Name: novel rna-binding proteins isolation by the rapid methodology
Uploaded: 2016-09-30T14:00:00
Description: Watch this Scientific Journal Video about Novel RNA-Binding Proteins Isolation by the RaPID Methodology at JoVE.com

Biz hızlı protokol kurma ve gerekli plazmitleri sağlayarak onların yararlı tavsiyeler Prof. Jeff Gerst ve Boris Slobodin teşekkür ederiz. Biz de LC-MS / MS analizi ile ona yardım için Smoler Proteomiks Merkezi&#39;nden bu protokolü Dr. Tamar Ziv kurulması ona yardım için Dr. Avigail Atir-Lande teşekkür ederim. Biz YEF3 antikor Prof. TG Kinzy (Rutgers) teşekkür ederim. Bu çalışma Binational Bilim Vakfı hibe 2011013 tarafından desteklenmiştir.

Not: 12 MS2 bağlayıcı siteleri kapsayan bir dizi takın (MS2 döngüler; MS2L) istenen genomik içine, genellikle, açık okuma çerçevesinin (ORF) 3 &#39;UTR arasındadır. Bu entegrasyon için ayrıntılı bir protokol yerde 22 sağlanır. PCR, kuzey analizi veya RT-PCR 20,23 tarafından uygun ekleme ve ifade doğrulayın. Entegrasyon 3&#39;UTR&#39;sinden sentezi ile müdahale etmedi doğrulamak için önemlidir. Buna ek olarak, indüklenebilir bir promotör (metiyonin tükenmesi) ekspresyonu...

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Çeşitli yöntemler ilişkili proteinleri 11,34 35 tanımlamak için belirli bir mRNA izolasyonunu kullanımı. Bu yöntemler, bir RNA-protein etkileşimlerini incelemek için in vitro ve in vivo stratejileri geçerlidir. In vitro yöntemler, dışsal RBPS yakalamak ve RNP kompleksleri 36,37 izole etmek için hücre lizatı ile RNA transkripsiyonu inkübe edin. Bu tip bir etkili bir yaklaşım, bir düzenleyici RNA motifi 18 bağlanan yeni ...

RNA bağlayıcı proteinler (RBPS) S. yaklaşık% 10&#39;unu temsil cerevisiae proteinleri 1,2 ve memeli proteinlerinin 3-5 arasında yaklaşık% 15. Bunlar mRNA transkripsiyon sonrası işleme ve düzenleme, çeviri, ribozom biogenezinin, tRNA Aminoacylation ve modifikasyon, kromatin yeniden ve daha fazlası gibi birçok hücresel süreçlere de karışırlar. RBPS önemli bir alt grubu, mRNA bağlayıcı proteinler (mRNPs) 6,7 olan. MRNA olgunlaşma sürecinde, farklı RBPS transkript bağlamak ve nükleus, hücresel lokalizasyonu, çeviri ve bozulma 6-8 dışarı nükleer işleme, ihracat aracılık eder. Böylece, herhangi bir zaman noktasında belirli bir transkript bağlı RBPS ayrı set onun işleme ve sonuçta onun kaderi belirler. Bir mRNA ile ilişkili RBPS belirlenmesi önemli ölçüde transkripsiyon sonrası düzenleme altında yatan süreçlerin anlayışımızı artırabilir. genetik çeşitlilikMikroskobik, biyokimyasal ve biyoinformatik yöntemleri (9-11 gözden) mRNA düzenlenmesinde rol proteinleri tespit etmek için kullanılmıştır. Bununla birlikte, bu yöntem sadece birkaç özel bir hedef mRNA ile ilişkili proteinlerin belirlenmesini mümkün kılmaktadır. Dikkat çekici bir şekilde, maya hücreleri bir ekspresyon kolleksiyonunu taramak için yem olarak ilgili mRNA&#39;nın kullanır maya üç hibrid sistemi (Y3H) &#39;dir. Pozitif klonlar genellikle büyüme seçimi ya da muhabir ifade 12-14 ile gözlenir. Bu yöntemin en önemli avantajı, hücre ortamında ve RNA-protein etkileşiminin gücünü ölçmek için yeteneği taranabilir proteinlerin büyük bir sayıdır. Dezavantajları nedeniyle spesifik olmayan yanlış pozitif sonuç, görece olarak yüksek ve füzyon proteini, av veya yem RNA yanlış katlanma kısmen hatalı negatif sonuçlar, yüksek bir potansiyel bulunmaktadır. Genetik yaklaşım alternatif afinite purifi olanilişkili proteinler RNA katyonudur. Poli A ihtiva eden mRNA oligo dT sütun kullanılarak izole edilebilir, ve bunların ilişkili proteinler kütle spektrometrisi ile tespit edilir. RNA-protein etkileşimi, kısa menzilli kovalent bağlar yapan çapraz bağlama suretiyle, hücresel bir çerçevede, muhafaza edilir. Oligo dT sütunun kullanılması mRNA 3,5,15 A-içeren herhangi bir poli ile ilişkili tüm proteom küresel bir görünüm verir. Bununla birlikte, belirli bir mRNA ile ilişkili proteinlerin bir listesi sağlamaz. Çok az metotlar, bir kimlik gerçekleştirmek için kullanılabilir. ÇİFT yöntemi hedefe mRNA 16,17 ile tamamlayıcılık ile nükleik asidin transfeksiyon gerektirir. nükleik asit, aynı zamanda etkileşim bölgesi yakın çevresinde RBPS için çapraz bağlama sağlayan bir peptit eklenir. çapraz bağlama sonrasında RBP-peptid-nükleik asit izole edilebilir ve proteomik analizine tabi tutulmuştur. Son zamanlarda, bir aptamer tabanlı metodoloji oldubaşarılı bir şekilde, memeli hücre hatları 18 alıntılar uygulanır. streptavidin geliştirilmiş afiniteye sahip bir RNA aptamer geliştirilmiş ve (bu durumda AU zengin elemanı (ARE)) ilgi konusu bir sekansına kaynaştırılır. Aptamer RNA streptavidin boncuk bağlanmış ve hücre lizatı ile karıştırıldı. Sırasına ilişkili proteinler arıtıldı ve kütle spektrometrisi (MS) ile tespit edilmiştir. Bu yöntem hücresel ayarları (örneğin, in vitro) dışında meydana dernekler bulgulamışsak da, mRNA ile ilişkili proteinlerin izolasyonu genomuna aptamers vermek ve bu şekilde mümkün kılacak şekilde, gelecekte değişecektir olduğu süre hücresel ortam (yani, in vivo). Genetik manipülasyonlar iyi kurulmuş maya, olarak, (Prof. Dr. Jeff Gerst laboratuarında geliştirilen) hızlı yöntem, in vivo dernekler 19 bir görünüm sağlar. Rapid (özel ve MS2 kat proteinin bağlanma güçlü MS2- birleştirirVe) streptavidin bağlama alanı (SBP MS2 RNA dizisine CP) konjuge boncuk streptavidin. Bu streptavidin boncuk ile MS2 etiketli mRNA&#39;ların etkili arınma sağlar. Ayrıca, MS2 döngüler 12 kopya sentezleme RNA eş zamanlı olarak bağlanan ve izolasyon verimini geliştirmek için, altı MS2-CP izin verir. Bu protokol, bu nedenle elüt örnekler kütle spektrometrisi ile proteomik analizine tabi kez yeni mRNA ilişkili proteinlerin belirlenmesini sağlamak için önerilmiştir. Son zamanlarda, maya PMP1 mRNA 20 ile bağlantılı yeni proteinleri belirlemek için hızlı kullanmıştır. PMP1 mRNA daha önce ER membran ile ilişkili olduğu ve 3 &#39;çevrilmemiş bölgesi (UTR) Bu ilişki 21 önemli bir belirleyici olduğu bulunmuştur gördü. Bu nedenle, PMP1 3 &#39;UTR bağlanan RBPS lokalizasyonu önemli bir rol oynaması olasıdır. HIZLI sıvı kromatografı ve ardındany-kütle spektrometrisi / kütle spektrometrisi (LC-MS / MS) PMP1 20 ile etkileşim çok sayıda yeni protein da tespit ettik. Bu yazıda, Rapid metodoloji ayrıntılı bir protokol sağlar, gerek önemli kontroller yapılır ve verim ve özgüllüğünü artırabilir teknik ipuçları edilecek.

<table border="0" cellpadding="0" cellspacing="0" width="632">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Tris</td>
			<td style="width:107px;">sigma</td>
			<td style="width:119px;">T1503</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SDS</td>
			<td>bio-lab</td>
			<td align="right">1981232300</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DTT</td>
			<td>sigma</td>
			<td>D9779</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Acidic Phenol (pH 4.3)</td>
			<td>sigma</td>
			<td>P4682</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Acidic Phenol: Chloroform (5:1, pH 4.3)</td>
			<td>sigma</td>
			<td>P1944</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Chloroform</td>
			<td>bio-lab</td>
			<td align="right">3080521</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Formaldehyde</td>
			<td>Frutarom</td>
			<td align="right">5551820</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glycine</td>
			<td>sigma</td>
			<td>G7126</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NP-40</td>
			<td>Calbiochem</td>
			<td align="right">492016</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Heparin</td>
			<td>Sigma</td>
			<td>H3393</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phenylmethylsulfonyl Flouride (PMSF)</td>
			<td>Sigma</td>
			<td>P7626</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Leupeptin</td>
			<td>Sigma</td>
			<td>L2884</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Aprotinin</td>
			<td>Sigma</td>
			<td>A1153</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Soybean Trypsin Inhibitor</td>
			<td>Sigma</td>
			<td>T9003</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pepstatin</td>
			<td>Sigma</td>
			<td>P5318</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DNase I</td>
			<td>Promega</td>
			<td>M610A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ribonuclease&#160; Inhibitor</td>
			<td>Takara</td>
			<td>2313A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glass Beads</td>
			<td>Sartorius</td>
			<td>BBI-8541701</td>
			<td>0.4-0.6mm diameter&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mini BeadBeater</td>
			<td>BioSpec</td>
			<td colspan="2">Mini BeadBeater 16</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Guanidinium</td>
			<td>Sigma</td>
			<td>G4505</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Avidin</td>
			<td>Sigma</td>
			<td>A9275</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Streptavidin Beads</td>
			<td>GE Healthcare&#160;</td>
			<td>17-5113-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bovine serum albumin (BSA)</td>
			<td>Sigma</td>
			<td>A7906</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Yeast tRNA</td>
			<td>Sigma</td>
			<td>R8508</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Biotin</td>
			<td>Sigma</td>
			<td>B4501</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Yeast extract</td>
			<td>Bacto</td>
			<td dir="LTR">288620</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">peptone</td>
			<td>Bacto</td>
			<td dir="LTR">211677</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glucose</td>
			<td>Sigma</td>
			<td>G8270</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1 x Phosphate-Buffered saline (PBS)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.2 M NaOH</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">4 x Laemmli Sample Buffer (LSB)</td>
			<td></td>
			<td></td>
			<td>0.2 M Tris-Hcl pH 6.8, 8% SDS, 0.4 M DTT, 40% glycerol, 0.04% Bromophenol-Blue.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hot phenol lysis buffer</td>
			<td></td>
			<td></td>
			<td>10 mM Tris pH 7.5, 10 mM EDTA, 0.5% SDS&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">3 M Sodium Acetate pH 5.2</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">100% and 70% Ethanol (EtOH)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RNase-free water</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">RaPID lysis buffer</td>
			<td></td>
			<td></td>
			<td>20 mM Tris pH 7.5, 150 mM NaCl, 1.8 mM MgCl2, 0.5% NP-40, 5 mg/ml Heparin, 1 mM Dithiothreitol (DTT), 1 mM Phenylmethylsulfonyl Flouride (PMSF), 10 &#181;g/ml Leupeptin, 10 &#181;g/ml Aprotinin, 10 &#181;g/ml Soybean Trypsin Inhibitor, 10 &#181;g/ml Pepstatin, 20 U/ml DNase I, 100 U/ml Ribonuclease&#160; Inhibitor.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2x Cross-linking reversal buffer</td>
			<td></td>
			<td></td>
			<td dir="LTR">100 mM Tris pH 7.4, 10 mM EDTA, 20 mM DTT, 2 % SDS.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RaPID wash buffer</td>
			<td></td>
			<td></td>
			<td>20 mM Tris-HCl pH 7.5,&#160; 300 mM NaCl, 0.5% NP-40</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.5 M EDTA pH 8</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Silver Stain Plus Kit</td>
			<td>Bio-Rad&#160;</td>
			<td>161-0449</td>
			<td>For detecting proteins in polyacrylamide gels</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SD selective medium&#160;</td>
			<td></td>
			<td></td>
			<td>1.7 g/l Yeast nitrogen base with out amino acids and ammonium sulfate, 5 g/l Ammonium sulfate, 2% glucose, 350 mg/l Threonine, 40 mg/l Methionine, 40 mg/l Adenine, 50 mg/l Lysine, 50 mg/l Tryptophan, 20 mg/l Histidine, 80 mg/l Leucine, 30 mg/l Tyrosine, 40 mg/l Arginine</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-eEF3 (EF3A,YEF3)</td>
			<td colspan="2">Gift from Kinzy TG. (UMDNJ Robert Wood Johnson Medical School)</td>
			<td>1:5,000</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti GFP antibody</td>
			<td>Santa Cruz</td>
			<td>sc-8334</td>
			<td>1:3,000</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti rabbit IgG-HRP conjugated</td>
			<td>SIGMA</td>
			<td>A9169</td>
			<td>1:10,000</td>
		</tr>
	</tbody>
</table>

novel rna-binding proteins isolation by the rapid methodology

RNA-binding proteins (RBPs) play important roles in every aspect of RNA metabolism and regulation. Their identification is a major challenge in modern biology. Only a few in vitro and in vivo methods enable the identification of RBPs associated with a particular target mRNA. However, their main limitations are the identification of RBPs in a non-cellular environment (in vitro) or the low efficiency isolation of RNA of interest (in vivo). An RNA-binding protein purification and identification (RaPID) methodology was designed to overcome these limitations in yeast and enable efficient isolation of proteins that are associated in vivo. To achieve this, the RNA of interest is tagged with MS2 loops, and co-expressed with a fusion protein of an MS2-binding protein and a streptavidin-binding protein (SBP). Cells are then subjected to crosslinking and lysed, and complexes are isolated through streptavidin beads. The proteins that co-purify with the tagged RNA can then be determined by mass spectrometry. We recently used this protocol to identify novel proteins associated with the ER-associated PMP1 mRNA. Here, we provide a detailed protocol of RaPID, and discuss some of its limitations and advantages.

Rapid ilişkili proteinler ile spesifik bir hedef RNA&#39;nın izolasyonu sağlar. başarısı için kritik böylece proteinin yeterli miktarda elde edilmesi mümkün olduğu kadar sağlam RNA tutmak. RNA izolasyonu etkinliğini ve kalitesini saptamak için northern analizi (Şekil 1A) gerçekleştirilir. Kuzey analizi doğrudan Rapid verimliliğini ve kalitesini raporlama avantajına sahiptir. Bu nedenle, tam uzunluktaki ve bozunma ürünlerinin nispi miktarları tek bi...

Watch this Scientific Journal Video about Novel RNA-Binding Proteins Isolation by the RaPID Methodology at JoVE.com

Novel RNA-Binding Proteins Isolation by the RaPID Methodology

RNA-protein interactions lie at the heart of many cellular processes. Here, we describe an in vivo method to isolate specific RNA and identify novel proteins that are associated with it. This could shed new light on how RNAs are regulated in the cell.

Rapid Metodoloji tarafından Roman RNA Bağlayıcı Proteinler İzolasyon

RNA-binding proteins (RBPs) play important roles in every aspect of RNA metabolism and regulation. Their identification is a major challenge in modern ...

Research

JoVE Journal

Genetics

Conjugative Mating Assays for Sequence-specific Analysis of Transfer Proteins Involved in Bacterial Conjugation

Bakteriyel Konjugasyon ilgilendim Transferi Proteinlerin Dizi özgü Analiz konjugatif Çiftleşme Tahliller

The transfer of genetic material by bacterial conjugation is a process that takes place via complexes formed by specific transfer proteins. In Escherichia ...

Kinetics of Lagging-strand DNA Synthesis In Vitro by the Bacteriophage T7 Replication Proteins

Kinetics of Lagging-strand DNA Synthesis In Vitro by the Bacteriophage T7 Replication Proteins

Kaplama DNA sentezinin kinetiği<em&gt; İn Vitro</em&gt; Bakteriyofaj T7 çoğaltma Proteinler tarafından

Here we provide protocols for the kinetic examination of lagging-strand DNA synthesis in vitro by the replication proteins of bacteriophage T7. The T7 ...

Bidirectional Retroviral Integration Site PCR Methodology and Quantitative Data Analysis Workflow

Çift Yönlü Retroviral Entegrasyon Sitesi PCR Metodolojisi ve Kantitatif Veri Analizi İş Akışı

Integration Site (IS) assays are a critical component of the study of retroviral integration sites and their biological significance. In recent retroviral ...

A Standard Methodology to Examine On-site Mutagenicity As a Function of Point Mutation Repair Catalyzed by CRISPR/Cas9 and SsODN in Human Cells

Yerinde mutagenisitesinin CRISPR/Cas9 ve SsODN insan hücrelerinde tarafından katalize nokta mutasyon onarım bir fonksiyonu olarak incelemek için standart bir metodoloji

Combinatorial gene editing using CRISPR/Cas9 and single-stranded oligonucleotides is an effective strategy for the correction of single-base point ...

Methodology for Accurate Detection of Mitochondrial DNA Methylation

Mitokondrial DNA metilasyonu doğru algılanması için metodoloji

Quantification of DNA methylation can be achieved using bisulfite sequencing, which takes advantage of the property of sodium bisulfite to convert ...

Novel Sequence Discovery by Subtractive Genomics

Eksiltici genomik tarafından roman sıra bulma

Subtractive genomics can be used in any research where the goal is to identify the sequence of a gene, protein, or general region that is embedded in a ...

Sequencing of mRNA from Whole Blood using Nanopore Sequencing

Nanopore sıralamayı kullanarak tüm kan mRNA sıralaması

Sequencing in remote locations and resource-poor settings presents unique challenges. Nanopore sequencing can be successfully used under such conditions, ...

Mass Spectrometry-Based Proteomics Analyses Using the OpenProt Database to Unveil Novel Proteins Translated from Non-Canonical Open Reading Frames

Kütle spektrometresi tabanlı proteomik analizleri roman proteinler açıklayacak OpenProt veritabanı kullanarak kanonik olmayan açık okuma çerçevesi tercüme

Genome annotation is central to today's proteomic research as it draws the outlines of the proteomic landscape. Traditional models of open reading frame ...

An Assay for Quantifying Protein-RNA Binding in Bacteria

Bakterilerde protein RNA bağlayıcı ölçümü için bir tahlil

In the initiation step of protein translation, the ribosome binds to the initiation region of the mRNA. Translation initiation can be blocked by binding ...

Exploring Sequence Space to Identify Binding Sites for Regulatory RNA-Binding Proteins

Düzenleyici RNA Bağlayıcı Proteinler için Bağlayıcı Siteleri Belirlemek için Sıra Alanını Keşfetmek

Gene regulation plays an important role in all cells. Transcriptional, post-transcriptional (or RNA processing), translational, and post-translational ...