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.

Floresan primer uzatma TSP- veya ikincil bir RNA işleme tanımlanması için iki, RNA&#39;ların 5 &#39;uçlarını belirlemek için basit ve hızlı bir yöntemdir. Dolayı floresan primerlerin kullanımı ile reaksiyonlar, ayarlanabilir ve (radyoaktif olarak etiketlenmiş primerler aksine olarak) ilave güvenlik önlemleri olmadan çalışabilir. Numuneler floresan ile tespit edilir elektroforez X-ışını filmleri yaygın olarak kullanılan radyoaktif yöntemlerle karşılaştırıldığında hızlı bir analizine ...

Primer uzatma 1 bir taban çözünürlüğe kadar özel bir RNA moleküllerinin 5 &#39;ucunun saptanması için moleküler bir yöntemdir. 5&#39;-RACE (cDNA uçlarının hızlı yükseltilmesi) gibi diğer yöntemlerin bir avantajı, hızlı bir geri dönüş süresi ve kolay bir RNA moleküllerinin farklı uzunluklarının bir karışımı analiz yeteneğidir. Bu yöntem, RNA molekülleri, belirli uzunluktaki cDNA fragmanlarının üretilmesi, özel bir flüoresan primerler kullanılarak transkripsiyon reaksiyonlarını ters tabi tutarak çalışır. Bu cDNA molekülleri denatüre edici poliakrilamid jelleri üzerinde geleneksel Sanger dizileme reaksiyonlarının 2 yanına uygulanır ve bağlı floresan etiketli primerlerin kullanımı ile fluoresansları ile tespit edilebilir. CDNA fragmanlarının uzunlukları 5 &#39;RNA uçlarının eşleşmesini sağlayan bir sıralama merdiven karşılaştırılarak değerlendirilir. Geleneksel olarak, primer uzatma reaksiyonları bağlantılı olarak kullanılanradyoaktif izotoplar X-ray filmlerde cDNA molekülleri tespit etmek. Onların duyarlılığı biraz daha düşük de olsa dolayı sağlık tehlikeleri, atık bertaraf sorunları ve kullanım kolaylığı, yeni protokoller, otomatik dizicilerle ile primer uzatma tespiti için floresan kullanmaktadır. Floresan primerler (bizim elimizde bir yıldan daha fazla), uzun bir süre için kararlı olduğu gibi, floresanla işaretlenmiş primerler kullanılarak, tekrar eden radyo-etiketleme prosedürü, ihmal edilebilir. Burada açıklandığı gibi bir yöntem otomatik bir jel sekanslayıcı kullanır, ancak küçük değişiklikler yapılarak, kapillerli da cDNA ayrılması ve keşfi 3 için kullanılabilir. Jel analizi paralel yapısı mümkün RNA bölünme ve işlem daha az miktarda tespit edilebilir. Diğer bir avantajı tespit edilebilir terminali bölünme ya da bir bazın işleme gibi, bu yöntemin yüksek çözünürlüktür. RNA bölünme ya da işlem, t tespiti ile ilgili olarakprimer uzantıları ypically iki farklı tür ayırt edilir. Diğer bir durumda, işlem, in vivo olarak yapılması ve sonuçta elde edilen RNA saflaştırılmıştır ise Bir durumda, olan enzimatik işlem, saflaştırılmış RNA ve arıtılmış enzim kullanılarak in vitro olarak yapılır. Bir primer uzatma in vitro olarak, her iki durumda da RNA RNA kaynağına bağlı olarak, ancak, tabi tutulur, bu yöntem, bir in vitro veya in vivo olarak, primer genişlemesini, adlandırılır ya. Burada mevcut protokol, bunların kullanım kolaylığı (herhangi bir saflaştırılmış protein için) ve aynı zamanda kopyalama başlangıç ​​noktaları ve belirlemek için olasılığı, sadece, in vivo bir primer uzatma odaklanır. Bununla birlikte in vitro primer uzantıları ile aynı şekilde ayarlanır, prensip olarak ve bu protokol, bir başlangıç ​​noktası olarak görev yapabilir. Burada gösterilen yöntem sürece yüksek giriş oranlı gibi pek çok bakteri türleri uygulanabilirNükleik asitlerin saflığı ve yüksek verimli hazırlanması. Laboratuarımızda araştırma toksin antitoksin (TA-) sistemler 4,5, primer uzatma yöntemi yaygın olarak kullanıldığı bir alanın düzenleme kapsamında odaklanmaktadır. TA-sistemleri istikrarlı ve endojen aktif toksik protein ve toksisite 6,7 zıt bir çoğunlukla kararsız protein ya da RNA antitoksin oluşmaktadır prokaryotik genomları mevcut küçük genetik unsurlardır. Toksin aktivitesi bazen RNaz etkinliğinin 8,9 En sık replikasyonu, hücre çeperi sentezi veya başka mekanizmalar inhibisyonu tarafından uygulanan, ancak. Tipik haliyle, RNaz özgüllük primer uzatma yöntemdir biri farklı testler, iletken ile belirlenir. Parçalanır ve tam uzunlukta parçalarının bir karışımı, aynı zamanda, 5 &#39;ucunun saptanması için analiz edilebilir Primer uzatma reaksiyonları, bu uygulama için uygundur. In vitro ve in vivo primeri uzantılarında bir karışımını kullanarak,spesifik toksin RNaz ayrılma, örneğin, sekans özgüllük 10-13 belirlenebilir. <img alt="Şekil 1," fo:content-width="6in" src="/files/ftp_upload/52134/52134fig1highres.jpg" width="600" /> . Primer uzatma prosedürünün 1. Genel Bakış Şekil Bakteriyel kültürler deneysel ihtiyaçlarına göre kuluçkaya ve tedavi edilir. Toplam RNA, hücre cDNA elde edildi hedefe özgü floresan DNA primerler kullanılarak bir ters transkripsiyon reaksiyonu, DNA izlerini ortadan kaldırmak için DNase I ile işleme tabi tutuldu ve tabi elde edilir. Genomik DNA ya da plazmidler ekstre edilmiş ve daha sonra cDNA fragmanları ile boyut karşılaştırma için floresan Sanger dizileme reaksiyonları için kullanılır. Primer uzatma ürünleri denatüre üre poliakrilamid jel üzerinde Sanger dizileme ürünlerin yanında çalıştırmak ve otomatik lazer ve mikroskop ile analiz edilir. La cDNA bandı ile çizgiler kadar olan sıralama bazSt olarak 5 &#39;cDNA ucu (mavi ok) tabanı. Fekete daha fazla bilgi, vd. 3 <a href="https://www.jove.com/files/ftp_upload/52134/52134fig1highres.jpg" target="_blank">Bu rakamın büyük bir versiyonunu görmek için lütfen buraya tıklayınız.</a> Bütün primer uzatma işlemi ile ilgili bilgi, Şekil 1 &#39;de bulunabilir. Kısaca, bakteri hücreleri, hasat edilir, kültürlenir, hücre topağı eritilir ve RNA ekstre edildi. Arındırılmış RNA daha sonra ters transkriptaz için şablon olarak hareket edebilir DNA moleküllerinin izlerini ortadan kaldırmak için DNase I ile işleme tabi tutulur. Özel flüoresan primerler ilgi bölgeye hibridize ve daha sonra tek sarmallı Tamamlayıcı DNA (cDNA) &#39;de elde edilen, ters transkribe edilmiş RNA ilave edilir. Bir sıralama merdiven floresan primer kullanılarak, geleneksel Sanger dizileme ile oluşturulan ve primer uzatma cDNA parçaları ile birlikte bir denatüre edici poliakrilamid jeli üzerinde ayrılır. Elde edilenJel ilgi 5 &#39;uçlarının tanımlanmasına izin veren, floresan bantları karşılaştırılarak analiz edilir. Transkripsiyonal başlangıç ​​noktaları ve işleme siteleri daha sonra sırası karşılaştırmaları tek tek değerlendirilir.

<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

Flüoresan göre primer uzatma (FPE) transkripsiyonel başlangıç ​​noktalarının ya da RNA moleküllerinin işlenmesi bölgelerini belirlemek için moleküler bir yöntemdir. Bu özel floresanla işaretlenmiş primerler ve poliakrilamid jel elektroforezi ile denaturasyon ile elde edilen cDNA parçaları daha sonra analizi kullanılarak, ilgi konusu RNA&#39;nın ters transkripsiyonu ile elde edilir. Aynı zamanda, geleneksel bir Sanger dizileme Reaksiyon bunların tam karşılık gelen tabanlarına cDNA fragmanlarının uçları eşlemek için jel üzerinde çalıştırılır. Ürün klonlanmış olması ve çok sayıda aday dizilenmiştir 5&#39;-RACE (cDNA uçlarının hızlı büyütülmesi), aksine, primer uzama yolu ile üretilen bir cDNA fragmanlarının toplu eşzamanlı olarak bir jel vadede tespit edilebilir. Buna ek olarak, (sonuçların son analizde, ters transkripsiyon ile ilgili) bütün işlem bir iş günü içinde tamamlanabilir. Floresanla işaretlenmiş primerler kullanılarak, tehlikeli radyoaktif izotop kullanımı reaktifler etiketliönlenebilir ve ürünler elektroforez işlemi sırasında tespit edilebilir işlem süreleri azaltılır. Aşağıdaki protokol, güvenilir ve hızlı S. (örneğin, toksin antitoksin sistem bileşenleri ile) bir başlangıç ​​noktası ve RNA işleme the transkripsiyonel anlamak için RNA&#39;ların 5 &#39;uçlarını tespit etmek için, bir in vivo floresan primer uzatma yöntemi tarif S. aureus, E. E. coli ve diğer bakteriler.

Şekil 6&#39;da gösterildiği gibi, bir primer uzatma reaksiyonu ilgi transkript transkripsiyonel başlangıç ​​noktalarını belirlemek için kullanılabilir ve (tipik olarak -10 ila -35 elemanlar tarafından tanımlanan) hızlandırıcı bölgeler anlamak için yardımcı olabilir. En üstteki (uzun) cDNA parçası, mRNA 5 &#39;ucunu temsil eder ve bu sıralama merdiven göre kolayca eşlenebilir. <img alt="Şekil 6," src="/files/ftp_upload/52134/52134fig6highres.jpg"...

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.

Floresan Bazlı Astar Uzatma Tekniği Transkripsiyonal Başlangıç ​​Noktalarını ve RNases yarılması Siteleri belirleme<em&gt; In Vivo</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

27.0K Görüntüleme.  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.

Video: 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.

Gerçek zamanlı analiz bağlama, transkripsiyon, çeviri ve ciro hücresel etkinleştirme sırasında küresel olayları görüntülemek üzere transkripsiyon faktörü

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

Genetik

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.

Tahmin ve Gen Düzenleme Elementlerin Doğrulama Retinoik Asit Kaynaklı Embriyonik sırasında Aktif Hücre Farklılaşma Kök

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

Gelişim Biyolojisi

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.

Çeviri başlatma karmaşık oluşumu memeli mRNA'ların Toeprinting analizi

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

Biyokimya

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.

Diseksiyon eş transkripsiyon RNA Işleme etkinlikleri için suni RNA polimeraz II uzama kompleksler

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 Kullanarak Yüksek Zamansal Çözünürlükte Protein-RNA Etkileşim Dinamiğinin İn Vivo İzlenmesi

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

Practical Aspects of Sample Preparation and Setup of 1H R1&#961; Relaxation Dispersion Experiments of RNA

RNA is a highly flexible biomolecule, wherein changes in structures play crucial roles in the functions that RNA molecules execute as cellular messengers and modulators. While these dynamic states remain hidden to most structural methods, R1&#961; relaxation dispersion (RD) spectroscopy allows the study of conformational dynamics in the micro- to millisecond regime at atomic resolution. The use of 1H as the observed nucleus further expands the time regime covered and gives direct access to hydrogen bonds and base pairing.
The challenging steps in such a study are high-purity and high-yield sample preparation, potentially 13C- and 15N-labeled, as well as setup of experiments and fitting of data to extract population, exchange rate, and secondary structure of the previously invisible state. This protocol provides crucial hands-on steps in sample preparation to ensure the preparation of a suitable RNA sample and setup of 1H R1&#961; experiments with both isotopically labeled and unlabeled RNA samples.

We present a protocol to measure micro- to millisecond dynamics on 13C/15N-labeled and unlabeled RNA with 1H R1&#961; relaxation dispersion nuclear magnetic resonance (NMR) spectroscopy. The focus of this protocol lies in high-purity sample preparation and setup of NMR experiments.

RNA'nın 1 H R1φ Gevşeme Dispersiyon Deneylerinin Numune Hazırlama ve Kurulumunun Pratik Yönleri

RNA is a highly flexible biomolecule, wherein changes in structures play crucial roles in the functions that RNA molecules execute as cellular messengers ...

A Reporter Based Cellular Assay for Monitoring Splicing Efficiency

During gene expression, the vital step of pre-mRNA splicing involves accurate recognition of splice sites and efficient assembly of spliceosomal complexes to join exons and remove introns prior to cytoplasmic export of the mature mRNA. Splicing efficiency can be altered by the presence of mutations at splice sites, the influence of trans-acting splicing factors, or the activity of therapeutics. Here, we describe the protocol for a cellular assay that can be applied for monitoring the splicing efficiency of any given exon. The assay uses an adaptable plasmid encoded 3-exon/2-intron minigene reporter, which can be expressed in mammalian cells by transient transfection. Post-transfection, total cellular RNA is isolated, and the efficiency of exon splicing in the reporter mRNA is determined by either primer extension or semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR). We describe how the impact of disease associated 5&#8242; splice-site mutations can be determined by introducing them in the reporter; and how the suppression of these mutations can be achieved by co-transfection with U1 small nuclear RNA (snRNA) construct carrying compensatory mutations in its 5&#8242; region that basepairs with the 5&#8242;-splice sites at exon-intron junctions in pre-mRNAs. Thus, the reporter can be used for the design of therapeutic U1 particles to improve recognition of mutant 5&#8242; splice-sites. Insertion of cis-acting&#160;regulatory sites, such as splicing enhancer or silencer sequences, into the reporter can also be used to examine the role of U1 snRNP in regulation mediated by a specific alternative splicing factor. Finally, reporter expressing cells can be incubated with small molecules to determine the effect of potential therapeutics on constitutive pre-mRNA splicing or on exons carrying mutant 5&#8242; splice sites. Overall, the reporter assay can be applied to monitor splicing efficiency in a variety of conditions to study fundamental splicing mechanisms and splicing-associated diseases.

This protocol describes a minigene reporter assay to monitor the impact of 5&#180;-splice site mutations on splicing and develops suppressor U1 snRNA for the rescue of mutation-induced splicing inhibition. The reporter and suppressor U1 snRNA constructs are expressed in HeLa cells, and splicing is analyzed by primer extension or RT-PCR.

Birleştirme Verimliliğini İzlemek için Muhabir Tabanlı Hücresel Test

During gene expression, the vital step of pre-mRNA splicing involves accurate recognition of splice sites and efficient assembly of spliceosomal complexes ...

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-Klip - RNA Cilt Proteinler Transcriptome geniş Cilt Siteleri Belirlemek Amacıyla Bir Yöntem

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

Biyoloji

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 - Bireysel Nükleotid Çözünürlük protein-RNA Etkileşimleri Transcriptome geniş Haritalama

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.

Tek Yaşayan Hücreler mRNA Transkripsiyon Kinetik Ölçüm

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

Floresan Bazlı Astar Uzatma Tekniği Transkripsiyonal Başlangıç Noktalarını ve RNases yarılması Siteleri belirleme

Özet

Daha Fazla Video Keşfet

Gerçek zamanlı analiz bağlama, transkripsiyon, çeviri ve ciro hücresel etkinleştirme sırasında küresel olayları görüntülemek üzere transkripsiyon faktörü

Tahmin ve Gen Düzenleme Elementlerin Doğrulama Retinoik Asit Kaynaklı Embriyonik sırasında Aktif Hücre Farklılaşma Kök

Çeviri başlatma karmaşık oluşumu memeli mRNA'ların Toeprinting analizi

Diseksiyon eş transkripsiyon RNA Işleme etkinlikleri için suni RNA polimeraz II uzama kompleksler

χCRAC Kullanarak Yüksek Zamansal Çözünürlükte Protein-RNA Etkileşim Dinamiğinin İn Vivo İzlenmesi

RNA'nın 1 H R_1φ Gevşeme Dispersiyon Deneylerinin Numune Hazırlama ve Kurulumunun Pratik Yönleri

Birleştirme Verimliliğini İzlemek için Muhabir Tabanlı Hücresel Test

PAR-Klip - RNA Cilt Proteinler Transcriptome geniş Cilt Siteleri Belirlemek Amacıyla Bir Yöntem

iCLIP - Bireysel Nükleotid Çözünürlük protein-RNA Etkileşimleri Transcriptome geniş Haritalama

Tek Yaşayan Hücreler mRNA Transkripsiyon Kinetik Ölçüm

Floresan Bazlı Astar Uzatma Tekniği Transkripsiyonal Başlangıç ​​Noktalarını ve RNases yarılması Siteleri belirleme

Özet

Daha Fazla Video Keşfet

Gerçek zamanlı analiz bağlama, transkripsiyon, çeviri ve ciro hücresel etkinleştirme sırasında küresel olayları görüntülemek üzere transkripsiyon faktörü

Tahmin ve Gen Düzenleme Elementlerin Doğrulama Retinoik Asit Kaynaklı Embriyonik sırasında Aktif Hücre Farklılaşma Kök

Çeviri başlatma karmaşık oluşumu memeli mRNA'ların Toeprinting analizi

Diseksiyon eş transkripsiyon RNA Işleme etkinlikleri için suni RNA polimeraz II uzama kompleksler

χCRAC Kullanarak Yüksek Zamansal Çözünürlükte Protein-RNA Etkileşim Dinamiğinin İn Vivo İzlenmesi

RNA'nın 1 H R1φ Gevşeme Dispersiyon Deneylerinin Numune Hazırlama ve Kurulumunun Pratik Yönleri

Birleştirme Verimliliğini İzlemek için Muhabir Tabanlı Hücresel Test

PAR-Klip - RNA Cilt Proteinler Transcriptome geniş Cilt Siteleri Belirlemek Amacıyla Bir Yöntem

iCLIP - Bireysel Nükleotid Çözünürlük protein-RNA Etkileşimleri Transcriptome geniş Haritalama

Tek Yaşayan Hücreler mRNA Transkripsiyon Kinetik Ölçüm

Floresan Bazlı Astar Uzatma Tekniği Transkripsiyonal Başlangıç Noktalarını ve RNases yarılması Siteleri belirleme

RNA'nın 1 H R_1φ Gevşeme Dispersiyon Deneylerinin Numune Hazırlama ve Kurulumunun Pratik Yönleri