Podcasts about argonaute

The Aedes aegypti mosquito transmits deadly viruses like Zika, chikungunya, and dengue, but doesn't actually get sick from the diseases it carries. George Dimopoulos of the Johns Hopkins Malaria Research Institute talks with Stephanie Desmon about a new discovery of a protein, Argonaute 2, that plays a key role in the mosquito's immune system, and how genetically modifying mosquitos could make them vulnerable to the viruses they carry. They also discuss how much of an impact killing off large numbers of mosquitos would have, both on the burden of disease and larger ecological balance.

Si torna a parlare di contrabbando in Valposchiavo grazie alla seconda edizione di s-Confini, rassegna culturale che riflette su un periodo storico che ha significato molto per le comunità situate - appunto - sul confine.Il Festival transfrontaliero propone fino al 7 maggio un ricco programma (s-confini.eu) che spazia dalla mostra dal titolo “Il contrabbando visto da Bernardo Lardi”, al monologo teatrale di Chiara Balsarini, sino alla presentazione del giallo-storico di Andrea Paganini “Le indagini imperfette” e del romanzo storico "Dal Bernina al Naviglio” a firma di Massimo Lardi. Ci saranno escursioni lungo i sentieri un tempo battuti dai contrabbandieri, visite guidate alle torrefazioni ancora in attività a Brusio e a Poschiavo dove sarà possibile inebriarsi di quel profumo che un tempo aleggiava in tutta la valle.Si parlerà anche dell'universo femminile con “Donne – altre vite, stesso cuore”, una mostra fotografica dell'Associazione Argonaute di Sondrio che si inserisce nel lavoro di recupero della memoria della storia sociale di Valtellina e Valchiavenna portata avanti da tempo dall'associazione. E sempre grazie alle Argonaute si terrà una conferenza dal titolo "Percorsi sconfinanti - donne migranti nel secondo dopoguerra”.L'iniziativa è promossa dal Centro culturale Casa Besta e dal MET Museo Etnografico Tiranese, in collaborazione con Valposchiavo Turismo e Consorzio turistico Media Valtellina.

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.01.01.522328v1?rss=1 Authors: Naeli, P., Zhang, X., Harris Snell, P., Chatterjee, S., Kamran, M., Ladak, R. J., Orr, N., Duchaine, T., Sonenberg, N., Jafarnejad, S. M. Abstract: microRNAs (miRNAs) inhibit mRNA translation initiation by recruiting the GIGYF2/4EHP translation repressor complex to the mRNA 5' cap structure. Viruses utilise miRNAs to impair the host antiviral immune system and facilitate viral infection by expressing their own miRNAs or co-opting cellular miRNAs. We recently reported that the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) encoded non-structural protein 2 (NSP2) interacts with GIGYF2. This interaction is critical for blocking translation of the Ifn1-b mRNA that encodes the cytokine Interferon-beta, and thereby impairs the host antiviral immune response. However, it is not known whether NSP2 also affects miRNA-mediated silencing. Here, we demonstrate the pervasive augmentation of the miRNA-mediated translational repression of cellular mRNAs by NSP2. We show that NSP2 interacts with Argonaute 2, the core component of the miRNA-Induced Silencing Complex (miRISC) and enhances the translational repression mediated by natural miRNA binding sites in the 3' UTR of cellular mRNAs. Our data reveal an additional layer of the complex mechanism by which SARS-CoV-2 and likely other coronaviruses manipulate the host gene expression program through co-opting the host miRNA-mediated silencing machinery. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Kotaro Nakanishi, associate professor of biochemistry, studies Argonaute proteins and how they join with microRNA to form complexes in cells. Errors in this process can lead to many different types of diseases, including cancer and autism.

dessanをゲストに迎え、CRISPR–Cas9やTALENなどのゲノム編集技術とその背後にある遺伝学について話しました。Show notes The Nobel Prize in Chemistry 2020…The Nobel Prize in Chemistry 2020 was awarded jointly to Emmanuelle Charpentier and Jennifer A. Doudna “for the development of a method for genome editing.” Scientifc Background on the Nobel Prize in Chemistry 2020 A TOOL FOR GENOME EDITING…ノーベル財団による詳細なCRISPR研究のレビュー、そしてなぜDoudnaとCharpentierの二人が受賞に値するのかについて説明している。 Emmanuelle Charpentier…DoudnaとCharpentierは2011年に学会で知り合い、そこからノーベル賞につながる共同研究を開始した。 Jennifer Doudna…CharpentierとともにCRISPRの仕組みを明らかにし、その後のゲノム編集そしてCasタンパクを用いた核酸検出など多大な貢献をした。 2. An emerging technology is always not perfect…始まって間もない第二回でもざっくりとCRISPR–Cas9について解説しました。 dessanが出た過去回のResearchat.fmのエピソード…ep37, ep38, ep51, ep58 37. Biological Enigma … 今回のep67はep37の続編という位置付けなので、分子細胞生物学に興味がある方はep37を聴いてからep76を聴いていただけるとより理解が深まります。 ゲノム編集とはなにか　「ＤＮＡのハサミ」クリスパーで生命科学はどう変わるのか (ブルーバックス) (Amazon) 遺伝学 (Wikipedia) 遺伝学的スクリーニング…逆遺伝学と順遺伝学について。 トランスジェニック動物 表現型 (Wikipedia) Thomas Hunt Morgan (Wikipedia) 遺伝子の必須性は細胞の進化可能性にリンクした定量的特性である…必須遺伝子などについて Cell type (Wikipedia)…日本語ではセルタイプあるいは細胞種 部位特異的ヌクレアーゼを基盤とするゲノム編集技術…ZFNやFok1などについての山本先生らによる日本語解説論文。 72. The Hitchhiker’s Guide to the Enzyme Galaxy…エピソード72では制限酵素の多様な世界について紹介しています。 TALEN (Wikipedia) Golden Gate Assembly法…Type IISと呼ばれる制限酵素を用いることで、複数のDNA断片を一度に連結させることのできるクローニング技術。 ガイドRNA…guide RNAあるいはsingle-guide RNAとも呼ばれるcrRNAとtracrRNAを一つのRNAとして連結させたRNA分子。 crRNA…CRISPR RNA tracrRNA…trans-activating crRNA A Programmable Dual-RNA–Guided DNA Endonuclease in Adaptive Bacterial Immunity. Jinek et al Science 2012.…ノーベル賞の受賞に直接的に関連するDoudnaとCharpentierらによる論文。この仕事によってCas9によるゲノム編集の仕組みの大枠が解明された。 Ribozyme (Wikipedia) Argonaute (Ago) Editorial notes 実はdessanと初めて話しました。そしてまだ対面では会ったことはないのちょっと面白くないですか？(soh) ノーベルのおかげで、急遽参加させていただきました！(dessan) ~亜米利加男塾~ 王TALEN 「切断確認」という冗談はさておき、リトアニアのVirginijus Siksnys先生についても話したかったですね。(tadasu) dessan相変わらずの分かりやすさでした！何かに例える事のいい側面と失われる情報の側面が悩ましい。(coela)

現存する伝説の武器について話しました。Shownotes 前回の武器回 researchat.fm ep30 Battle Aura 草薙剣 … 別名天叢雲剣 三種の神器 … 八咫鏡、草薙剣、八尺瓊勾玉。三種の神器といえばKOFを思い出しますね。 一向一揆 THE CELL … 分子生物学における代表的な鈍器の一つ 放射性炭素年代測定 … C14の存在比率で年代測定をする手法 空海 熱田神宮 … 草薙剣の実物が祀られている 三種の神器と贈与税 … 非課税 壇ノ浦の戦い … 草薙剣の形代が一度失われた 聖骸布 古事記 千と千尋の神隠し ポールアレン 十握剣 … 日本神話に出てくる剣。特定の剣を指す言葉ではなく、10束の長さの剣の総称。 天之尾羽張剣 … イザナギが使用した十握剣。カグツチを斬った。 天羽々斬剣 … スサノオが使用した十握剣。ヤマタノオロチを斬った。 八咫烏 ミスティルテイン … 古ノルド語・アイスランド語、スウェーデン語・デンマーク語、ノルウェー語でヤドリギの木を差す。北欧神話でバルドルを殺すのに使用された。 バルドル … 世界中の生物・無生物から傷付けられないはずだったが、ヤドリギの木だけは例外だった。 グラディウス … 古代ローマで使用された剣。特殊の剣を指す言葉ではない。同名の名作シューティングゲームもあります。 ツヴァイハンダー … 剣の種類、特定の剣を指す言葉ではない。 クレイモア … 剣の種類、特定の剣を指す言葉ではない。実は2種類ある。余談ですが同名の漫画もオススメです。 ショーテル … 剣の種類、特定の剣を指す言葉ではない。 ファイナルファンタジーシリーズ … いろんな伝説の武器が登場 ヴァルキリープロファイル … 北欧神話を題材としたプレイステーションの名作ゲーム。フレイ様の「まったく褒められたものではないわね」はトラウマ。リンクはスマホ移植版。 刀剣乱舞 天下五剣　… 童子切・鬼丸・三日月・大典太・数珠丸 映画刀剣乱舞-継承- Fateシリーズ 村正 … 具体的な日本刀の名前ではなく刀工の名前 干将莫耶 … 中国の名剣、並びに製作者の夫婦の名前 ジョワユーズ … シャルルマーニュ伝説でシャルルマーニュが所持していた剣。ルーブル美術館にあります！ カール大帝 … 初代神聖ローマ皇帝。フランス語でシャルルマーニュ。シャルルマーニュ関連は伝説と史実の両方があるのでややこしい。 アーサー王 … 円卓の騎士、エクスカリバーなどが有名なアーサー王伝説に登場する王様。 エクスカリバー … 語る事が無いぐらい有名な剣 死海文書の偽物 デュランダル … フランスの叙事詩ローランの歌に登場する英雄・ローランが持つ剣。イーリアスに登場するヘクトールが所持していた剣と同一のものとされることもある。ロカマドールの岩に今も刺さっているという話やそれはレプリカであるという話があるらしい。 ローラン … シャルルマーニュに仕えた聖騎士 イーリアス … 最古期の古代ギリシア詩作品 ヘクトール … ギリシャ神話の英雄 トロイア戦争 … トロイの木馬で有名 天の逆鉾 … 日本の中世神話に登場する矛。レプリカが高千穂山頂部に刺さっている(オリジナルの柄は地中にあるらしい)。坂本龍馬が引っこ抜いた話が有名。 イタリアの修道院に刺さってる剣　…　サン・ガルガーノ修道院には岩に刺さった剣が保管されている。 カーテナ … イギリス王家に代々伝わる剣。剣の先が折れたような形をしており無先刀、無鋒剣、慈悲の剣などと呼ばれる。戴冠宝器の一つ。ピューリタン革命で失われたが後に作り直された。現在ロンドン塔の宝物館に保管されている。アーサー王伝説に登場するトリスタンが使用したとされる伝承がある。 トリスタン … 「トリスタンとイゾルデ」や「アーサー王物語」に登場する騎士。 連合王国の戴冠宝器 … 141個ある。中には聖界正義の剣（Sword of Spiritual Justice）、俗界正義の剣（Sword of Temporal Justice）、献納の宝剣（Jewelled Sword of Offering）、国剣（Great Sword of State）などカッチョイイ名前の剣がある。 ロンドン塔の宝物館 カリナン … ロンドン塔で展示されている世界最大のダイヤモンド。530.20カラット。 4C… 宝飾用ダイヤモンドの品質を評価する国際基準。色（color）、透明度（clarity）、重さ（carat）、研磨（cut）の4点。 甲斐駒ケ岳の二本剣　　… 甲斐駒ケ岳に刺さっている二本の剣 七支刀 蜻蛉切 … 本田忠勝が使用した槍。天下名三槍の一つ。 ダマスカス … 都市の名前並びにそこで造られていた鋼。 ガンダリウム合金 … 詳細は省くがガンダムに使われている合金 ティソーナ … スペインの叙事詩「わがシッドの歌」の主人公(実在の人物ロドリーゴ・ディアス・デ・ビバール)が所持していた二振りの剣のうちの一本。ブルゴスの博物館(スペイン)で展示されている。ダマスカス鋼製。 わがシッドの歌 根津美術館 弓取式 越王勾践剣 方天画戟 … よくゲームなどで呂布が持っている武器。これも厳密には特定の武器を指す言葉ではない。 青龍偃月刀 … よくゲームなどで関羽が持っている武器。これも厳密には特定の武器を指す言葉ではない。 蛇矛 … よくゲームなどで張飛が持っている武器。これも厳密には特定の武器を指す言葉ではない。 イージス … アイギス、エイジスとも。ギリシャ神話で女神アテナが持つ防具。 イージス艦 ペルセウス … ギリシャ神話の英雄。ゴルゴーン(メデューサ)を退治など様々なエピソードがある。 テセウス … ギリシャ神話の英雄。ミノタウルス退治などのエピソードが有名。 テセウスの船 … パラドックスの一つ。 アルゴナウタイ … ギリシャ神話でアルゴー船に搭乗した英雄の総称。英語ではアルゴノーツ。 Argonaut … RNAサイレンシングに関わるタンパク質。RNAサイレンシングに関してはep45でも話してます！r リチャードドーキンス The Extended Phenotype UMA タキタロウ … 釣りキチ三平にO池の滝太郎として登場 倉谷滋 キングダム 終末のワルキューレ … 今回のおすすめ漫画 Editorial notes 古事記をまた読み返したくなりました (soh) このshownotesの熱量を見ればcoelaさんがどれだけ頑張っていたかがわかると思います。ただ、あまりにも何を話しているのかわからなくて泣きました。他分野の学会に参加した気分でした。ヨイショすらできなかったので面白いと感じた方はおたよりかTwitterでcoelaさんを褒めていただくとcoelaさんも喜ぶと思います。そうすると第三回もすぐあるかもしれません。(tadasu) 第三回はもう少しみんなに興味持って貰えそうなネタ考えとくわ！！(coela)

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.08.03.235127v1?rss=1 Authors: Haskell, D., Zinovyeva, A. Abstract: microRNAs (miRNAs) and RNA binding proteins (RBPs) regulate gene expression at the post-transcriptional level, but the extent to which these key regulators of gene expression coordinate and the precise mechanisms of their coordination are not well understood. RNA binding proteins often have recognizable RNA binding domains that correlate with specific protein function. Recently, several RBPs containing K Homology (KH) RNA binding domains were shown to work with miRNAs to regulate gene expression, raising the possibility that KH domains may be important for coordinating with miRNA pathways in gene expression regulation. To ascertain whether additional KH domain proteins functionally interact with miRNAs during Caenorhabditis elegans development, we knocked down twenty-four genes encoding KH-domain proteins in several miRNA sensitized genetic backgrounds. Here, we report that a majority of the KH domain-containing genes genetically interact with multiple miRNAs and Argonaute alg-1. Interestingly, two KH domain genes, predicted splicing factors sfa- and asd-2, genetically interacted with all of the miRNA mutants tested, while other KH domain genes exhibited functional interactions only with specific miRNAs. Our domain architecture and phylogenetic relationship analyses of the C. elegans KH domain-containing proteins revealed potential groups that may share both structure and function. Collectively, we show that many C. elegans KH domain RBPs functionally interact with miRNAs, suggesting direct or indirect coordination between these two classes of post-transcriptional gene expression regulators. Copy rights belong to original authors. Visit the link for more info

Dr Carolyn Lam: Welcome to Circulation on the Run, your weekly podcast summary and backstage pass to the journal and its editors. I'm Dr Carolyn Lam, associate editor from the National Heart Center and Duke National University of Singapore. Dr Greg Hundley: And I'm Dr Greg Hundley, associate editor, Director of the Pauley Heart Center from VCU Health in Richmond, Virginia. Dr Carolyn Lam: Our feature paper today discusses trans-ethnic genome-wide association studies and the insights in the genetic architecture and heritability of long QT syndrome, a massive study that we will be digging into, but only after we talk a little bit about the other papers in this week's issue. And I'm going to start, Greg. Are you ready with your coffee? Dr Greg Hundley: I am. Dr Carolyn Lam: The first original paper really represents seminal work, showing that the endothelium can directly regulate obesity and insulin resistance. Now, as obesity develops, there is a decline in adipose tissue vascularity, which seems counterintuitive, and an increase in fibrosis. So authors, led by Dr Chen from the Irell and Manella Graduate School of Biological Sciences in the City of Hope, speculated that the reduction in vascularity in this adipose tissue might have an adverse effect on adipose tissue function. Now, these authors previously identified Argonaute-1, or AG01, a key component of microRNA-induced silencing complex, as a crucial regulator in hypoxia-induced angiogenesis. So in the current study, they aim to determine the AG01-mediated endothelial cell transcriptome, the functional importance of AG01-regulated endothelial function in vivo, and the relevance to adipose tissue function and obesity. A new mouse model with genetic deletion of AG01 in the endothelium was useful to investigate the importance of endothelial regulation of adipose tissue function. The findings were that in mice fed high fat, high sucrose diet, the suppression of endothelial AG01 promoted adipose tissue browning, and led to an anti-obesity phenotype. Endothelial cell AG01 thrombospondin-1 pathway was induced in the endothelium from human donors with insulin resistance. In total, this study suggests a novel mechanism, by which endothelial cells through AG01 thrombospondin-1 pathway controls vascularization and function of adipose tissues, insulin sensitivity, and whole-body metabolic state. Dr Greg Hundley: Interesting, Carolyn. So tell me about this clinically. Where do we take this from here? Dr Carolyn Lam: I thought you would ask. So endothelial dysfunction, per se, can cause metabolic dysregulation, rendering targeting dysfunctional endothelium, a potential therapeutic strategy to counteract obesity, and metabolic disorders. So this study really opens a door to that. Dr Greg Hundley: Very nice. Well, I've got another basic science paper, and it evaluates single-cell RNA sequencing to dissect the immunological network of autoimmune myocarditis. And it comes from Dr Jiangping Song from the State Key Laboratory of Cardiovascular Disease of Fuwai Hospital, and the National Center for Cardiovascular Disease, Chinese Academy of Medical Sciences, and Peking Union Medical College. So Carolyn, the study aimed to investigate the immunological network during the transition from myocarditis to cardiomyopathy, and to identify the genes contributing to the inflammatory response to myocarditis. So mice were treated with myosin heavy chain alpha-peptides to generate an experimental autoimmune myocarditis model. The investigators performed single-cell RNA sequencing analysis of CD45 plus cells extracted from mouse hearts during different experimental autoimmune myocarditis phases, including normal control, acute inflammation, subacute inflammation, and then in the myopathy phase. Also, human heart tissues were collected from surgically removed hearts of patients who had undergone heart transplantation. Dr Carolyn Lam: So what did they find, Greg? Dr Greg Hundley: Well, Carolyn, a comparison of the single-cell RNA sequencing data from different experimental autoimmune myocarditis phases suggested that some cell clusters, such as macrophage cluster 2 and Th17 cells, were associated with the inflammatory response in the experimental autoimmune myocarditis model. The HIF1A expression level correlated with the extent of the inflammatory response, and PX-478, a HIF1A inhibitor, alleviated the inflammation during the different experimental autoimmune myocarditis phases. Immunohistochemical staining revealed that HIF1A expression was upregulated in autoimmune myocarditis from the tissue samples from the explanted hearts. Thus, the HIF1A inhibitor alleviated inflammatory cell infiltration, and that may serve as a potential therapeutic target in clinical practice. Dr Carolyn Lam: Wow. That is some serious clinical implications. Well, my next paper is really the first systematic echocardiographic evaluation of consecutive patients requiring hospitalization due to COVID-19, and it comes from Dr Topilsky and colleagues from Tel Aviv Medical Center. Dr Greg Hundley: So Carolyn, what did they find in this series? Dr Carolyn Lam: So among a hundred consecutive patients diagnosed with COVID-19 infection who underwent complete echocardiographic evaluation, within 24 hours of admission, only 32% had a normal echocardiogram at baseline. The most frequent abnormality was right ventricular dilatation or dysfunction. Among patients developing clinical deterioration during follow-up, which were 20% of these hospitalized patients, repeated echocardiograms showed further deterioration of the right ventricular parameters, probably related to increased pulmonary resistance. Five of these patients had deep vein thrombosis. Dr Greg Hundley: Carolyn, my next study comes from Dr Stephen Fremes, and it's a modeling study out of the University of Toronto. It modeled TAVR versus SAVR valve durability to determine the effects on life expectancy across a broad range of age. Dr Carolyn Lam: Interesting. And what were the results? Dr Greg Hundley: Well, based on their simulation models, the durability of TAVR valves must be 70% shorter than that of surgically replaced valves to result in reduced life expectancy in patients with similar demographics to recent trials. However, in younger patients, the threshold for TAVR valve durability was substantially higher. In younger patients, life expectancy was reduced when TAVR durability was 30%, 40% and 50% shorter than surgical valves in 40, 50 or 60-year-old patients, respectively. So Carolyn, these findings suggest that durability concerns should not influence the initial treatment decision regarding TAVR versus SAVR in older low-risk patients, based on current evidence supporting TAVR valve durability. However, in younger low-risk patients, valve durability must be weighed against other patient factors, such as life expectancy. Dr Carolyn Lam: Thanks Greg, for that summary. Well, let me tell you about other papers in this issue. There are a pair of letters to the editor by Dr Opotowsky, and a response by Dr Goldberg regarding the paper results of the Fontan Udenafil Exercise Longitudinal, or FUEL trial. There's a research letter by Dr Strik, Validating QT-Interval Measurement Using the Apple Watch ECG to Enable Remote Monitoring During the COVID-19 Pandemic. There are two On My Mind papers, the first, Telemedicine and Forgotten America by Dr Julien, and the second, The COVID-19 Pandemic: Ethical and Scientific Imperatives for "Natural" Experiments by Dr Lewis. Dr Greg Hundley: Very nice. Well, Carolyn, I've got a research letter evaluating the effect of evolocumab on atherogenic lipoproteins during the peri and early post-infarction period. It's a placebo-controlled randomized trial from Dr Gary Gerstenblith. Sarah Cuddy also worked through a tough case of cardiac amyloid when a fat biopsy was negative, but imaging studies of the heart suggested cardiac amyloid. Carolyn, I've also got an On My Mind piece, and it's entitled, Can Old Ally Defeat a New Enemy? And it's by Dr Paul Gurbel, and he discusses the use of inhaled aspirin to treat patients with COVID-19. And then finally, Carolyn, I have a prospective piece from Dr Robert Lefkowitz who discusses β-arrestin-biased angiotensin II receptor agonists for treatment of COVID-19. Well, Carolyn, what a great issue, and let's get onto that feature discussion. Dr Carolyn Lam: Yay. Let's go, Greg. Dr Greg Hundley: Well, listeners. Now we're turning to our feature discussion, and we are very fortunate to have Professor Connie Bezzina from Amsterdam University Medical Center to talk to us about her paper related to long QT syndrome. Welcome, Connie. And I was wondering, before we get started in discussing your paper, could you tell us a little bit about the background in this area? And then, what was the hypothesis that you wanted to address? Prof Connie Bezzina: So over the last 20 to 30 years, we've learned a lot about the genetic underpinnings of inherited cardiac disorders associated with sudden cardiac arrest. And basically, we've learned a lot about mutations in specific genes that co-segregate with these disorders within families. However, two outstanding features have remained unresolved. Essentially, the first unresolved issue is the fact that we observe, oftentimes, a low disease penetrance and variable disease expression within families, which means that not everybody within a family that carries a familial mutation is affected by the disorder. But two, so among those that are affected, some are affected more severely than others. So some people would have only the ECG abnormality, whereas other people, for instance, would have the ECG abnormality and arrhythmic events. And you could also have individuals, indeed, who don't even manifest any disease manifestations. This is one of the outstanding challenges. The other outstanding challenge is the fact that, despite extensive genetic testing of the known genes in some probands and some families, they remain genetically lucid, in that we don't find a likely genetic defect in a minority of families. And of course, that hinders genetic testing and implementation of genetic testing in such families. Dr Greg Hundley: What was the question you were going to answer with your study? And tell us a little bit about your study design and your study population. Prof Connie Bezzina: Yeah, so essentially, we figured that assigning these disorders to one large genetic defect might be an oversimplification of biological phenomenon. So we hypothesized that even in these Mendelian disorders, the inheritance of additional genetic factors alongside the familial mutation could contribute to risk. Of course, there will be other factors such as environmental factors, which we did not tackle in the study. The central hypothesis of the study was that common genetic variation, which is present in the germ population, could modulate the effect of the familial genetic defect of the Mendelian mutation. So in order to do this, we assembled a large consortium of investigators from multiple centers in Europe, in North America and Japan, worldwide, to bring together about 1700 probands with the long QT syndrome. So we tested this hypothesis in the long QT syndrome because we figured, among the rare inherited rhythm disorders, it's one of the more common disorders. Also, because each individual center has too few patients. To do this locally, we put this group of investigators together to come up with 1700 probands. The study design was a genome-wide association study with a case-control design, where we tested the association of millions of SNPs littered across the genome with susceptibility for the disorder. So this led us to identify three single-nucleotide polymorphisms that are associated with susceptibility for the long QT syndrome. What we immediately saw is that, actually, these three SNPs, perhaps not surprisingly at all, had been previously associated with the extent of the QT interval, with QT interval in the general population. This is not surprising, of course, because repolarization is a central part of physiological mechanism in the long QT syndrome. So this basically indicated overlap between genetic control of the QT interval in the germ population and susceptibility to the long QT syndrome. So the fact that the three SNPs that we identified as long QT syndrome susceptibility SNPs had been associated with QT interval duration in the germ population, we felt that that was pointing to assure genetic underpinnings between these two phenotypes. So we went on to investigate that by looking at the correlation between the odds ratio for long QT syndrome susceptibility and the effect that these SNPs have on QT interval in the germ population. And in fact, we found a very high correlation between those. So essentially, this pointed to sure genetic factors between QT interval in the germ population and long QT syndrome susceptibility. Of course, we wanted to look for disease variability. The next thing we wanted to do was whether these SNPs could actually explain disease variability. Now, this was perhaps the most disappointing part of the study, because when we constructed a polygenic risk score based on SNPs that impact on the QT in the germ population, we found no relation to QT interval among patients, and also no relation to life-threatening arrhythmic events among the patients. We think that this is because our patients... or probrands. They're primarily probands, so they are all more sick. So we didn't have enough variability in our patient set to identify an association with disease variability. And in fact, this is at variance with previous studies that tested individual SNPs, and even our own studies with smaller polygenic risk scores that did find an association between a polygenic risk score based on QT SNPs and QT prolongation and events among patients. So we think that this is certainly something to study further in the future, in larger patient sets where we not only have the probands, but also their relatives, their mutation-carrying relatives, which will give us a bigger variability to actually test this hypothesis. So we think that looking at probands actually was a very good design to find susceptibility variance but was not maybe a good design to find SNPs or polygenic risk scores to test their effect on disease variability. Dr Greg Hundley: It sounds like you've found certain gene low PSI that indicate a predilection for prolongation of the QT interval, but not necessarily are those gene low PSI consistent with who's going to experience an adverse cardiovascular event as a result of their genetic constitution. Is that a fair statement? Prof Connie Bezzina: Well, I think that the setting, because we had probands, they were the most sick people in their families. I think to have stronger conclusions on that, we need to test the polygenic risk scores in families where there are people who are differentially affected. Dr Greg Hundley: I see. I- Prof Connie Bezzina: We had too-narrow of a variability in a probands-only design, as opposed to a study where we would have probands who are severely affected and mutation-carrying relatives who are less severely affected. Dr Greg Hundley: Very nice. So that puts that clearly into context. This was a massive effort. You have quite a list of investigators, and you mentioned you had to gather so many sites. How would you conduct that next study? Would you need another large collection of individuals and many sites to take that on? Prof Connie Bezzina: Yes. I'm a geneticist, and geneticists always want larger, larger numbers, and I'm also one of those. So I'm interested in explaining as much as possible into individual variability. And I think to do that properly, I think we should go preferably for a similar design where we will approach the same centers. And hopefully, we can organize the next study, which will have these probands and their relatives. Dr Greg Hundley: Now, just quickly, for us working in the clinic, how should we approach genetic testing in patients with long QT? Prof Connie Bezzina: At the moment, I think our findings don't have an immediate impact. I think our findings tell us about the genetic architecture of the disorder. And actually, one thing I haven't gone into yet is the fact that what we also found is that patients who do not have mutations in the no-long QT genes, which were called mutation-negative, which are about 20% of all long QT syndrome probands, actually have a higher burden of these common variants that prolong the QT interval. So we think, actually, that mutation-negative long QT syndrome probands will not have a Mendelian large effect variant but will have perhaps a higher burden of these QT-prolonging alleles. Therefore, I think this has direct implications for clinical genetics of these patients, because if you have a proband in whom you don't find a mutation in the known genes, you could think that maybe it is not monogenic, which has implications because you don't have a single genetic defect to test on that family. One would need to keep follow-up of more family members until we understand more about the genetics of those individuals. Dr Greg Hundley: So Connie, this has been just a wonderful discussion. Any additional studies examining the genetic architecture of individuals that we need to think about for the future? Prof Connie Bezzina: Sure. So for long QT syndrome in particular, as additional SNPs that modulate the QT interval in the germ population are identified, it will be very important to incorporate these into larger polygenic risk scores, and see whether we could have a better discriminative capacity of such polygenic risk scores in discriminating between severely affected and less severely affected people, or who is more at risk for an arrhythmic event. Outside of long QT syndrome, I think there's a lot of work to be done with respect to the likely complex inheritance of many of these disorders that we previously considered to be Mendelian. So for instance, ongoing work in our group concerns Brugada syndrome, where we're seeing the same kind of thing, and hypertrophic cardiomyopathy, where we're seeing the same kind of inheritance. Dr Greg Hundley: Well listeners, on behalf of both Carolyn and myself, we look forward to catching you on the run next week. Take care. This program is copyright the American Heart Association 2020.

kyonさんをゲストに迎え、生涯学習、トランスポゾンを抑制するpiRNA研究の最前線の動向、ハムスターなど動物を用いた研究の醍醐味とその難しさ、コーヒー豆の焙煎、モノクローナル抗体の自作、美しい実験結果を求める姿勢などについて話しました。（ゲスト：kyon）Show notes The ALLPATHS-LG assembly of Golden Hamster (Mesocricetus auratus) (ハムスターゲノム)… MIT (Broad institute) でシークエンス、アセンブルされたゲノム情報。結構高品質に繋がっているが、約20%がギャップ (N) であるため解析には向いていない。kyonは共同研究を通してゲノムを決め直している。 CHO細胞…Chinese Hamster Ovary (CHO)細胞はタンパク質の発現などで頻繁に用いられる細胞株の一つ。 SUZUKI DRZ400SM ヤマハ・ビラーゴ/YAMAHA Virago250 生涯学習 Harvard extension school…生涯学習向けのハーバードの講座。様々なレベルの授業が用意されている。多くの授業がオンラインで受けられる。 アメリカのChild careの価格…これはボストン周辺の価格であり、他の都市では異なる。例えば2-3歳児については週5日、フルタイムで預けると月$2,135、23万円程度である。 2-body problem…夫婦などのパートナーがともに科学者としてのキャリアを形成する際、同じ場所（大学や研究所など）でともに職を得ることが非常に難しい場合が往々にしてあり、これを2-body problemと呼ぶ。日本ではほとんど聞かないが、北米などでは夫婦でアプライできるPIポジションなどがある。 制限酵素の自動販売機…写真のように酵素が自販機で買えるの羨ましい。 やなか珈琲…今日はここのエチオピア シダモ・アラングァディの浅煎り (ミディアムロースト) と深煎り (フレンチロースト) の豆を頂いた。次回にレビューします。 コーヒーの保存法 藤本健二…北朝鮮の最高指導者・金正日の元・専属料理人。今はピョンヤンに日本料理屋があるらしい… フィッシャーの正確確率検定 ゴールデンハムスター… ハム太郎はゴールデンハムスター。 ジャンガリアン ハム太郎 PIWIタンパク質…生殖細胞特異的に発現し、piRNAとpiRISC複合体を形成してRNAサイレンシングに寄与する。主な標的はトランスポゾン。ちなみにPiwi遺伝子を人工的に欠損させると不妊の表現型を示す。 piRNA…昔はrasiRNA (Repeat associated small interfering RNA) と呼ばれていた。2001年にハエの生殖細胞でトランスポゾンをRNAサイレンシングする因子として最初に見つかった。論文: Double-stranded RNA-mediated silencing of genomic tandem repeats and transposable elements in the D. melanogaster germline. Current Biology 2001 RNAサイレンシング機構…小さなRNA (20-30塩基) とArgonauteタンパク質から成る複合体 (RISC) が中核となって起こる遺伝子発現制御機構 PIWI-Interacting RNA: Its Biogenesis and Functions. Annual Review of Biochemistry 2015 miRNA トランスポゾン バーバラ・マクリントック…トランスポゾンの発見で1983年にノーベル賞受賞。染色体・遺伝学分野における女性研究者の草分け的存在。 ヘテロクロマチン ヒストンの機構について ハエOSC細胞 (Ovarian Somatic Cells, Saito et al., Nature 2009 モノクローナル抗体 (Wikipedia) ハイブリドーマ…kyonはこのハイブリドーマ(細胞)からモノクローナル抗体を産生させ、精製することで実験に使用することができた。 免疫沈降法 イエロールーム フリーザ Liquid-Liquid Phase Separation in Biology…最近流行りの細胞内におけるphase separation (相分離)について。 不完全性周期…マウスは４～５日の短い性周期をもつため不完全性周期動物に分類されます。一方、ヒトは３～４週の長い周期のため完全性周期動物に分類されます。ハムスターは4日です。 ウェスタンブロット法 MAX鈴木…大食い系最強YouTuber、フードファイター。 今日ヤバいやつにあった (Youtube)…kyon紹介のYoutuber Eric Lander (Wikipedia) Constructing and Screening a Recombinant DNA Library. MIT 7.01SC Fundamentals of Biology. Eric Lander (Youtube)… Eric Lander先生による超楽しいベーシックなMIT分子生物学の学部の授業。板書の美しさ、英語の聞き取りやすさ、ジョーク、小さな実験からライブラリを用いた大規模実験までを一気通貫で説明するストーリー、どれをとっても素晴らしいのでぜひ見てほしいです。 Basic Mechanisms of Cloning, excerpt 1, MIT 7.01SC Fundamentals of Biology. Eric Lander (Youtube)… こちらはクローニングの基礎編。制限酵素やライゲースの説明。おなじみのNEBのカタログも登場する。こんな授業を受けたかったなー。 NEBのカタログ…素晴らしいリソース。NEBが売り出している酵素やそのスペックを眺めているだけで楽しいし、知らない酵素や何気なく使っているバッファーに詳しくなれるし、新しい実験を思いつくヒントになるかもしれない。 Editorial notes お声掛けいただいてありがとうございました。初めてのゲスト出演、しかもトップバッター。とっても嬉しかったです！身構えてカンペ作ったけど、皆さんが話題を広げてくれて助かりました。ちなみにちょうど来週、学位審査の中間発表があるのでとても良い練習になりましたｗ (kyon) ゲスト回も楽しく収録できてよかったのと二人以上で収録する際、オーディオのルーティングの知見が溜まってよかった (soh) kyonさんありがとうございます、とても勉強になりました！これを機にyoutuberさんの動画を観てみようと思う (coela) 初期ドラえもんは尻尾を引っ張ると透明になっていたことを思い出しました。そしてcoelaさんがいきなりマウスのシメ方を話し始めて本当にサイコパスで、怖いなと思いました (tadasu)

Raul Andino joins Vincent and Amy to talk about the finding that a cricket paralysis virus protein restricts RNA-based immunity in insects by regulating the activity and stability of the Argonaute protein. Hosts: Vincent Racaniello and Amy Rosenfeld Guest: Raul Andino Subscribe (free): iTunes, Google Podcasts, RSS, email Become a patron of TWiV! Links for this episode TWiV 138: RISCy business with Raul Andino TWiV 33: Live in Philly Viral protein restricts insect immunity (Cell Host Micr) Timestamps by Jolene. Thanks! Weekly Science Picks Amy - Insectropolis Vincent- Genome-edited baby Intro music is by Ronald Jenkees. Send your virology questions and comments to twiv@microbe.tv

The TWiV Council explores the finding that facial appearance affects science communication, and evidence that RNA interference confers antiviral immunity in mammalian cells. Hosts: Vincent Racaniello, Dickson Despommier, Alan Dove, and Kathy Spindler Become a patron of TWiV! Links for this episode JMBE SciComm Issue Facial appearance affects science communication (PNAS) Your face matters to science (virology blog) RNA based antiviral immunity in mammals (Immunity) How mice say nodavirus (TWiV 245) Press release on science meeting gender disparity The Traditional Lecture is Not Dead. I Would Know – I’m A Professor (virology blog) Sound of Silence by Disturbed (YouTube) Letters read on TWiV 449 Weekly Science Picks Kathy - ASV 2017 Virolympics Crossword (pdf) Dickson - The Visible Mouse Alan - Is profitable publishing bad for science? Vincent - Locally Sourced Science Listener Pick Neva - Michael Summers interview and Virus coloring book Intro music is by Ronald Jenkees. Send your virology questions and comments to twiv@microbe.tv

Hosts: Vincent Racaniello, Dickson Despommier, Alan Dove, and Kathy Spindler The lovely TWiV team explore evolution of our fecal virome, and the antiviral RNA interference response in the nematode C. elegans.  Become a patron of TWiV! Links for this episode ASM Clinical Virology Symposium Evolution of the human fecal virome (PNAS) Antiviral RNAi response in C. elegans (Curr Biol) Orsay virus (TWiV 123) Broad VSV host range (J Comp Neurol) Image credit Letters read on TWiV 433 This episode is brought to you by Blue Apron. Blue Apron is the #1 fresh ingredient and recipe delivery service in the country. See what’s on the menu this week and get your first 3 meals free with your first purchase – WITH FREE SHIPPING – by going to blueapron.com/twiv Weekly Science Picks Alan - Contact information for Senators and Representatives Kathy - How Tumor Virology Transformed Oncology Dickson - Seven new species of Peacock Spider Vincent - Proposed NIH budget cut and Expensive Oxford Comma Listener Pick Kim - Pigeon Fashion Week (TWiEVO 7) Richard - aeroMorph and Understanding Molecular EvolutionJohnye - Ice Instruments and Fahrenheit and Celsius Intro music is by Ronald Jenkees. Send your virology questions and comments to twiv@microbe.tv

Hosts: Vincent Racaniello, Dickson Despommier, Alan Dove, Rich Condit, and Kathy Spindler The TWiVerinoes discuss the potential for prion spread by plants, global circulation patterns of influenza virus, and the roles of Argonautes and a viral protein in RNA silencing in plants. Links for this episode Prions in plants (Cell Rep) 11:35 Global influenza virus circulation (Nature) 20:40 Rough patch for plant RNA silencing (Lab Times) 29:50 Role of Argonaute proteins in plant viral defense (PLoS Path) 32:15 Rub-inoculation (YouTube) 42:45 Image credit Letters read on TWiV 343 4:00, 1:11:45 Timestamps by Jennifer. Thank you! Weekly Science Picks 1:41:45 Dickson - Global rainfall and snowfall map and A Week in the Life of RainAlan - Automated patch clampKathy - More Women in Science mini LegosRich - The Martian trailerVincent - It's a bug's life Listener Pick of the Week May - p53 by Sue Armstrong Send your virology questions and comments (email or mp3 file) to twiv@twiv.tv

RNA interference (RNAi) is a highly conserved process of gene silencing in which Argonaute family proteins are guided by small RNA molecules to complementary targets. In the fission yeast Schizosaccharomyces pombe, RNAi is required for heterochromatin formation at centromeres. Although it seems counterintuitive, pericentromeric heterochromatin in fission yeast is transcribed. The transcripts are processed by RNAi machinery, which is in turn guided back to the pericentromeric repeats by sequence complementarity of the Argonaute-bound small interfering RNA (siRNA) and the nascent transcript. This generates a positive-feedback loop of siRNA amplification that recruits factors required for the assembly of heterochromatin. Previously, it was suggested that a fission yeast class of Dicer-independent small RNAs called primal small RNAs (priRNAs) initiates the positive-feedback loop of siRNA generation and heterochromatin assembly. However, the biogenesis of priRNAs as well as of Dicer-independent small RNAs from other organisms was not well understood. The results presented here identify Triman, a novel 3’-5’ exonuclease that is involved in the final step of biogenesis of both priRNAs and siRNAs in fission yeast. It was observed that Argonaute binds longer priRNA and siRNA precursors from the total RNA fraction. This is followed by the recruitment of Triman to trim 3’ ends of Argonaute-bound small RNAs to the mature size. The final trimming of priRNAs and siRNAs is required for de novo heterochromatin formation at centromeres and the mating-type locus as well as for the maintenance of facultative heterochromatin islands. Furthermore, it was shown that in cells lacking Rrp6, a nuclease subunit of the exosome, RNAi targets various genes across the yeast genome. This demonstrated that the exosome protects the genome against aberrant RNAi. Spurious RNAi targeting in rrp6∆ cells at majority of loci occurs via accumulation of antisense transcripts that are processed into priRNAs in a Triman-dependent manner. These results suggest that Argonaute association with cellular degradation products which are processed into priRNAs might serve as a surveillance mechanism to guard the genome against invading genomic elements (Marasovic et al. 2013).

Hosts: Vincent Racaniello, Dickson Despommier, Alan Dove, Rich Condit, and Kathy Spindler Guest: Lee Feinman Lee joins the TWiV team to discuss the value of post-doctoral training, and how a cellular microRNA assists in the replication of hepatitis C virus. Links for this episode Spin by Robert Charles Wilson Immunization of preterm infants (Dev Period Med) miR-122 alters HCV translation-replication balance (Cell Host Micr) Holding a miR to HCV (TWiV 180) Treating hepatitis C by blocking miR-122 (virology blog) Click chemistry (Wikipedia) Image credit Ebolavirus sitrep (WHO) Letters read on TWiV 324 Weekly Science Picks Lee - Furthering America's Research and Upgoer five and sixAlan - Best snow shovelRich - Ripple tankKathy - FractalsDickson - World Press Photo Contest 2015Vincent - What to do about antivax politicians and physicians Listener Pick of the Week Patricia - Future of Bioscience Graduate & Postdoc trainingPaul - This is my son Griffin, and he may have measles Send your virology questions and comments (email or mp3 file) to twiv@twiv.tv

Hosts: Vincent Racaniello, Dickson Despommier, Alan Dove, Rich Condit, and Kathy Spindler Vincent, Dickson, Alan, Rich, and Kathy review two papers that present evidence for RNA interference as an antiviral immunity mechanism in mammals. Links for this episode: World Polio Day Mole Day John Holland's publications RNAi is antiviral in mammals (Science) Antiviral RNA interference in mammals (Science) RNAi, antiviral after all (Science) Is RNA interference antiviral in mammals? (Cell Host Microbe) Nodamura virus (Nature) Ebolavirus proteins suppress RNAi (J Virol) Illustrated is an siRNA (orange), dicer (top right), and argonaute (bottom) Letters read on TWiV 256 Weekly Science Picks Dickson - Wildlife Photographers of the Year 2013Kathy - John Holland's Emerging Infectious Disease lecture (YouTube)Alan - The worst part is notRich - The Universe in a Single Atom by Dalai Lama (Mind and Life Institute)Vincent - The Truth about T. Rex by Brian Switek Listener Pick of the Week Stephen - International Institute for Species Exploration (Top 10 species choice)  Send your virology questions and comments (email or mp3 file) to twiv@twiv.tv

Hosts: Vincent Racaniello and Rich Condit Guests: Robert Krug and Christopher Sullivan Vincent and Rich visit the University of Texas at Austin and meet up with Bob and Chris to talk about their work on influenza virus and microRNAs. Links for this episode: Virus-encoded microRNAs (PLoS Path) microRNA targetomes of polyomavirus (J Virol) Innate and RNAi reciprocal inhibition (Cell) ISG15 pathway (Trends Micro) Role of viral NS1 protein (Virology) Cap-snatching (Cell) Weekly Science Picks Rich - Unraveling BoleroVincent - New botulinum toxin, DURC implications, and inconvenient truths Send your virology questions and comments (email or mp3 file) to twiv@twiv.tv

Hosts: Vincent Racaniello, Alan Dove, and Rich Condit Vincent, Alan, and Rich review association of an interferon-induced protein with severe influenza, and stabilization of HCV RNA by a microRNA. Links for this episode: IFITM3 and severe influenza (Nature) Genetics of flu susceptibiligy (EurekAlert!) Stabilization of HCV RNA by Ago2-miR-122 (PNAS) Clinical trial of anti-mIR-122 TWiV on Facebook Letters read on TWiV 180 Weekly Science Picks Alan - Micro Empire (Vimeo)Rich - Census of marine lifeVincent - Pinterest Listener Pick of the Week Mark - The Secret Life of Plankton (YouTube)

Vincent meets up with Raul Andino in San Francisco to discuss the RNAi-based antiviral defense system of Drosophila, the fruit fly, and how it is antagonized by viruses.

Wed, 6 Apr 2011 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/13021/ https://edoc.ub.uni-muenchen.de/13021/1/Stoehr_Julia.pdf Stöhr, Julia Regina ddc:540, ddc:500, Fakultät für Chemie und Pharmazie

Thu, 3 Feb 2011 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/12656/ https://edoc.ub.uni-muenchen.de/12656/1/ruedel_sabine.pdf Rüdel, Sabine ddc:540, ddc:500, Fakultät für Chemie und Pharmazie

Thu, 17 Dec 2009 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/12419/ https://edoc.ub.uni-muenchen.de/12419/1/ender_christine.pdf Ender, Christine ddc:540, ddc:500, Fakultät für Chemie und Pharm

microRNAs (miRNAs) are small non-coding RNAs of 21-24 nt in size, which are endogenously expressed in higher eukaryotes and play important roles in processes such as tissue development and stress response and in several diseases including cancers. In mammals, miRNAs guide proteins of the Argonaute family (Ago proteins) to partially complementary sequences typically located in the 3’-untranslated regions (3’-UTRs) of specific target mRNAs, leading to translational repression or mRNA degradation. To gain further insight into the function of human miRNAs, we analyzed the protein as well as the RNA composition of miRNA-Ago protein complexes in molecular detail. To identify novel Ago-interacting proteins, we isolated Ago complexes and investigated them by mass spectrometry and co-immunoprecipitation experiments. We found that trinucleotide repeat-containing 6B (TNRC6B), Moloney leukemia virus 10 (MOV10), RNA binding motif protein 4 (RBM4) and Importin 8 (Imp8) interact with human Ago proteins. Moreover, using RNA interference and EGFP and dual luciferase reporter assays, we demonstrated that these factors are required for miRNA function, indicating that we have identified new components of the miRNA pathway. Intriguingly, depletion of Imp8 does not affect the levels of mature miRNAs or the interaction of miRNAs with Ago proteins, but is required for efficient association of Ago-miRNA complexes with their target mRNAs. Thus, Imp8 is the first factor acting at the level of target mRNA binding, establishing a novel layer of regulation for the miRNA pathway. Imp8 is an Importin-β-like protein, which has previously been implicated in nuclear import of substrate proteins. In line with these results, we demonstrated that a detectable fraction of Ago2 localizes to the nucleus of human cells. Moreover, knockdown of Imp8 by RNAi reduces the nuclear signal of Ago2, suggesting that Imp8 affects the nuclear localization of Ago2. Therefore, our data suggest that Imp8 has a dual function both in the cytoplasmic miRNA pathway and in nuclear transport of Ago proteins. To identify small RNAs, which associate with human Ago proteins, we isolated, cloned and sequenced small RNAs bound to Ago1 and Ago2 complexes. In addition to known miRNAs, we found several small RNAs, which derive from small nucleolar RNAs (snoRNAs). We therefore investigated the function of one particular small RNA, which is derived from the snoRNA ACA45 and showed that it functions like a miRNA. Interestingly, this small RNA is processed by the miRNA maturation factor Dicer, but does not require the microprocessor complex that is essential for processing of primary miRNA transcripts. Thus, we have identified a novel biogenesis pathway of a new class of small RNAs that can function like miRNAs. To experimentally identify mRNAs that are stably associated with miRNA-Ago protein complexes, we isolated and analyzed Ago1 and Ago2-bound mRNAs by cloning and sequencing and by microarray hybridization techniques. Using dual luciferase reporter assays, we demonstrated that many Ago-associated mRNAs are indeed miRNA targets. Therefore, we have developed a method allowing for the identification of miRNA target mRNAs from cell lines or tissues of interest independently of computational predictions. In a project that was independent of our studies on Ago protein complexes, we investigated structural and functional requirements for the activity of small interfering RNAs (siRNAs). siRNAs are small double-stranded RNAs of appr. 21 nt in size, which trigger the sequence-specific endonucleolytic degradation of perfectly complementary target transcripts upon binding to Ago2. However, both single strands of a siRNA duplex can potentially have unwanted “off-target effects” by repressing partially complementary target mRNAs through binding to their 3’-UTRs. We therefore developed a method to selectively inhibit the activity of the siRNA strand that is dispensable for target silencing (“passenger strand”) through chemical modification of its 5’-end. This method could be a useful tool for the design of highly specific siRNAs. Taken together, we have analyzed the composition of Ago-miRNA protein complexes by a variety of methods and identified novel protein factors of the miRNA pathway, a novel class of small RNAs as well as a panel of previously unknown miRNA target mRNAs. The techniques for the purification and the analysis of Ago complexes that were developed in this study will provide useful tools for future analyses of miRNA pathway factors, small RNAs and miRNA target mRNAs from any tissue or cell line of interest.