イノベーション ジョン r. エヴァンス リーダー基金 (35017)、キャンパス アルバータ州革新プログラムおよびアルバータ州省の自然科学と工学研究会のカナダ発見助成 (RGPIN-2017-05463)、カナダの財団によって資金が供給されたこの作品経済発展と貿易。

注: RNA はリボヌクレアーゼ (RNases) による劣化に非常に敏感です。RNA のままを維持する標準的な予防策を講じる。手袋を頻繁に変更します。プロトコルのすべての手順でフィルター処理されたピペット チップ、ヌクレアーゼ フリー容器、ヌクレアーゼ フリー化学物質を使用します。ヌクレアーゼ フリーの使用またはピロ炭酸ジエチルエステル (DEPC)-すべてのソリューションのための水を処理します。
1. 溶液の調製
<ol><li>Toeprinting バッファーを準備: 20 mM トリス-HCl (pH 7.6)、100 mM KOAc、2.5 mM Mg(OAc)2, 5% (w/v) スクロース, 2 mM ジチオトレイトール (DTT) と 0.5 mM スペルミジン。<ol>
<li>凍結融解の繰り返しを避けるために-20 ° C で使い捨ての因数で DTT とスペルミジンを保存します。 注: DTT とスペルミジン使用の直前に toeprinting バッファーに追加する必要があります。DTT とスペルミジンを欠けているソリューションは、-20 ° C で保存できます。</li>
	</ol>
</li>
	<li>85 mM 5'-グアニル imidodiphosphate (GMP PNP) と 91 mM アデノシン三リン酸 (ATP) の因数を準備します。因数-20 ° C で保存します。</li>
	<li>6% ポリアクリルアミド-7 M 尿素ゲル ミックス 450 mL を準備: 67.5 mL 40 %acrylamide:bis-アクリルアミド (19:1)、尿素、112.5 x TBE (トリス、ホウ酸塩、thylenediaminetetraacetic 酸 (EDTA)) 5 mL と 120 mL の水の 189 g。37 ° C の水浴または攪拌しながらホット プレート上に温暖化によって、尿素を溶かします。ソリューションをフィルター (e.g。、0.2 μ m 硝酸セルロース真空フィルター)。 注: ソリューションは 4 ° C で少なくとも 1 か月間保存できます。 注意: アクリルアミド単量体は皮膚を通して吸収される神経毒です。皮膚への接触を避けるために大きい心配を取る (すなわち。 保護メガネ、手袋、白衣、)。ポリアクリルアミドは重合 100% 完了するまで続行できません、注意して処理も必要があります。</li>
	<li>ホルムアミド ローディングの染料の準備: 95% ホルムアミド、18 ミリメートルの EDTA、0.025% (w/v) ナトリウム dodecyl 硫酸塩 (SDS)、0.025% (w/v) ブロモフェノールの青、0.025% (w/v) キシレンシアノール。-20 ° C にてストア</li>
	<li>プライマー (5' CTCGATATGTGCATCTGTA; 5' IRDye 800 付いた端) の 1 nmol を 100 μ L の 10 pmol/μ L の作業濃度の水に溶解します。光から保護使い捨て因数 (約 10 μ L) で-20 ° C で保存します。</li>
</ol>2. mRNA の準備
<ol><li>適切なテンプレートからポリメラーゼ連鎖反応 (PCR) による mRNA の合成 DNA テンプレートの増幅 (すなわち。、ゲノム DNA やプラスミド DNA、必要に応じて)。高忠実度の製造元の指示に従って DNA ポリメラーゼを使用し次の反応条件 (35 サイクル): 溶融、98 ° C、10 s;アニール、53 ° C、20 s;拡張、72 ° C、30 秒。 注: 前方のプライマー (5' AAGCTTAATACGACTCACTATAG) RNA のトランスクリプション; のように T7 プロモーターが組み込まれています逆のプライマー (5' T51GAATTCGGATCCGACCGTGG) 51 チミン残基には、転写の RNA のポリ A の尾を提供が含まれます。RNA生体外でプラスミド DNA から転写もあることに注意してください。</li>
	<li>体外に適切な転写キットを使用は、IRES 含む RNA または DNA のテンプレートから頂いた RNA に議事録します。製造元の指示に従ってください。標準の 20 μ L 反応ボリュームの RNA のサンプルを準備します。37 ° C で 30 分間、RNase フリーの DNase の 2 ユニットと新しく合成した RNA を扱う</li>
	<li>110 に DNase 処理 RNA を希釈ヌクレアーゼ フリー水 μ L は、室温で 20,000 × g で 110 μ L 酸フェノール、渦 5 s、3 分間遠心を追加。新しい 1.5 mL および microfuge の管に水相の 100 μ L を削除 10 μ L の 3 M 酢酸ナトリウムと渦 2 s 追加、100% エタノール, 渦 5 s の 3 つのボリュームを追加して、一晩-20 ° C で RNA を沈殿させます。</li>
	<li>遠心分離機 > 4 ° C と上清を捨て、30 分 20,000 × g。冷たい 70% (v/v) エタノール 500 μ l 添加の餌を洗浄し、遠心分離を繰り返します。限り可能と風乾清吸引 5 ~ 10 分のペレットは、ペレットが外れないように注意してください。</li>
	<li>ヌクレアーゼ フリー水 0.5 μ g/μ L の作業濃度を生成するための適切な量の RNA を再懸濁します。これは-80 ° C で保存または、すぐに使用できます。</li>
</ol>3. Toeprinting 反応
<ol><li>ウサギ網状赤血球ライセート (RRL、ヌクレアーゼ扱われない)、RNase 阻害剤、91 mM ATP (最終 1.82 mM) の 1 μ L と 85 mM GMP PNP (1.7 mM 最終) 1.5 mL および microfuge の管の 1 μ L の 1 μ L (40 台) のミックス 15 μ L。30 の ° c で 5 分間インキュベートします。 注: RRL は、凍結融解の繰り返しを避けるために単一の使用のため検体必要があります。重要な制御は、RRL、RNA の二次構造のため逆のトランスクリプションの自然な休止時間を明らかにするために不足している反応です。このコントロールの RRL を交換する toeprinting バッファーを追加します。</li>
	<li>RNA の 0.5 μ g (1 μ L) を追加し、30 の ° c で 5 分間インキュベートします。</li>
	<li>Toeprinting バッファーの 22 μ L を追加し、30 ° C、3 分で孵化させなさい。</li>
	<li>0.5 μ L (5 pmol) IRDye 標識プライマーを追加し、氷上で 10 分間インキュベートします。</li>
	<li>25 mM の dNTPs の 2 μ L を追加 (最終濃度: 1 mM)、2 Toeprinting バッファーの 3.5 μ、1 μ L の鳥 Myeloblastosis ウイルス逆転写酵素 (RT-AMV) 100 mM Mg(OAc)2μ L。最終巻は 50 μ L です。</li>
	<li>45 分 30 ° C で反応を孵化させなさい。</li>
	<li>ヌクレアーゼ フリー水の 200 μ L を追加し、25:24:1 フェノール: クロロホルム: イソアミル アルコール (pH 約 8.0) の 250 μ L を即座に抽出します。渦 5 s と 20,000 × g で室温で 3 分間遠心します。新しい 1.5 mL および microfuge の管に水相を削除 100% エタノール, 渦 5 s の 3 つのボリュームを追加して、-20 ° C で一晩沈殿します。</li>
	<li>遠心分離機 > 4 ° C と上清を捨て、30 分 20,000 × g。冷たい 70% (v/v) エタノール 500 μ L の DNA の餌を洗います。遠心分離機 > 4 ° C で 15 分間 20,000 × gくらい上清を吸引と 5 ~ 10 分のペレット、ペレットが外れないように気をつけて乾燥した空気。</li>
	<li>ヌクレアーゼ フリー水 6 μ l 中でペレットを溶解し、ホルムアミド ローディングの染料の 3 μ L を追加します。</li>
</ol>4. シーケンス反応
<ol><li>チェーンの終端文字として dideoxynucleotides (ddNTPs) を使用して、標準的な配列の反作用のための 2.1 からの DNA のテンプレートそして 3.4 の手順から IRDye 標識プライマーを使用します。該当の DNA シーケンス キットを使用し、製造元の指示に従ってください。</li>
	<li>ホルムアミド ローディングの染料の 3 μ L の各シーケンス反応のミックス 6 μ L は。</li>
</ol>5. ゲル法と電気泳動の配列の作製
メモ: このプロトコル ゲル ベースの蛍光イメージャと 21 cm × 23 cm × 0.2 mm のゲルを使用してが必要な場合、他のシーケンサーやゲル-サイズ、合わせることができます。
<ol><li>0.2 mm スペーサー ショート (23 × 25 cm)、100% エタノールで長い (30 × 25 cm) ガラス板を徹底的に掃除します。空気が乾燥。</li>
	<li>6% ポリアクリルアミド-7 M 尿素ゲルのミックス 30 mL を混ぜて 10% (w/v) 過硫酸アンモニウム (AP) の 200 μ L、テトラメチルエチレンジアミン (TEMED) の 20 μ L。泡を避けるために世話、ゲルを注ぎなさい、' サメの歯 ' ゲル櫛を挿入します。室温で 1 h の重合にゲルを許可します。</li>
	<li>シーケンス装置を組み立てるし、1 で貯水池を埋める x TBE。</li>
	<li>中古 1500 V で実行 15 分の 55 の ° C の温度を達成するまで。</li>
	<li>配列のゲルの上に 5 分負荷 1 μ L 85 ° C まで (から 3.9 または 4.2 の手順) サンプル/読み込み染料混合物を加熱します。</li>
	<li>8 h の 1500 V でゲルを実行します。マシンは、リアルタイムでバンドを読み取ります。</li>
	<li>装置を分解し、アクリルアミドゲルと連続したバッファーを破棄します。</li>
</ol>

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著者は開示する利害の対立があります。

Toeprinting は、高度に制御された状況下での翻訳開始複合体の形成を支援する目的の RNA の能力を直接測定する強力な手法です。このプロトコルは toeprinting の簡略化手法を記述する哺乳類の Rna。ウサギ網状赤血球ライセート (RRL)、リボソーム、eIFs、イニシエーターの便利なソースとして使用されます、tRNA、IRESトランス-要因 (ITAFs) の演技します。実験者は選択の彼らの RNA を提供し、自...

翻訳は、ほとんどの携帯電話のエネルギーを消費すると厳しく規制された1を翻訳には理にかなって。逆に、翻訳の制御不全- とタンパク質出力の結果として変化-がん1,2などのストレス応答と疾患の状態が多くみられます。翻訳調節の主な利点は、細胞が様々 な刺激3に対応するため、タンパク質の出力を変更することが速度です。翻訳の規制はこのように細胞の生存と死1,2,3に影響を与えることができる重要なメカニズムを表します。翻訳の手順の開始はほとんど高度規制で複雑な3です。簡単に言えば、真核生物の Mrna には翻訳のために不可欠であるほとんど常に 5' m7G キャップが含まれています。真核生物の開始因子 eIF4E、eIF4A、eIF4G 帽子依存した開始が必要です (キャップ認識複合体) の mRNA の 5' 端と対話します。43 細胞リボソーム複雑、eIF2 バインド イニシエーターを含む tRNA および eIF3、mRNAを介してeIF3 と eIF4G の相互作用の 5' 端に募集しています。開始前複合体は eIF4A 援用 mRNA をスキャンすると思った (RNA ヘリカーゼ) 開始コドン (AUG) が見つかるまで。48 s 開始複合体が形成されその後、tRNA はリボソームの P サイトに配信。最後に、60 s と 40 s リボソームのサブユニットは翻訳伸長1,3,4に続いて 80 年代開始複合体の形成に統一されています。対照的に、内部リボソームのエントリ サイト (沢のカキツバタ) は、開始コドン3に直接 subunit 40 代を募集して 5' キャップの要件をバイパスします。生理的ストレス条件キー一般的な真核生物の開始因子 (eIFs) の変更による mRNA 翻訳を減衰させます。しかし、ストレス応答タンパク質をエンコードよく特定の Mrna の選択的な翻訳を可能にする非正規翻訳開始機構、癌のような病気の状態で非正規翻訳開始の破綻が関与して1,2。 細胞の沢のカキツバタなど、控えめな RNA 規制要素32,などの Mrna の翻訳を可能にします。
翻訳調節の 1 つの特に興味深い側面は、正規と非正規の特定の mRNA の翻訳のメカニズムを理解することです。Toeprinting は、特定の Rnaの生体外の翻訳開始機構の詳細な解明ができるテクニックです。Toeprinting の全体的な目標はどのシーケンス、構造要素またはアクセサリーを決定するために翻訳開始さまざまな条件下でリボソーム複合体の形成を核に関心の RNA の能力を評価するには要因は、効率的な翻訳開始に必要です。たとえば、リボソーム募集を 5' キャップの不在で妨げが、IRES の存在によって刺激があります。
技術の原理は体外にある開始複合体フォーム、し、逆に書き写すように翻訳のコンポーネント (または浄化されたコンポーネント) を含む細胞抽出物の存在下でそれを孵化、関心の RNA を転写特定のプライマー RNA。安定 RNA リボソーム複合体にはリボソームのいわゆる 'toeprint'5,6、7の 3' 端に失速する逆のトランスクリプションが原因となります。
このプロトコル、ribosomal 亜単位、eIFs、Trna、IRESトランスで-因子 (ITAFs) は便利なウサギ網状赤血球ライセート (RRL) に寄贈します。このプロトコルの別の利点は、標識プライマーではなく、蛍光標識したプライマーとジェル ベースの蛍光イメージャの使用です。これは、プライマーを標識化と同様にゲルを乾燥増に公開するなど、追加の手順を排除します。蛍光バンドより高い解像度を可能にするゲルの実行中に、リアルタイムに記録されます。アポトーシス蛋白 (XIAP) IRES RNA の上限なし X リンク阻害剤は、キャップ Mrna はこのテクニック8でも分析できますがここでは、例として使用されます。
セル lysates の翻訳プロセスの最終的な出力を測定する西部の分析とは異なり toeprinting、RNA の翻訳開始複合体形成を測定するための in vitroアプローチです。この還元主義的アプローチは、基板や翻訳開始因子の非常に詳細な研究 (e.g、キャップまたは国連おおわれた mRNA IRES の構造、有無、ポリ A の尾、特定の蛋白質の要因の規定の。など)。したがって、翻訳8のさまざまなモードや mRNA の構造の沢のカキツバタなどタンパク質合成9,10の効果を研究する toeprinting を使用できます。

<table><tbody><tr><td>DEPC (Diethyl pyrocarbonate)</td><td>Sigma</td><td>D5758-100ML</td><td></td></tr><tr><td>TRIS base, Ultrapure</td><td>JT Baker</td><td>4109-01</td><td></td></tr><tr><td>KOAc (Potassium acetate)</td><td>Bio Basic</td><td>PB0438</td><td></td></tr><tr><td>Mg(OAc)&lt;sub&gt;2&lt;/sub&gt; (Magnesium acetate tetrahydrate)</td><td>Bio Basic</td><td>MB0326</td><td></td></tr><tr><td>Sucrose, molecular biology grade</td><td>Calbiochem</td><td>573113-1KG</td><td></td></tr><tr><td>Spermidine</td><td>Sigma</td><td>85558</td><td></td></tr><tr><td>GMP-PNP (Guanosine 5&amp;prime;-[&amp;beta;,&amp;gamma;-imido]triphosphate trisodium salt hydrate) 0.1 M solution</td><td>Sigma</td><td>G0635</td><td></td></tr><tr><td>ATP (Adenosine 5&amp;prime;-triphosphate) disodium salt, 100 mM solution</td><td>Sigma</td><td>A6559</td><td></td></tr><tr><td>19:1 Acrylamide:bis-acrylamide, 40%</td><td>Bio Basic</td><td>A0006</td><td></td></tr><tr><td>Urea</td><td>Bio Basic</td><td>UB0148</td><td></td></tr><tr><td>500mL bottle top filtration units, 0.2 &amp;micro;m</td><td>Sarstedt</td><td>83.1823.101</td><td></td></tr><tr><td>Formamide</td><td>Sigma</td><td>F9037-100ML</td><td></td></tr><tr><td>EDTA (disodium salt, dihydrate)</td><td>Bio Basic</td><td>EB0185</td><td></td></tr><tr><td>SDS</td><td>Bio Basic</td><td>SB0485</td><td></td></tr><tr><td>Bromophenol blue</td><td>Bio Basic</td><td>BDB0001</td><td></td></tr><tr><td>Xylene cyanol FF</td><td>Bio Basic</td><td>XB0005</td><td></td></tr><tr><td>MEGAshortscript T7 transcription kit</td><td>Ambion</td><td>AM1354</td><td></td></tr><tr><td>mMESSAGE mMACHINE T7 transcription kit</td><td>Ambion</td><td>AM1344</td><td></td></tr><tr><td>Acid Phenol:Chloroform (5:1)</td><td>Ambion</td><td>AM9722</td><td></td></tr><tr><td>25:24:1 Phenol:Chloroform:Isoamyl Alcohol</td><td>Invitrogen</td><td>15593-049</td><td></td></tr><tr><td>Rabbit Reticulocyte Lysate (RRL). Should NOT be nuclease-treated.</td><td>Green Hectares, USA</td><td></td><td>Contact Green Hectares, ask for 1:1 RRL:water</td></tr><tr><td>RiboLock RNase Inhibitor (40 U/&amp;micro;L)</td><td>Thermo Fisher</td><td>E00382</td><td></td></tr><tr><td>100 mM dNTPs</td><td>Invitrogen</td><td>56172, 56173, 56174, 56175</td><td>Mix equal parts for a stock of 25 mM each.</td></tr><tr><td>AMV-RT, 10 U/&amp;micro;L</td><td>Promega</td><td>M5101</td><td></td></tr><tr><td>Sequenase Version 2.0 DNA Sequencing Kit</td><td>Thermo Fisher</td><td>707701KT</td><td></td></tr><tr><td>Model 4200 IR2 DNA analyzer</td><td>LI-COR</td><td></td><td>Product has been discontinued</td></tr><tr><td>APS (Ammonium Persulfate)</td><td>Bio Basic</td><td>AB0072</td><td></td></tr><tr><td>TEMED</td><td>Bio Basic</td><td>TB0508</td><td></td></tr><tr><td>Phusion High Fidelity Polymerase</td><td>New England Biolabs</td><td>M0530</td><td></td></tr><tr><td>Turbo Dnase</td><td>Thermo Fisher</td><td>AM2238</td><td></td></tr></tbody></table>

toeprinting analysis of translation initiation complex formation on mammalian mrnas

翻訳開始は、タンパク質合成の律速段階で細胞でタンパク質出力を調節する重要なポイントを表します。タンパク質合成の調節は、細胞のストレス応答するキーと不全、癌などの多くの病気の状態の中心。例えば、帽子独立した方法で細胞ストレスは減衰キャップ依存主導によるグローバル翻訳の阻害につながるが、特定のストレス応答タンパク質群選択的に変換されます。これらの特定の Mrna の翻訳のため、細胞内部リボソームのエントリ サイト (沢のカキツバタ) などの控えめな RNA 規制要素を許可します。このような Mrna の同定とそれらの調節機構の解析、分子生物学の主要な領域をされています。翻訳開始に関連した、Toeprinting、RNA の構造と機能の研究の方法です。Toeprinting の目標は体外の能力を評価するためにどのシーケンス、構造要素、またはアクセサリー要因がリボソームに関与しているを決定するためにさまざまな条件下でリボソームと安定な錯体を形成する RNA を転写バインド-効率的な翻訳開始前のカーソル。西部の分析やポリソームのプロファイリングなどの他の技術と一緒に翻訳開始制御機構の堅牢な特性の toeprinting ことができます。

8,10体外帽子独立した翻訳開始をサポートする XIAP IRES の能力は前述しました。Toeprinting は XIAP IRES 開始複合体のメカニズムの詳細を調査する重要な技術です。XIAP IRES (図 1A) を含む mRNA は DNA コンストラクトだったの in vitro転写、toeprinting 分析を受けます。ここで使用される IRE...

Watch this Scientific Journal Video about Toeprinting Analysis of Translation Initiation Complex Formation on Mammalian mRNAs at JoVE.com

Toeprinting Analysis of Translation Initiation Complex Formation on Mammalian mRNAs

哺乳類の Mrna の翻訳開始複合体形成の Toeprinting 分析

体外の能力を計測する Toeprinting の目的は、リボソームさまざまな条件の下でフォームの翻訳開始複合体に RNA を転写しました。このプロトコルは toeprinting 哺乳類の RNA のための方法を記述してキャップ依存、IRES 駆動の両方の翻訳を勉強する使用ことができます。

Translation initiation is the rate-limiting step of protein synthesis and represents a key point at which cells regulate their protein output. Regulation ...

toeprinting-analysis-translation-initiation-complex-formation-on

Universidad de Cartagena UDEC

Research

JoVE Journal

Biochemistry

12.4K ビュー.  University of Lethbridge. 体外の能力を計測する Toeprinting の目的は、リボソームさまざまな条件の下でフォームの翻訳開始複合体に RNA を転写しました。このプロトコルは toeprinting 哺乳類の RNA のための方法を記述してキャップ依存、IRES 駆動の両方の翻訳を勉強する使用ことができます。

ビデオ: Toeprinting Analysis of Translation Initiation Complex Formation on Mammalian mRNAs

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

Artificial RNA Polymerase II Elongation Complexes for Dissecting Co-transcriptional RNA Processing Events

Eukaryotic mRNA synthesis is a complex biochemical process requiring transcription of a DNA template into a precursor RNA by the multi-subunit enzyme RNA polymerase II and co-transcriptional capping and splicing of the precursor RNA to form the mature mRNA. During mRNA synthesis, the RNA polymerase II elongation complex is a target for regulation by a large collection of transcription factors that control its catalytic activity, as well as the capping, splicing, and 3&#8217;-processing enzymes that create the mature mRNA. Because of the inherent complexity of mRNA synthesis, simpler experimental systems enabling isolation and investigation of its various co-transcriptional stages have great utility.
In this article, we describe one such simple experimental system suitable for investigating co-transcriptional RNA capping. This system relies on defined RNA polymerase II elongation complexes assembled from purified polymerase and artificial transcription bubbles. When immobilized via biotinylated DNA, these RNA polymerase II elongation complexes provide an easily manipulable tool for dissecting co-transcriptional RNA capping and mechanisms by which the elongation complex recruits and regulates capping enzyme during co-transcriptional RNA capping. We anticipate this system could be adapted for studying recruitment and/or assembly of proteins or protein complexes with roles in other stages of mRNA maturation coupled to the RNA polymerase II elongation complex.

Here, we describe the assembly of RNA polymerase II (Pol II) elongation complexes requiring only short synthetic DNA and RNA oligonucleotides and purified Pol II. These complexes are useful for studying mechanisms underlying co-transcriptional processing of transcripts associated with the Pol II elongation complex.

共転写RNA処理イベントを解剖するための人工RNAポリメラーゼII伸長複合体

Eukaryotic mRNA synthesis is a complex biochemical process requiring transcription of a DNA template into a precursor RNA by the multi-subunit enzyme RNA ...

遺伝学

An In Vitro Single-Molecule Imaging Assay for the Analysis of Cap-Dependent Translation Kinetics

Cap-dependent protein synthesis is the predominant translation pathway in eukaryotic cells. While various biochemical and genetic approaches have allowed extensive studies of cap-dependent translation and its regulation, high resolution kinetic characterization of this translation pathway is still lacking. Recently, we developed an in vitro assay to measure cap-dependent translation kinetics with single-molecule resolution. The assay is based on fluorescently labeled antibody binding to nascent epitope-tagged polypeptide. By imaging the binding and dissociation of antibodies to and from nascent peptide&#8211;ribosome&#8211;mRNA complexes, the translation progression on individual mRNAs can be tracked. Here, we present a protocol for establishing this assay, including mRNA and PEGylated slide preparations, real-time imaging of translation, and analysis of single molecule trajectories. This assay enables tracking of individual cap-dependent translation events and resolves key translation kinetics, such as initiation and elongation rates. The assay can be widely applied to distinct translation systems and should broadly benefit in vitro studies of cap-dependent translation kinetics and translational control mechanisms.

An In Vitro Single-Molecule Imaging Assay for the Analysis of Cap-Dependent Translation Kinetics

Tracking individual translation events allows for high-resolution kinetic studies of cap-dependent translation mechanisms. Here we demonstrate an in vitro single-molecule assay based on imaging interactions between fluorescently labeled antibodies and epitope-tagged nascent peptides. This method enables single-molecule characterization of initiation and peptide elongation kinetics during active in vitro cap-dependent translation.

キャップ依存翻訳キネティクス解析のための インビトロ 単分子イメージングアッセイ

Cap-dependent protein synthesis is the predominant translation pathway in eukaryotic cells. While various biochemical and genetic approaches have allowed ...

In vivo Interrogation of Central Nervous System Translatome by Polyribosome Fractionation

Multiple processes are involved in gene expression including transcription, translation and stability of mRNAs and proteins. Each of these steps are tightly regulated, affecting the final dynamics of protein abundance. Various regulatory mechanisms exist at the translation step, rendering mRNA levels alone an unreliable indicator of gene expression. In addition, local regulation of mRNA translation has been particularly implicated in neuronal functions, shifting &#39;translatomics&#39; to the focus of attention in neurobiology. The presented method can be used to bridge transcriptomics and proteomics.
Here we describe essential modifications to the technique of polyribosome fractionation, which interrogates the translatome based on the association of actively translated mRNAs to multiple ribosomes and their differential sedimentation in sucrose gradients. Traditionally, working with in vivo samples, particularly of the central nervous system (CNS), has proven challenging due to the restricted amounts of material and the presence of fatty tissue components. In order to address this, the described protocol is specifically optimized for use with minimal amount of CNS material, as demonstrated by the use of single mouse spinal cord and brain. Briefly, CNS tissues are extracted and translating ribosomes are immobilized on mRNAs with cycloheximide. Myelin flotation is then performed to remove lipid rich components. Fractionation is performed on a sucrose gradient where mRNAs are separated according to their ribosomal loading. Isolated fractions are suitable for a range of downstream assays, including new genome wide assay technologies.

In vivo Interrogation of Central Nervous System Translatome by Polyribosome Fractionation

This protocol illustrates essential modifications of polyribosome fractionation in order to study the translatome of in vivo CNS samples. It allows global assessment of translation and transcription regulation through the isolation and comparison of total RNA to ribosome bound RNA fractions.

ポリリボソーム画によって中枢神経系Translatomeの生体尋問中

Multiple processes are involved in gene expression including transcription, translation and stability of mRNAs and proteins. Each of these steps are ...

神経科学

Defining the Program of Maternal mRNA Translation during In vitro Maturation using a Single Oocyte Reporter Assay

Events associated with oocyte nuclear maturation have been well described. However, much less is known about the molecular pathways and processes that take place in the cytoplasm in preparation for fertilization and acquisition of totipotency. During oocyte maturation, changes in gene expression depend exclusively on the translation and degradation of maternal messenger RNAs (mRNAs) rather than on transcription. Execution of the translational program, therefore, plays a key role in establishing oocyte developmental competence to sustain embryo development. This paper is part of a focus on defining the program of maternal mRNA translation that takes place during meiotic maturation and at the oocyte-to-zygote transition. In this method paper, a strategy is presented to study the regulation of translation of target mRNAs during in vitro oocyte maturation. Here, a Ypet reporter is fused to the 3&#39; untranslated region (UTR) of the gene of interest and then micro-injected into oocytes together with polyadenylated mRNA encoding for mCherry to control for injected volume. By using time-lapse microscopy to measure reporter accumulation, translation rates are calculated at different transitions during oocyte meiotic maturation. Here, the protocols for oocyte isolation and injection, time-lapse recording, and data analysis have been described, using the Ypet/interleukin-7 (IL-7)-3&#39; UTR reporter as an example.

This protocol describes a reporter assay to study the regulation of mRNA translation in single oocytes during in vitro maturation.

単一の卵母細胞レポーターアッセイを用いたインビトロ成熟時の母体母体翻訳プログラムの定義

Events associated with oocyte nuclear maturation have been well described. However, much less is known about the molecular pathways and processes that ...

発生生物学

Isolation of Translating Ribosomes Containing Peptidyl-tRNAs for Functional and Structural Analyses

Recently, structural and biochemical studies have detailed many of the molecular events that occur in the ribosome during inhibition of protein synthesis by antibiotics and during nascent polypeptide synthesis. Some of these antibiotics, and regulatory nascent polypeptides mostly in the form of peptidyl-tRNAs, inhibit either peptide bond formation or translation termination1-7. These inhibitory events can stop the movement of the ribosome, a phenomenon termed "translational arrest". Translation arrest induced by either an antibiotic or a nascent polypeptide has been shown to regulate the expression of genes involved in diverse cellular functions such as cell growth, antibiotic resistance, protein translocation and cell metabolism8-13. Knowledge of how antibiotics and regulatory nascent polypeptides alter ribosome function is essential if we are to understand the complete role of the ribosome in translation, in every organism.
Here, we describe a simple methodology that can be used to purify, exclusively, for analysis, those ribosomes translating a specific mRNA and containing a specific peptidyl-tRNA14. This procedure is based on selective isolation of translating ribosomes bound to a biotin-labeled mRNA. These translational complexes are separated from other ribosomes in the same mixture, using streptavidin paramagnetic beads (SMB) and a magnetic field (MF). Biotin-labeled mRNAs are synthesized by run-off transcription assays using as templates PCR-generated DNA fragments that contain T7 transcriptional promoters. T7 RNA polymerase incorporates biotin-16-UMP from biotin-UTP; under our conditions approximately ten biotin-16-UMP molecules are incorporated in a 600 nt mRNA with a 25% UMP content. These biotin-labeled mRNAs are then isolated, and used in in vitro translation assays performed with release factor 2 (RF2)-depleted cell-free extracts obtained from Escherichia coli strains containing wild type or mutant ribosomes. Ribosomes translating the biotin-labeled mRNA sequences are stalled at the stop codon region, due to the absence of the RF2 protein, which normally accomplishes translation termination. Stalled ribosomes containing the newly synthesized peptidyl-tRNA are isolated and removed from the translation reactions using SMB and an MF. These beads only bind biotin-containing messages.
The isolated, translational complexes, can be used to analyze the structural and functional features of wild type or mutant ribosomal components, or peptidyl-tRNA sequences, as well as determining ribosome interaction with antibiotics or other molecular factors 1,14-16. To examine the function of these isolated ribosome complexes, peptidyl-transferase assays can be performed in the presence of the antibiotic puromycin1. To study structural changes in translational complexes, well established procedures can be used, such as i) crosslinking to specific amino acids14 and/or ii) alkylation protection assays1,14,17.

A major impediment to biochemical analyses of ribosomes containing nascent peptidyl-tRNAs has been the presence of other ribosomes in the same samples, ribosomes not involved in the translation of the specific mRNA sequence being analyzed. We developed a simple methodology to purify, exclusively, the ribosomes containing the nascent peptidyl-tRNA of interest.

機能と構造解析のためにペプチジル- tRNAを含む翻訳リボソームの分離

Recently, structural and biochemical studies have detailed many of the molecular events that occur in the ribosome during inhibition of protein synthesis ...

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. ...

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.

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.

ポリソームプロファイリングを用いたマウス脳の発達における翻訳の解析

The proper development of the mammalian brain relies on a fine balance of neural stem cell proliferation and differentiation into different neural cell ...

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 ...

哺乳類の Mrna の翻訳開始複合体形成の Toeprinting 分析

要約

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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

共転写RNA処理イベントを解剖するための人工RNAポリメラーゼII伸長複合体

キャップ依存翻訳キネティクス解析のためのインビトロ単分子イメージングアッセイ

ポリリボソーム画によって中枢神経系Translatomeの生体尋問中

単一の卵母細胞レポーターアッセイを用いたインビトロ成熟時の母体母体翻訳プログラムの定義

機能と構造解析のためにペプチジル- tRNAを含む翻訳リボソームの分離

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を用いたマウス脳の発達における翻訳の解析

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

哺乳類の Mrna の翻訳開始複合体形成の Toeprinting 分析

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

共転写RNA処理イベントを解剖するための人工RNAポリメラーゼII伸長複合体

キャップ依存翻訳キネティクス解析のための インビトロ 単分子イメージングアッセイ

ポリリボソーム画によって中枢神経系Translatomeの生体尋問中

単一の卵母細胞レポーターアッセイを用いたインビトロ成熟時の母体母体翻訳プログラムの定義

機能と構造解析のためにペプチジル- tRNAを含む翻訳リボソームの分離

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を用いたマウス脳の発達における翻訳の解析

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

キャップ依存翻訳キネティクス解析のためのインビトロ単分子イメージングアッセイ

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