SV NIH K22AI077353 ve Landenberger Vakfı&#39;nın finansman desteği için minnettar olduğunu. KR minnetle NIH (5SC1GM083754) fon kabul eder.

<ol>
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Yazarlar ifşa hiçbir şey yok.

HeLa hücre nükleer özütlerinde veya bu ekstrelerden parçalandı bölme faktörleri ile gerçekleştirilen in vitro pre-mRNA 3 &#39;bölünme reaksiyonu, çekirdek bölünmesi faktörler ve ana komplekslerinin 16-21 tanımlanmasını sağladı. Bu faktörlerle ilişkili daha birçok proteinlerin son zamanlarda 22 tespit edilmiştir ve in vitro reaksiyon bu proteinlerin reaksiyonu nasıl katkıda ışık tutacak devam edebilir. In vivo reaksiyon cotranscriptional ...

En olgun ökaryotik mesajı RNA&#39;lar (mRNAlarının) biyosentezi gibi kapatma, yapıştırma ve poliadenilasyonun gibi çeşitli transkripsiyon sonrası değişiklikler gerektirir. Bu değişiklikler genel olarak doğru işlem 1 yürütülmesini sağlamak ve güçlü bir mRNA&#39;nın stabilitesini arttırmak kuple edilir. 3 &#39;, memeli ön mRNA uç oluşumu 5&#39;e adenilat tortularının ilave edildi ve ardından yeni oluşan RNA endonükleolitik yarılmasıyla üretilir&#39; poly (A) polimeraz (PAP) 2-4 ürünü ayrıldı. Memelilerde, bölünme özel ön-RNA dizileri üzerinde toplanır, yaklaşık 800 kDa, ve, bir çok-bileşenli protein kompleksi ile gerçekleştirilir. Poli (A) sinyal sekansı, yüksek oranda korunmuş bir kanonik hexanucleotide sekansı AAUAAA, yaklaşık 10-30 nt alt bölünme sitesi yönlendirir. Bu site spesifik yarılma ve poliadenilasyon özgüllük faktörü (CPSF) ve 73 kD alt ünitesi tarafından tanınanCSPF endonükleaz aktivitesi içerir. Bölünme uyarım faktörü (CSTF) alt poli (A) Alanı daha dejenere GU-ya da U-zengin sekansı element bağlar. Ayrıca memeli bölünme faktörü I (CFIm) ve memeli bölünme faktörü II (CFII) bölünmesi için gerekli olan. CFIm gen bir dizi için tanımlandığı gibidir ve önemli fizyolojik işlemleri 5-8 dahil olmak gibi olmuştur üst dizisi elemanları (kullanım) spesifik UGUA (N) sitelerine bağlanır. İn vitro olarak, RNA işleme tepkimelerinin genel olarak radyo-etiketli RNA alt tabakaların 9-12 kullanımı ile analiz edilmiştir. Bunlar, bakteriyofaj promotör T7 ya da SP6 ikinci run-off transkripsiyonu ile sentezlenebilir. Daha önce karakterize edilmemiş bir poliadenilasyon sitesi incelenirken, önemli alt dizileri cDNA içinde mevcut olmayabilir gibi, RNA alt tabakanın üretilmesi için, genomik DNA yerine cDNA kullanmak gereklidir. En az 150 nt upstrea dahil tasarım yüzeylerm ve 50 nt aşağısında olgun mRNA üzerinde yarılma sitesi / ucundan. Alt-tabaka bölünme oluşumu daha hızlı göç eder; Diğer parçalar spesifik olmayan nükleaz hareketiyle oluşturulmuş olabilir, çünkü ancak, reaksiyonun özgüllüğü doğru işlem sinyal dizileri üzerinde bağımlılığı ile doğrulanmalıdır. Bu nedenle, AAUAAA dizisi (örneğin, AAGAAA) bir nokta mutasyonu ile alt-tabakalar RNA bölünme reaksiyonu için bir negatif kontrol olarak hizmet etmektedir. Radyo işaretli RNA küçük bir miktar bölünme reaksiyonları için kullanıldığı göz önüne alındığında, çoğu nükleer özütlerinde de yüksek bolca mevcut RNazlar sorunlu olabilir ve özü hazırlanması için başlangıç ​​malzemesinin seçimi sınırlayabilir. HeLa hücreleri, endojen RNases düşük seviyelerini ihtiva edebilmekte ve bu yüzden, bu deneylerde iyi bir performans göstermektedir. In vivo ve in vitro RNA substratların endonükleolitik klivaj hemen poli (A) ekleme, Per izlemektedirs bölünmüş ara tespit edilebilir miktarlarda mevcut değildir. Bu nedenle, belirli bir RNA dizisini ya da bir bölünme reaksiyonunda yer alan proteinleri ya da çalışma için, deneyler meydana gelen poliadenilasyon önlemek koşullarda yapılır. Bir parçalanma reaksiyonunu zarar vermeden poliadenilasyona durdurmak böylece hiçbir poliadenilasyonunun üzerinde bölünme bağımlılığı, ya da tam tersi vardır. Bu nedenle, ATP tek bir nükleotid poli (A) bölgesinde dahil edilebilir ve sadece bir RNA klivaj tespit edilebilir, böylece 3 &#39;hidroksil grubuna sahip olmayan bir zincir sonlandırma analogu ile değiştirilir. Karmaşıklığı ve tahlil bu tür yüksek derecede belirli özellikler göz önüne alındığında, in vitro mRNA öncülerinin ayırma / Poli (A) makine ile endonükleolitik bölünme incelemek için ayrıntılı bir video protokol açıklar. Biz, yetkili nükleer özleri hazırlamak radyoetiketlenmiş RNA alt tabakalar oluşturmak, parçalanma reaksiyonunu gerçekleştirmek ve sonucu analiz etme ve yorumlama nasıl tarifing ürünleri. Şekil 1, bir bölme deneyi için kullanılacak olan HIV-1 ön-mRNA öğeleri içerisinde 3 &#39;ucunda kodlama için alt-tabaka RNA&#39;ların bir örneğini göstermektedir. HIV RNA genomunun 3 &#39;ucu, örneğin poli (A) sitesi, G+ U zengin bölge ve bütün viral mRNA transkriptlerinin 13 etkin bir şekilde olgunlaşması için gerekli olan USE elemanı olarak çok önemli bir düzenleyici sekansların oluşmaktadır . Bu örnekte giriş RNA substrat 338 nt ve bölünme 237 üzerine nt olması beklenir. Poliadenilasyon oluşmasına izin verilir ise, ürün bir yayma 237 ve 437 nt arasında anlamlı olacaktır.

<table><tbody><tr><td>1 L Celstir flask</td><td>Wheaton</td><td>356884</td><td>Different sizes available</td></tr><tr><td>4 position slow speed stirrer</td><td>VWR</td><td>12621-076</td><td></td></tr><tr><td>Swinging bucket centrifuge</td><td>Beckman Coulter</td><td></td><td>Allegra x-15R with SX4500 Rotor</td></tr><tr><td>Ultracentrifuge</td><td>Beckman Coulter</td><td></td><td>Optima L-100 XP Ultra with SW41 Rotor</td></tr><tr><td>Table top centrifuge 5417R</td><td>Eppendorf</td><td></td><td>Refridgerated</td></tr><tr><td>Thermomixer incubator</td><td>Eppendorf</td><td></td><td></td></tr><tr><td>250 ml conical tubes</td><td>Corning</td><td>430776</td><td></td></tr><tr><td>50 ml conical tubes</td><td>BD Falcon</td><td>352098</td><td></td></tr><tr><td>Ultraclear centrifuge tubes</td><td>Beckman Coulter</td><td>344059</td><td></td></tr><tr><td>JOKLIK modified MEM</td><td>Lonza</td><td>04-719Q</td><td></td></tr><tr><td>Fetal bovine serum</td><td>Atlas</td><td>F-0500-A</td><td>Heat inactivated</td></tr><tr><td>L-glut:pen:strep</td><td>Gemini Bio-Products</td><td>400-110</td><td></td></tr><tr><td>1 M Tris-HCl pH 8.0</td><td>Mediatech</td><td>46-031-CM</td><td></td></tr><tr><td>Magnesium chloride</td><td>Fisher</td><td>BP214-500</td><td></td></tr><tr><td>Potassium chloride</td><td>MP Biomedicals</td><td>194844</td><td></td></tr><tr><td>HEPES</td><td>Fisher</td><td>BP310-500</td><td></td></tr><tr><td>DTT</td><td>Alexis Biomedicals</td><td>280-001-G-010</td><td></td></tr><tr><td>Glycerol</td><td>Fisher</td><td>BP229-4</td><td></td></tr><tr><td>5 M sodium chloride solution</td><td>Mediatech</td><td>46-032-CV</td><td></td></tr><tr><td>EDTA 0.5 M solution</td><td>Sigma-Aldrich</td><td>E7889-100ml</td><td></td></tr><tr><td>EDTA</td><td>Fisher</td><td>BP120-500</td><td></td></tr><tr><td>PMSF</td><td>Thermo Scientific</td><td>36978</td><td></td></tr><tr><td>Ammonium sulfate</td><td>Fisher</td><td>A702-500</td><td></td></tr><tr><td>Complete EDTA-free protease inhibitor cocktail tablets</td><td>Roche</td><td>04 693 132 001</td><td></td></tr><tr><td>Slide-A-Lyzer Dialysis Cassettes Kit</td><td>Thermo Scientific</td><td>66372</td><td>MWCO 7,000 0.5 ml-3 ml</td></tr><tr><td>15 ml Dounce tissue grinder set</td><td>Sigma-Aldrich</td><td>D9938-1SET</td><td>Different sizes available</td></tr><tr><td>Expand high fidelity PCR kit</td><td>Roche</td><td>11 732 650 001</td><td></td></tr><tr><td>10 mM dNTP Mix</td><td>Invitrogen</td><td>Y02256</td><td>10 mM each nucleotide</td></tr><tr><td>MaxiScript SP6/T7 kit</td><td>Ambion</td><td>AM1322</td><td></td></tr><tr><td>m7G(5&#039;)ppp(5&#039;) G RNA cap</td><td>New England Biolabs</td><td>S1404S</td><td></td></tr><tr><td>Century Marker Template Plus</td><td>Ambion</td><td>AM7782</td><td></td></tr><tr><td>Easytides UTP [&amp;alpha;-&lt;sup&gt;32&lt;/sup&gt;P] 250 &amp;mu;Ci</td><td>Perkin Elmer</td><td>BLU507H250UC</td><td></td></tr><tr><td>Gel loading buffer II</td><td>Ambion</td><td>8546G</td><td></td></tr><tr><td>DEPC treated water</td><td>Ambion</td><td>AM9906</td><td></td></tr><tr><td>10x TAE</td><td>Fisher</td><td>BP13354</td><td></td></tr><tr><td>10x TBE</td><td>Ameresco Life Sciences</td><td>0658-4L</td><td></td></tr><tr><td>10x PBS</td><td>Fisher</td><td>BP399-20</td><td></td></tr><tr><td>Urea</td><td>Fisher</td><td>BP169-212</td><td></td></tr><tr><td>Ammonium persulfate</td><td>Bio-Rad</td><td>161-0700</td><td></td></tr><tr><td>TEMED</td><td>Fisher</td><td>BP150-20</td><td></td></tr><tr><td>Ammonium acetate</td><td>Fisher</td><td>A637-500</td><td></td></tr><tr><td>40% 19:1 acrylamide:bis-acrylamide</td><td>Bio-Rad</td><td>161-0144</td><td></td></tr><tr><td>Glycogen</td><td>Roche</td><td>10 901 393 001</td><td></td></tr><tr><td>100% absolute ethanol 200 proof</td><td>Acros</td><td>61509-0040</td><td></td></tr><tr><td>Acid phenol:chloroform</td><td>Ambion</td><td>9720</td><td>For RNA</td></tr><tr><td>Scintilation fluid</td><td>Fisher</td><td>SX18-4</td><td></td></tr><tr><td>Rnase inhibitor</td><td>Promega</td><td>N261B</td><td></td></tr><tr><td>Polyvinyl alcohol- PVA</td><td>Sigma-Aldrich</td><td>P8136-250G</td><td></td></tr><tr><td>Creatine phosphate</td><td>Calbiochem</td><td>2380</td><td></td></tr><tr><td>100 mM dATP</td><td>Fisher</td><td>BP2560-4</td><td></td></tr><tr><td>SDS</td><td>Acros</td><td>23042-5000</td><td></td></tr><tr><td>Proteinase K</td><td>Fisher</td><td>BP1700-100</td><td></td></tr><tr><td>Adjustable sequencing unit</td><td>Sigma-Aldrich</td><td>Z351881-1EA</td><td></td></tr><tr><td>Binder clips</td><td>Office Depot</td><td>838-056</td><td></td></tr><tr><td>20 cm x 42 cm glass plates</td><td>Sigma-Aldrich</td><td>Z352543</td><td>1 SET</td></tr><tr><td>20 cm x 22 cm glass plates</td><td>Sigma-Aldrich</td><td>Z35252-7</td><td>1 SET</td></tr><tr><td>20 cm x 42 cm aluminum cooling plates</td><td>Sigma-Aldrich</td><td>Z352667</td><td>1 EA</td></tr><tr><td>0.4 mm x 22 cm spacers</td><td>Sigma-Aldrich</td><td>Z35230-6</td><td>1 SET</td></tr><tr><td>0.4 mm x 42 cm spacers</td><td>Sigma-Aldrich</td><td>Z352314-1</td><td>1 SET</td></tr><tr><td>8-well comb</td><td>Sigma-Aldrich</td><td>Z35195-4</td><td>1 EA</td></tr><tr><td>16-well comb</td><td>Sigma-Aldrich</td><td>Z351962</td><td>1 EA</td></tr><tr><td>32-well comb</td><td>Sigma-Aldrich</td><td>Z351970</td><td>1 EA</td></tr><tr><td>Gel repel coating</td><td>C.B.S. Scientific</td><td>SGR-0401</td><td>or SGR-0101 for individual bottle</td></tr><tr><td>Gel loading tips</td><td>Rainin</td><td>GT-10-4</td><td>0.1-10 &amp;mu;l</td></tr><tr><td>Sequencing PowerPac HV</td><td>Bio-Rad</td><td>PowerPac HV</td><td>5,000 V/500 mA/400 W</td></tr><tr><td>Gel Dryer Model 583</td><td>Bio-Rad</td><td>Model 583</td><td></td></tr><tr><td>Hydrotech vacuum pump for gel dryer</td><td>Bio-Rad</td><td></td><td></td></tr><tr><td>Glogos II Glow-in-the-dark markers</td><td>Agilent</td><td>420201</td><td></td></tr><tr><td>Film 8 x 10</td><td>Midsci</td><td>BX810</td><td></td></tr><tr><td>Film 14 x 17</td><td>Phenix</td><td>F-BX1417</td><td></td></tr><tr><td>Autoradiography cassette</td><td>Fisher</td><td>FBCA 1417</td><td>8 x 10 size available</td></tr></tbody></table>

analysis of rna processing reactions using cell free systems: 3' end cleavage of pre-mrna substrates in vitro

Memeli mRNA&#39;ların 3 &#39;ucu RNA polimeraz II (RNPII) ile transkripsiyonun sonlandırılması ile ani oluşmuş değildir. Bunun yerine, RNPII olgun RNA&#39;lar ötesinde ön-mRNA sentezler ve endonükleaz aktivitesi olan bir süreç belirli bir site gereklidir. Öncü RNA bölünmesi normalde CA dinükleotidleri sonra konsensüs polyA bölgesi (AAUAAA) aşağı nt 10-30 oluşur. Yarılma kompleks, yaklaşık olarak 800 kDa&#39;lık çok faktörlü bir protein kompleksi, ikinci proteinler, bu özel nükleaz aktivitesi gerçekleştirmek. Belirli RNA sekansları, yukarı akış ve poliA site alt bölünme kompleksinin işe kontrol eder. Hemen bölünmesinden sonra, pre-mRNA stabil olgun RNA mesajları üretmek için poli A polimerazı (PAP) ile poliadenile edilmiştir. Bir RNA transkriptinin 3 &#39;ucunun radyo-etiketli RNA Processing özel alt-tabakalar ile hücre nükleer özler kullanılarak incelenebilir. Özetle, uzun bir 32 &lt;/ Sup&gt; P-etiketli Parçalanmamış öncü RNA, in vitro olarak nükleer ekstreler ile inkübe edilir, ve bölünme jel elektroforez ve otoradyografi ile değerlendirilir. Uygun bölünme oluştuğunda, 5 &#39;kısa bir ürün olarak algılanır ve ölçülür ayrıldı. Burada, bir örnek olarak, kullanılarak ayrıntılı olarak HIV-1 mRNA&#39;ların 3 &#39;ucu işleme bölünme tahlili tarif eder.

RNA poli (A) HIV-1 siteleri (Şekil 2) bir bölünme tahlili temsil edici sonuçları. Bu jelin üstünde yavaş göç eden bir bant Parçalanmamış RNA alt tabakanın, gözlemleyebiliriz. Belirli ürün, daha hızlı parçalanan beklenen boyutta jelde çalışan en yoğun kısa fragman bandı, ve poliA sekansı (mutPolyA) RNA, bir nokta mutasyon ihtiva mutpoly (A) kontrolü yarılma tahlilinden özellikle yoktur alt-tabaka. Giriş substratın degradasyon ürünleri, bazen gözlemlenebilir. Yarılma ak...

Watch this Scientific Journal Video about Analysis of RNA Processing Reactions Using Cell Free Systems: 3' End Cleavage of Pre-mRNA Substrates in vitro at JoVE.com

Analysis of RNA Processing Reactions Using Cell Free Systems: 3' End Cleavage of Pre-mRNA Substrates in vitro

RNA polimeraz II olgun mRNA&#39;nın 3 &#39;ucunun ötesine uzanan bir ön-madde RNA sentezler. Olgun RNA uç yarılma kompleksinin endonükleaz etkinliği ile, RNA dizileri tarafından dikte bir bölgede, cotranscriptionally oluşturulur. Burada, detay in vitro bölünme reaksiyonları çalışma yöntemi biz.

Pre-mRNA Substratlarm 3 &#39;End Bölünme: Hücre Ücretsiz Sistemlerini Kullanma RNA İşleme Reaksiyonları Analizi<em&gt; In vitro</em

The 3&#8217; end of mammalian mRNAs is not formed by abrupt termination of transcription by RNA polymerase II (RNPII). Instead, RNPII synthesizes ...

analysis-rna-processing-reactions-using-cell-free-systems-3-end

Research

JoVE Journal

Biology

Analysis of RNA Processing Reactions Using Cell Free Systems: 3' End Cleavage of Pre-mRNA Substrates in vitro

12.8K Görüntüleme.  The Scripps Research Institute.  RNA polimeraz II olgun mRNA'nın 3 'ucunun ötesine uzanan bir ön-madde RNA sentezler. Olgun RNA uç yarılma kompleksinin endonükleaz etkinliği ile, RNA dizileri tarafından dikte bir bölgede, cotranscriptionally oluşturulur. Burada, detay in vitro bölünme reaksiyonları çalışma yöntemi biz. 

Video: Analysis of RNA Processing Reactions Using Cell Free Systems: 3' End Cleavage of Pre-mRNA Substrates in vitro

Adapting 3' Rapid Amplification of CDNA Ends to Map Transcripts in Cancer

Maturation of eukaryotic mRNAs involves 3&#39; end formation, which involves the addition of a poly(A) tail. In order to map the 3&#39; end of a gene, the traditional method of choice is 3&#39; rapid amplification of cDNA ends (3&#39; RACE). Protocols for 3&#39; RACE require the careful design and selection of nested primers within the 3&#39; untranslated region (3&#39; UTR) of the target gene of interest. However, with a few modifications the protocol can be used to include the entire 3&#39; UTR and sequences within the open reading frame (ORF), providing a more comprehensive picture of the relationship between the ORF and the 3&#39; UTR. This is in addition to identification of the polyadenylation signal (PAS), as well as the cleavage and polyadenylation site provided by conventional 3&#39; RACE. Expanded 3&#39; RACE can detect unusual 3&#39; UTRs, including gene fusions within the 3&#39; UTR, and the sequence information can be used to predict potential miRNA binding sites as well as AU rich destabilizing elements that may affect the stability of the transcript.

The two different 3&#39; rapid amplification of cDNA ends (3&#39; RACE) protocols described here make use of two different DNA polymerases to map sequences that include a segment of the open reading frame (ORF), the stop codon, and the entire 3&#39; UTR of a transcript using RNA obtained from different cancer cell lines.

3' adapte CDNA hızlı amplifikasyon transkript kanser eşlemek için sona erer

Maturation of eukaryotic mRNAs involves 3&#39; end formation, which involves the addition of a poly(A) tail. In order to map the 3&#39; end of a gene, the ...

Genetik

Transcriptome Analysis of Single Cells

Many gene expression analysis techniques rely on material isolated from heterogeneous populations of cells from tissue homogenates or cells in culture.1,2,3 In the case of the brain, regions such as the hippocampus contain a complex arrangement of different cell types, each with distinct mRNA profiles. The ability to harvest single cells allows for a more in depth investigation into the molecular differences between and within cell populations. We describe a simple and rapid method for harvesting cells for further processing. Pipettes often used in electrophysiology are utilized to isolate (using aspiration) a cell of interest and conveniently deposit it into an Eppendorf tube for further processing with any number of molecular biology techniques. Our protocol can be modified for the harvest of dendrites from cell culture or even individual cells from acute slices.
We also describe the aRNA amplification method as a major downstream application of single cell isolations. This method was developed previously by our lab as an alternative to other gene expression analysis techniques such as reverse-transcription or real-time polymerase chain reaction (PCR).4,5,6,7,8 This technique provides for linear amplification of the polyadenylated RNA beginning with only femtograms of material and resulting in microgram amounts of antisense RNA. The linearly amplified material provides a more accurate estimation than PCR exponential amplification of the relative abundance of components of the transcriptome of the isolated cell. The basic procedure consists of two rounds of amplification. Briefly, a T7 RNA polymerase promoter site is incorporated into double stranded cDNA created from the mRNA transcripts. An overnight in vitro transcription (IVT) reaction is then performed in which T7 RNA polymerase produces many antisense transcripts from the double stranded cDNA. The second round repeats this process but with some technical differences since the starting material is antisense RNA. It is standard to repeat the second round, resulting in three rounds of amplification. Often, the third round in vitro transcription reaction is performed using biotinylated nucleoside triphosphates so that the antisense RNA produced can be hybridized and detected on a microarray.7,8

In this article we describe a simple method for the harvesting of single cells from rat primary neuronal cultures and subsequent transcriptome analysis using aRNA amplification. This approach is generalizable to any cell type.

Tek Hücre Transcriptome Analizi

Many gene expression analysis techniques rely on material isolated from heterogeneous populations of cells from tissue homogenates or cells in ...

Nörobilim

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 ...

Single-step Purification of Macromolecular Complexes Using RNA Attached to Biotin and a Photo-cleavable Linker

For many years, the exceptionally strong and rapidly formed interaction between biotin and streptavidin has been successfully utilized for partial purification of biologically important RNA/protein complexes. However, this strategy suffers from one major disadvantage that limits its broader utilization: the biotin/streptavidin interaction can be broken only under denaturing conditions that also disrupt the integrity of the eluted complexes, hence precluding their subsequent functional analysis and/or further purification by other methods. In addition, the eluted samples are frequently contaminated with the background proteins that nonspecifically associate with streptavidin beads, complicating the analysis of the purified complexes by silver staining and mass spectrometry. To overcome these limitations, we developed a variant of the biotin/streptavidin strategy in which biotin is attached to an RNA substrate via a photo-cleavable linker and the complexes immobilized on streptavidin beads are selectively eluted to solution in a native form by long wave UV, leaving the background proteins on the beads. Shorter RNA binding substrates can be synthesized chemically with biotin and the photo-cleavable linker covalently attached to the 5&#39; end of the RNA, whereas longer RNA substrates can be provided with the two groups by a complementary oligonucleotide. These two variants of the UV-elution method were tested for purification of the U7 snRNP-dependent processing complexes that cleave histone pre-mRNAs at the 3&#39; end and they both proved to compare favorably to other previously developed purification methods. The UV-eluted samples contained readily detectable amounts of the U7 snRNP that was free of major protein contaminants and suitable for direct analysis by mass spectrometry and functional assays. The described method can be readily adapted for purification of other RNA binding complexes and used in conjunction with single- and double-stranded DNA binding sites to purify DNA-specific proteins and macromolecular complexes.

RNA/protein complexes purified using botin-streptavidin strategy are eluted to solution under denaturing conditions in a form unsuitable for further purification and functional analysis. Here, we describe a modification of this strategy that utilizes a photo-cleavable linker in RNA and a gentle UV-elution step, yielding native and fully functional RNA/protein complexes.

Tek adım arıtma RNA bağlı Biotin ve fotoğraf yarilabilir bir bağlayıcı kullanarak makromoleküllerin kompleksleri

For many years, the exceptionally strong and rapidly formed interaction between biotin and streptavidin has been successfully utilized for partial ...

Biyokimya

Studying RNA Interactors of Protein Kinase RNA-Activated during the Mammalian Cell Cycle

Protein kinase RNA-activated (PKR) is a member of the innate immune response proteins and recognizes the double-stranded secondary structure of viral RNAs. When bound to viral double-stranded RNAs (dsRNAs), PKR undergoes dimerization and subsequent autophosphorylation. Phosphorylated PKR (pPKR) becomes active and induces phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF2&#945;) to suppress global translation. Increasing evidence suggests that PKR can be activated under physiological conditions such as during the cell cycle or under various stress conditions without infection. However, our understanding of the RNA activators of PKR is limited due to the lack of a standardized experimental method to capture and analyze PKR-interacting dsRNAs. Here, we present an experimental protocol to specifically enrich and analyze PKR bound RNAs during the cell cycle using HeLa cells. We utilize the efficient crosslinking activity of formaldehyde to fix PKR-RNA complexes and isolate them via immunoprecipitation. PKR co-immunoprecipitated RNAs can then be further processed to generate a high-throughput sequencing library. One major class of PKR-interacting cellular dsRNAs is mitochondrial RNAs (mtRNAs), which can exist as intermolecular dsRNAs through complementary interaction between the heavy-strand and the light-strand RNAs. To study the strandedness of these duplex mtRNAs, we also present a protocol for strand-specific qRT-PCR. Our protocol is optimized for the analysis of PKR-bound RNAs, but it can be easily modified to study cellular dsRNAs or RNA-interactors of other dsRNA binding proteins.

We present experimental approaches for studying RNA-interactors of double-stranded RNA binding protein kinase RNA-activated (PKR) during the mammalian cell cycle using HeLa cells. This method utilizes formaldehyde to crosslink RNA-PKR complexes and immunoprecipitation to enrich PKR-bound RNAs. These RNAs can be further analyzed through high-throughput sequencing or qRT-PCR.

RNA Interactors Protein kinaz memeli hücre döngüsü sırasında RNA-harekete geçirmek, eğitim

Protein kinase RNA-activated (PKR) is a member of the innate immune response proteins and recognizes the double-stranded secondary structure of viral ...

Extremely Rapid and Specific Metabolic Labelling of RNA In Vivo with 4-Thiouracil (Ers4tU)

The nucleotide analogue, 4-thiouracil (4tU), is readily taken up by cells and incorporated into RNA as it is transcribed in vivo, allowing isolation of the RNA produced during a brief period of labelling. This is done by attaching a biotin moiety to the incorporated thio group and affinity purifying, using streptavidin coated beads. Achieving a good yield of pure, newly synthesized RNA that is free of pre-existing RNA makes shorter labelling times possible and permits increased temporal resolution in kinetic studies. This is a protocol for very specific, high yield purification of newly synthesized RNA. The protocol presented here describes how RNA is extracted from the yeast Saccharomyces cerevisiae. However, the protocol for purification of thiolated RNA from total RNA should be effective using RNA from any organism once it has been extracted from the cells. The purified RNA is suitable for analysis by many widely used techniques, such as reverse transcriptase-qPCR, RNA-seq and SLAM-seq. The specificity, sensitivity and flexibility of this technique allow unparalleled insights into RNA metabolism.

Extremely Rapid and Specific Metabolic Labelling of RNA In Vivo with 4-Thiouracil Ers4tU

The use of thiolated uracil to sensitively and specifically purify newly transcribed RNA from the yeast Saccharomyces cerevisiae.

RNA In Vivo'nun 4-Thiouracil (Ers4tU) ile Son Derece Hızlı ve Spesifik Metabolik Etiketlemesi

The nucleotide analogue, 4-thiouracil (4tU), is readily taken up by cells and incorporated into RNA as it is transcribed in vivo, allowing isolation of ...

DNAzyme-dependent Analysis of rRNA 2&#8217;-O-Methylation

Guide box C/D small nucleolar RNAs (snoRNAs) catalyze 2&#8217;-O-methylation of ribosomal and small nuclear RNA. However, a large number of snoRNA in higher eukaryotes may promiscuously recognize other RNA species and 2&#8217;-O-methylate multiple targets. Here, we provide step-by-step guide for the fast and non-expensive analysis of the site-specific 2&#8217;-O-methylation using a well-established method employing short DNA oligonucleotides called DNAzymes. These DNA fragments contain catalytic sequences which cleave RNA at specific consensus positions, as well as variable homology arms directing DNAzyme to its RNA targets. DNAzyme activity is inhibited by 2-&#8217;O-methylation of the nucleotide adjacent to the cleavage site in the RNA. Thus, DNAzymes, limited only by the consensus of the cleaved sequence, are perfect tools for the quick analysis of snoRNA-mediated RNA 2&#8217;-O-methylation. We analyzed snoRNA snR13- and snR47-guided 2&#8217;-O-methylation of 25S ribosomal RNA in Saccharomyces cerevisiae to demonstrate the simplicity of the technique and to provide a detailed protocol for the DNAzyme-dependent assay.

Here we present a protocol for DNAzyme-dependent cleavage of RNA. This enables fast and site-dependent analysis of RNA 2&#8217;-O-methylation. This approach can be used for the preliminary or major assessment of snoRNA activity.

RRNA 2'-O-Metilasyonun DNAzyme bağımlı analizi

Guide box C/D small nucleolar RNAs (snoRNAs) catalyze 2&#8217;-O-methylation of ribosomal and small nuclear RNA. However, a large number of snoRNA in ...

Gelişim Biyolojisi

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 ...

Substrate Generation for Endonucleases of CRISPR/Cas Systems

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

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

CRISPR / Cas Sistemleri Endonükleazlar için Substrat Üretimi

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

Biyoloji

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

Fluorescence based primer extension (FPE) is a molecular method to determine transcriptional starting points or processing sites of RNA molecules. This is achieved by reverse transcription of the RNA of interest using specific fluorescently labeled primers and subsequent analysis of the resulting cDNA fragments by denaturing polyacrylamide gel electrophoresis. Simultaneously, a traditional Sanger sequencing reaction is run on the gel to map the ends of the cDNA fragments to their exact corresponding bases. In contrast to 5&#39;-RACE (Rapid Amplification of cDNA Ends), where the product must be cloned and multiple candidates sequenced, the bulk of cDNA fragments generated by primer extension can be simultaneously detected in one gel run. In addition, the whole procedure (from reverse transcription to final analysis of the results) can be completed in one working day. By using fluorescently labeled primers, the use of hazardous radioactive isotope labeled reagents can be avoided and processing times are reduced as products can be detected during the electrophoresis procedure.
In the following protocol, we describe an in vivo fluorescent primer extension method to reliably and rapidly detect the 5&#39; ends of RNAs to deduce transcriptional starting points and RNA processing sites (e.g., by toxin-antitoxin system components) in S. aureus, E. coli and other bacteria.

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 ...

Pre-mRNA Substratlarm 3 'End Bölünme: Hücre Ücretsiz Sistemlerini Kullanma RNA İşleme Reaksiyonları Analizi

Özet

Daha Fazla Video Keşfet

Bu videodaki bölümler

3' adapte CDNA hızlı amplifikasyon transkript kanser eşlemek için sona erer

Tek Hücre Transcriptome Analizi

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

Tek adım arıtma RNA bağlı Biotin ve fotoğraf yarilabilir bir bağlayıcı kullanarak makromoleküllerin kompleksleri

RNA Interactors Protein kinaz memeli hücre döngüsü sırasında RNA-harekete geçirmek, eğitim

RNA In Vivo'nun 4-Thiouracil (Ers4tU) ile Son Derece Hızlı ve Spesifik Metabolik Etiketlemesi

RRNA 2'-O-Metilasyonun DNAzyme bağımlı analizi

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

CRISPR / Cas Sistemleri Endonükleazlar için Substrat Üretimi

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

Pre-mRNA Substratlarm 3 'End Bölünme: Hücre Ücretsiz Sistemlerini Kullanma RNA İşleme Reaksiyonları Analizi

Özet

Daha Fazla Video Keşfet

Bu videodaki bölümler

3' adapte CDNA hızlı amplifikasyon transkript kanser eşlemek için sona erer

Tek Hücre Transcriptome Analizi

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

Tek adım arıtma RNA bağlı Biotin ve fotoğraf yarilabilir bir bağlayıcı kullanarak makromoleküllerin kompleksleri

RNA Interactors Protein kinaz memeli hücre döngüsü sırasında RNA-harekete geçirmek, eğitim

RNA In Vivo'nun 4-Thiouracil (Ers4tU) ile Son Derece Hızlı ve Spesifik Metabolik Etiketlemesi

RRNA 2'-O-Metilasyonun DNAzyme bağımlı analizi

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

CRISPR / Cas Sistemleri Endonükleazlar için Substrat Üretimi

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

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