA の領域はタンパク質とゲノムのレベルで翻訳の定量化に変換されます、精度の高い識別ができる強力な方法として浮上しています。ここでは、出芽酵母リボ Seq を使用して翻訳のゲノムワイドな定量化のための一般的なプロトコルを提案する.また、同時に何千もの同じサンプルの mrna の翻訳効率を定量化し、比較実験への応答でこれらのパラメーターの変更を可能にリボ Seq データ mRNA 豊富な測定と組み合わせること操作または異なる生理学的状態に。ヌクレアーゼ消化、ショ糖勾配分画法によるフット プリントとはそのままリボソーム複合体の分離および DNA ライブラリの準備を使用して適切なと共にディープ シーケンスのリボソーム足跡の生成のための詳しいプロトコルについて述べる生体内で翻訳の正確な分析を確保するために必要な品質を制御します。

詳細なプロファイルデータ リボソームの構造バイオ情報解析パイプラインをされている8,9で説明しました。さらに、いくつかの研究グループの差動遺伝子発現解析とシーケンス データをプロファイリング方法10、11,12 リボソームに固有の処理のためのバイオ...

Watch this Scientific Journal Video about Genome-wide Quantification of Translation in Budding Yeast by Ribosome Profiling at JoVE.com

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

翻訳制御蛋白質量の制御に重要な役割を果たしています。ここでは、我々 は出芽酵母出芽酵母における翻訳の定量分析のための高スループット方法をについて説明します。

リボソームのプロファイリングによる出芽酵母における翻訳のゲノムの定量化

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

Universidad San Sebastian

Research

JoVE Journal

Biochemistry

11.4K ビュー.  Boston University School of Medicine. 翻訳制御蛋白質量の制御に重要な役割を果たしています。ここでは、我々 は出芽酵母出芽酵母における翻訳の定量分析のための高スループット方法をについて説明します。

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

Quantitative Immunofluorescence to Measure Global Localized Translation

The mechanisms regulating mRNA translation are involved in various biological processes, such as germ line development, cell differentiation, and organogenesis, as well as in multiple diseases. Numerous publications have convincingly shown that specific mechanisms tightly regulate mRNA translation. Increased interest in the translation-induced regulation of protein expression has led to the development of novel methods to study and follow de novo protein synthesis in cellulo. However, most of these methods are complex, making them costly and often limiting the number of mRNA targets that can be studied. This manuscript proposes a method that requires only basic reagents and a confocal fluorescence imaging system to measure and visualize the changes in mRNA translation that occur in any cell line under various conditions. This method was recently used to show localized translation in the subcellular structures of adherent cells over a short period of time, thus offering the possibility of visualizing de novo translation for a short period during a variety of biological processes or of validating changes in translational activity in response to specific stimuli.

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.

グローバルを測定する定量的蛍光ローカライズ翻訳

The mechanisms regulating mRNA translation are involved in various biological processes, such as germ line development, cell differentiation, and ...

生化学

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

Global defects in RNA polymerase II transcription might be overlooked by transcriptomic studies analyzing steady-state RNA. Indeed, the global decrease in mRNA synthesis has been shown to be compensated by a simultaneous decrease in mRNA degradation to restore normal steady-state levels. Hence, the genome-wide quantification of mRNA synthesis, independently from mRNA decay, is the best direct reflection of RNA polymerase II transcriptional activity. Here, we discuss a method using non-perturbing metabolic labeling of nascent RNAs in Saccharomyces cerevisiae (S. cerevisiae). Specifically, the cells are cultured for 6 min with a uracil analog, 4-thiouracil, and the labeled newly transcribed RNAs are purified and quantified to determine the synthesis rates of all individual mRNA. Moreover, using labeled Schizosaccharomyces pombe cells as internal standard allows comparing mRNA synthesis in different S. cerevisiae strains. Using this protocol and fitting the data with a dynamic kinetic model, the corresponding mRNA decay rates can be determined.

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

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.

酵母4 チオウラシルの定量化とラベリング代謝新しく合成された mRNA RNA ポリメラーゼ II の活性のためのプロキシとして

Global defects in RNA polymerase II transcription might be overlooked by transcriptomic studies analyzing steady-state RNA. Indeed, the global decrease in ...

遺伝学

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

The ribosome profiling technique (RIBO-seq) is currently the most effective tool for studying the process of protein synthesis in vivo. The advantage of this method, in comparison to other approaches, is its ability to monitor translation by precisely mapping the position and number of ribosomes on a mRNA transcript.
In this article, we describe the consecutive stages of sample collection and preparation for RIBO-seq method in bacteria, highlighting the details relevant to the planning and execution of the experiment.
Since the RIBO-seq relies on intact ribosomes and related mRNAs, the key step is rapid inhibition of translation and adequate disintegration of cells. Thus, we suggest filtration and flash-freezing in liquid nitrogen for cell harvesting with an optional pretreatment with chloramphenicol to arrest translation in bacteria. For the disintegration, we propose grinding frozen cells with mortar and pestle in the presence of aluminum oxide to mechanically disrupt the cell wall. In this protocol, sucrose cushion or a sucrose gradient ultracentrifugation for monosome purification is not required. Instead, mRNA separation using polyacrylamide gel electrophoresis (PAGE) followed by the ribosomal footprint excision (28-30 nt band) is applied and provides satisfactory results. This largely simplifies the method as well as reduces the time and equipment requirements for the procedure. For library preparation, we recommend using the commercially available small RNA kit for Illumina sequencing from New England Biolabs, following manufacturer&#39;s guidelines with some degree of optimization.
The resulting cDNA libraries present appropriate quantity and quality required for next generation sequencing (NGS). Sequencing of the libraries prepared according to the described protocol results in 2 to 10 mln uniquely mapped reads per sample providing sufficient data for comprehensive bioinformatic analysis. The protocol we present is quick and relatively easy and can be performed with standard laboratory equipment.

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.

細菌におけるRIBO-seq:NGSシーケンシングのためのサンプル収集とライブラリ調製プロトコル

The ribosome profiling technique (RIBO-seq) is currently the most effective tool for studying the process of protein synthesis in vivo. The advantage of ...

Polysome Profiling without Gradient Makers or Fractionation Systems

Polysome fractionation by sucrose density gradient centrifugation is a powerful tool that can be used to create ribosome profiles, identify specific mRNAs being translated by ribosomes, and analyze polysome associated factors. While automated gradient makers and gradient fractionation systems are commonly used with this technique, these systems are generally expensive and can be cost-prohibitive for laboratories that have limited resources or cannot justify the expense due to their infrequent or occasional need to perform this method for their research. Here, a protocol is presented to reproducibly generate polysome profiles using standard equipment available in most molecular biology laboratories without specialized fractionation instruments. Moreover, a comparison of polysome profiles generated with and without a gradient fractionation system is provided. Strategies to optimize and produce reproducible polysome profiles are discussed. Saccharomyces cerevisiae is utilized as a model organism in this protocol. However, this protocol can be easily modified and adapted to generate ribosome profiles for many different organisms and cell types.

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

グラジエントメーカーまたは分画システムを使用しないポリソームプロファイリング

Polysome fractionation by sucrose density gradient centrifugation is a powerful tool that can be used to create ribosome profiles, identify specific mRNAs ...

Eukaryotic Polyribosome Profile Analysis

Protein synthesis is a complex cellular process that is regulated at many levels. For example, global translation can be inhibited at the initiation phase or the elongation phase by a variety of cellular stresses such as amino acid starvation or growth factor withdrawal. Alternatively, translation of individual mRNAs can be regulated by mRNA localization or the presence of cognate microRNAs. Studies of protein synthesis frequently utilize polyribosome analysis to shed light on the mechanisms of translation regulation or defects in protein synthesis. In this assay, mRNA/ribosome complexes are isolated from eukaryotic cells.  A sucrose density gradient separates mRNAs bound to multiple ribosomes known as polyribosomes from mRNAs bound to a single ribosome or monosome. Fractionation of the gradients allows isolation and quantification of the different ribosomal populations and their associated mRNAs or proteins. Differences in the ratio of polyribosomes to monosomes under defined conditions can be indicative of defects in either translation initiation or elongation/termination. Examination of the mRNAs present in the polyribosome fractions can reveal whether the cohort of individual mRNAs being translated changes with experimental conditions. In addition, ribosome assembly can be monitored by analysis of the small and large ribosomal subunit peaks which are also separated by the gradient. In this video, we present a method for the preparation of crude ribosomal extracts from yeast cells, separation of the extract by sucrose gradient and interpretation of the results.  This procedure is readily adaptable to mammalian cells.

This article describes a protocol for the extraction of translating ribosomes from eukaryotic cells. Once extracted, ribosomes are separated into monosomes and polyribosomes by sucrose gradient fractionation to allow different ribosomal populations to be analyzed. As such, this method is the gold standard for examining the regulation of translation.

真核生物のポリリボソームプロファイル解析

Protein synthesis is a complex cellular process that is regulated at many levels. For example, global translation can be inhibited at the initiation phase ...

生物学

Analysis of Translation Initiation During Stress Conditions by Polysome Profiling

Precise control of mRNA translation is fundamental for eukaryotic cell homeostasis, particularly in response to physiological and pathological stress. Alterations of this program can lead to the growth of damaged cells, a hallmark of cancer development, or to premature cell death such as seen in neurodegenerative diseases. Much of what is known concerning the molecular basis for translational control has been obtained from polysome analysis using a density gradient fractionation system. This technique relies on ultracentrifugation of cytoplasmic extracts on a linear sucrose gradient. Once the spin is completed, the system allows fractionation and quantification of centrifuged zones corresponding to different translating ribosomes populations, thus resulting in a polysome profile. Changes in the polysome profile are indicative of changes or defects in translation initiation that occur in response to various types of stress. This technique also allows to assess the role of specific proteins on translation initiation, and to measure translational activity of specific mRNAs. Here we describe our protocol to perform polysome profiles in order to assess translation initiation of eukaryotic cells and tissues under either normal or stress growth conditions.

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.

ポリソームプロファイリングによってストレス状態の間に翻訳開始の分析

Precise control of mRNA translation is fundamental for eukaryotic cell homeostasis, particularly in response to physiological and pathological stress. ...

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

mRNA translation plays a central role in the regulation of gene expression and represents the most energy consuming process in mammalian cells. Accordingly, dysregulation of mRNA translation is considered to play a major role in a variety of pathological states including cancer. Ribosomes also host chaperones, which facilitate folding of nascent polypeptides, thereby modulating function and stability of newly synthesized polypeptides. In addition, emerging data indicate that ribosomes serve as a platform for a repertoire of signaling molecules, which are implicated in a variety of post-translational modifications of newly synthesized polypeptides as they emerge from the ribosome, and/or components of translational machinery. Herein, a well-established method of ribosome fractionation using sucrose density gradient centrifugation is described. In conjunction with the in-house developed &#8220;anota&#8221; algorithm this method allows direct determination of differential translation of individual mRNAs on a genome-wide scale. Moreover, this versatile protocol can be used for a variety of biochemical studies aiming to dissect the function of ribosome-associated protein complexes, including those that play a central role in folding and degradation of newly synthesized polypeptides.

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.

ゲノム規模で哺乳類Translatomesのソーム分画および分析

mRNA translation plays a central role in the regulation of gene expression and represents the most energy consuming process in mammalian cells. ...

Assessment of Selective mRNA Translation in Mammalian Cells by Polysome Profiling

Regulation of protein synthesis represents a key control point in cellular response to stress. In particular, discreet RNA regulatory elements were shown to allow to selective translation of specific mRNAs, which typically encode for proteins required for a particular stress response. Identification of these mRNAs, as well as the characterization of regulatory mechanisms responsible for selective translation has been at the forefront of molecular biology for some time. Polysome profiling is a cornerstone method in these studies. The goal of polysome profiling is to capture mRNA translation by immobilizing actively translating ribosomes on different transcripts and separate the resulting polyribosomes by ultracentrifugation on a sucrose gradient, thus allowing for a distinction between highly translated transcripts and poorly translated ones. These can then be further characterized by traditional biochemical and molecular biology methods. Importantly, combining polysome profiling with high throughput genomic approaches allows for a large scale analysis of translational regulation.

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.

ポリソームプロファイリングによる哺乳動物細胞における選択的mRNAの翻訳の評価

Regulation of protein synthesis represents a key control point in cellular response to stress. In particular, discreet RNA regulatory elements were shown ...

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

Rapid responses involving fast redistribution of messenger(m)RNA and alterations of mRNA translation are pertinent to ongoing homeostatic adjustments of the cells. These adjustments are critical to eukaryotic cell survivability and &#39;damage control&#39; during fluctuating nutrient and salinity levels, temperature, and various chemical and radiation stresses. Due to the highly dynamic nature of the RNA-level responses, and the instability of many of the RNA:RNA and RNA:protein intermediates, obtaining a meaningful snapshot of the cytoplasmic RNA state is only possible with a limited number of methods. Transcriptome-wide, RNA-seq-based ribosome profiling-type experiments are among the most informative sources of data for the control of translation. However, absence of a uniform RNA and RNA:protein intermediate stabilization can lead to different biases, particularly in the fast-paced cellular response pathways. In this article, we provide a detailed protocol of rapid fixation applicable to eukaryotic cells of different permeability, to aid in RNA and RNA:protein intermediate stabilization. We further provide examples of isolation of the stabilized RNA:protein complexes based on their co-sedimentation with ribosomal and poly(ribo)somal fractions. The separated stabilized material can be subsequently used as part of ribosome profiling-type experiments, such as in Translation Complex Profile sequencing (TCP-seq) approach and its derivatives. Versatility of the TCP-seq-style methods has now been demonstrated by the applications in a variety of organisms and cell types. The stabilized complexes can also be additionally affinity-purified and imaged using electron microscopy, separated into different poly(ribo)somal fractions and subjected to RNA sequencing, owing to the ease of the crosslink reversal. Therefore, methods based on snap-chilling and formaldehyde fixation, followed by the sedimentation-based or other type of RNA:protein complex enrichment, can be of particular interest in investigating finer details of rapid RNA:protein complex dynamics in live cells.

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

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.

真核細胞からのトランスレーショナル複合体の迅速な インビボ 固定と分離

Rapid responses involving fast redistribution of messenger(m)RNA and alterations of mRNA translation are pertinent to ongoing homeostatic adjustments of ...

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

In recent years, it has become evident that ribosomes not only decode our mRNA but also guide the emergence of the polypeptide chain into the crowded cellular environment. Ribosomes provide the platform for spatially and kinetically controlled binding of membrane-targeting factors, modifying enzymes, and folding chaperones. Even the assembly into high-order oligomeric complexes, as well as protein-protein network formation steps, were recently discovered to be coordinated with synthesis.
Here, we describe Selective Ribosome Profiling, a method developed to capture co-translational interactions in vivo. We will detail the various affinity purification steps required for capturing ribosome-nascent-chain complexes together with co-translational interactors, as well as the mRNA extraction, size exclusion, reverse transcription, deep-sequencing, and big-data analysis steps, required to decipher co-translational interactions in near-codon resolution.

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.

選択的リボソームプロファイリングによる共翻訳相互作用ネットワークのグローバル同定

In recent years, it has become evident that ribosomes not only decode our mRNA but also guide the emergence of the polypeptide chain into the crowded ...

リボソームのプロファイリングによる出芽酵母における翻訳のゲノムの定量化

要約

さらに動画を探す

この動画の章

グローバルを測定する定量的蛍光ローカライズ翻訳

酵母4 チオウラシルの定量化とラベリング代謝新しく合成された mRNA RNA ポリメラーゼ II の活性のためのプロキシとして

細菌におけるRIBO-seq:NGSシーケンシングのためのサンプル収集とライブラリ調製プロトコル

グラジエントメーカーまたは分画システムを使用しないポリソームプロファイリング

真核生物のポリリボソームプロファイル解析

ポリソームプロファイリングによってストレス状態の間に翻訳開始の分析

ゲノム規模で哺乳類Translatomesのソーム分画および分析

ポリソームプロファイリングによる哺乳動物細胞における選択的mRNAの翻訳の評価

真核細胞からのトランスレーショナル複合体の迅速なインビボ固定と分離

選択的リボソームプロファイリングによる共翻訳相互作用ネットワークのグローバル同定

リボソームのプロファイリングによる出芽酵母における翻訳のゲノムの定量化

要約

さらに動画を探す

この動画の章

グローバルを測定する定量的蛍光ローカライズ翻訳

酵母4 チオウラシルの定量化とラベリング代謝新しく合成された mRNA RNA ポリメラーゼ II の活性のためのプロキシとして

細菌におけるRIBO-seq:NGSシーケンシングのためのサンプル収集とライブラリ調製プロトコル

グラジエントメーカーまたは分画システムを使用しないポリソームプロファイリング

真核生物のポリリボソームプロファイル解析

ポリソームプロファイリングによってストレス状態の間に翻訳開始の分析

ゲノム規模で哺乳類Translatomesのソーム分画および分析

ポリソームプロファイリングによる哺乳動物細胞における選択的mRNAの翻訳の評価

真核細胞からのトランスレーショナル複合体の迅速な インビボ 固定と分離

選択的リボソームプロファイリングによる共翻訳相互作用ネットワークのグローバル同定

真核細胞からのトランスレーショナル複合体の迅速なインビボ固定と分離