Najmeh Nikpour and Fiona Alum provided suggestions and edited this manuscript. Department of Biochemistry at McGill University supported HM and VM with the CIHR Training Initiative in Chemical Biology. This work was supported by Canadian Institute of Health Research (CIHR) grant 119464 (to R. S.).

以下のプロトコルは、蛍光ベースのRNA編集アッセイを実施するための手順について説明します。アッセイは、96ウェルまたは384ウェルプレートを実験の範囲に応じて、単一PCRチューブで行うことができる。続いて、蛍光シグナルは、適切なリアルタイムPCR検出システムで読み取ることができる。ここでアッセイは、384ウェルプレートの文脈で説明されている。 1。培養T.トリパノソーマ細胞<ol><li> Tの増殖培地を準備しますトリパノソーマプロサイクリック型細胞。培地1Lの場合： <ol><li> 800ミリリットルミリQ水に25.4グラムSDM-79粉末を溶解。 </li><li> 10 M NaOHで7.3にNaHCO 3およびpHを2グラムを加える。 </li><li> 900ミリリットル、濾過滅菌の最終容量にナノ純水を追加します。 </li><li> 10％の最終濃度までウシ胎児血清（FBS）、ペニシリン - ストレプトマイシン溶液及びヘミン（2.5 mg / mlで）を加える（v / v）で、100 U / mlおよび7.5 mg / Lであった。 </li></ol></li><li> &lt;Tの300ミリリットルを育てる1.5×10 7細胞/ mlの密度に70rpmで振盪em&gt;のトリパノソーマ1.7A野生型（プロサイクリック型）28℃での細胞。注：これは600の編集反応に十分の全タンパク質の約0.5 mgのアクティブeditosomeの3ミリリットルを生成する必要があります。 </li><li> 4℃で10分間、6,000×gの遠心分離により細胞を収穫</li><li>冷却したPBSG緩衝液（10mMのNa 2 HPO 4、10mMのNaH 2 PO 4、145mMのNaCl、および6 mMグルコース）50mlでペレットを洗浄し、10分間10,000 xgでの遠心分離によって細胞を再度スピンダウン4℃ </li></ol>原油ミトコンドリアの2。分離注：すべてのステップがeditosome活性を保つために氷の上で、または4℃で行われるべきである。 <ol><li> DTE緩衝液（1mMのトリス-HCl、8.0および1mM EDTA）30ml中に回収した細胞を再懸濁する。アップなでる上下氷上に少なくとも10倍の細胞膜を破壊する40ミリリットルの無菌ダウンスホモジナイザー（予備冷却）を使用します。 </李&gt; <li>優先的にミトコンドリアをダウンさせるために、4℃で10分間15800×gで0.25メートル遠心機の最終濃度にホモジネートに、すぐに4.3ミリリットル60％ショ糖（ すなわち 1.75 M W / V）を追加します。 </li><li> STM緩衝液（20mMトリス-HCl、pH8.0、250mMのスクロースおよび2mMのMgCl 2）4.6mlのミトコンドリアペレットを再懸濁。私はそれぞれ0.3mmであり、9 U / mlの、最終濃度を0.1MのCaCl 2の13.8μlのRNaseフリーのDNaseの4を添加する。氷上で1時間混合物をインキュベートする。 </li><li> 4℃で10分間15,800 xgで遠心I. DNアーゼを不活性化するSTE緩衝液（20mMのTris-HCl pH8.0、250 mMスクロースおよび2mM EDTA）4.6 mlを加え</li><li> 400溶解緩衝液（10mMのトリス-HCl、pH7.2、10mMのMgCl 2、100mMのKCl、1μg/ mlのペプスタチン、1mMのDTT、および1×完全EDTAフリープロテアーゼ阻害剤）とへの転送中にペレットを再懸濁マイクロチューブ。 </li><li> 10％トリトンX-100、1％aの最終濃度に追加ndはチューブローテーター上、4℃で15分間、溶解物をインキュベートする。 </li><li> 4℃で15分間17000×gで2回遠心分離することにより、ミトコンドリアの溶解液をクリア。クリア上澄みを毎回保持。 </li></ol> 3。Editosome精製<ol><li>ミリモルのMg（OAcを含む）20、2×HHE勾配緩衝液（40mMのHEPES pHが7.9を使用して、超遠心分離管中の10 mlの10％〜30％（v / v）の線形グリセロール勾配（ 表1）を注ぐ2、100mMのKCl、および2 mMのEDTA）および取扱説明書に従うことによって、勾配メーカー。 </li><li>慎重にグリセロール勾配の上部から溶液500μlを除去し、静かにクリアされ、ミトコンドリア溶解物500μLをロードします。超遠心機を用いて、4℃で6時間178000×gでスピン。 </li><li> 4℃で、勾配の上から下へ順に500μlの画分を収集その後の使用まで-80℃で液体窒素とストアを使用して分画を凍結スナップ。 </li></ol><ol><li> 3分間90℃で加熱することにより、1:1のモル比でT7プロモーターオリゴヌクレオチド（5&#39;-TAATACGACTCACTATAGGG-3 &#39;）を用いてT7プロモーター配列（ 表2）に相補的な配列を含有し、RTに冷却し、それぞれのDNA鋳型をアニール少なくとも10分間。 </li><li>取扱説明書に従って、 インビトロ転写キットを用いてRNAを転写する。 </li><li> 7 M尿素染料（7 M尿素、0.05％キシレンCynol、および0.05％ブロモフェノールブルー）を等量加えることで転写反応を停止します。 9％のポリアクリルアミドゲル（10％アクリルアミド、7 M尿素、1X TBE）を変性滅菌フィルター上で実行されます。 </li><li>それぞれのRNAを見つけて切除する短波UVランプでシャドーイング紫外線（UV）を使用します。微量遠心チューブに切り出したゲル片を置き、ゲル溶出緩衝液（20mMトリス-HCl、pH7.5、250mMのNaOAcを、1mMのEDTAおよび0.25％SDS）400μlを添加する。チューブローテーター上で室温で一晩溶出する。 </li><li> PRECI冷100％エタノール1mlを添加し、30分間または-20°Cで一晩-80℃のいずれかでインキュベートすることによって溶出したRNAをpitate。 </li><li> RNAをダウンペレットを4℃で30分間16,000×gで遠心分離、。 </li><li> 75％エタノール1mlでペレットを洗浄。 4℃で20分間16,000 xgで遠心分離</li><li> 表2に示すように、所望の濃度を達成するのRNaseフリー水で適宜RNAペレットを再懸濁する。 </li></ol> 5。蛍光ベースのRNA編集アッセイ<ol><li>単一反応のため、preA6Rbzやマイクロチューブ内gA6Rbz（1:2.5モル比）の2.5ピコモル（1μL）の1ピコモル（1μl）を組み合わせて、3分間70ºCでインキュベートし、時のために、それが室温で放置少なくとも10分。 </li><li>一方、1×HHE緩衝液（25mM HEPES pH7.9の、10mMののMg（OAc）として保護2、50mMのKCl及び10mM EDTA）、1mMの含有校正反応のために、preA6RbzとgA6Rbzことなく、 表3を用いて、マスターミックスを調製ATP、5mMのCaCl 2、トルラ酵母RNA、0.1％トリトンX-100、および精製editosomeの16 ngの/μlの。 </li><li>マスターミックスを完成するためにアニールpreA6RbzとgA6Rbzを追加します。 </li><li> RNaseフリー水（無化合物を有するウェル）または200μMの化学化合物の2液の2液のいずれかを含むウェルにマスターミックス（ 表3）の18μLを分注すると、図5によると、プレート内のコントロールサンプルが含まれています。 </li><li>プレートシーラーでプレートをシールし、気泡を除去するために、下にプレートを回転。 4時間28℃でインキュベートする。 </li><li>各ウェルにgA6Rbzのライバルの25ピコモル（2μL）を追加します。新鮮なシーラーを置き、下にプレートを回転し、リアルタイムPCR装置の上に置きます。以下の実験をプログラムする： ステップ1：5分間85℃;ステップ2：10分間24℃;ステップ3：停止します。 </li><li> 23μLの最終容量に各ウェルに、FRET基質の15ピコモル（1μL）を追加します。新鮮なシーラーでプレートをシール。すぐにプレートを回転し、リアルタイムPCR装置の上に戻って配置します。 </li><li>プログラムは、以下の手順を使用して新しい実験： ステップ1：1分間37°C;ステップ2：読み出し;ステップ3：1、40回に進み;ステップ4：停止します。 </li><li>読書を必要とし、FAMフィルターを選択し、全てのウェルを選択することで設定プレート。入力23μL、ボリュームとファイル名を指定して実行を開始します。 </li><li>分析のためのバーグラフに傾きをプロットすることによって動力学的測定値を得るために、各ウェル/試料から得られた値の傾きを算出する。 NOTE：運動読取サンプルと背景との間の信号対雑音比を改善する;バックグラウンドサンプルはゼロに近い傾斜を有する場合と同じです。終点の読みはより高いバックグラウンドノイズを有する。 </li></ol>

<ol>
	<li>Benne, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=transcript+of+the+frameshifted+coxII+gene+from+trypanosome+mitochondria+contains+four+nucleotides+that+are+not+encoded+in+the+DNA.">transcript of the frameshifted coxII gene from trypanosome mitochondria contains four nucleotides that are not encoded in the DNA.</a> Cell. 46, 819-826 (1986).</li><li>Hajduk, S., Ochsenreiter, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+editing+in+kinetoplastids.">RNA editing in kinetoplastids.</a> RNA biology. 7, 229-236 (2010).</li><li>Aphasizhev, R., Aphasizheva, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Uridine+insertion/deletion+editing+in+trypanosomes:+a+playground+for+RNA-guided+information+transfer.">Uridine insertion/deletion editing in trypanosomes: a playground for RNA-guided information transfer.</a> Wiley interdisciplinary reviews RNA. 2, 669-685 (2011).</li><li>Seiwert, S. D., Stuart, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+editing:+transfer+of+genetic+information+from+gRNA+to+precursor+mRNA+in+vitro.">RNA editing: transfer of genetic information from gRNA to precursor mRNA in vitro.</a> Science. 266, 114-117 (1994).</li><li>Weng, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Guide+RNA-binding+complex+from+mitochondria+of+trypanosomatids.">Guide RNA-binding complex from mitochondria of trypanosomatids.</a> Molecular. 32, 198-209 (2008).</li><li>Hashimi, H., Zikova, A., Panigrahi, A. K., Stuart, K. D., Lukes, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TbRGG1,+an+essential+protein+involved+in+kinetoplastid+RNA+metabolism+that+is+associated+with+a+novel+multiprotein+complex.">TbRGG1, an essential protein involved in kinetoplastid RNA metabolism that is associated with a novel multiprotein complex.</a> RNA. 14, 970-980 (2008).</li><li>Ammerman, M. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Architecture+of+the+trypanosome+RNA+editing+accessory+complex,+MRB1.">Architecture of the trypanosome RNA editing accessory complex, MRB1.</a> Nucleic Acids Res. 40, 5637-5650 (2012).</li><li>Huang, C. E., O'Hearn, S. F., Sollner-Webb, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assembly+and+function+of+the+RNA+editing+complex+in+Trypanosoma+brucei+requires+band+III+protein.">Assembly and function of the RNA editing complex in Trypanosoma brucei requires band III protein.</a> Molecular and cellular biology. 22, 3194-3203 (2002).</li><li>Carnes, J., Trotter, J. R., Peltan, A., Fleck, M., Stuart, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+editing+in+Trypanosoma+brucei+requires+three+different+editosomes.">RNA editing in Trypanosoma brucei requires three different editosomes.</a> Molecular and cellular biology. 28, 122-130 (2008).</li><li>Hearn, S. F., Huang, C. E., Hemann, M., Zhelonkina, A., Sollner-Webb, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trypanosoma+brucei+RNA+editing+complex:+band+II+is+structurally+critical+and+maintains+band+V+ligase,+which+is+nonessential.">Trypanosoma brucei RNA editing complex: band II is structurally critical and maintains band V ligase, which is nonessential.</a> Molecular and cellular biology. 23, 7909-7919 (2003).</li><li>Schnaufer, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+RNA+ligase+essential+for+RNA+editing+and+survival+of+the+bloodstream+form+of+Trypanosoma+brucei.">An RNA ligase essential for RNA editing and survival of the bloodstream form of Trypanosoma brucei.</a> Science. 291, 2159-2162 (2001).</li><li>Tarun, S. Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=KREPA6+is+an+RNA-binding+protein+essential+for+editosome+integrity+and+survival+of+Trypanosoma+brucei.">KREPA6 is an RNA-binding protein essential for editosome integrity and survival of Trypanosoma brucei.</a> RNA. 14, 347-358 (2008).</li><li>Croft, S. L., Barrett, M. P., Urbina, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemotherapy+of+trypanosomiases+and+leishmaniasis.">Chemotherapy of trypanosomiases and leishmaniasis.</a> Trends in parasitology. 21, 508-512 (2005).</li><li>Denise, H., Barrett, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Uptake+and+mode+of+action+of+drugs+used+against+sleeping+sickness.">Uptake and mode of action of drugs used against sleeping sickness.</a> Biochemical pharmacology. 61, 1-5 (2001).</li><li>Seiwert, S. D., Heidmann, S., Stuart, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+visualization+of+uridylate+deletion+in+vitro+suggests+a+mechanism+for+kinetoplastid+RNA+editing.">Direct visualization of uridylate deletion in vitro suggests a mechanism for kinetoplastid RNA editing.</a> Cell. 84, 831-841 (1996).</li><li>Igo, R. P., Palazzo, S. S., Burgess, M. L., Panigrahi, A. K., Stuart, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Uridylate+addition+and+RNA+ligation+contribute+to+the+specificity+of+kinetoplastid+insertion+RNA+editing.">Uridylate addition and RNA ligation contribute to the specificity of kinetoplastid insertion RNA editing.</a> Molecular and cellular biology. 20, 8447-8457 (2000).</li><li>Igo, R. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+uridylate-specific+exoribonuclease+activity+in+Trypanosoma+brucei+RNA+editing.">Role of uridylate-specific exoribonuclease activity in Trypanosoma brucei RNA editing.</a> Eukaryotic cell. 1, 112-118 (2002).</li><li>Wang, B., Salavati, R., Heidmann, S., Stuart, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+hammerhead+ribozyme+substrate+and+reporter+for+in+vitro+kinetoplastid+RNA+editing.">A hammerhead ribozyme substrate and reporter for in vitro kinetoplastid RNA editing.</a> RNA. 8, 548-554 (2002).</li><li>Moshiri, H., Salavati, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+fluorescence-based+reporter+substrate+for+monitoring+RNA+editing+in+trypanosomatid+pathogens.">A fluorescence-based reporter substrate for monitoring RNA editing in trypanosomatid pathogens.</a> Nucleic Acids Res. 38, e138 (2010).</li><li>Jenne, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+identification+and+characterization+of+hammerhead-ribozyme+inhibitors+using+fluorescence-based+technology.">Rapid identification and characterization of hammerhead-ribozyme inhibitors using fluorescence-based technology.</a> Nature. 19, 56-61 (2001).</li><li>Hartig, J. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein-dependent+ribozymes+report+molecular+interactions+in+real+time.">Protein-dependent ribozymes report molecular interactions in real time.</a> Nature. 20, 717-722 (2002).</li><li>Famulok, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Allosteric+aptamers+and+aptazymes+as+probes+for+screening+approaches.">Allosteric aptamers and aptazymes as probes for screening approaches.</a> Current opinion in molecular therapeutics. 7, 137-143 (2005).</li><li>Teng, B., Burant, C. F., Davidson, N. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+cloning+of+an+apolipoprotein+B+messenger+RNA+editing+protein.">Molecular cloning of an apolipoprotein B messenger RNA editing protein.</a> Science. 260, 1816-1819 (1993).</li><li>Jurica, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Searching+for+a+wrench+to+throw+into+the+splicing+machine.">Searching for a wrench to throw into the splicing machine.</a> Nature chemical biology. 4, 3-6 (2008).</li><li>Spahn, C., Prescott, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Throwing+a+spanner+in+the+works:+antibiotics+and+the+translation+apparatus.">Throwing a spanner in the works: antibiotics and the translation apparatus.</a> Journal of molecular medicine. 74, 423-439 (1996).</li></ol>

The authors have nothing to disclose.

トリパノソーマのRNA編集複合体に対する阻害剤を同定するための新規のハイスループットスクリーニング方法はtrypanosomatidsによって引き起こされる疾患に対抗する創薬のための新しいツールを提供し、提示された。 FRETベースのリボザイムアッセイは広範囲に異なる目的のために20〜22に使用されている;しかしながら、我々は、RNA編集アクティビティ19 のインビトロ監?...

RNA編集、mRNAの転写後修飾のプロセスは、第1 trypanosomatidsに発見された。それ以来、かなりの作業がトリパノソーマ 2,3におけるRNA編集のメカニズムを研究してなされた。一連の酵素反応において、editosome、約20のタンパク質のコア複合体は、エネルギー発生の酸化的リン酸化系の複数のコンポーネントのための成熟したミトコンドリアmRNAを作成する。触媒のイベントの順序は、ガイドRNA（gRNAs）によって指示されるように、ヌクレオチド鎖切断、ウリジレート（U）追加や削除、および連結される4。 コアeditosome複合タンパク質に加えて、補因子の数も5-7同定されている。これらのタンパク質は、主に独立した複合体にグループ化が見られる。しかし、コアeditosome複合体におけるタンパク質集合の順序や小物とのコア複合体の相互作用パターン複合体は、まだ決定されている。 trypanosomatidsにおけるRNA編集処理をターゲットとすると、そのeditosome複合体の構築と機能を研究する際の補助化学解剖器具を提供することがあります。さらに、いくつかのeditosomeタンパク質に関する機能的研究は、薬剤が8月12日を対象として、その可能性を示す、様々なライフステージ全体の本質を示している。したがって、editosome見出さの阻害剤はまた、trypanosomatidsに対するリード化合物として作用することができる。トリパノソーマによって引き起こされる疾患に対して、現在利用可能な薬剤は、毒性が非効率的で13,14高価ですので、これは、タイムリーである。 効率的で便利なin vitroアッセイは、RNA編集をブロックする特異的阻害剤の化学の世界を探求することが必要である。 3つのアッセイが開発されeditosome活動を監視するために使用されている： インビトロ RNA編集アッセイ15（a） において 、フルラウンド、（b）は、 インビトロ RNA編集アッセイ16,17 に予め切断されND（C）ハンマーヘッドリボザイム（HHR）ベースのアッセイ18。最初の2つのアッセイは、放射能の助けを借りて編集された製品（ATPアーゼ6 mRNA）の直接可視化に依存しています。 HHRベースのアッセイは、編集時リボザイムとして動作するようにモデル化されているATPアーゼ6 mRNAの修正版を使用しています。機能的なリボザイムはその後、具体的レポーターとして、放射性標識されたRNA基質を切断する。最近、母子里らは、「ミックスおよび測定」を開発放射性標識RNA基質は、蛍光共鳴エネルギー移動（FRET）基板19で置換されているRNA編集を監視するために、インビトロレポーターアッセイで HHR系。このアッセイの原理の利点は、（a）は、アクティブリボザイムと基質切断の生産が低量（ すなわち 20μL）で同一チューブ内で同時に発生すると、迅速かつ便利なミックスと分析の指標の一種で、（B ）は、放射性標識物質の使用を回避し、（c）の感度すなわち蛍光マイクロタイタープレート形式で計測、および（d）は高い信号対雑音比によってfforded。このアッセイを用いて、精製されたeditosomeに対する既知のRNA編集リガーゼ阻害剤の効果は、19を確認した。この実験は、主にTから全体editosomesに対して、RNA編集酵素阻害剤の迅速な同定のためのアッセイを検証ブルーセイ 。 図1は、蛍光ベースのインビトロ RNA編集アッセイでの詳細なステップバイステップの概略図である。このプロトコルは、 インビトロで監視RNA編集のために使用することができるいずれかまたは容易に様々なスケールの化合物ライブラリをスクリーニングするために適合され得る。

<table border="0" cellpadding="0" cellspacing="0" width="979">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:391px;">Name of Material/ Equipment</td>
			<td style="width:203px;">Company</td>
			<td style="width:139px;">Catalog Number</td>
			<td style="width:248px;">Comments/Description</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">SDM-79 Medium</td>
			<td>Gibco by life technologies</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">Fetal Bovine Serum</td>
			<td>life technologies</td>
			<td style="width:139px;">12483-020</td>
			<td>heat inactivation at 550C for 1 h</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:391px;">Hemin, minimum 80%</td>
			<td style="width:203px;">Sigma</td>
			<td style="width:139px;">H5533-10G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:391px;">Penicillin-Streptomycin Sollution</td>
			<td style="width:203px;">Fisher Scientific</td>
			<td style="width:139px;">MT-30-002-CI</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:391px;">Dnase 1 recombinant, Rnase Free&#160;</td>
			<td style="width:203px;">Roche</td>
			<td style="width:139px;">4716728001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:391px;">T7 RiboMax Express Large&#160; scale RNA&#160; production system</td>
			<td style="width:203px;">Promega</td>
			<td style="width:139px;">P1320</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:391px;">Kimble Kontes Dounce Tissue Grinders&#160;&#160;</td>
			<td style="width:203px;">Fisher Scientific</td>
			<td style="width:139px;">K885300-0040&#160;&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:391px;">Gradient Master, ver 5.25&#160;</td>
			<td style="width:203px;">Biocomp</td>
			<td style="width:139px;">107-201M</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:391px;">Ultra Clear Tube, 13.2ML</td>
			<td style="width:203px;">Beckman Coulter</td>
			<td style="width:139px;">344059</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:391px;">Optima L-100XP&#160; Ultracentrifuge&#160;</td>
			<td style="width:203px;">Beckman Coulter</td>
			<td style="width:139px;">392052</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:391px;">SW 41 Ti ROTOR</td>
			<td style="width:203px;">Beckman Coulter</td>
			<td style="width:139px;">331336</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:391px;">MicroSeal &#39;B&#39; Seal, Seals</td>
			<td style="width:203px;">Biorad</td>
			<td style="width:139px;">MSB1001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:391px;">CFX 384 Touch Real-Time PCR Detection System</td>
			<td style="width:203px;">Biorad</td>
			<td style="width:139px;">185-5484</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:391px;">Acryl/Bis solution (19: 1), 40% (w/v)</td>
			<td style="width:203px;">Bio Basic</td>
			<td style="width:139px;">A0006-500ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:391px;">Urea, Molecular biology grade, 1Kg</td>
			<td style="width:203px;">life technologies</td>
			<td style="width:139px;">AM9902</td>
			<td style="width:248px;"></td>
		</tr>
	</tbody>
</table>

rna catalyst as a reporter for screening drugs against rna editing in trypanosomes

実質的な進歩は、トリパノソーマにおけるミトコンドリアRNA編集のメカニズムを決定する際になされたものである。同様に、かなりの進歩は、RNA編集を触媒editosome複合体の成分を同定してなされたもの。しかし、これらのタンパク質がどのように連携するか、まだ明らかではない。 editosomeに対するハイスループットスクリーニングから得られた化学化合物は、編集サイクルにおける1つ以上のステップをブロックするか、または影響を及ぼし得る。したがって、新規な化学化合物の同定はeditosome機能およびアセンブリを切開するための貴重な分子プローブを生成する。以前の研究において、 インビトロ編集アッセイは、放射標識RNAを用いて行った。これらのアッセイは、ハイスループット目的のために非効率的で時間のかかる不適当である。ここでは、均質な蛍光ベース「ミックスと測定」RNA編集を監視するためにハンマーヘッドリボザイムのin vitroレポーターアッセイを、提示されている。唯一のRNA編集の結果としてハンマーヘッド型リボザイム蛍光共鳴エネルギー移動（FRET）オリゴリボヌクレオチド基質は、切断を受ける。これは、順番に、それによって信号を生成するクエンチャーからのフルオロフォアの分離をもたらす。 editosome機能が阻害されると対照的に、蛍光シグナルをクエンチする。これは、 インビトロ RNA編集またはeditosomeの機能を阻害することができる化学物質のハイスループットスクリーニングで監視することが一般的に適用可能であるべきで高感度かつ簡単なアッセイである。

大型画面を設定するために必要な必要な手順を示すために、2〜5がアッセイの品質に関する代表対照実験である図 。これらは、スクリーニングの数日にわたって一貫性のあるアッセイのための、または異なる画面を比較するための不可欠な対照実験である。 蛍光信号対雑音比を評価すること 大規模なセットア?...

Watch this Scientific Journal Video about RNA Catalyst as a Reporter for Screening Drugs against RNA Editing in Trypanosomes at JoVE.com

RNA Catalyst as a Reporter for Screening Drugs against RNA Editing in Trypanosomes

A highly sensitive ribozyme-based assay, applicable to high-throughput screening of chemicals targeting the unique process of RNA editing in trypanosomatid pathogens, is described in this paper. Inhibitors can be used as tools for hypothesis-driven analysis of the RNA editing process and ultimately as therapeutics.

トリパノソーマにおけるRNA編集に対する薬剤をスクリーニングするためのレポーターとして、RNA触媒

Substantial progress has been made in determining the mechanism of mitochondrial RNA editing in trypanosomes. Similarly, considerable progress has been ...

rna-catalyst-as-reporter-for-screening-drugs-against-rna-editing

Research

JoVE Journal

Biology

8.4K ビュー.  McGill University. A highly sensitive ribozyme-based assay, applicable to high-throughput screening of chemicals targeting the unique process of RNA editing in trypanosomatid pathogens, is described in this paper. Inhibitors can be used as tools for hypothesis-driven analysis of the RNA editing process and ultimately as therapeutics.

ビデオ: RNA Catalyst as a Reporter for Screening Drugs against RNA Editing in Trypanosomes

Visualizing RNA Localization in Xenopus Oocytes

RNA localization is a conserved mechanism of establishing cell polarity. Vg1 mRNA localizes to the vegetal pole of Xenopus laevis oocytes and acts to spatially restrict gene expression of Vg1 protein. Tight control of Vg1 distribution in this manner is required for proper germ layer specification in the developing embryo. RNA sequence elements in the 3' UTR of the mRNA, the Vg1 localization element (VLE) are required and sufficient to direct transport. To study the recognition and transport of Vg1 mRNA in vivo, we have developed an imaging technique that allows extensive analysis of trans-factor directed transport mechanisms via a simple visual readout. 
To visualize RNA localization, we synthesize fluorescently labeled VLE RNA and microinject this transcript into individual oocytes. After oocyte culture to allow transport of the injected RNA, oocytes are fixed and dehydrated prior to imaging by confocal microscopy. Visualization of mRNA localization patterns provides a readout for monitoring the complete pathway of RNA transport and for identifying roles in directing RNA transport for cis-acting elements within the transcript and trans-acting factors that bind to the VLE (Lewis et al., 2008, Messitt et al., 2008). We have extended this technique through co-localization with additional RNAs and proteins (Gagnon and Mowry, 2009, Messitt et al., 2008), and in combination with disruption of motor proteins and the cytoskeleton (Messitt et al., 2008) to probe mechanisms underlying mRNA localization.

Visualizing RNA Localization in Xenopus Oocytes

Visualization of in vivo RNA transport is accomplished by microinjection of fluorescently labeled RNA transcripts into Xenopus oocytes, followed by confocal microscopy.

可視化RNAの局在にアフリカツメガエル卵母細胞

RNA localization is a conserved mechanism of establishing cell polarity.  Vg1 mRNA localizes to the vegetal pole of Xenopus laevis oocytes and acts to ...

生物学

In vitro Transcription and Capping of Gaussia Luciferase mRNA Followed by HeLa Cell Transfection

In vitro transcription is the synthesis of RNA transcripts by RNA polymerase from a linear DNA template containing the corresponding promoter sequence (T7, T3, SP6) and the gene to be transcribed (Figure 1A). A typical transcription reaction consists of the template DNA, RNA polymerase, ribonucleotide triphosphates, RNase inhibitor and buffer containing Mg2+ ions.
Large amounts of high quality RNA are often required for a variety of applications. Use of in vitro transcription has been reported for RNA structure and function studies such as splicing1, RNAi experiments in mammalian cells2, antisense RNA amplification by the "Eberwine method"3, microarray analysis4 and for RNA vaccine studies5. The technique can also be used for producing radiolabeled and dye labeled probes6. Warren, et al. recently reported reprogramming of human cells by transfection with in vitro transcribed capped RNA7. The T7 High Yield RNA Synthesis Kit from New England Biolabs has been designed to synthesize up to 180 &mu;g RNA per 20 &mu;l reaction. RNA of length up to 10kb has been successfully transcribed using this kit. Linearized plasmid DNA, PCR products and synthetic DNA oligonucleotides can be used as templates for transcription as long as they have the T7 promoter sequence upstream of the gene to be transcribed.
Addition of a 5' end cap structure to the RNA is an important process in eukaryotes. It is essential for RNA stability8, efficient translation9, nuclear transport10 and splicing11. The process involves addition of a 7-methylguanosine cap at the 5' triphosphate end of the RNA. RNA capping can be carried out post-transcriptionally using capping enzymes or co-transcriptionally using cap analogs. In the enzymatic method, the mRNA is capped using the Vaccinia virus capping enzyme12,13. The enzyme adds on a 7-methylguanosine cap at the 5' end of the RNA using GTP and S-adenosyl methionine as donors (cap 0 structure). Both methods yield functionally active capped RNA suitable for transfection or other applications14 such as generating viral genomic RNA for reverse-genetic systems15 and crystallographic studies of cap binding proteins such as eIF4E16.
In the method described below, the T7 High Yield RNA Synthesis Kit from NEB is used to synthesize capped and uncapped RNA transcripts of Gaussia luciferase (GLuc) and Cypridina luciferase (CLuc). A portion of the uncapped GLuc RNA is capped using the Vaccinia Capping System (NEB). A linearized plasmid containing the GLuc or CLuc gene and T7 promoter is used as the template DNA. The transcribed RNA is transfected into HeLa cells and cell culture supernatants are assayed for luciferase activity. Capped CLuc RNA is used as the internal control to normalize GLuc expression.

In vitro Transcription and Capping of Gaussia Luciferase mRNA Followed by HeLa Cell Transfection

This method describes high yield in vitro synthesis of both capped and uncapped mRNA from a linearized plasmid containing the Gaussia luciferase (GLuc) gene. The RNA is purified and a fraction of the uncapped RNA is enzymatically capped using the Vaccinia virus capping enzyme. In the final step, the mRNA is transfected into HeLa cells and cell culture supernatants are assayed for luciferase activity.

HeLa細胞のトランスフェクションに続いてGaussiaルシフェラーゼmRNA のインビトロ転写とキャッピングの

In vitro transcription is the synthesis of RNA transcripts by RNA polymerase from a linear DNA template containing the corresponding promoter sequence ...

Chromatin Isolation by RNA Purification (ChIRP)

Long noncoding RNAs are key regulators of chromatin states for important biological processes such as dosage compensation, imprinting, and developmental gene expression 1,2,3,4,5,6,7. The recent discovery of thousands of lncRNAs in association with specific chromatin modification complexes, such as Polycomb Repressive Complex 2 (PRC2) that mediates histone H3 lysine 27 trimethylation (H3K27me3), suggests broad roles for numerous lncRNAs in managing chromatin states in a gene-specific fashion 8,9. While some lncRNAs are thought to work in cis on neighboring genes, other lncRNAs work in trans to regulate distantly located genes. For instance, Drosophila lncRNAs roX1 and roX2 bind numerous regions on the X chromosome of male cells, and are critical for dosage compensation 10,11. However, the exact locations of their binding sites are not known at high resolution. Similarly, human lncRNA HOTAIR can affect PRC2 occupancy on hundreds of genes genome-wide 3,12,13, but how specificity is achieved is unclear. LncRNAs can also serve as modular scaffolds to recruit the assembly of multiple protein complexes. The classic trans-acting RNA scaffold is the TERC RNA that serves as the template and scaffold for the telomerase complex 14; HOTAIR can also serve as a scaffold for PRC2 and a H3K4 demethylase complex 13.
Prior studies mapping RNA occupancy at chromatin have revealed substantial insights 15,16, but only at a single gene locus at a time. The occupancy sites of most lncRNAs are not known, and the roles of lncRNAs in chromatin regulation have been mostly inferred from the indirect effects of lncRNA perturbation. Just as chromatin immunoprecipitation followed by microarray or deep sequencing (ChIP-chip or ChIP-seq, respectively) has greatly improved our understanding of protein-DNA interactions on a genomic scale, here we illustrate a recently published strategy to map long RNA occupancy genome-wide at high resolution 17. This method, Chromatin Isolation by RNA Purification (ChIRP) (Figure 1), is based on affinity capture of target lncRNA:chromatin complex by tiling antisense-oligos, which then generates a map of genomic binding sites at a resolution of several hundred bases with high sensitivity and low background. ChIRP is applicable to many lncRNAs because the design of affinity-probes is straightforward given the RNA sequence and requires no knowledge of the RNA's structure or functional domains.

Chromatin Isolation by RNA Purification ChIRP

ChIRP is a novel and rapid technique to map genomic binding sites of long noncoding RNAs (lncRNAs). The method takes advantage of the specificity of anti-sense tiling oligonucleotides to allow the enumeration of lncRNA-bound genomic sites.

RNA精製によってクロマチンの単離（チャープ）

Long noncoding RNAs are key regulators of chromatin states for important biological processes such as dosage compensation, imprinting, and developmental ...

Substrate Generation for Endonucleases of CRISPR/Cas Systems

The interaction of viruses and their prokaryotic hosts shaped the evolution of bacterial and archaeal life. Prokaryotes developed several strategies to evade viral attacks that include restriction modification, abortive infection and CRISPR/Cas systems. These adaptive immune systems found in many Bacteria and most Archaea consist of clustered regularly interspaced short palindromic repeat (CRISPR) sequences and a number of CRISPR associated (Cas) genes (Fig. 1) 1-3. Different sets of Cas proteins and repeats define at least three major divergent types of CRISPR/Cas systems 4. The universal proteins Cas1 and Cas2 are proposed to be involved in the uptake of viral DNA that will generate a new spacer element between two repeats at the 5' terminus of an extending CRISPR cluster 5. The entire cluster is transcribed into a precursor-crRNA containing all spacer and repeat sequences and is subsequently processed by an enzyme of the diverse Cas6 family into smaller crRNAs 6-8. These crRNAs consist of the spacer sequence flanked by a 5' terminal (8 nucleotides) and a 3' terminal tag derived from the repeat sequence 9. A repeated infection of the virus can now be blocked as the new crRNA will be directed by a Cas protein complex (Cascade) to the viral DNA and identify it as such via base complementarity10. Finally, for CRISPR/Cas type 1 systems, the nuclease Cas3 will destroy the detected invader DNA 11,12 .
These processes define CRISPR/Cas as an adaptive immune system of prokaryotes and opened a fascinating research field for the study of the involved Cas proteins. The function of many Cas proteins is still elusive and the causes for the apparent diversity of the CRISPR/Cas systems remain to be illuminated. Potential activities of most Cas proteins were predicted via detailed computational analyses. A major fraction of Cas proteins are either shown or proposed to function as endonucleases 4.
Here, we present methods to generate crRNAs and precursor-cRNAs for the study of Cas endoribonucleases. Different endonuclease assays require either short repeat sequences that can directly be synthesized as RNA oligonucleotides or longer crRNA and pre-crRNA sequences that are generated via in vitro T7 RNA polymerase run-off transcription. This methodology allows the incorporation of radioactive nucleotides for the generation of internally labeled endonuclease substrates and the creation of synthetic or mutant crRNAs. Cas6 endonuclease activity is utilized to mature pre-crRNAs into crRNAs with 5'-hydroxyl and a 2',3'-cyclic phosphate termini.

CRISPR/Cas systems mediate adaptive immunity in Bacteria and Archaea. Many Cas proteins are proposed to act as endoribonucleases acting on crRNA precursors of varying length. Here we illustrate three different approaches to generate pre-crRNA substrates for the biochemical analysis of Cas endonuclease activity.

CRISPR / CASシステムの酵素のための基質の生成

The  interaction of viruses and their prokaryotic hosts shaped the evolution of  bacterial and archaeal life. Prokaryotes developed several strategies to ...

Depletion of Ribosomal RNA for Mosquito Gut Metagenomic RNA-seq

The mosquito gut accommodates dynamic microbial communities across different stages of the insect's life cycle. Characterization of the genetic capacity and functionality of the gut community will provide insight into the effects of gut microbiota on mosquito life traits. Metagenomic RNA-Seq has become an important tool to analyze transcriptomes from various microbes present in a microbial community. Messenger RNA usually comprises only 1-3% of total RNA, while rRNA constitutes approximately 90%. It is challenging to enrich messenger RNA from a metagenomic microbial RNA sample because most prokaryotic mRNA species lack stable poly(A) tails. This prevents oligo d(T) mediated mRNA isolation. Here, we describe a protocol that employs sample derived rRNA capture probes to remove rRNA from a metagenomic total RNA sample. To begin, both mosquito and microbial small and large subunit rRNA fragments are amplified from a metagenomic community DNA sample. Then, the community specific biotinylated antisense ribosomal RNA probes are synthesized in vitro using T7 RNA polymerase. The biotinylated rRNA probes are hybridized to the total RNA. The hybrids are captured by streptavidin-coated beads and removed from the total RNA. This subtraction-based protocol efficiently removes both mosquito and microbial rRNA from the total RNA sample. The mRNA enriched sample is further processed for RNA amplification and RNA-Seq.

A ribosomal RNA (rRNA) depletion protocol was developed to enrich messenger RNA (mRNA) for RNA-seq of the mosquito gut metatranscriptome. Sample specific rRNA probes, which were used to remove rRNA via subtraction, were created from the mosquito and its gut microbes. Performance of the protocol can result in the removal of approximately 90-99% of rRNA.

モスキートガットメタゲノムRNA-seqのためのリボソームRNAの枯渇

The mosquito gut accommodates dynamic microbial communities across different stages of the insect's life cycle. Characterization of the genetic capacity ...

RNA In situ Hybridization in Whole Mount Embryos and Cell Histology Adapted for Marine Elasmobranchs

Marine elasmobranchs are valued animal models for biomedical and genomic studies as they are the most primitive vertebrates to have adaptive immunity and have unique mechanisms for osmoregulation 1-3. As the most primitive living jawed-vertebrates with paired appendages, elasmobranchs are an evolutionarily important model, especially for studies in evolution and development. Marine elasmobranchs have also been used to study aquatic toxicology and stress physiology in relationship to climate change 4. Thus, development and adaptation of methodologies is needed to facilitate and expand the use of these primitive vertebrates to multiple biological disciplines. Here I present the successful adaptation of RNA whole mount in situ hybridization and histological techniques to study gene expression and cell histology in elasmobranchs.
Monitoring gene expression is a hallmark tool of developmental biologists, and is widely used to investigate developmental processes 5. RNA whole mount in situ hybridization allows for the visualization and localization of specific gene transcripts in tissues of the developing embryo. The expression pattern of a gene's message can provide insight into what developmental processes and cell fate decisions a gene may control. By comparing the expression pattern of a gene at different developmental stages, insight can be gained into how the role of a gene changes during development.
While whole mount in situ's provides a means to localize gene expression to tissue, histological techniques allow for the identification of differentiated cell types and tissues. Histological stains have varied functions. General stains are used to highlight cell morphology, for example hematoxylin and eosin for general staining of nuclei and cytoplasm, respectively. Other stains can highlight specific cell types. For example, the alcian blue stain reported in this paper is a widely used cationic stain to identify mucosaccharides. Staining of the digestive tract with alcian blue can identify the distribution of goblet cells that produce mucosaccharides. Variations in mucosaccharide constituents on short peptides distinguish goblet cells by function within the digestive tract 6. By using RNA whole mount in situ's and histochemical methods concurrently, cell fate decisions can be linked to gene-specific expression.
Although RNA in situ's and histochemistry are widely used by researchers, their adaptation and use in marine elasmobranchs have met limited and varied success. Here I present protocols developed for elasmobranchs and used on a regular basis in my laboratory. Although further modification of the RNA in situ's hybridization method may be needed to adapt to different species, the protocols described here provide a strong starting point for researchers wanting to adapt the use of marine elasmobranchs to their scientific inquiries.

RNA In situ Hybridization in Whole Mount Embryos and Cell Histology Adapted for Marine Elasmobranchs

By combining methods for RNA whole mount in situ hybridization and histology, gene expression can be linked with cell fate decisions in the developing embryo. These methods have been adapted to marine elasmobranchs and facilitate the use of these animals as model organisms for biomedical, toxicology and comparative studies.

RNAを<em&gt;その場</emホールマウント胚および細胞組織学で&gt;ハイブリダイゼーションは、海洋軟骨魚類に適合した

Marine  elasmobranchs are valued animal models for biomedical and genomic studies as  they are the most primitive vertebrates to have adaptive immunity ...

A Chemical Screening Procedure for Glucocorticoid Signaling with a Zebrafish Larva Luciferase Reporter System

Glucocorticoid stress hormones and their artificial derivatives are widely used drugs to treat inflammation, but long-term treatment with glucocorticoids can lead to severe side effects. Test systems are needed to search for novel compounds influencing glucocorticoid signaling in vivo or to determine unwanted effects of compounds on the glucocorticoid signaling pathway. We have established a transgenic zebrafish assay which allows the measurement of glucocorticoid signaling activity in vivo and in real-time, the GRIZLY assay (Glucocorticoid Responsive In vivo Zebrafish Luciferase activitY). The luciferase-based assay detects effects on glucocorticoid signaling with high sensitivity and specificity, including effects by compounds that require metabolization or affect endogenous glucocorticoid production. We present here a detailed protocol for conducting chemical screens with this assay. We describe data acquisition, normalization, and analysis, placing a focus on quality control and data visualization. The assay provides a simple, time-resolved, and quantitative readout. It can be operated as a stand-alone platform, but is also easily integrated into high-throughput screening workflows. It furthermore allows for many applications beyond chemical screening, such as environmental monitoring of endocrine disruptors or stress research.

We describe the procedure and data analysis of a chemical screening system for glucocorticoid stress hormone signaling using zebrafish larvae: the Glucocorticoid Responsive In vivo Zebrafish Luciferase activitY (GRIZLY) assay. The assay sensitively and specifically detects effects on glucocorticoid signaling by compounds that require metabolization or affect endogenous glucocorticoid production.

ゼブラフィッシュ幼虫ルシフェラーゼレポーターシステムを用いたグルココルチコイドシグナル伝達のための化学スクリーニング法

Glucocorticoid stress hormones and their artificial derivatives are widely used drugs to treat inflammation, but long-term treatment with glucocorticoids ...

Detection of RNA-binding Proteins by In Vitro RNA Pull-down in Adipocyte Culture

RNA-binding proteins (RBPs) are emerging as a regulatory layer in the development and function of adipose. RBPs play a key role in the gene expression regulation at posttranscriptional levels by affecting the stability and translational efficiency of target mRNAs. RNA pull-down technique has been widely used to study RNA-protein interaction, which is necessary to elucidate the mechanism underlying RBPs&#39; as well as long non-coding RNAs&#39; (lncRNAs) function. However, the high lipid abundance in adipocytes poses a technical challenge in conducting this experiment. Here a detailed RNA pull-down protocol is optimized for primary adipocyte culture. An RNA fragment from androgen receptor&#39;s (AR) 3&#39; untranslated region (3&#39;UTR) containing an adenylate-uridylate-rich elementwas used as an example to demonstrate how to retrieve its RBP partner, HuR protein, from adipocyte lystate. The method described here can be applied to detect the interactions between RBPs and noncoding RNAs, as well as between RBPs and coding RNAs.

Detection of RNA-binding Proteins by In Vitro RNA Pull-down in Adipocyte Culture

An RNA pull-down protocol is optimized here for detection of interactions between RNA-binding proteins (RBPs) and noncoding as well as coding RNAs. An RNA fragment from androgen receptor (AR) was used as an example to demonstrate how to retrieve its RBP from lystate of primary brown adipocytes.

RNA結合タンパク質の検出によって、<em&gt;インビトロ</em脂肪細胞培養における&gt; RNAプルダウン

RNA-binding proteins (RBPs) are emerging as a regulatory layer in the development and function of adipose. RBPs play a key role in the gene expression ...

Practical Use of RNA Interference: Oral Delivery of Double-stranded RNA in Liposome Carriers for Cockroaches

RNA interference (RNAi) has been widely applied for uncovering the biological functions of numerous genes, and has been envisaged as a pest control tool operating by disruption of essential gene expression. Although different methods, such as injection, feeding, and soaking, have been reported for successful delivery of double-stranded RNA (dsRNA), the efficiency of RNAi through oral delivery of dsRNA is highly variable among different insect groups. The German cockroach, Blattella germanica, is highly sensitive to the injection of dsRNA, as shown by many studies published previously. The present study describes a method to demonstrate that the dsRNA encapsulated with liposome carriers is sufficient to retard the degradation of dsRNA by midgut juice. Notably, the continuous feeding of dsRNA encapsulated by liposomes significantly reduces the tubulin expression in the midgut, and led to the death of cockroaches. In conclusion, the formulation and utilization of dsRNA lipoplexes, which protect dsRNA against nucleases, could be a practical use of RNAi for insect pest control in the future.

This manuscript demonstrates the depletion of gene expression in the midgut of the German cockroach through oral ingestion of double-stranded RNA encapsulated in liposomes.

RNA 干渉法の活用: ゴキブリのリポソーム キャリアで二本鎖 RNA の経口デリバリー

RNA interference (RNAi) has been widely applied for uncovering the biological functions of numerous genes, and has been envisaged as a pest control tool ...

Imaging FITC-dextran as a Reporter for Regulated Exocytosis

Regulated exocytosis is a process by which cargo, which is stored in secretory granules (SGs), is released in response to a secretory trigger. Regulated exocytosis is fundamental for intercellular communication and is a key mechanism for the secretion of neurotransmitters, hormones, inflammatory mediators, and other compounds, by a variety of cells. At least three distinct mechanisms are known for regulated exocytosis: full exocytosis, where a single SG fully fuses with the plasma membrane, kiss-and-run exocytosis, where a single SG transiently fuses with the plasma membrane, and compound exocytosis, where several SGs fuse with each other, prior to or after SG fusion with the plasma membrane. The type of regulated exocytosis undertaken by a cell is often dictated by the type of secretory trigger. However, in many cells, a single secretory trigger can activate multiple modes of regulated exocytosis simultaneously. Despite their abundance and importance across cell types and species, the mechanisms that determine the different modes of secretion are largely unresolved. One of the main challenges in investigating the different modes of regulated exocytosis, is the difficulty in distinguishing between them as well as exploring them separately. Here we describe the use of fluorescein isothiocyanate (FITC)-dextran as an exocytosis reporter, and live cell imaging, to differentiate between the different pathways of regulated exocytosis, focusing on compound exocytosis, based on the robustness and duration of the exocytic events.

Here we detail a method for live cell imaging of regulated exocytosis. This method utilizes FITC-dextran, which accumulates in lysosome-related organelles, as a reporter. This simple method also allows distinguishing between different modes of regulated exocytosis in cells that are difficult to manipulate genetically.

規制エキソサイトーシスの記者として FITC デキストランをイメージング

Regulated exocytosis is a process by which cargo, which is stored in secretory granules (SGs), is released in response to a secretory trigger. Regulated ...