Name: fluorescence based primer extension technique to determine transcriptional starting points and cleavage sites of rnases in vivo
Uploaded: 2014-10-31T14:00:00.000+00:00
Duration: 10 min 51 s
Description: Watch this Scientific Journal Video about Fluorescence Based Primer Extension Technique to Determine Transcriptional Starting Points and Cleavage Sites of RNases In Vivo at JoVE.com

We thank Anne Wochele for her assistance in the laboratory and Vera Augsburger for help with the automated gel sequencer. We thank the Deutsche Forschungsgemeinschaft for funding by grants BE4038/2 and BE4038/5 within the &#8220;priority programmes&#8221; SPP1316 and SPP1617.

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The authors have nothing to disclose that would present a conflict of interest.

蛍光プライマー伸長はTSP-または二次RNA処理識別のためのいずれか、RNAの5 &#39;末端を決定するための簡単​​かつ迅速な方法である。原因蛍光プライマーの使用のために、反応が設定することができ、（放射性標識したプライマーの場合と異なり）追加のセキュリティ対策なしで実行されます。サンプルは、蛍光によって検出されるように、電気泳動、X線フィルムが一般的に使用される放射?...

プライマー伸長は、1つの基地解像度までの特定のRNA分子の5 &#39;末端を決定するための分子方法である。そのような5&#39;-RACE（cDNA末端の迅速増幅）のような他の方法の利点は、高速のターンアラウンドタイムと容易にRNA分子の異なる長さの混合物を分析する能力である。 この方法は、特定の長さのcDNA断片を生成する、特異的な蛍光プライマーを用いて逆転写反応するRNA分子を供することによって動作する。これらのcDNA分子は、変性ポリアクリルアミドゲル上で従来のサンガー配列決定反応2と一緒に実行される起因蛍光標識プライマーの使用に、それらの蛍光によって検出することができる。 cDNA断片の長さは、5 &#39;RNA末端のマッピングを可能にする、シークエンシングラダーとの比較によって評価される。 伝統的に、プライマー伸長反応は、組み合わせて使用​​される放射性同位体でX線フィルム上のcDNA分子を検出することができる。それらの感度が若干低いとはいえ起因する健康被害、廃棄物処理の問題や取り扱いの容易さのために、より新しいプロトコルは、自動化されたシーケンサーを持つプライマー伸長を検出するための蛍光を利用している。蛍光プライマーは（我々の手で一年以上）、長い​​間安定しているように、蛍光標識されたプライマーを用いて、定期的な放射性標識化手順は、省略することができる。 ここで記述する方法は、自動化されたゲルシーケンサーを利用したが、若干の変更を加えて、キャピラリーシーケンサーも、cDNAの分離および検出の3のために使用することができる。ゲル分析の並列性は、RNA切断または加工の小さな量を検出することが可能となる。別の利点は、検出することができる末端切断、あるいは一つの塩基の処理として、この方法は、高解像度である。 RNA切断または処理、tは検出に関してはypicallyプライマー拡張の2つのタイプが区別される。他の場合には、処理は、生体内で行われ、得られたRNAが精製されるのに対し、あるケースでは、酵素処理は、精製されたRNAおよび精製された酵素を用いてインビトロで行われる。プライマー伸長は、in vitroで実施する両方の場合において、RNAは、RNAの供給源に依存して、しかし、供され、この方法は、in vitroまたはin vivoでのプライマー伸長と呼ばれるか。ここで提示したプロトコルでは、使いやすさのために（無精製タンパク質必要）と同時に、転写開始点と処理を決定する可能性を、in vivoでのプライマー伸長のみに焦点を当てる。しかし、in vitroでのプライマー拡張は同じように設定する原則にあり、このプロトコルは、出発点として役立つことができる。 ここに示された方法は、それらが高に適しているように、多くの細菌種に適用することができ純度および核酸の高収率製剤。 私たちの研究室での研究は、毒素-抗毒素（TA-）システム4,5、プライマー伸長法が広く使用されているフィールドの規制範囲に焦点を当てています。 TA-システムが安定しており、内因的に積極的な毒性タンパク質や毒性6,7を打ち消す主に不安定なタンパク質またはRNA抗毒素で構成され、原核生物のゲノム中に存在する小さな遺伝的要素である。毒素活性は、しばしば複製の阻害、細胞壁合成、または他の機構によって作用するが、ほとんどの場合RNase活性8,9によってれる。一般的に、RNアーゼ特異性プライマー伸長法であるそのうちの一つの異なる試験を行うことによって決定される。切断された完全長断片の混合物が同時にそれらの5 &#39;末端を決定するために分析することができるように、プライマー伸長反応は、この用途に非常に適している。 、in vitroおよびin vivoプライマーエクステンション内のミックスを使用特定の毒素のRNase切断は、 例えば、配列特異性は、10-13を決定することができる。 <img alt="図1" fo:content-width="6in" src="/files/ftp_upload/52134/52134fig1highres.jpg" width="600" /> プライマー伸長手順の概要を図1。細菌培養実験のニーズに応じてインキュベートし、処理される。全RNAを、細胞から抽出したDNAの痕跡を除去するためにDNアーゼIで処理したcDNAを得た標的特異的蛍光DNAプライマーを用いて逆転写反応に供される。ゲノムDNAまたはプラスミドを抽出し、続いてcDNA断片とサイズ比較用蛍光サンガー配列決定反応のために使用される。プライマー伸長産物は、変性尿素ポリアクリルアミドゲル上サンガーシークエンシング産物と一緒に実行して、自動化されたレーザー顕微鏡で分析する。ラcDNAのバンドとラインアップしているシーケンシングベースSTの5 &#39;cDNAの端（青い矢印）のベース。 Feketeの中でより多くの情報、 ら 3 <a href="https://www.jove.com/files/ftp_upload/52134/52134fig1highres.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a> 全体のプライマー伸長手順の概要を図1に見ることができる。簡単に説明すると、細菌細胞を培養し、収穫し、細胞ペレットを溶解し、RNAを抽出した。精製されたRNAは、逆転写酵素のためのテンプレートとして作用し得るDNA分子の痕跡を除去するためにDNアーゼIで処理する。特異的な蛍光プライマーは、RNAに付加関心領域にハイブリダイズし、続いて一本鎖の相補的DNA（cDNA）の結果として、逆転写されている。配列決定ラダーは、蛍光プライマーを用いた従来のサンガー配列決定によって作成され、プライマー伸長cDNA断片の横に変性ポリアクリルアミドゲル上で分離する。結果として生じるゲルは、対象の5 &#39;末端の同定を可能にする、蛍光バンドを比較することによって分析される。転写開始点およびプロセシング部位は、次いで配列比較によって個別に評価される。

<table><tbody><tr><td>AMV Reverse Transcriptase (20-25 U/&amp;micro;l)</td><td>NEB / Roche</td><td>NEB: M0277-T / Roche: 10109118001</td><td></td></tr><tr><td>DNase I (RNase free)</td><td>Ambion (life technologies)</td><td>AM2222</td><td></td></tr><tr><td>FastPrep-24 Instrument&amp;nbsp;</td><td>MPBio</td><td>116004500</td><td></td></tr><tr><td>Fluorescently labeled primers</td><td>Biomers</td><td>n/a</td><td>5&amp;rsquo; DY-681 modification of &amp;ldquo;ordinary&amp;rdquo; DNA oligonucleotides. Compatible dyes such as the LICOR IRDye 700/800 are also available from other suppliers such as IDTdna.</td></tr><tr><td>Li-Cor 4200 Sequencer incl. ImagIR Data collection software</td><td>Li-Cor</td><td></td><td>Product discontinued</td></tr><tr><td>NanoDrop 2000</td><td>Thermo Scientific</td><td></td><td></td></tr><tr><td>Nuclease free water</td><td>Ambion (life technologies)</td><td>AM9915G</td><td></td></tr><tr><td>Plasmid mini preparation kit</td><td>QIAGEN</td><td>12125</td><td></td></tr><tr><td>RapidGel-XL-40% Concentrate</td><td>USB</td><td>US75863</td><td></td></tr><tr><td>RNA STORAGE BUFFER</td><td>Ambion (life technologies)</td><td>AM7000</td><td></td></tr><tr><td>Roti-Aqua-P/C/I</td><td>Carl Roth</td><td>X985.3</td><td>Alternative: &amp;ldquo;Acid-Phenol:Chloroform, pH 4.5 (with IAA, 125:24:1)&amp;rdquo; from Ambion (AM9720)</td></tr><tr><td>SUPERase&amp;bull;In RNase Inhibitor</td><td>Ambion (life technologies)</td><td>AM2696</td><td></td></tr><tr><td>Thermo Sequenase fluorescently labelled primer cycle sequencing kit with 7-deaza-dGTP</td><td>GE Healthcare</td><td>RPN2538</td><td></td></tr><tr><td>TRIzol reagent</td><td>life technologies</td><td>15596-026</td><td></td></tr><tr><td>Zirconia/Silica Beads 0.1 mm</td><td>BioSpec</td><td>11079101z</td><td></td></tr></tbody></table>

fluorescence based primer extension technique to determine transcriptional starting points and cleavage sites of rnases in vivo

蛍光ベースのプライマー伸長（FPE）は、転写開始点またはRNA分子のプロセシング部位を決定するための分子方法である。これは、特定の蛍光標識プライマーおよび変性ポリアクリルアミドゲル電気泳動によって得られたcDNA断片のその後の分析を使用して、目的のRNAを逆転写することによって達成される。同時に、従来のサンガー配列決定反応は、正確な対応するベースにcDNA断片の末端をマッピングするためにゲル上で実行される。生成物をクローン化しなければならず、複数の候補を配列決定5&#39;-RACE（cDNA末端の迅速増幅）とは対照的に、プライマー伸長によって生成されたcDNA断片の大部分は、同時に1つのゲルの実行を検出することができる。また、（逆転写の結果の最終分析に）全体の手順は、1営業日で完了することができる。蛍光標識されたプライマーを用いることによって、危険な放射​​性同位体の使用は、試薬を標識回避することができ、生成物は、電気泳動手順中に検出することができるように、処理時間が低減される。 以下のプロトコールでは、確実かつ迅速にS.（例えば、毒素-抗毒素システムコンポーネントによって）開始点及びRNAプロセッシング部位、転写を推定するRNAの5 &#39;末端を検出するためのin vivo蛍光プライマー伸長方法を記載球菌、E。 coliおよび他の細菌。

図6に示されるように、プライマー伸長反応は、目的の転写物の転写開始点を決定するために用いることができる（通常-10および-35要素によって識別される）のプロモーター領域を推定するのを助けることができる。一番上の（最長）のcDNA断片は、mRNAの5 &#39;末端を表し、従って、配列決定ラダーと比較した場合に容易にマッピングすることができる。 <img al...

Watch this Scientific Journal Video about Fluorescence Based Primer Extension Technique to Determine Transcriptional Starting Points and Cleavage Sites of RNases In Vivo at JoVE.com

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

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

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

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

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Research

JoVE Journal

Biology

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

26.9K ビュー.  University of Tübingen. We here describe a fluorescence based primer extension method to determine transcriptional starting points from bacterial transcripts and RNA processing in vivo using an automated gel sequencer.

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

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

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

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

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

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

遺伝学

Prediction and Validation of Gene Regulatory Elements Activated During Retinoic Acid Induced Embryonic Stem Cell Differentiation

Embryonic development is a multistep process involving activation and repression of many genes. Enhancer elements in the genome are known to contribute to tissue and cell-type specific regulation of gene expression during the cellular differentiation. Thus, their identification and further investigation is important in order to understand how cell fate is determined. Integration of gene expression data (e.g., microarray or RNA-seq) and results of chromatin immunoprecipitation (ChIP)-based genome-wide studies (ChIP-seq) allows large-scale identification of these regulatory regions. However, functional validation of cell-type specific enhancers requires further in vitro and in vivo experimental procedures. Here we describe how active enhancers can be identified and validated experimentally. This protocol provides a step-by-step workflow that includes: 1) identification of regulatory regions by ChIP-seq data analysis, 2) cloning and experimental validation of putative regulatory potential of the identified genomic sequences in a reporter assay, and 3) determination of enhancer activity in vivo by measuring enhancer RNA transcript level. The presented protocol is detailed enough to help anyone to set up this workflow in the lab. Importantly, the protocol can be easily adapted to and used in any cellular model system.

In this work we provide an experimental workflow of how active enhancers can be identified and experimentally validated.

レチノイン酸誘導された胚性幹細胞分化の間に活性化された予測と遺伝子調節要素の検証

Embryonic development is a multistep process involving activation and repression of many genes. Enhancer elements in the genome are known to contribute to ...

発生生物学

Toeprinting Analysis of Translation Initiation Complex Formation on Mammalian mRNAs

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

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

哺乳類の Mrna の翻訳開始複合体形成の Toeprinting 分析

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

生化学

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

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

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

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

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

Monitoring Protein-RNA Interaction Dynamics In Vivo at High Temporal Resolution Using &#967;CRAC

The interaction between RNA-binding proteins (RBPs) and their RNA substrates exhibits fluidity and complexity. Within its lifespan, a single RNA can be bound by many different RBPs that will regulate its production, stability, activity, and degradation. As such, much has been done to understand the dynamics that exist between these two types of molecules. A particularly important breakthrough came with the emergence of &#8216;cross-linking and immunoprecipitation&#8217; (CLIP). This technique allowed stringent investigation into which RNAs are bound by a particular RBP. In short, the protein of interest is UV cross-linked to its RNA substrates in vivo, purified under highly stringent conditions, and then the RNAs covalently cross-linked to the protein are converted into cDNA libraries and sequenced. Since its conception, many derivative techniques have been developed in order to make CLIP amenable to particular fields of study. However, cross-linking using ultraviolet light is notoriously inefficient. This results in extended exposure times that make the temporal study of RBP-RNA interactions impossible. To overcome this issue, we recently designed and built much-improved UV irradiation and cell harvesting devices. Using these new tools, we developed a protocol for time-resolved analyses of RBP-RNA interactions in living cells at high temporal resolution: Kinetic CRoss-linking and Analysis of cDNAs (&#967;CRAC). We recently used this technique to study the role of yeast RBPs in nutrient stress adaptation. This manuscript provides a detailed overview of the &#967;CRAC method and presents recent results obtained with the Nrd1 RBP.

Kinetic cross-linking and analysis of cDNA is a method that allows investigation of the dynamics of protein-RNA interactions in living cells at high temporal resolution. Here the protocol is described in detail, including the growth of yeast cells, UV cross-linking, harvesting, protein purification, and next generation sequencing library preparation steps.

χCRACを用いたin vivoでのタンパク質-RNA相互作用ダイナミクスの高時間分解能モニタリング

The interaction between RNA-binding proteins (RBPs) and their RNA substrates exhibits fluidity and complexity. Within its lifespan, a single RNA can be ...

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

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

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

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

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

生物学

iCLIP - Transcriptome-wide Mapping of Protein-RNA Interactions with Individual Nucleotide Resolution

The unique composition and spatial arrangement of RNA-binding proteins (RBPs) on a transcript guide the diverse aspects of post-transcriptional regulation1. Therefore, an essential step towards understanding transcript regulation at the molecular level is to gain positional information on the binding sites of RBPs2.
Protein-RNA interactions can be studied using biochemical methods, but these approaches do not address RNA binding in its native cellular context. Initial attempts to study protein-RNA complexes in their cellular environment employed affinity purification or immunoprecipitation combined with differential display or microarray analysis (RIP-CHIP)3-5. These approaches were prone to identifying indirect or non-physiological interactions6. In order to increase the specificity and positional resolution, a strategy referred to as CLIP (UV cross-linking and immunoprecipitation) was introduced7,8. CLIP combines UV cross-linking of proteins and RNA molecules with rigorous purification schemes including denaturing polyacrylamide gel electrophoresis. In combination with high-throughput sequencing technologies, CLIP has proven as a powerful tool to study protein-RNA interactions on a genome-wide scale (referred to as HITS-CLIP or CLIP-seq)9,10. Recently, PAR-CLIP was introduced that uses photoreactive ribonucleoside analogs for cross-linking11,12.
Despite the high specificity of the obtained data, CLIP experiments often generate cDNA libraries of limited sequence complexity. This is partly due to the restricted amount of co-purified RNA and the two inefficient RNA ligation reactions required for library preparation. In addition, primer extension assays indicated that many cDNAs truncate prematurely at the crosslinked nucleotide13. Such truncated cDNAs are lost during the standard CLIP library preparation protocol. We recently developed iCLIP (individual-nucleotide resolution CLIP), which captures the truncated cDNAs by replacing one of the inefficient intermolecular RNA ligation steps with a more efficient intramolecular cDNA circularization (Figure 1)14. Importantly, sequencing the truncated cDNAs provides insights into the position of the cross-link site at nucleotide resolution. We successfully applied iCLIP to study hnRNP C particle organization on a genome-wide scale and assess its role in splicing regulation14.

The spatial arrangement of RNA-binding proteins on a transcript is a key determinant of post-transcriptional regulation. Therefore, we developed individual-nucleotide resolution UV crosslinking and immunoprecipitation (iCLIP) that allows precise genome-wide mapping of the binding sites of an RNA-binding protein.

iCLIP - トランスクリプトーム全体の個々のヌクレオチド分解能タンパク質- RNA相互作用のマッピング

The unique composition and spatial arrangement of RNA-binding proteins (RBPs) on a transcript guide the diverse aspects of post-transcriptional ...

Measuring the Kinetics of mRNA Transcription in Single Living Cells

The transcriptional activity of RNA polymerase II (Pol II) is a dynamic process and therefore measuring the kinetics of the transcriptional process in vivo is of importance. Pol II kinetics have been measured using biochemical or molecular methods.1-3 In recent years, with the development of new visualization methods, it has become possible to follow transcription as it occurs in real time in single living cells.4 Herein we describe how to perform analysis of Pol II elongation kinetics on a specific gene in living cells.5, 6 Using a cell line in which a specific gene locus (DNA), its mRNA product, and the final protein product can be fluorescently labeled and visualized in vivo, it is possible to detect the actual transcription of mRNAs on the gene of interest.7, 8 The mRNA is fluorescently tagged using the MS2 system for tagging mRNAs in vivo, where the 3'UTR of the mRNA transcripts contain 24 MS2 stem-loop repeats, which provide highly specific binding sites for the YFP-MS2 coat protein that labels the mRNA as it is transcribed.9 To monitor the kinetics of transcription we use the Fluorescence Recovery After Photobleaching (FRAP) method. By photobleaching the YFP-MS2-tagged nascent transcripts at the site of transcription and then following the recovery of this signal over time, we obtain the synthesis rate of the newly made mRNAs.5 In other words, YFP-MS2 fluorescence recovery reflects the generation of new MS2 stem-loops in the nascent transcripts and their binding by fluorescent free YFP-MS2 molecules entering from the surrounding nucleoplasm. The FRAP recovery curves are then analyzed using mathematical mechanistic models formalized by a series of differential equations, in order to retrieve the kinetic time parameters of transcription.

RNA polymerase II transcriptional kinetics are measured on specific genes in living cells. mRNAs transcribed from the gene of interest are fluorescently tagged and using Fluorescence Recovery After Photobleaching (FRAP) the in vivo kinetics of transcriptional elongation are obtained.

単一生細胞におけるmRNAの転写の動態を測定

The transcriptional activity of RNA polymerase II (Pol II) is a dynamic process and therefore measuring the kinetics of the transcriptional process in ...

In Situ Hybridization for the Precise Localization of Transcripts in Plants

With the advances in genomics research of the past decade, plant biology has seen numerous studies presenting large-scale quantitative analyses of gene expression. Microarray and next generation sequencing approaches are being used to investigate developmental, physiological and stress response processes, dissect epigenetic and small RNA pathways, and build large gene regulatory networks1-3. While these techniques facilitate the simultaneous analysis of large gene sets, they typically provide a very limited spatiotemporal resolution of gene expression changes. This limitation can be partially overcome by using either profiling method in conjunction with lasermicrodissection or fluorescence-activated cell sorting4-7. However, to fully understand the biological role of a gene, knowledge of its spatiotemporal pattern of expression at a cellular resolution is essential. Particularly, when studying development or the effects of environmental stimuli and mutants can the detailed analysis of a gene's expression pattern become essential. For instance, subtle quantitative differences in the expression levels of key regulatory genes can lead to dramatic phenotypes when associated with the loss or gain of expression in specific cell types. 
Several methods are routinely used for the detailed examination of gene expression patterns. One is through analysis of transgenic reporter lines. Such analysis can, however, become time-consuming when analyzing multiple genes or working in plants recalcitrant to transformation. Moreover, an independent validation to ensure that the transgene expression pattern mimics that of the endogenous gene is typically required. Immunohistochemical protein localization or mRNA in situ hybridization present relatively fast alternatives for the direct visualization of gene expression within cells and tissues. The latter has the distinct advantage that it can be readily used on any gene of interest. In situ hybridization allows detection of target mRNAs in cells by hybridization with a labeled anti-sense RNA probe obtained by in vitro transcription of the gene of interest. 
Here we outline a protocol for the in situ localization of gene expression in plants that is highly sensitivity and specific. It is optimized for use with paraformaldehyde fixed, paraffin-embedded sections, which give excellent preservation of histology, and DIG-labeled probes that are visualized by immuno-detection and alkaline-phosphatase colorimetric reaction. This protocol has been successfully applied to a number of tissues from a wide range of plant species, and can be used to analyze expression of mRNAs as well as small RNAs8-14.

In Situ Hybridization for the Precise Localization of Transcripts in Plants

The in situ hybridization protocol described here allows a direct localization of mRNA and small RNA expression at the cellular level with high sensitivity and specificity. The procedure is optimized for paraffin-embedded plant tissue sections, is applicable to a wide range of plants and tissues, and can be completed within ten days.

植物の転写産物の正確な位置のためのin situハイブリダイゼーション

With the advances in genomics research of the past decade, plant biology has seen numerous studies presenting large-scale quantitative analyses of gene ...

A Fluorescence-based Exonuclease Assay to Characterize DmWRNexo, Orthologue of Human Progeroid WRN Exonuclease, and Its Application to Other Nucleases

WRN exonuclease is involved in resolving DNA damage that occurs either during DNA replication or following exposure to endogenous or exogenous genotoxins. It is likely to play a role in preventing accumulation of recombinogenic intermediates that would otherwise accumulate at transiently stalled replication forks, consistent with a hyper-recombinant phenotype of cells lacking WRN. In humans, the exonuclease domain comprises an N-terminal portion of a much larger protein that also possesses helicase activity, together with additional sites important for DNA and protein interaction. By contrast, in Drosophila, the exonuclease activity of WRN (DmWRNexo) is encoded by a distinct genetic locus from the presumptive helicase, allowing biochemical (and genetic) dissection of the role of the exonuclease activity in genome stability mechanisms. Here, we demonstrate a fluorescent method to determine WRN exonuclease activity using purified recombinant DmWRNexo and end-labeled fluorescent oligonucleotides. This system allows greater reproducibility than radioactive assays as the substrate oligonucleotides remain stable for months, and provides a safer and relatively rapid method for detailed analysis of nuclease activity, permitting determination of nuclease polarity, processivity, and substrate preferences.

Exonucleases play critical roles in ensuring genome stability. Loss of WRN exonuclease function results in premature aging. Studying substrates and other requirements of the nuclease in vitro can help elucidate its role in vivo. Here we demonstrate a rapid and reproducible fluorescence-based assay to measure its nuclease activity.

WRN exonuclease is involved in resolving DNA damage that occurs either during DNA replication or following exposure to endogenous or exogenous genotoxins. ...

転写開始点を決定するための蛍光に基づくプライマー伸長技術とのRNaseの切断サイト

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