我々は、迅速なプロトコルを設定し、必要なプラスミドを提供することで、それらの有益な助言のための教授ジェフGerstとボリスSlobodinに感謝します。また、LC-MS / MS分析と彼女の助けをSmolerプロテオミクスセンターからこのプロトコルと博士タマルジブを確立する上で彼女の助けのために博士Avigail ATIR-ランデに感謝します。我々はYEF3抗体について教授TG Kinzy（ラトガーズ）をお願いいたします。この作品は、国間科学財団からの助成金2011013によってサポートされていました。

注：12 MS2結合部位からなる配列を挿入（MS2はループ; MS2L）を希望のゲノム遺伝子座に、通常のオープンリーディングフレーム（ORF）および3 &#39;UTRの間。この統合のための詳細なプロトコルは、他の場所22に設けられています。 PCR、ノーザン分析またはRT-PCR 20,23により適切な挿入および発現を確認してください。統合が3&#39;UTRの合成に介入しなかったことを確認することが重要です。また、誘導性プロモーター（メチオニン枯渇）の発現下SBPに融合プラスミド発現MS2-CPはまた、細胞24に導入されるべきです。導入MS12ループを除く同一の株は、非特異的なシグナルを検出するための対照として使用されるべきです。 1.迅速な精製<ol><li> MS2タグmRNAの20を発現する （目的の遺伝子の発現レベルに応じて酵母細胞の250-1,000 mlで使用）、酵母細胞を500mlの成長と適切な増殖培地（ 例えば 、合成デキストロース（SD）選択培地）中で30°Cの時に1.0 - MS2-CP-GFP-SBP融合タンパク質24は 600 0.8 ODまで。 </li><li> RTで4分間、3000×gで遠心分離細胞と上清を捨てます。 </li><li> MS2の発現を誘導するために30°Cの時60分- 1×リン酸洗浄細胞は、生理食塩水（PBS）、（ステップ1.2）以前のように遠心分離、メチオニンなしのSD等量の（ステップ1.1）で再懸濁し、45インキュベートをバッファリング-CP-GFP-SBP融合タンパク質。 注：長い誘導時間は、GFPの凝集を引き起こす可能性があります。 </li><li>細胞の脇に10ミリリットルを設定し、単純な「ホットフェノール」の方法25を使用して、このサンプルからRNAを抽出します。注意：フェノールは非常に有毒であり、その安全上の指示に従って処理してください。 注：このRNAが単離されたRNA（ 図1A）の品質のために良いの参照です。 </li><li> 500ミリリットルの細胞については、最終的なCに1.35ミリリットル37％ホルムアルデヒドを追加RNA-タンパク質複合体を架橋するために、0.1％のoncentration。 10分間30°Cのインキュベーションを続けます。注意：ホルムアルデヒドは非常に有毒であり、その安全上の指示に従って処理してください。 </li><li> 0.125 Mの最終濃度を2.5 26.5 mlのMグリシンを追加し、架橋を停止する3分間インキュベートします。上のこの段階から、RNA分解を最小限に抑えるために氷の上にすべてをかけます。 </li><li> 4°Cので4分間、3000×gで遠心分離細胞と45ミリリットルの冷1×PBSで洗浄します。 </li><li>最初の細胞培養の100ミリリットル当たり1ミリリットル急速な溶解緩衝液中で細胞を再懸濁し。 </li><li>スクリューキャップマイクロチューブに500μlのアリコートに分割し、各アリコート（約フル0.2ミリリットルのPCRチューブ）に冷却されたガラスビーズの〜400ミリグラムを追加します。 </li><li>ビーズにおける溶解細胞を3分間ビーター。直ちに氷上に溶解液を入れました。 注：RNA分解の主な原因である細胞溶解リリース細胞のRNaseを、。迅速な溶解緩衝液は、RNアーゼINHIを高濃度に含んでいますこのような非特異的RNase阻害剤ヘパリンと特異的阻害剤としてbitors、。素早く作業と冷たいすべてを維持することも推奨されます。 </li><li>新しいチューブにライセートを転送します。ピアースホット針（0.8ミリメートル×40 mm）の各マイクロチューブの底に小さな穴と、15 mlチューブの上にマイクロチューブを配置します。マイクロチューブや15ミリリットルチューブ間のアダプタとして5ミリリットル注射器のヘッドを使用してください。 4 O℃で1分間、3,000×gでチューブアセンブリを遠心</li><li>細胞の破片をクリアするには、4 O℃で10分間万×gで新しい1.5 mlチューブと遠心分離機にフロースルー15mlチューブからの転送。 </li><li>それぞれのプールシングル15ミリリットルチューブにすべての1.5ミリリットルチューブからの上清、および脇RNAとタンパク質を単離するための溶解液の1/50と1/100量を設定し、。 注：これらは、その後の分析のための「入力」のサンプル（ 図1）として機能します。 </li><li>私の500ミリリットル当たりアビジン300μgのを追加します。nitial酵母培養し、一定の圧延（10回転）の下で4°Cの時に（これは10分に短縮することができます）30分間インキュベートします。 注：ストレプトアビジンビーズへの結合からアビジンブロックビオチンおよびビオチン化タンパク質。 </li><li>細胞溶解物とのインキュベーション前に、ストレプトアビジンビーズを事前に洗います。 <ol><li> 1.5ミリリットルマイクロ遠心チューブにストレプトアビジンビーズスラリーの約300μLを移し、上部の上清を除去します。これは、ビーズを250μlになります。迅速な溶解用緩衝液1mlで2回ビーズを洗浄してください。各ステップの間に4 O Cで2分間、660×gでストレプトアビジンビーズを遠心します。 </li><li>迅速な溶解緩衝液0.5mlを、10 mg / mlでBSAおよび10mg / mlの酵母tRNAの10μlを0.5mlで1時間インキュベートすることによってビーズを遮断します。必要に応じて、ビーズを回転させながら4°Cのでブロッキング溶液中でO / Nままにすることができます。 </li><li>急速な溶解緩衝液1mlで二回洗浄します。 注：磁気ビーズは、時間とbackgrを減少させるためにも使用することができますound。これらは、しかしながら、セファロースビーズより低い容量を有します。 </li></ol></li><li> （ステップ1.14から）アビジン含有溶解液に予め洗浄したストレプトアビジンビーズの250μlを添加して、一定の回転（10回転）と4℃で1時間インキュベートします。 注：このインキュベーション時間は、十分な結合時間を提供し、RNA分解の可能性を最小限に抑えるとの間の妥協です。 </li><li>ストレプトアビジンビーズを消去し、新しい15mlチューブに上清を移す4 O℃で2分間、660×gで遠心分離します。それぞれ、脇RNAとタンパク質を単離するための溶解液の1/50と1/100量を設定します。 注：これらは、「未結合」のサンプルになります。 </li><li>急速な溶解緩衝液1mlでビーズを洗浄し、軽く振って、新しい1.5 mlのマイクロ遠心チューブに移し、ステップ1.17のように遠心分離。この手順を3回繰り返します。 </li><li> 4℃で5分間、1迅速な洗浄緩衝液のmlであり、この時間回転（10回転）でビーズを洗浄します。二回、この手順を繰り返します。 </l私&gt; <li>上記のように1×PBSと遠心1mlでビーズを洗浄します。 </li><li> 1×PBS120μlの再びビーズを洗浄します。遠心分離は、上記のように、そして「ウォッシュ」サンプルとしてRNAとタンパク質抽出のための全体上清を取ります。 注：この最後の洗浄サンプルは、洗浄のストリンジェンシーを示しますし、溶出サンプルと同じボリュームである必要があります。これは、その後のアッセイの処理およびロードを簡素化します。 </li><li>カラムからRNAおよびRNA結合タンパク質を溶出するために、1×PBSで6 mMのビオチンの120μlを添加し、一定の回転（10回転）で4℃で45分間インキュベートします。 </li><li>遠心分離機は、ビーズのように、上記と新しい1.5 mlチューブに溶出された物質を移します。再び溶出された試料をスピンし、ビーズのないキャリーオーバーを確保しないように新しい1.5 mlチューブに上相を転送します。 </li><li> RNAまたはタンパク質抽出のためのサンプルを取ります。 注：撮影したボリュームは、目的のRNAまたはタンパク質の以下のアッセイおよび発現レベルに依存すべき。ガイドラインとしては、ノーザン分析26のために、新しいタンパク質を発見するために29ウエスタンブロット23,27またはRT-PCR 28のために、20％を取り、質量分析のために、サンプルの80％を取る、全体のサンプルを取ります。 </li></ol> 2. RNA抽出<ol><li> 2倍架橋反転バッファーを等量加え、65℃で45分間インキュベートします。 RNA抽出のために直接進みます。 注：架橋反転バッファが大幅にRNA抽出20を向上させるエチレンジアミン四酢酸（EDTA）が含まれています。 </li><li> 「入力」と「未結合」のサンプル（1.13と1.17ステップ）からRNAを抽出するためにクロロホルム法26：標準のフェノールを使用してください。 注：これらのサンプルは、アッセイを利用し、その後の逆転写酵素（RT）を阻害することができるRNase阻害剤ヘパリンを多量に含む（ 例えば 、RT-PCR）、したがって、LiClを沈殿30は、それを取り除くことが推奨されます。 「WASH」と「溶出」サンプル（1.21と1.24ステップ）は、RNAの低量が含まれており、次のような工程を経て沈殿させます。 </li><li> 「洗浄」と「溶出」サンプルに8Mグアニジン塩酸（のGuHCl）と100％の2倍容量のエタノールを等量加えます。少なくとも2時間、-80℃でインキュベートします。 注：グアニジンを効率的にタンパク質を変性し、それによって試料中のRNaseおよびプロテアーゼを阻害します。 </li><li> 30分20,000×gで、4℃で遠心分離し、上清を捨てます。 20,000×gで再び冷たい80％エタノールおよび遠心分離機で15分間、4°Cをペレットを洗浄。 </li><li> RNaseを含まない水400μlの中でRNAペレットを溶解し、残ったのGuHClまたは他の汚染物質を除去するために再び沈殿します。 3 M酢酸ナトリウム、pH 5.2および100％エタノール800μlの40μlのを追加し、少なくとも1時間、-20℃でインキュベートします。 </li><li>遠心分離し、ステップ2.4のように洗浄し、10μlのチップですべてのエタノールを除去。 RNAペルを再懸濁し10μlのRNaseフリー水でら。 RNAペレットは通常表示されないように細心の注意を払ってください。 注：23（ 図1）で説明したように試料をノーザン分析のために使用することができます。 </li></ol> 3.タンパク質ウエスタンブロットまたは質量分析のための準備<ol><li>タンパク質試料に4倍のLaemmliサンプルバッファー（LSB）の1/3量を加え、RNAとタンパク質の架橋を逆に65℃で45分間インキュベートします。 注：サンプルは-20℃で保存することができます。 </li><li> SDSポリアクリルアミドゲル（SDS-PAGE）31上のタンパク質を実行します。分析すべきタンパク質のサイズに応じてゲルの割合を調整します。 注：10％アクリルアミドゲルは、分離の広い範囲を提供し、第一近似として良いです。 </li><li>分離効率（ 図 1C）の制御のための標準的なイムノブロット分析27のように膜にタンパク質を移します。クマシーブルーRのいずれかで染色質量分析の前に、250または銀染色。 注：それはより敏感で、より良い検出（ 図1B）ことができますので、シルバーステインは好ましい選択です。染色は手動で調製した溶液または市販のキットのいずれかで行うことができます。最終的な染料溶液中のゲルのインキュベーション時間は、実験的に決定されるべきです。長いインキュベーションは、低濃度タンパク質を明らかにすることができるが、このようなMS2-CP-SBP-GFPのような高度に発現されたタンパク質は、上で染色すると、その周辺でタンパク質を取り巻く隠すことが発生する可能性があります。さらに、高いバックグラウンドが発生することがあります。慎重に反応を追跡し、適切な時に停止液を追加します。 </li><li>ゲルからバンドを切り取り、1.5 mlチューブに移します。 -20℃で保存サンプルは、またはLC-MS / MS分析32に続いてタンパク質抽出及びトリプシン消化のために送信します。 </li><li>ゲル分離工程を除外するための代替プロテオミクスアプローチとして、そうでステップ1.24から溶出された材料を消化LC-MS / MS分析33にトリプシンと対象を使用してリューション。 注：SDS-PAGE分離を省略すると、プロトコルが単純化され、歩留まりを向上させます。ただし、サンプルは、洗剤NP-40、質量分析を阻害するの痕跡が含まれている可能性があります。この問題を克服するために、次の手順を実行します。 <ol><li> 15分- 10のためにSDS-PAGE 31上のタンパク質を実行します。 </li><li>レーンの全体部分を切り取り（ すなわち 、タンパク質が入っています）。 注：タンパク質試料が少量のタンパク質のシグナルを不明瞭にすることができるMS2-CP-SBPの有意な量のタンパク質が含まれる場合があります。 </li></ol></li></ol>

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The authors declare no competing financial interests.

様々な方法は、関連するタンパク質11,34 35を識別するために、特定のmRNAの単離を使用します。これらの方法は、RNA-タンパク質相互作用を調べるために、in vitroおよびin vivo戦略に適用する。 インビトロ方法は、外因RBPsを捕捉し、RNP複合体36,37を単離するために細胞溶解物を用いてRNAを転写インキュベートします。このタイプの効果的?...

RNA結合タンパク質（RBPs）はSの約10％を占めセレビシエタンパク質1,2および哺乳動物タンパク質3-5の約15％。これらは、mRNAの転写後プロセシングおよび調節、翻訳、リボソーム生合成、tRNAのアミノアシル化と修正、クロマチンリモデリング、およびより多くのような多くの細胞プロセスに関与しています。 RBPsの重要なサブグループは、mRNA結合タンパク質（mRNPs）6,7です。 mRNAの成熟の過程において、異なるRBPsは、転写物と結合し、その核処理、核のうち輸出、細胞内局在、翻訳および劣化6-8を媒介します 。したがって、任意の時点で特定の転写産物に結合しているRBPsの別個のセットは、その処理し、最終的にその運命を決定します。 mRNAと関連RBPsの識別が大幅に転写後調節の基礎をなす過程の理解を向上させることができます。遺伝的多様顕微鏡、生化学およびバイオインフォマティクス方法は、（9-11に概説）mRNAの調節に関与するタンパク質を同定するために使用されてきました。しかしながら、これらの方法のほんの数は、特定の標的mRNAと関連するタンパク質の同定を可能にします。注目すべきは、酵母細胞における発現ライブラリーをスクリーニングするために餌として関心のmRNAを利用した酵母スリーハイブリッドシステム（Y3H）、です。陽性クローンは、通常の成長の選択またはレポーター発現12-14を通して観察されます。この方法の重要な利点は、細胞環境およびRNA-タンパク質相互作用の強さを測定する能力に走査することができる多数のタンパク質です。欠点は、融合タンパク質の餌または餌RNAのミスフォールディングに、部分的に、非特異的結合に起因する偽陽性の結果の比較的大きい数、により偽陰性の結果のために高い可能性を含みます。 遺伝的アプローチに代わるものは親和性purifiですその関連タンパク質とRNAのカチオン。ポリAを含むmRNAは、オリゴdTカラムの使用によって分離することができ、およびそれらの関連タンパク質は、質量分析によって検出されます。 RNA-タンパク質相互作用は、短距離共有結合を作る架橋によってその細胞コンテキストに保存されています。オリゴdTカラムの使用は、任意のポリA-含むmRNA 3,5,15に関連付けられているプロテオーム全体のグローバルな視点をもたらします。しかし、これは、特定のmRNAに関連付けられているタンパク質のリストを提供していません。非常に少数の方法は、このような識別を達成するために利用可能です。 PAIRの方法は、標的mRNAの16,17に対する相補性を有する核酸のトランスフェクションを伴います。核酸はまた、相互作用部位に近接してRBPsに架橋可能ペプチドに付着されます。架橋後、RBP-ペプチド核酸を単離することができ、プロテオミクス分析に供​​しました。最近、アプタマーベースの方法論がありました正常哺乳動物細胞株18からの抽出物に適用されます。ストレプトアビジンへの改善された親和性を有するRNAアプタマーを開発し、（この場合はAUリッチエレメント（ARE））目的の配列に融合させました。アプタマーARE RNAをストレプトアビジンビーズに付着し、細胞溶解物と混合しました。 ARE配列に関連するタンパク質を精製し、質量分析（MS）により同定しました。この方法は、細胞の設定（ すなわち 、 インビトロ ）の外側に発生する関連付けを検出しているが、mRNAに関連するタンパク質の単離をゲノムにアプタマーを導入し、従って、可能なように、将来的に修飾される可能性が高いにある間細胞環境（ すなわち 、in vivoで ）。遺伝子操作は、十分に確立された酵母では、（教授ジェフGerstの研究室で開発された）迅速法は、in vivo団体19のビューを提供します。ラピッド（MS2コートタンパク質の特異的かつ強力な結合を兼ね備えMS2-MS2 RNA配列に、ストレプトアビジン結合ドメイン（SBP）のストレプトアビジン結合ビーズにCP）。これは、ストレプトアビジンビーズを介してMS2-タグ付けされたmRNAの効率的な精製を可能にします。また、MS2は、ループの12のコピーの発現は、RNAに同時に結合し、その分離の効率を高めるために6 MS2-のCPまで使用できます。このプロトコルは、従って、溶出された試料を質量分析によってプロテオミクス分析に供​​された後、新規のmRNA結合タンパク質の同定を可能にするために示唆されました。 我々は最近、酵母PMP1 mRNAを20に関連付けられた新規タンパク質を同定するための迅速を利用した。PMP1 mRNAが以前にER膜と関連することが示され、その3 &#39;非翻訳領域（UTR）は、この関連21の主要な決定要因であることが判明しました。したがって、PMP1 3 &#39;UTRに結合RBPsその局在に重要な役割を果たす可能性があります。 RAPIDは、液体クロマトグラフが続きますY字型質量分析/質量分析（LC-MS / MS）は、PMP1 20と相互作用する多数の新規タンパク質の同定をもたらしました。ここで、我々は急速な方法論、実行する必要がある重要なコントロール、および収量と特異性を向上させることができる技術的なヒントの詳細なプロトコルを提供します。

<table border="0" cellpadding="0" cellspacing="0" width="632">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Tris</td>
			<td style="width:107px;">sigma</td>
			<td style="width:119px;">T1503</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SDS</td>
			<td>bio-lab</td>
			<td align="right">1981232300</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DTT</td>
			<td>sigma</td>
			<td>D9779</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Acidic Phenol (pH 4.3)</td>
			<td>sigma</td>
			<td>P4682</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Acidic Phenol: Chloroform (5:1, pH 4.3)</td>
			<td>sigma</td>
			<td>P1944</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Chloroform</td>
			<td>bio-lab</td>
			<td align="right">3080521</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Formaldehyde</td>
			<td>Frutarom</td>
			<td align="right">5551820</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glycine</td>
			<td>sigma</td>
			<td>G7126</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NP-40</td>
			<td>Calbiochem</td>
			<td align="right">492016</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Heparin</td>
			<td>Sigma</td>
			<td>H3393</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phenylmethylsulfonyl Flouride (PMSF)</td>
			<td>Sigma</td>
			<td>P7626</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Leupeptin</td>
			<td>Sigma</td>
			<td>L2884</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Aprotinin</td>
			<td>Sigma</td>
			<td>A1153</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Soybean Trypsin Inhibitor</td>
			<td>Sigma</td>
			<td>T9003</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pepstatin</td>
			<td>Sigma</td>
			<td>P5318</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DNase I</td>
			<td>Promega</td>
			<td>M610A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ribonuclease&#160; Inhibitor</td>
			<td>Takara</td>
			<td>2313A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glass Beads</td>
			<td>Sartorius</td>
			<td>BBI-8541701</td>
			<td>0.4-0.6mm diameter&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mini BeadBeater</td>
			<td>BioSpec</td>
			<td colspan="2">Mini BeadBeater 16</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Guanidinium</td>
			<td>Sigma</td>
			<td>G4505</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Avidin</td>
			<td>Sigma</td>
			<td>A9275</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Streptavidin Beads</td>
			<td>GE Healthcare&#160;</td>
			<td>17-5113-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bovine serum albumin (BSA)</td>
			<td>Sigma</td>
			<td>A7906</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Yeast tRNA</td>
			<td>Sigma</td>
			<td>R8508</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Biotin</td>
			<td>Sigma</td>
			<td>B4501</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Yeast extract</td>
			<td>Bacto</td>
			<td dir="LTR">288620</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">peptone</td>
			<td>Bacto</td>
			<td dir="LTR">211677</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glucose</td>
			<td>Sigma</td>
			<td>G8270</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1 x Phosphate-Buffered saline (PBS)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.2 M NaOH</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">4 x Laemmli Sample Buffer (LSB)</td>
			<td></td>
			<td></td>
			<td>0.2 M Tris-Hcl pH 6.8, 8% SDS, 0.4 M DTT, 40% glycerol, 0.04% Bromophenol-Blue.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hot phenol lysis buffer</td>
			<td></td>
			<td></td>
			<td>10 mM Tris pH 7.5, 10 mM EDTA, 0.5% SDS&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">3 M Sodium Acetate pH 5.2</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">100% and 70% Ethanol (EtOH)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RNase-free water</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">RaPID lysis buffer</td>
			<td></td>
			<td></td>
			<td>20 mM Tris pH 7.5, 150 mM NaCl, 1.8 mM MgCl2, 0.5% NP-40, 5 mg/ml Heparin, 1 mM Dithiothreitol (DTT), 1 mM Phenylmethylsulfonyl Flouride (PMSF), 10 &#181;g/ml Leupeptin, 10 &#181;g/ml Aprotinin, 10 &#181;g/ml Soybean Trypsin Inhibitor, 10 &#181;g/ml Pepstatin, 20 U/ml DNase I, 100 U/ml Ribonuclease&#160; Inhibitor.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2x Cross-linking reversal buffer</td>
			<td></td>
			<td></td>
			<td dir="LTR">100 mM Tris pH 7.4, 10 mM EDTA, 20 mM DTT, 2 % SDS.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RaPID wash buffer</td>
			<td></td>
			<td></td>
			<td>20 mM Tris-HCl pH 7.5,&#160; 300 mM NaCl, 0.5% NP-40</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.5 M EDTA pH 8</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Silver Stain Plus Kit</td>
			<td>Bio-Rad&#160;</td>
			<td>161-0449</td>
			<td>For detecting proteins in polyacrylamide gels</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SD selective medium&#160;</td>
			<td></td>
			<td></td>
			<td>1.7 g/l Yeast nitrogen base with out amino acids and ammonium sulfate, 5 g/l Ammonium sulfate, 2% glucose, 350 mg/l Threonine, 40 mg/l Methionine, 40 mg/l Adenine, 50 mg/l Lysine, 50 mg/l Tryptophan, 20 mg/l Histidine, 80 mg/l Leucine, 30 mg/l Tyrosine, 40 mg/l Arginine</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-eEF3 (EF3A,YEF3)</td>
			<td colspan="2">Gift from Kinzy TG. (UMDNJ Robert Wood Johnson Medical School)</td>
			<td>1:5,000</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti GFP antibody</td>
			<td>Santa Cruz</td>
			<td>sc-8334</td>
			<td>1:3,000</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti rabbit IgG-HRP conjugated</td>
			<td>SIGMA</td>
			<td>A9169</td>
			<td>1:10,000</td>
		</tr>
	</tbody>
</table>

novel rna-binding proteins isolation by the rapid methodology

RNA-binding proteins (RBPs) play important roles in every aspect of RNA metabolism and regulation. Their identification is a major challenge in modern biology. Only a few in vitro and in vivo methods enable the identification of RBPs associated with a particular target mRNA. However, their main limitations are the identification of RBPs in a non-cellular environment (in vitro) or the low efficiency isolation of RNA of interest (in vivo). An RNA-binding protein purification and identification (RaPID) methodology was designed to overcome these limitations in yeast and enable efficient isolation of proteins that are associated in vivo. To achieve this, the RNA of interest is tagged with MS2 loops, and co-expressed with a fusion protein of an MS2-binding protein and a streptavidin-binding protein (SBP). Cells are then subjected to crosslinking and lysed, and complexes are isolated through streptavidin beads. The proteins that co-purify with the tagged RNA can then be determined by mass spectrometry. We recently used this protocol to identify novel proteins associated with the ER-associated PMP1 mRNA. Here, we provide a detailed protocol of RaPID, and discuss some of its limitations and advantages.

RAPIDは、その関連タンパク質との特異的標的RNAの単離を可能にします。その成功のために重要なことにより、タンパク質の十分な量を得る、可能な限り無傷のRNAを保っています。 RNAの単離効率と品質を決定するために、ノーザン分析は（ 図1A）が行われます。ノーザン分析は、直接迅速の効率と品質を報告するという利点を有します。したがって、完全?...

Watch this Scientific Journal Video about Novel RNA-Binding Proteins Isolation by the RaPID Methodology at JoVE.com

Novel RNA-Binding Proteins Isolation by the RaPID Methodology

RNA-protein interactions lie at the heart of many cellular processes. Here, we describe an in vivo method to isolate specific RNA and identify novel proteins that are associated with it. This could shed new light on how RNAs are regulated in the cell.

急速な手法による新規RNA結合タンパク質の単離

RNA-binding proteins (RBPs) play important roles in every aspect of RNA metabolism and regulation. Their identification is a major challenge in modern ...

novel-rna-binding-proteins-isolation-by-the-rapid-methodology

Research

JoVE Journal

Genetics

8.9K ビュー.  Technion - Israel Institute of Technology. RNA-protein interactions lie at the heart of many cellular processes. Here, we describe an in vivo method to isolate specific RNA and identify novel proteins that are associated with it. This could shed new light on how RNAs are regulated in the cell. 

ビデオ: Novel RNA-Binding Proteins Isolation by the RaPID Methodology

Conjugative Mating Assays for Sequence-specific Analysis of Transfer Proteins Involved in Bacterial Conjugation

The transfer of genetic material by bacterial conjugation is a process that takes place via complexes formed by specific transfer proteins. In Escherichia coli, these transfer proteins make up a DNA transfer machinery known as the mating pair formation, or DNA transfer complex, which facilitates conjugative plasmid transfer. The objective of this paper is to provide a method that can be used to determine the role of a specific transfer protein that is involved in conjugation using a series of deletions and/or point mutations in combination with mating assays. The target gene is knocked out on the conjugative plasmid and is then provided in trans through the use of a small recovery plasmid harboring the target gene. Mutations affecting the target gene on the recovery plasmid can reveal information about functional aspects of the target protein that result in the alteration of mating efficiency of donor cells harboring the mutated gene. Alterations in mating efficiency provide insight into the role and importance of the particular transfer protein, or a region therein, in facilitating conjugative DNA transfer. Coupling this mating assay with detailed three-dimensional structural studies will provide a comprehensive understanding of the function of the conjugative transfer protein as well as provide a means for identifying and characterizing regions of protein-protein interaction.

Here, we present a protocol to knockout a gene of interest involved in plasmid conjugation and subsequently analyze the impact of its absence using mating assays. The function of the gene is further explored to a specific region of its sequence using deletion or point mutations.

細菌接合に関与する転写タンパク質の配列特異的分析のための接合交配アッセイ

The transfer of genetic material by bacterial conjugation is a process that takes place via complexes formed by specific transfer proteins. In Escherichia ...

遺伝学

Kinetics of Lagging-strand DNA Synthesis In Vitro by the Bacteriophage T7 Replication Proteins

Here we provide protocols for the kinetic examination of lagging-strand DNA synthesis in vitro by the replication proteins of bacteriophage T7. The T7 replisome is one of the simplest replication systems known, composed of only four proteins, which is an attractive feature for biochemical experiments. Special emphasis is placed on the synthesis of ribonucleotide primers by the T7 primase-helicase, which are used by DNA polymerase to initiate DNA synthesis. Because the mechanisms of DNA replication are conserved across evolution, these protocols should be applicable, or useful as a conceptual springboard, to investigators using other model systems. The protocols described here are highly sensitive and an experienced investigator can perform these experiments and obtain data for analysis in about a day. The only specialized piece of equipment required is a rapid-quench flow instrument, but this piece of equipment is relatively common and available from various commercial sources. The major drawbacks of these assays, however, include the use of radioactivity and the relative low throughput.

Kinetics of Lagging-strand DNA Synthesis In Vitro by the Bacteriophage T7 Replication Proteins

We describe sensitive, gel-based discontinuous assays to examine the kinetics of lagging-strand initiation using the replication proteins of bacteriophage T7.

遅行本鎖DNA合成の動態<em&gt;インビトロ</em&gt;バクテリオファージT7複製タンパク質によって

Here we provide protocols for the kinetic examination of lagging-strand DNA synthesis in vitro by the replication proteins of bacteriophage T7. The T7 ...

Bidirectional Retroviral Integration Site PCR Methodology and Quantitative Data Analysis Workflow

Integration Site (IS) assays are a critical component of the study of retroviral integration sites and their biological significance. In recent retroviral gene therapy studies, IS assays, in combination with next-generation sequencing, have been used as a cell-tracking tool to characterize clonal stem cell populations sharing the same IS. For the accurate comparison of repopulating stem cell clones within and across different samples, the detection sensitivity, data reproducibility, and high-throughput capacity of the assay are among the most important assay qualities. This work provides a detailed protocol and data analysis workflow for bidirectional IS analysis. The bidirectional assay can simultaneously sequence both upstream and downstream vector-host junctions. Compared to conventional unidirectional IS sequencing approaches, the bidirectional approach significantly improves IS detection rates and the characterization of integration events at both ends of the target DNA. The data analysis pipeline described here accurately identifies and enumerates identical IS sequences through multiple steps of comparison that map IS sequences onto the reference genome and determine sequencing errors. Using an optimized assay procedure, we have recently published the detailed repopulation patterns of thousands of Hematopoietic Stem Cell (HSC) clones following transplant in rhesus macaques, demonstrating for the first time the precise time point of HSC repopulation and the functional heterogeneity of HSCs in the primate system. The following protocol describes the step-by-step experimental procedure and data analysis workflow that accurately identifies and quantifies identical IS sequences.

This manuscript describes the experimental procedure and software analysis for a bidirectional integration site assay that can simultaneously analyze upstream and downstream vector-host junction DNA. Bidirectional PCR products can be used for any downstream sequencing platform. The resulting data are useful for a high-throughput, quantitative comparison of integrated DNA targets.

双方向レトロウイルスの統合サイトPCRの方法論と定量的データ解析ワークフロー

Integration Site (IS) assays are a critical component of the study of retroviral integration sites and their biological significance. In recent retroviral ...

A Standard Methodology to Examine On-site Mutagenicity As a Function of Point Mutation Repair Catalyzed by CRISPR/Cas9 and SsODN in Human Cells

Combinatorial gene editing using CRISPR/Cas9 and single-stranded oligonucleotides is an effective strategy for the correction of single-base point mutations, which often are responsible for a variety of human inherited disorders. Using a well-established cell-based model system, the point mutation of a single-copy mutant eGFP gene integrated into HCT116 cells has been repaired using this combinatorial approach. The analysis of corrected and uncorrected cells reveals both the precision of gene editing and the development of genetic lesions, when indels are created in uncorrected cells in the DNA sequence surrounding the target site. Here, the specific methodology used to analyze this combinatorial approach to the gene editing of a point mutation, coupled with a detailed experimental strategy to measuring indel formation at the target site, is outlined. This protocol outlines a foundational approach and workflow for investigations aimed at developing CRISPR/Cas9-based gene editing for human therapy. The conclusion of this work is that on-site mutagenesis takes place as a result of CRISPR/Cas9 activity during the process of point mutation repair. This work puts in place a standardized methodology to identify the degree of mutagenesis, which should be an important and critical aspect of any approach destined for clinical implementation.

This protocol outlines the workflow of a CRISPR/Cas9-based gene editing system for the repair of point mutations in mammalian cells. Here, we use a combinatorial approach to gene editing with a detailed follow-on experimental strategy for measuring indel formation at the target site&#8212;in essence, analyzing onsite mutagenesis.

点突然変異修復 CRISPR/Cas9 とひと細胞での SsODN によって触媒の機能として敷地内変異原性を調べるための標準方法論

Combinatorial gene editing using CRISPR/Cas9 and single-stranded oligonucleotides is an effective strategy for the correction of single-base point ...

Methodology for Accurate Detection of Mitochondrial DNA Methylation

Quantification of DNA methylation can be achieved using bisulfite sequencing, which takes advantage of the property of sodium bisulfite to convert unmethylated cytosine into uracil, in a single-stranded DNA context. Bisulfite sequencing can be targeted (using PCR) or performed on the whole genome and provides absolute quantification of cytosine methylation at the single base-resolution. Given the distinct nature of nuclear- and mitochondrial DNA, notably in the secondary structure, adaptions of bisulfite sequencing methods for investigating cytosine methylation in mtDNA should be made. Secondary and tertiary structure of mtDNA can indeed lead to bisulfite sequencing artifacts leading to false-positives due to incomplete denaturation poor access of bisulfite to single-stranded DNA. Here, we describe a protocol using an enzymatic digestion of DNA with BamHI coupled with bioinformatic analysis pipeline to allow accurate quantification of cytosine methylation levels in mtDNA. In addition, we provide guidelines for designing the bisulfite sequencing primers specific to mtDNA, in order to avoid targeting undesirable NUclear MiTochondrial segments (NUMTs) inserted into the nuclear genome.

Here, we present a protocol to allow accurate quantification of mitochondrial DNA (mtDNA) methylation. In this protocol, we describe an enzymatic digestion of DNA with BamHI coupled with a bioinformatic analysis pipeline which can be used to avoid overestimation of mtDNA methylation levels caused by the secondary structure of mtDNA.

ミトコンドリア DNA のメチル化の正確な検出のための方法論

Quantification of DNA methylation can be achieved using bisulfite sequencing, which takes advantage of the property of sodium bisulfite to convert ...

Novel Sequence Discovery by Subtractive Genomics

Subtractive genomics can be used in any research where the goal is to identify the sequence of a gene, protein, or general region that is embedded in a larger genomic context. Subtractive genomics enables a researcher to isolate a target sequence of interest (T) by comprehensive sequencing and subtracting out known genetic elements (reference, R). The method can be used to identify novel sequences such as mitochondria, chloroplasts, viruses, or germline restricted chromosomes, and is particularly useful when T cannot be easily isolated from R. Beginning with the comprehensive genomic data (R + T), the method uses Basic Local Alignment Search Tool (BLAST) against a reference sequence, or sequences, to remove the matching known sequences (R), leaving behind the target (T). For subtraction to work best, R should be a relatively complete draft that is missing T. Since sequences remaining after subtraction are tested through quantitative Polymerase Chain Reaction (qPCR), R does not need to be complete for the method to work. Here we link computational steps with experimental steps into a cycle that can be iterated as needed, sequentially removing multiple reference sequences and refining the search for T. The advantage of subtractive genomics is that a completely novel target sequence can be identified even in cases in which physical purification is difficult, impossible, or expensive. A drawback of the method is finding a suitable reference for subtraction and obtaining T-positive and negative samples for qPCR testing. We describe our implementation of the method in the identification of the first gene from the germline-restricted chromosome of zebra finch. In that case computational filtering involved three references (R), sequentially removed over three cycles: an incomplete genomic assembly, raw genomic data, and transcriptomic data.

The purpose of this protocol is to use a combination of computational and bench research to find novel sequences that cannot be easily separated from a co-purifying sequence, which may be only partially known.

減法混色のゲノム解析による新規シーケンスの発見

Subtractive genomics can be used in any research where the goal is to identify the sequence of a gene, protein, or general region that is embedded in a ...

Sequencing of mRNA from Whole Blood using Nanopore Sequencing

Sequencing in remote locations and resource-poor settings presents unique challenges. Nanopore sequencing can be successfully used under such conditions, and was deployed to West Africa during the recent Ebola virus epidemic, highlighting this possibility. In addition to its practical advantages (low cost, ease of equipment transport and use), this technology also provides fundamental advantages over second-generation sequencing approaches, particularly the very long read length, ability to directly sequence RNA, and real-time availability of data. Raw read accuracy is lower than with other sequencing platforms, which represents the main limitation of this technology; however, this can be partially mitigated by the high read depth generated. Here, we present a field-compatible protocol for sequencing of the mRNAs encoding for Niemann-Pick C1, which is the cellular receptor for ebolaviruses. This protocol encompasses extraction of RNA from animal blood samples, followed by RT-PCR for target enrichment, barcoding, library preparation, and the sequencing run itself, and can be easily adapted for use with other DNA or RNA targets.

Nanopore sequencing is a novel technology that allows cost-effective sequencing in remote locations and resource-poor settings. Here, we present a protocol for sequencing of mRNAs from whole blood that is compatible with such conditions.

ナノポアシーケンシングを用いた全血からの mRNA の配列決定

Sequencing in remote locations and resource-poor settings presents unique challenges. Nanopore sequencing can be successfully used under such conditions, ...

Mass Spectrometry-Based Proteomics Analyses Using the OpenProt Database to Unveil Novel Proteins Translated from Non-Canonical Open Reading Frames

Genome annotation is central to today's proteomic research as it draws the outlines of the proteomic landscape. Traditional models of open reading frame (ORF) annotation impose two arbitrary criteria: a minimum length of 100 codons and a single ORF per transcript. However, a growing number of studies report expression of proteins from allegedly non-coding regions, challenging the accuracy of current genome annotations. These novel proteins were found encoded either within non-coding RNAs, 5' or 3' untranslated regions (UTRs) of mRNAs, or overlapping a known coding sequence (CDS) in an alternative ORF. OpenProt is the first database that enforces a polycistronic model for eukaryotic genomes, allowing annotation of multiple ORFs per transcript. OpenProt is freely accessible and offers custom downloads of protein sequences across 10 species. Using OpenProt database for proteomic experiments enables novel proteins discovery and highlights the polycistronic nature of eukaryotic genes. The size of OpenProt database (all predicted proteins) is substantial and need be taken in account for the analysis. However, with appropriate false discovery rate (FDR) settings or the use of a restricted OpenProt database, users will gain a more realistic view of the proteomic landscape. Overall, OpenProt is a freely available tool that will foster proteomic discoveries.

OpenProt is a freely accessible database that enforces a polycistronic model of eukaryotic genomes. Here, we present a protocol for the use of OpenProt databases when interrogating mass spectrometry datasets. Using OpenProt database for analysis of proteomic experiments allows for discovery of novel and previously undetectable proteins.

非正規の開いたリーディング ・ フレームから翻訳された蛋白質を発表する OpenProt データベースを利用した質量分析を用いたプロテオミクス解析

Genome annotation is central to today's proteomic research as it draws the outlines of the proteomic landscape. Traditional models of open reading frame ...

An Assay for Quantifying Protein-RNA Binding in Bacteria

In the initiation step of protein translation, the ribosome binds to the initiation region of the mRNA. Translation initiation can be blocked by binding of an RNA binding protein (RBP) to the initiation region of the mRNA, which interferes with ribosome binding. In the presented method, we utilize this blocking phenomenon to quantify the binding affinity of RBPs to their cognate and non-cognate binding sites. To do this, we insert a test binding site in the initiation region of a reporter mRNA and induce the expression of the test RBP. In the case of RBP-RNA binding, we observed a sigmoidal repression of the reporter expression as a function of RBP concentration. In the case of no-affinity or very low affinity between binding site and RBP, no significant repression was observed. The method is carried out in live bacterial cells, and does not require expensive or sophisticated machinery. It is useful for quantifying and comparing between the binding affinities of different RBPs that are functional in bacteria to a set of designed binding sites. This method may be inappropriate for binding sites with high structural complexity. This is due to the possibility of repression of ribosomal initiation by complex mRNA structure in the absence of RBP, which would result in lower basal reporter gene expression, and thus less-observable reporter repression upon RBP binding.

In this method, we quantify the binding affinity of RNA binding proteins (RBPs) to cognate and non-cognate binding sites using a simple, live, reporter assay in bacterial cells. The assay is based on repression of a reporter gene.

細菌におけるタンパク質-RNA結合を定量するためのアッセイ

In the initiation step of protein translation, the ribosome binds to the initiation region of the mRNA. Translation initiation can be blocked by binding ...

Exploring Sequence Space to Identify Binding Sites for Regulatory RNA-Binding Proteins

Gene regulation plays an important role in all cells. Transcriptional, post-transcriptional (or RNA processing), translational, and post-translational steps are used to regulate specific genes. Sequence-specific nucleic acid-binding proteins target specific sequences to control spatial or temporal gene expression. The binding sites in nucleic acids are typically characterized by mutational analysis. However, numerous proteins of interest have no known binding site for such characterization. Here we describe an approach to identify previously unknown binding sites for RNA-binding proteins. It involves iterative selection and amplification of sequences starting with a randomized sequence pool. Following several rounds of these steps-transcription, binding, and amplification-the enriched sequences are sequenced to identify a preferred binding site(s). Success of this approach is monitored using in vitro binding assays. Subsequently, in vitro and in vivo functional assays can be used to assess the biological relevance of the selected sequences. This approach allows identification and characterization of a previously unknown binding site(s) for any RNA-binding protein for which an assay to separate protein-bound and unbound RNAs exists.

Sequence specificity is critical for gene regulation. Regulatory proteins that recognize specific sequences are important for gene regulation. Defining functional binding sites for such proteins is a challenging biological problem. An iterative approach for identification of a binding site for an RNA-binding protein is described here and is applicable to all RNA-binding proteins.

調節RNA結合タンパク質の結合部位を同定するための配列空間の探索

Gene regulation plays an important role in all cells. Transcriptional, post-transcriptional (or RNA processing), translational, and post-translational ...