Name: analysis of termination of transcription using brutp-strand-specific transcription run-on (tro) approach
Uploaded: 2017-03-12T15:00:00.000+00:00
Duration: 12 min 12 s
Description: Watch this Scientific Journal Video about Analysis of Termination of Transcription Using BrUTP-strand-specific Transcription Run-on TRO Approach at JoVE.com

Research in Ansari lab was supported by the research grant (No. MCB 1020911) from National Science Foundation.

注：このメソッドはMedler、Sおよびアンサリ、A 2で報告された研究論文で使用されました。 1.培養および収穫細胞<ol><li>新たに画線サッカロマイセス・セレビシエプレートからYPD培地中の細胞の5mLの培養を開始する（を10g / L酵母抽出物が20g / Lペプトン、2％デキストロース）。 </li><li> 30℃、250 rpmでオービタルシェーカーで一晩細胞を増殖させます。 </li><li> 250mLのバッフル付きフラスコ中のYPD培地中で100mLの最終容量まで一晩成長させた培養物を100倍に希釈します。 注：文化の100ミリリットルの合計は、TROの反応ごとに必要とされています。複数の反応が必要な場合は、文化の大容量を使用する必要があります。 </li><li> 600 nmの（ - 1.0 600〜0.8）で1.0から0.8の光学密度に細胞を増殖させます。 </li><li>滅菌した50 mLのコニカルチューブ中で4℃で5分間、820×gでの遠心分離によって細胞を回収。 </li><li>氷冷TMN府の10 mLの細胞を再懸濁ffer（10mMのトリス-HCl pH7.5で、5mMのMgCl 2、100mMのNaCl）と氷上で5分間インキュベートします。 </li><li>細胞をペレット化するために4℃で5分間、820×gで遠心分離します。静かに吸引して上清を除去します。 </li></ol> 2.透過性にして、Run-上のアッセイのための細胞を準備<ol><li>切り捨てられたP 千チップを用いて穏やかに上下にピペッティングし、0.6％のサルコシル溶液1mLで細胞を再懸濁します。チルド1.5 mLのマイクロ遠心チューブに細胞懸濁液を転送します。 </li><li>プラットフォームシェーカー上で25分間、4℃で穏やかに細胞を振ります。 注：微量遠心チューブは、この段階でプロモーターで生じる新しい開始事象の可能性を回避し、低温を維持するために氷で50 mLコニカルチューブに充填されるべきです。 </li><li>卓上冷却遠心機を用いて4℃で6分間、1200×gで細胞を遠心。 </li><li>静かあたりから余分な液体を除去するために上清を吸引細胞ペレットをmeabilized。 </li></ol> 3.転写ランオンの反応<ol><li>転写ランオンバッファの150μLを加える（50 mMトリス-塩酸pHが7.5、100のKCl、10mMのMgCl 2を、2 mMのDTT（ジチオスレイトール）、0.75 mMのATP、CTP、GTP、およびBrUTP、200 UのRNaseの各透過性細胞ペレットに対する阻害剤）。 </li><li>ピペッティングによって切り捨てられたP 200チップを用いてダウン穏やかに混合します。 注：複数のサンプルを処理する場合は、すべてのサンプルが準備が整うまで氷上にすべてを保持し、次のステップに進みます。 </li><li>水浴中で30℃で5分間インキュベートします。 注：新生RNAにBrUTPの最適な取り込みを確実にするために1〜5分の間のインキュベーション時間を調整します。 </li><li>冷たいすぐに使用できるRNA単離試薬500μLを加えて反応を停止します。室温で5分間インキュベートします。 </li></ol> 4. RNA抽出<ol><li>セルsuspensiに酸洗浄ガラスビーズの200μLを追加（ステップ3.4）に、ボルテックスミキサーで4℃で20分間激しく振ります。 </li><li>ホット22 G針を使用して、マイクロチューブの底に穴を開け、15-mLの滅菌コニカルチューブの上に置きます。 </li><li>細胞溶解物を収集するために4℃で1分間、300×gで遠心分離します。 1.5 mLのマイクロ遠心チューブに細胞溶解物を転送します。 </li><li>細胞溶解物に冷たいRNA単離試薬とクロロホルムの200μLの500μLを追加します。 4℃で20分間、16168×gでボルテックスミキサーや遠心分離機上のコンテンツを混ぜます。 </li><li>新しいマイクロチューブに上部の水相を転送します。 </li><li> pHが4-5コールドフェノール/クロロホルム/イソアミルアルコールを等量の（：：24 1 125）を追加します。 4℃で15分間、16168×gでボルテックスミキサーや遠心分離機に激しく振ります。 </li><li>新しいマイクロチューブにRNAを含む上部の水相を転送します。繰り返しステップ4.6をさらに2回。 </li><li>最終的なRNAを含む水相に、5 MのNaCl STOCを追加0.3 Mの最終濃度は、RNAを沈殿させるために冷エタノールの3倍のボリュームを追加取得するにはKです。 <ol><li> 1時間または一晩-20℃でインキュベートします。 注： - 抗BrdUビーズは（ステップ5.3参照）、ブロッキングインキュベートされている間に4.11のステップ4.9を実行します。 </li></ol></li><li> 4℃で20分間、16168×gで遠心分離します。上清を除去し、DEPC（ジエチル）100μLの水でRNAペレットを再懸濁します。 </li><li>取り込まれていないBrUTPヌクレオチドからRNAを分離するために、RNA単離ミニキットを使用してください。 RNaseフリー水100μLを用いてカラムからRNAを溶出。 </li><li> 5分間65℃の水浴中でRNAをインキュベートした後、少なくとも2分間氷上に移します。 </li></ol> BrUTP標識RNAの5アフィニティ精製<ol><li> 3MのNaCl、0.2 MのNaH 2 PO 4：抗BrdUは、バッファ（20×SSPEを結合0.25×SSPE（ナトリウム生理食塩水リン酸EDTA）の500μLを追加し、ビーズを定住の25μLへ</sub&gt;、0.02 M EDTA、pHが7.4、1 mMのEDTA、0.05％のTween、37.5 mMのNaCl）で。 4℃で30秒、200×gで遠心分離し、上清を除去します。 </li><li>繰り返しステップ5.1 3回。 </li><li>ビーズを緩衝液（1×結合緩衝液、0.1％ポリビニルピロリドン、1mgの超高純度ウシ血清アルブミン（BSA））をブロッキングの500μLを加え、1穏やかに振る - プラットフォームシェーカー上で4℃で2時間。 </li><li>ステップ5.1で説明したように、結合緩衝液500μLでビーズをさらに2回洗浄します。 </li><li>ビーズに結合緩衝液400μLを加えます。 </li><li>ビーズに直接RNAの100μL（ステップ4.11から）を転送します。プラットフォームシェーカー上で4℃で2時間 - 穏やかが1振盪しながらインキュベートします。 </li><li>結合緩衝液500μL、低塩緩衝液の500μL（0.2×のSSPE、1 mMのEDTA、0.05％のTween）、高塩緩衝液500μL（0.25×SSPE、1 mMのEDTA、0.05％Tweenでビーズを順番に洗って、で回転100mMのNaCl）、および二回TET緩衝液（1×TE、0.05％のTween）の500μLと、各洗浄の間に4℃で30秒間、200×gで。 </li><li> 150μlので二回、順次ビーズからBrUTP標識したRNAを溶出し、一回の溶出緩衝液（20mMのDTT、150mMのNaCl、50mMのトリス-HCl pH7.5で、1mMのEDTA、0.1％SDS）200μlで、インキュベートすることにより清からビーズを分離するために、短いスピンに続いて水浴中で42℃で4分間。 </li></ol> BrUTP標識RNAの6降水<ol><li> BrUTPは、RNAを標識したアフィニティー精製に5 - pHが4冷たいフェノール/クロロホルム/イソアミルアルコール（：：24 1 125）の500μLを追加します。 4℃で15分間16168×gでボルテックスミキサーや遠心分離機に混ぜます。 </li><li>新しいマイクロチューブに上部の水相を転送します。 </li><li> RNAを沈殿させ、0.3Mの最終濃度と冷エタノールの3倍の量を取得するために、5 M NaCl貯蔵を追加します。 </li><li> -20℃で一晩インキュベートします。 </li><li> 4℃で30分間16168×gで遠心分離することによって沈殿したRNAを収集します。&lt;/李&gt; <li>上清を除去し、10分間空気乾燥さペレットを許可します。 </li><li> DEPC水の26μLでRNAペレットを再懸濁します。 </li><li>分光光度計を用いて260nmの吸光度を測定することにより、最終RNA濃度を決定します。また、260/280比を決定します。 〜2の比率は、純粋なRNAサンプルを示します。 </li><li> cDNA合成のために必要とされるまで-80℃でアリコート中のRNAサンプルを保管してください。 </li></ol> BrUTP標識RNAの7.逆転写<ol><li> DEPC水を用いて100 ngの/μLにRNA濃度を調整します。各cDNA合成反応のためのRNA0.5μgのを使用してください。 注：RNA鋳型の濃度が最適なcDNA合成を達成するために、0.5から1.0μgのに変化してもよいです。 </li><li> 図3Aに示すように、ポリ（A）部位（終結部位）の下流に設計された複数のリバースプライマーを用いてRNAを逆転写。 </li><li>各逆転写反応には、RNAのtemplatμlの5を追加電子（0.5μgの）、1μLのdNTPミックス（各10mM）、リバースプライマーC、D、E、FまたはGの2μL（50μMずつ）とヌクレアーゼフリー水（ 図3Aを参照してください）。各チューブの最終反応容量は13μLです。 注：逆転写反応の数は、cDNA合成のために使用されるリバースプライマーの数に依存します。たとえば、ASC1のために5リバースプライマーを使用したため、5逆転写反応を設定しました。 cDNA合成のために使用される逆方向プライマーに基づいてC、D、E、F及びGのような管をラベル。 </li><li>任意の二次構造配座を除去するために5分間65℃でサーモサイクラー中でRNA鋳型、dNTP混合物およびプライマーを含有するチューブをインキュベートし、少なくとも2分間、4℃に冷却します。 </li><li> 1μLを加えるRNA鋳型とし、プライマー（ステップ7.4）を含む各チューブに転写酵素（200 U /μL）、4μLのバッファーと0.1 M DTTの2μLを逆転。上下静かにピペッティングで。 </li><li> 60分間42℃でサーモサイクラーで反応混合物をインキュベートすることによって、逆転写を実行します。 </li><li> 20分間65℃で逆転写酵素を不活性化します。 </li><li> -20℃でのcDNAを保存したり、次のステップに進みます。 </li></ol> cDNAの8.増幅<ol><li>ステップ7.8、1.0μLのフォワードプライマーB（10μM）、1.0μLから3.0μLのcDNAテンプレートを次のようにステップ7.8から（ASC1ためのC、D、E、FおよびG標識）各cDNA調製の増幅のためのPCR反応を行いますリバースプライマーCまたはDまたはEまたはFまたはG（10μM）、2.5μLの10×ポリメラーゼPCR緩衝液、0.5μLdNTPミックス（各10mM）、1.0μLのMgCl 2（2.5 mM）の、15.5μLのPCRグレードの水、および0.5 μL熱安定性ポリメラーゼ酵素。 注：5 1：1の範囲のcDNA鋳型の種々の希釈液100は、PCR産物の収率を最適化するためにテストされるべきです。単一のプロモーター近位フォワードプライマーは、cDNAを増幅するために使用されます異なるリバースプライマーを用いて得られました。例えば、プロモーターの近くに位置する単一の順方向プライマーBはASC1ためのCからGへの5逆方向プライマーと組み合わせて使用​​しました。 BC、BD、BE、BFおよびBGは、図3（a）に示すように、それぞれC、D、E、FおよびGとラベル付けチューブに使用しました。 </li><li>以下の熱サイクルパラメータを用いてPCRを実行します。1サイクルで2分間95°C（ポリメラーゼの活性化のためのホットスタート）、30から40サイクル30秒（変性）、15秒間54℃、95℃（アニーリング） 5分（最終伸長）のために、1分（伸長）のために68°C、および1サイクル68℃。 注：アニーリング温度は、使用する特定のプライマーに応じて変化し、伸長時間は、予想される生成物の大きさに依存して変化します。 </li><li>生成物の予想される大きさに応じて0.8％、1.5％、2％アガロースゲル上での電気泳動によりPCR産物を分離します。 </li><li>エルKaderi らに記載のようにPCR産物を定量化します。 、2012 11。 注：別の方法として、新生転写物の量を定量化するために、リアルタイムRT-qPCRの戦略を使用します。 </li></ol>

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The authors have nothing to disclose.

ここで使用される本鎖特異的TROプロトコルは、哺乳動物細胞12にGRO-のSeq（グローバルランオンシークエンシング）分析のために使用されるプロトコルから適応されました。我々は成功し、出芽酵母において新生転写を研究するためのプロトコルを変更しました。具体的には転写終結欠陥を分析するために、我々は、RNAの加水分解工程を取り除くことにより、更なるプロトコ?...

真核生物の転写サイクルは、次の3つの主要手順で構成されています。開始、伸長および終結。 RNAポリメラーゼIIによる転写の終結は、2つの異なる、相互に依存する手順3で構成されています。最初のステップは、テンプレートからの切断、ポリアデニル化およびmRNAの放出を伴う、とすぐにテンプレートからのポリメラーゼの離脱によってマーク第二段階が続きます。適切な終端は、転写の開始/再開時のポリメラーゼのリサイクルのため、下流の遺伝子の転写との干渉を防止するため、および広汎非コードRNA 4、5の転写を制限することによって、チェックで異常な転写を維持するために不可欠です。最近の進歩にもかかわらず、終了ははるかにRNAポリメラーゼII転写サイクルの少なくとも理解のステップです。信頼性の高い終端アッセイは、メカニズムを研究するために不可欠であるunderlyinin vivoでの RNAポリメラーゼIIによる転写のグラム終結。 実験的アプローチの数は、生理的条件下で活発に転写遺伝子に終止欠陥を検出するために使用されます。これらには、ノーザンブロット、終結因子チップ（クロマチン免疫沈降）、伝統的なTROアッセイが含まれます。これらの技術のそれぞれは、いくつかの利点と欠点を有します。伝統的なTROアッセイは、 インビボ 1 における転写終結の欠陥を検出するための最も信頼性の高い敏感なアプローチに使用されます。このアッセイは、細胞内の遺伝子の異なる領域に転写活性のポリメラーゼ分子を局在化します。通常の条件下では、アッセイは、厳密遺伝子（ 図1A）のプロモーター及びターミネーター領域との間で分配転写係合ポリメラーゼ分子を示します。不良終了時に、しかし、ポリメラーゼは、終了信号を読み取ることができません遺伝子（ 図1B）の3 &#39;末端の下流の領域で検出されます。 転写終結欠陥がプロモーター近位領域から開始センス転写ポリメラーゼの存在下で明らかにし、終了信号を読み取ることができないされ、 図に示すように、遺伝子の3 &#39;末端の下流の領域を転写続けます2A。真核生物のゲノムおよび遺伝子6、7、8、9、10の周りに圧倒的な普及意味で転写、ならびにアンチセンス方向を示す実現して、それはすぐに従来のTROアッセイは検出できないことが明らかになったポリメラーゼIF信号遺伝子の下流には、プロモーター、開始転写産物（ 図2A）またはSOMを開始した異常なRNAを表し、遺伝子またはターミネーター要素（ 図2B）、または遺伝子（ 図2C）の3 &#39;末端からアンチセンス方向のポリメラーゼ転写の下流の途中でewhere。観察されたリードスルーポリメラーゼ信号がプロモーターで開始さ拡張センスmRNAまたは検査中の遺伝子の下流を開始したアンチセンス転写物を表す場合は使用してBrUTPは、ここで説明する本鎖特異TROアッセイは、区別することができます。我々は成功した酵母2出芽における転写の終結でKin28キナーゼの役割を実証するために、このアプローチを使用しています。ここでのアプローチを採用我々はRna14は出芽酵母におけるASC1の転写の終結のために必要とされていることを示しています。

<table><tbody><tr><td>Phenol-chloroform-isoamyl alcohol mixture</td><td>Sigma-Aldrich</td><td>77619</td><td>pH 4-5</td></tr><tr><td>TRIzol reagent</td><td>Life technologies</td><td>15596-026</td><td></td></tr><tr><td>RNase Inhibitor, human placenta</td><td>New England Biolabs</td><td>M0307S</td><td></td></tr><tr><td>Anti-BrdU beads</td><td>Santa Cruz Biotechnology</td><td>sc-32323AC</td><td></td></tr><tr><td>Protoscript II</td><td>New England Biolabs</td><td>M03368L</td><td>or an equivalent enzyme</td></tr><tr><td>Advantage 2 polymerase Mix</td><td>Clontech</td><td>639201</td><td>or an equivalent enzyme</td></tr><tr><td>5-Bromouridine 5&#039; triphosphate sodium salt</td><td>Santa Cruz Biotechnology</td><td>sc-214314A</td><td></td></tr><tr><td>RNeasy Mini kit</td><td>Qiagen</td><td>74104</td><td></td></tr><tr><td>ATP</td><td>New England Biolabs</td><td>N0451AA</td><td></td></tr><tr><td>CTP</td><td></td><td>N0454AA</td><td></td></tr><tr><td>GTP</td><td></td><td>N0452AA</td><td></td></tr><tr><td>Ultra-pure bovine serum albumin (BSA)</td><td>MC Labs</td><td>UBSA-100</td><td></td></tr><tr><td>Asc1 B</td><td></td><td></td><td>AGATTCGTCGGTCACAAGTCC</td></tr><tr><td>Asc1 C</td><td></td><td></td><td>GAACTTTATACATATTCTTAGTT&lt;br/&gt; AGCAGTC</td></tr><tr><td>Asc1 D</td><td></td><td></td><td>TGTACATATGTATTTTCGCAGCA</td></tr><tr><td>Asc1 E</td><td></td><td></td><td>GCCAAGGAGACTGAATTTAATG</td></tr><tr><td>Asc1 F</td><td></td><td></td><td>CTATGGAATGGGGGTTTTAAG</td></tr><tr><td>Asc1 G</td><td></td><td></td><td>GGTTATGGCAGACATGCCAC</td></tr><tr><td>5s cDNA</td><td></td><td></td><td>AGATTGCAGCACCTGAGT</td></tr><tr><td>5&amp;rsquo; 5s reverse</td><td></td><td></td><td>GGTTGCGGCCATATCTAC</td></tr><tr><td>3&amp;rsquo; 5s reverse</td><td></td><td></td><td>TGAGTTTCGCGTATGGTC</td></tr></tbody></table>

analysis of termination of transcription using brutp-strand-specific transcription run-on (tro) approach

この原稿は、 インビボで転写終結欠陥を検出するためのプロトコルを記述しています。 BrUTPを使用して本鎖特異的TROプロトコルは、ここで説明する生理学的条件下で転写終結欠陥を分析するための強力な実験的アプローチです。伝統的なTROアッセイと同様に、転写終結欠陥1の指標として遺伝子の3 &#39;末端を越えて転写的に活性なポリメラーゼの存在に依存しています。これは、従来のTROアッセイで遭遇する二つの主要な問題を克服します。終結シグナルを読んポリメラーゼがプロモーター近位領域からの転写を開始したものであれば第一に、それを検出することができ、またはそれは単にどこか身体または3 &#39;内から非特異的に開始した広範囲に転写ポリメラーゼを表している場合遺伝子の末端。第二に、超えた場合に転写活性ポリメラーゼ信号を区別することができますターミネーター領域は、真に読み通しセンスmRNAの転写ポリメラーゼまたはターミネーターが開始非コードアンチセンスRNAの信号です。簡単に言えば、プロトコルは、精製された新生RNAを転写し、使用してcDNAを増幅する逆、指数関数的に増殖する酵母細胞を透過性BrUTPヌクレオチドの存在下で伸長するためにインビボで開始される転写を可能にする、親和性アプローチによってBrUTP標識RNAを精製することを含みますプロモーターと遺伝子2のターミネーター領域に隣接する鎖特異的プライマー。

転写終結欠陥を検出する際にBrUTP鎖特異TRO手続きの適用性を実証するために、我々はrna14-1と呼ばれるRNA14の終端不良、温度感受性変異株を使用していました。 RNAポリメラーゼIIによる転写の終結でRna14の役割が選択された遺伝子1のターミネーター近位領域でRNAを検出した伝統的なTROアッセイを用いて実証しました。非許容温度でrna14-1...

Watch this Scientific Journal Video about Analysis of Termination of Transcription Using BrUTP-strand-specific Transcription Run-on TRO Approach at JoVE.com

Analysis of Termination of Transcription Using BrUTP-strand-specific Transcription Run-on TRO Approach

We describe a basic experimental approach for analysis of termination of transcription by RNA polymerase II in vivo using BrUTP by the strand-specific transcription run-on (TRO) approach in budding yeast. This protocol can be extended to study transcription termination by other RNA polymerases both in yeast and higher eukaryotes.

BrUTP本鎖特異的転写ランオン（TRO）のアプローチを使用した転写の終結の分析

This manuscript describes a protocol for detecting transcription termination defect in vivo. The strand-specific TRO protocol using BrUTP described here ...

analysis-termination-transcription-using-brutp-strand-specific

Universidad San Sebastian

Research

JoVE Journal

Genetics

Analysis of Termination of Transcription Using BrUTP-strand-specific Transcription Run-on (TRO) Approach

9.8K ビュー.  Wayne State University. We describe a basic experimental approach for analysis of termination of transcription by RNA polymerase II in vivo using BrUTP by the strand-specific transcription run-on (TRO) approach in budding yeast. This protocol can be extended to study transcription termination by other RNA polymerases both in yeast and higher eukaryotes. 

ビデオ: Analysis of Termination of Transcription Using BrUTP-strand-specific Transcription Run-on TRO Approach

Real-time Analysis of Transcription Factor Binding, Transcription, Translation, and Turnover to Display Global Events During Cellular Activation

Upon activation, cells rapidly change their functional programs and, thereby, their gene expression profile. Massive changes in gene expression occur, for example, during cellular differentiation, morphogenesis, and functional stimulation (such as activation of immune cells), or after exposure to drugs and other factors from the local environment. Depending on the stimulus and cell type, these changes occur rapidly and at any possible level of gene regulation. Displaying all molecular processes of a responding cell to a certain type of stimulus/drug is one of the hardest tasks in molecular biology. Here, we describe a protocol that enables the simultaneous analysis of multiple layers of gene regulation. We compare, in particular, transcription factor binding (Chromatin-immunoprecipitation-sequencing (ChIP-seq)), de novo transcription (4-thiouridine-sequencing (4sU-seq)), mRNA processing, and turnover as well as translation (ribosome profiling). By combining these methods, it is possible to display a detailed and genome-wide course of action.
Sequencing newly transcribed RNA is especially recommended when analyzing rapidly adapting or changing systems, since this depicts the transcriptional activity of all genes during the time of 4sU exposure (irrespective of whether they are up- or downregulated). The combinatorial use of total RNA-seq and ribosome profiling additionally allows the calculation of RNA turnover and translation rates. Bioinformatic analysis of high-throughput sequencing results allows for many means for analysis and interpretation of the data. The generated data also enables tracking co-transcriptional and alternative splicing, just to mention a few possible outcomes.
The combined approach described here can be applied for different model organisms or cell types, including primary cells. Furthermore, we provide detailed protocols for each method used, including quality controls, and discuss potential problems and pitfalls.

This protocol describes the combinatorial use of ChIP-seq, 4sU-seq, total RNA-seq, and ribosome profiling for cell lines and primary cells. It enables tracking changes in transcription-factor binding, de novo transcription, RNA processing, turnover and translation over time, and displaying the overall course of events in activated and/or rapidly changing cells.

転写因子の結合、細胞活性中に事象を表示する転写、翻訳、回転率のリアルタイム解析

Upon activation, cells rapidly change their functional programs and, thereby, their gene expression profile. Massive changes in gene expression occur, for ...

遺伝学

Toeprinting Analysis of Translation Initiation Complex Formation on Mammalian mRNAs

Translation initiation is the rate-limiting step of protein synthesis and represents a key point at which cells regulate their protein output. Regulation of protein synthesis is the key to cellular stress-response, and dysregulation is central to many disease states, such as cancer. For instance, although cellular stress leads to the inhibition of global translation by attenuating cap-dependent initiation, certain stress-response proteins are selectively translated in a cap-independent manner. Discreet RNA regulatory elements, such as cellular internal ribosome entry sites (IRESes), allow for the translation of these specific mRNAs. Identification of such mRNAs, and the characterization of their regulatory mechanisms, have been a key area in molecular biology. Toeprinting is a method for the study of RNA structure and function as it pertains to translation initiation. The goal of toeprinting is to assess the ability of in vitro transcribed RNA to form stable complexes with ribosomes under a variety of conditions, in order to determine which sequences, structural elements, or accessory factors are involved in ribosome binding&#8212;a pre-cursor for efficient translation initiation. Alongside other techniques, such as western analysis and polysome profiling, toeprinting allows for a robust characterization of mechanisms for the regulation of translation initiation.

Toeprinting aims to measure the ability of in vitro transcribed RNA to form translation initiation complexes with ribosomes under a variety of conditions. This protocol describes a method for toeprinting mammalian RNA and can be used to study both cap-dependent and IRES-driven translation.

哺乳類の Mrna の翻訳開始複合体形成の Toeprinting 分析

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

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 ...

Mapping the Structure-Function Relationships of Disordered Oncogenic Transcription Factors Using Transcriptomic Analysis

Many cancers are characterized by chromosomal translocations which result in the expression of oncogenic fusion transcription factors. Typically, these proteins contain an intrinsically disordered domain (IDD) fused with the DNA-binding domain (DBD) of another protein and orchestrate widespread transcriptional changes to promote malignancy. These fusions are often the sole recurring genomic aberration in the cancers they cause, making them attractive therapeutic targets. However, targeting oncogenic transcription factors requires a better understanding of the mechanistic role that low-complexity, IDDs play in their function. The N-terminal domain of EWSR1 is an IDD involved in a variety of oncogenic fusion transcription factors, including EWS/FLI, EWS/ATF, and EWS/WT1. Here, we use RNA-sequencing to investigate the structural features of the EWS domain important for transcriptional function of EWS/FLI in Ewing sarcoma. First shRNA-mediated depletion of the endogenous fusion from Ewing sarcoma cells paired with ectopic expression of a variety of EWS-mutant constructs is performed. Then RNA-sequencing is used to analyze the transcriptomes of cells expressing these constructs to characterize the functional deficits associated with mutations in the EWS domain. By integrating the transcriptomic analyses with previously published information about EWS/FLI DNA binding motifs, and genomic localization, as well as functional assays for transforming ability, we were able to identify structural features of EWS/FLI important for oncogenesis and define a novel set of EWS/FLI target genes critical for Ewing sarcoma. This paper demonstrates the use of RNA-sequencing as a method to map the structure-function relationship of the intrinsically disordered domain of oncogenic transcription factors.

Intrinsically disordered domains are important for oncogenic fusion transcription factor function. To therapeutically target these proteins, a more detailed understanding of the regulatory mechanisms employed by these domains is required. Here, we use transcriptomics to map important structural features of the intrinsically disordered EWS domain in Ewing sarcoma.

トランスクリプトーム解析を用いた無秩序発がん性転写因子の構造機能関係のマッピング

Many cancers are characterized by chromosomal translocations which result in the expression of oncogenic fusion transcription factors. Typically, these ...

がん研究

A Rapid High-throughput Method for Mapping Ribonucleoproteins (RNPs) on Human pre-mRNA

Sequencing RNAs that co-immunoprecipitate (co-IP) with RNA binding proteins has increased our understanding of splicing by demonstrating that binding location often influences function of a splicing factor.  However, as with any sampling strategy the chance of identifying an RNA bound to a splicing factor is proportional to its cellular abundance.  We have developed a novel in vitro approach for surveying binding specificity on otherwise transient pre-mRNA. This approach utilizes a specifically designed oligonucleotide pool that tiles across introns, exons, splice junctions, or other pre-mRNA.  The pool is subjected to some kind of molecular selection. Here, we demonstrate the method by separating the oligonucleotide into a bound and unbound fraction and utilize a two color array strategy to record the enrichment of each oligonucleotide in the bound fraction. The array data generates high-resolution maps with the ability to identify sequence-specific and structural determinates of ribonucleoprotein (RNP) binding on pre-mRNA.  A unique advantage to this method is its ability to avoid the sampling bias towards mRNA associated with current IP and SELEX techniques, as the pool is specifically designed and synthesized from pre-mRNA sequence. The flexibility of the oligonucleotide pool is another advantage since the experimenter chooses which regions to study and tile across, tailoring the pool to their individual needs.  Using this technique, one can assay the effects of polymorphisms or mutations on binding on a large scale or clone the library into a functional splicing reporter and identify oligonucleotides that are enriched in the included fraction.  This novel in vitro high-resolution mapping scheme provides a unique way to study RNP interactions with transient pre-mRNA species, whose low abundance makes them difficult to study with current in vivo techniques.  



A Rapid High-throughput Method for Mapping Ribonucleoproteins RNPs on Human pre-mRNA

Due to the transient nature of pre-mRNA, it can be difficult to isolate and study in vivo. Here, we present a novel in vitro approach to investigate RNA-protein interactions using a synthetic oligo pool that tiles across selected regions of pre-mRNA.

ヒトmRNA前駆体上にマッピングリボ核の迅速なハイスループット法（RNPS）

Sequencing RNAs that co-immunoprecipitate (co-IP) with RNA binding proteins has increased our understanding of splicing by demonstrating that binding ...

生物学

PAR-CliP - A Method to Identify Transcriptome-wide the Binding Sites of RNA Binding Proteins

RNA transcripts are subjected to post-transcriptional gene regulation by interacting with hundreds of RNA-binding proteins (RBPs) and microRNA-containing ribonucleoprotein complexes (miRNPs) that are often expressed in a cell-type dependently. To understand how the interplay of these RNA-binding factors affects the regulation of individual transcripts, high resolution maps of in vivo protein-RNA interactions are necessary1.
A combination of genetic, biochemical and computational approaches are typically applied to identify RNA-RBP or RNA-RNP interactions. Microarray profiling of RNAs associated with immunopurified RBPs (RIP-Chip)2 defines targets at a transcriptome level, but its application is limited to the characterization of kinetically stable interactions and only in rare cases3,4 allows to identify the RBP recognition element (RRE) within the long target RNA. More direct RBP target site information is obtained by combining in vivo UV crosslinking5,6 with immunoprecipitation7-9 followed by the isolation of crosslinked RNA segments and cDNA sequencing (CLIP)10. CLIP was used to identify targets of a number of RBPs11-17. However, CLIP is limited by the low efficiency of UV 254 nm RNA-protein crosslinking, and the location of the crosslink is not readily identifiable within the sequenced crosslinked fragments, making it difficult to separate UV-crosslinked target RNA segments from background non-crosslinked RNA fragments also present in the sample.
We developed a powerful cell-based crosslinking approach to determine at high resolution and transcriptome-wide the binding sites of cellular RBPs and miRNPs that we term PAR-CliP (Photoactivatable-Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation) (see Fig. 1A for an outline of the method). The method relies on the incorporation of photoreactive ribonucleoside analogs, such as 4-thiouridine (4-SU) and 6-thioguanosine (6-SG) into nascent RNA transcripts by living cells. Irradiation of the cells by UV light of 365 nm induces efficient crosslinking of photoreactive nucleoside-labeled cellular RNAs to interacting RBPs. Immunoprecipitation of the RBP of interest is followed by isolation of the crosslinked and coimmunoprecipitated RNA. The isolated RNA is converted into a cDNA library and deep sequenced using Solexa technology. One characteristic feature of cDNA libraries prepared by PAR-CliP is that the precise position of crosslinking can be identified by mutations residing in the sequenced cDNA. When using 4-SU, crosslinked sequences thymidine to cytidine transition, whereas using 6-SG results in guanosine to adenosine mutations. The presence of the mutations in crosslinked sequences makes it possible to separate them from the background of sequences derived from abundant cellular RNAs.
Application of the method to a number of diverse RNA binding proteins was reported in Hafner et al.18

RNA transcripts are subject to extensive posttranscriptional regulation that is mediated by a multitude of trans-acting RNA-binding proteins (RBPs). Here we present a generalizable method to identify precisely and on a transcriptome-wide scale the RNA binding sites of RBPs.

PAR -クリップ - RNA結合タンパク質のトランスクリプトーム全体の結合部位を同定する方法

RNA transcripts are subjected to post-transcriptional gene regulation by interacting with hundreds of RNA-binding proteins (RBPs) and microRNA-containing ...

Analysis of RNA Processing Reactions Using Cell Free Systems: 3' End Cleavage of Pre-mRNA Substrates in vitro

The 3&#8217; end of mammalian mRNAs is not formed by abrupt termination of transcription by RNA polymerase II (RNPII). Instead, RNPII synthesizes precursor mRNA beyond the end of mature RNAs, and an active process of endonuclease activity is required at a specific site. Cleavage of the precursor RNA normally occurs 10-30 nt downstream from the consensus polyA site (AAUAAA) after the CA dinucleotides. Proteins from the cleavage complex, a multifactorial protein complex of approximately 800 kDa, accomplish this specific nuclease activity. Specific RNA sequences upstream and downstream of the polyA site control the recruitment of the cleavage complex. Immediately after cleavage, pre-mRNAs are polyadenylated by the polyA polymerase (PAP) to produce mature stable RNA messages.
Processing of the 3&#8217; end of an RNA transcript may be studied using cellular nuclear extracts with specific radiolabeled RNA substrates. In sum, a long 32P-labeled uncleaved precursor RNA is incubated with nuclear extracts in vitro, and cleavage is assessed by gel electrophoresis and autoradiography. When proper cleavage occurs, a shorter 5&#8217; cleaved product is detected and quantified. Here, we describe the cleavage assay in detail using, as an example, the 3&#8217; end processing of HIV-1 mRNAs.

Analysis of RNA Processing Reactions Using Cell Free Systems: 3' End Cleavage of Pre-mRNA Substrates in vitro

RNA polymerase II synthesizes a precursor RNA that extends beyond the 3' end of the mature mRNA. The end of the mature RNA is generated cotranscriptionally, at a site dictated by RNA sequences, via the endonuclease activity of the cleavage complex. Here, we detail the method to study cleavage reactions in vitro.

事前のmRNA基質の3 &#39;末端切断：無細胞系を用いたRNAプロセシング反応の解析<em&gt; in vitroで</em

The 3&#8217; end of mammalian mRNAs is not formed by abrupt termination of transcription by RNA polymerase II (RNPII). Instead, RNPII synthesizes ...

Fluorescence Based Primer Extension Technique to Determine Transcriptional Starting Points and Cleavage Sites of RNases In Vivo

Fluorescence based primer extension (FPE) is a molecular method to determine transcriptional starting points or processing sites of RNA molecules. This is achieved by reverse transcription of the RNA of interest using specific fluorescently labeled primers and subsequent analysis of the resulting cDNA fragments by denaturing polyacrylamide gel electrophoresis. Simultaneously, a traditional Sanger sequencing reaction is run on the gel to map the ends of the cDNA fragments to their exact corresponding bases. In contrast to 5&#39;-RACE (Rapid Amplification of cDNA Ends), where the product must be cloned and multiple candidates sequenced, the bulk of cDNA fragments generated by primer extension can be simultaneously detected in one gel run. In addition, the whole procedure (from reverse transcription to final analysis of the results) can be completed in one working day. By using fluorescently labeled primers, the use of hazardous radioactive isotope labeled reagents can be avoided and processing times are reduced as products can be detected during the electrophoresis procedure.
In the following protocol, we describe an in vivo fluorescent primer extension method to reliably and rapidly detect the 5&#39; ends of RNAs to deduce transcriptional starting points and RNA processing sites (e.g., by toxin-antitoxin system components) in S. aureus, E. coli and other bacteria.

Fluorescence Based Primer Extension Technique to Determine Transcriptional Starting Points and Cleavage Sites of RNases In Vivo

We here describe a fluorescence based primer extension method to determine transcriptional starting points from bacterial transcripts and RNA processing in vivo using an automated gel sequencer.

転写開始点を決定するための蛍光に基づくプライマー伸長技術とのRNaseの切断サイト<em&gt;インビボ</em

Fluorescence based primer extension (FPE) is a molecular method to determine transcriptional starting points or processing sites of RNA molecules. This is ...

Strand-Specific Analysis of Proteins at Replicating DNA Strands by Enrichment and Sequencing of Protein-Associated Nascent DNA Method

Genome duplication is orchestrated by replisome proteins, including helicases, which unwind double-stranded DNA into individual antiparallel single strands, each requiring distinct modes of replication: continuous (leading) and discontinuous (lagging). Understanding the interactions of chromatin-associated proteins with replicating DNA strands in vivo is crucial for elucidating the mechanisms of chromatin assembly and DNA repair coupled to DNA replication. This protocol presents the enrichment and sequencing of protein-associated nascent DNA (eSPAN) method, designed to quantify relative protein levels on nascent leading and lagging DNA strands at replication forks. The eSPAN procedure starts with chromatin immunoprecipitation (ChIP, in yeast) or cleavage under targets and tagmentation (CUT&amp;Tag; in mammalian cells) of a protein of interest, followed by enrichment of the protein-associated nascent DNA by bromodeoxyuridine (BrdU) immunoprecipitation. Strand-specific next-generation sequencing is applied to isolated ssDNA. This technique can be used to determine whether a protein is enriched at leading or lagging replication forks. The eSPAN provides genome-wide strand-preference of chromatin-associated proteins, including histones at replication forks.

Enrichment and sequencing of protein-associated nascent DNA (eSPAN) was developed to detect the relative abundance of a chromatin-associated protein on two replicating DNA strands, thereby revealing molecular insight into chromatin replication and its coupled processes. This protocol describes eSPAN procedures in yeast and mouse embryonic stem (ES) cells.

Genome duplication is orchestrated by replisome proteins, including helicases, which unwind double-stranded DNA into individual antiparallel single ...

Transcription Attenuation in Prokaryotes

Transcriptional attenuation occurs when&#160;RNA transcription is prematurely terminated due to the formation of a terminator mRNA hairpin structure.&#160; Bacteria use these hairpins to regulate the transcription process and control the synthesis of several amino acids including histidine, lysine, threonine, and phenylalanine. Transcription attenuation takes place in the non-coding regions of mRNA.
There are several different mechanisms used to attenuate transcription. In ribosome mediated transcriptional attenuation, the movement of a ribosome on the transcript is stalled or proceeds forward depending upon the availability of the tRNAs charged with a specific amino acid. High amino acid concentrations allow the ribosome to move forward leading to the formation of the terminator structure; deficiency of the amino acid stalls the ribosome and causes formation of the anti-terminator structure. The trp operon in E. coli, discussed below, is a good example of this type of mechanism. tRNA mediated transcriptional attenuation, as observed in trp operon of Lactococcus lactis, depends on an RNA-RNA interaction. When uncharged tRNAs are present in sufficient numbers, they directly bind to the mRNA and stabilize the anti-terminator structure. Transcriptional attenuation is also known to be mediated by proteins as found in bgl (beta-glucoside) operon in E. coli.&#160; This involves an RNA&#8212;protein interaction where a protein binds to the transcript and regulates the formation of an anti-terminator structure. More recently, another transcriptional attenuation mechanism was discovered where small metabolites like thiamine were observed to regulate transcription by directly binding to the non-coding mRNA segments, also known as riboswitches. Riboswitches can form a terminator or an anti-terminator structure depending upon the concentration and nature of a metabolite.&#160;
Trp Operon
The trp operon in E. coli contains a 140 nucleotide leader sequence before its first structural gene. This leader sequence has four distinct segments &#8211; 1 through 4&#8211; and regulates the transcription of the downstream structural genes. Segment&#160; 1 can form a hairpin structure with segment&#160; 2. This 1-2 hairpin structure is known as a pause structure, as during transcription, it stalls RNA polymerase until the ribosome binds the newly transcribed RNA. This synchronizes transcription and translation in bacteria.&#160; When tryptophan concentrations are low, a hairpin structure forms between segments 2 and 3, known as the anti-terminator structure. This anti-terminator structure allows the continuous transcription of the downstream genes which produce enzymes for tryptophan synthesis. In contrast, when tryptophan concentrations are sufficient, a hairpin structure forms between segments 3 and 4, called the terminator structure. Along with a&#160; series of uracil bases that follow, the terminator structure causes RNA polymerase to dissociate from the RNA and template DNA strands, resulting in the termination of the transcription.

BrUTP本鎖特異的転写ランオン（TRO）のアプローチを使用した転写の終結の分析

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