We are grateful to the past and present members of the laboratory, in particular Urszula Liwak, for critical discussion regarding polysome profiling protocols and sharing of data. This work was supported by operating grants from the Canadian institutes of Health Research, Cancer Research Society, and the National Science and Engineering Research Council of Canada to MH. MDF was supported by the Vanier Canada Graduate Scholarship Doctoral Award and the Ontario Graduate Scholarship, and this work forms part of her Ph.D. dissertation.

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The authors have nothing to disclose.

Polysomal profil farklı tedavi koşulları altında belirli transkript çeviri devletin ölçümü için olanak sağlayan güçlü bir tekniktir. Prensip çok basit bir tekniktir ama iyi Polizom profilleri üretmek için kritik bazıları yürütme birkaç adımları gerektirir. 1) RNazsız kimyasallar ve plastik malzeme kullanılarak, 2) (iyi sakaroz geçişlerini hazırlamak bizim deneyim% 10-50 sakaroz geçişlerini verimli bağlı ribozomlarla 40 / 60S alt birimleri ve mRNA&#39;lan) çözümlemek için en iyi iş, ve 3: Bu tuş adımlar ) en yüksek çeviri oranlarını yakalamak için katlanarak büyüyen hücreleri ve fazla% 80 konfluent kullanarak. Bu tür gen demonte veya aşırı ekspresyonuna sürece hücre tedavileri, yaparken plaka hücre miktarı hücreler son ucunda birleştirilebilmeli ne kadar bir fonksiyonu olacaktır, böylece bu ikinci aşaması dikkate alınmalıdır. Sonuçlarda Konusuted burada, Polizom profili analizi IRES&#39;e-barındıran mRNAlar XIAP ve Bcl-xL 12 çeviri düzenlenmesinde PDCD4 rol değerlendirmek için önemli bir araç oldu. Biz PDCD4 knock-down (Şekil 1B) üzerine genel Polizom profillerinde herhangi bir değişiklik dikkat etmedi aslında bize PDCD4 deney koşullarında küresel hücresel çeviri zarar vermedi sonucuna izin verdi. Önümüzdeki PDCD4 veya kontrol siRNA&#39;lara ile tedavi hücrelerde XIAP ve Bcl-xL mRNA&#39;ların dağılımını belirlemek için RT-qPCR analizi kullanılmıştır. Bu mRNA&#39;nın dağılımının niceliksel yerine Yarı-nicel analiz için ve tedaviler arasında çeviri verimliliği ince değişimlerin saptanması için sağlar, çünkü QPCR, standart RT-PCR veya Northern Blot karşıt olarak, polisom profilleri analiz için tercih edilen bir yöntemdir. Bu tekniği kullanarak, XIAP ve Bcl-xL mRNA&#39;ların dağılımı (gr edilen toplam hedef mRNA&#39;nın bir yüzdesi olarak ifade edilen bu gösterebilmişlerdiradient) belirgin ve böylece, bu mRNA çevirisinde bir artışla, PDCD4 knock-down (Şekil 1D, e) bunlarla ilişkili ribozom çok sayıda) ağır poliribozomların (mRNA içine kaydırılmıştır. Bu da XIAP ve Bcl-xL protein düzeyinde değil, bunların kararlı durum RNA düzeyi (Şekil 1F, G), bir artış ile teyit edilmiştir. Önemli bir şekilde, XIAP olmayan IRES varyantının polisom dağılımı tedavi edilen ve edilmeyen hücreleri (Şekil 1C) arasında aynı idi. Seçenek olarak ise, dönüşüm etkinliğini 14 (genellikle 2-4 fraksiyonlar) monosomes karşı polizom mRNA içeriği olan bir oranda (genellikle 5-10 fraksiyon) olarak sunulabilir. 254 nm absorbans okuma gözlenen zirveleri kendi ribozomal RNA (deterjan isnat edilemez olarak protokol boyunca kontrolleri kullanımı sonucu profilleme herhangi Polizom doğrulayarak önemlidirörneğin Triton X-100 gibi ler, kuvvetli) spektrumun bu bölgede absorbe ve gradyan üstündeki 40 / 60S tepe gizleyebilir. Lisatı olmayan ve / veya lisat tamponu ile Boş gradyanı, 254 nm&#39;de abzorbans, tamponlar nispi katkısının belirlenmesi için çalıştırılması gerekir. Artefactual yüksek mertebeden yapılar da aslında hiçbiri (örneğin &quot;sözde Polisomlar&quot; 20) vardır polizom hatalı yorumlara yol açabilir. Magnezyum Ayrılan iki değerlikli katyon kenetleme maddesi EDTA (80S ribozomlar stabilize etmek için işlem boyunca kullanılan) büyük (60S bileşen küçük (40S) içine ribozom ayrılmasına neden olacaktır lizatlarına ve gradyan tamponu (15 dakika için 20 mM) eklendi ) alt birimi. 260 nm&#39;de kaydedilen absorbans pikleri polisomlann gerçekten de Dolayısıyla, eğer EDTA muamelesiyle tek bir maksimum mRNA ve ribozomal birimleri serbest tekabül gradyanının üst kısmında görülen edilecek şekilde profili çökecek (veriler gösterilmemiştir). RastlayanProfilin EDTA kaynaklı çökmesiyle birlikte, qPCR analiz belirli hedeflerin hepsi şimdi degrade üstünde bulunmalıdır. Bir mRNA ile ilişkili ribozomların sayısı her zaman belirli bir mRNA tercüme ediliyor nasıl verimli bir göstergesi değildir. Nitekim, polisomlar aktif çeviri okuyucu farkında olmalıdır 21,22 durdu hale geldiği özel bağlamları vardır. Transkript aktif EDTA aksine, &#39;run-off&#39; aktif bir mRNA boyunca translocating olan ribozomların neden Puromycin ile örnek, tedavi) bir tarafından yapılabilir çeviri makine ile meşgul, veya b) örnek tedavisi olsun ya da olmasın belirlenmesi Yine herhangi bir aşağı translokasyon Ribozomların &#39;run-off&#39; neden başlangıç ​​kodonu sadece ilk ribozomun translokasyonu inhibe homoharringtonin&#39;le. QPCR analizi için kontrol transkriptleri kullanılması da önemlidir. SelecBu tür XIAP IRES olmayan IRES varyant bazı temizlik genin (GAPDH, aktin, tubulin, Nucleolin), ribozomal mRNA (18S, RPL13A) halinde ya da bu durumda, farklı tedavi koşullarında etkilenmez transkript tion, önemlidir doğrulama çeviri üzerinde tedavinin etkisinin spesifik ya da genel olup olmadığını. Bu kontrol transkriptleri burada yapılan gibi analiz mRNA&#39;ların dağılımını normalize etmek için kullanılabilir (XIAP ve Bcl-xL mRNA&#39;lar, GAPDH mRNA için normalize edildi ve her fraksiyon mRNA içeriğinin toplamı ile temsil edilen toplam mRNA&#39;nın içeriğinin yüzdesi olarak ifade edildi ) ya da seçenek olarak, hedef ve kontrol mRNA&#39;lar, mutlak değerler olarak ifade edilen ve ayrı olarak gösterilebilir. giriş lizatlan (toplam hacmi genellikle,% 10) olarak QPCR analizi belirli mRNA&#39;ların dağıtımı için kontrol edecek bir adımdır. İdeal olarak, giriş lizat belirli transkriptin mRNA içeriği analiz 10 fraksiyonları mRNA muhtevası toplamının ile karşılaştırılabilir nitelikte olmalıdır. NihayetBir isteğe bağlı aşama fenol kontrol etmek için: kloroform ile ekstre etme aşaması (kedi, kloramfenikol asetil transferaz gibi) ardından qPCR sayısal olarak ve hatta kullanılabilir normalleştirmek için bir in vitro transkribe raportör mRNA ile her polisom fraksiyonu başak etmektir Veriler 14. Herhangi bir teknik olduğu gibi, polisom profil bazı sınırlamalar sunulur. Genellikle 10 kesirler bir asgari güzel Polizom dağılımları olması için toplanması gerekmektedir (çeviri verimliliğinde daha ince kaymalar 20 veya daha fazla gerekebilir) olması bu adımı 4 örneklerinin sadece maksimum olabilir anlamda, sınırlayıcı yapar Bir anda analiz rahat bir kerede RNA ekstraksiyon ve 40 örnekleri ve 4 giriş kontrolleri QPCR analizini idare edebilmek için. Örneklem büyüklüğü kolayca QPCR yapmak çoğaltır kaç transkript analiz edilecek ve kaç ile ekleyebilirsiniz, ve bu pahalıya mal olabilir. QPCR tabanlı polysomal profil a diğer kısıtlılığınalysis sadece (transkript önceden bilinen analiz edilmesi) ve çeviri genom analizi için tatbik edilemez bir aday temelli yaklaşım yapılabilir olmasıdır. Ancak, 21. yüzyılın başında, araştırmacılar DNA kullanılarak bir genomik düzeyde polysomal RNA sorguya başladı nesil dizileme 16,17 ile daha yakın zamanda 15 ve mikroarray&#39;ler. Daha çok ayrı ayrı fraksiyonların QPCR analizi yapan daha haricinde bu protokolde tarif edildiği gibi, bu güçlü bir yaklaşım olarak, polysomal profil gerçekleştirilir, genel çeviri kısımlar (genellikle fraksiyon 2-4) ve polizom parçacıklar (genellikle kısımlar) toplanır ve varyasyonları analiz monosomes DNA mikro-dizisi ya da tüm genom RNA sekansları vasıtası ile. Dolayısıyla bu yaklaşım ile, bu değerlendirmek mümkündür tüm olan dağıtım kaymalar polisomlar dan monosomes için (çeviri azalma) ya da tam tersi (çeviri artış) belirli bir tedavi üzerine mRNAlarment. Belirli mRNA Polisomlar dağıtım Doğrulama ve miktar sonra qPCR yapılabilir. Polysomal profil ilkesinin daha da güçlü kullanım ribozom profillemesidir. Bu tekniğin daha yüksek verimlilik uzatma Polizom fraksiyonların 23 yüzlerce eşzamanlı izleme sağlayan son zamanlarda bildirildi. Mutant koleksiyonları veya büyük ölçekli RNAi ekranlarında öteleme verimliliği sondalama Bu açık bir avantaj sağlamaktadır. Ribozom profil protein sentezinde 24,25 yapan ribozomlar (ribozom ayak) tarafından korunan RNA parçaları eşleştirmek için yeni nesil dizileme yararlanır bir tekniktir. Bu teknikte, ribozom translokasyon sikloheksimid (geçici) ya da emetin (geri dönüşümsüz) bir ribozom ayak izi bir popülasyonunu üretmek üzere hücre lizizi ve RNaz sindirimi ile başlık ile inhibe edilebilir. Ribozomlar, zenginleştirilmiş total RNA izole edilir ve kirletici ribozomal ve mitokondriyal ribozomal RNA çıkarılır.Yaklaşık 26-28 nükleotid RNA ayak izi izole bir cDNA kütüphanesine dönüştürülmüştür, transkripsiyonu ve 26 dizilenmiştir ters. Standart Polizom profilleme aksine, bu yaklaşım sadece translationally düzenlenir belirli mRNA&#39;lan tanımlayan değil aynı zamanda belirli bir transkript boyunca tam işgal ve ribozomların yoğunluğu gibi kodon çözünürlükte mRNA özgü bilgileri verir. O nerede oluştuğunu transkript ribozom-istihdamına hakkında daha fazla bilgi verebilir Bu, özellikle, bu tür IRES-yataklık mRNA&#39;lanmn gibi çeviri özel modları ile mRNA&#39;larL için ilginçtir.

Protein sentezi (translasyon) düzenlenmesi, iyice hücre hayatta kalma ile bağlantılı önemli bir hücresel süreçtir. Çeviri hücrenin enerji% 50&#39;sinden fazlasını tüketir göz önüne alındığında, bu çeviri sıkı bir şekilde düzenlenmiştir ki ve çeviri tedirginlikler iyi tolere edilmez olması şaşırtıcı değildir. Tersine, bir çok sapkın hücresel süreçlerle nakil mekanizması ve çeviri çıkış (yani proteom) değiştirilmek üzere olmasını gerektirir. Bu genellikle, çeşitli stres cevabı ve hastalık durumlarında da olduğu, ve muhtemelen en iyi kanser örneklenmiştir. Translasyon kontrolünde önemli bir avantajı, hızlıca, çeşitli iç ve dış tetikleyicilere karşılık olarak protein çıkışı yeniden programlamak hücrelerin yeteneği, ve böylece ince ayar mekanizması olduğunu şunun hücre yaşamı ve ölümüyle 1 arasındaki denge. Öteleme kontrolü iki yönü özellikle ilginçtir; düzenleyici mec doğa anlayışıilk yerde seçici çeviri ortaya, özel tetikle idare hücresel yanıt olarak katılmak hanism (ler) ve kontrol belli bir çeviri izin elemanları, ve spesifik mRNA tespiti ve bunların protein ürünleri. Polizom profil Her iki yöntemin etkin çalışma sağlayan bir tekniktir. Polizom profil tekniğinin genel amacı eğitim ve farklı hücresel şartlar altında belirli mRNA&#39;lann çeviri durumunu ölçmek için. Tekniğin temel ilkesi, böylece, bir sükroz gradyanı üzerinde ultra-santrifüj ile elde edilen poliribozomların &#39;dondurulması farklı kopyalar üzerinde aktif çeviri ribozomlar ve ayrı&#39; (birkaç ribozom bağlayıcı) son derece çevrilmiş transkriptler arasında bir ayrım için izin vererek mRNA dönüşümüyle yakalamak için kötü (bir veya iki ribozomlar bağlı) olanlar çevrilmiştir. Bu özel protokol, antibiyotik sikloheksimidin bağlandığım60S ribozomal alt birim ve bloklar ribozom E sitesinden 2 deasetillenmiş tRNA serbest, ribozom translokasyon inhibe ve çeviri mRNA üzerinde ribozomlar durak için kullanılır. Ancak, aynı zamanda blok yapıldı uzama 3, kullanılabilirler, örneğin, emetin gibi bloke edici maddeler. Western blotting ve çeviri işleminin son çıkış ölçmek 35S-metionin etiketleme teknikleri farklı olarak, bu nedenle, farklı koşullar altında mekanizmalarından ilişkin daha ayrıntılı bir çalışma için izin veren, profil aktif çevrilmemiş mRNA ile ribozom ilişki ölçüm avantajına sahiptir polisom. Bu yüzden, tekniği kolayca özel çeviri 4-6 farklı modlar incelemek için adapte edilebilir, protein faktörleri çeviri düzenleme, 7-9, protein sentezini 1,10,11 ya da üst baş yangınlar ya da mRNA yapıları etkilerini etkileyen, gerilimli şartlar yer Protein synth açık okuma çerçeveleriagenezis 12-14. Günümüzde, polisom profil uygulama daha geniş polisomlann ilişkili mRNA&#39;ları analiz etmek için, DNA mikrodizilerinin 15 ya da yeni nesil dizileme 16,17 kullanımı ile bulunmaktadır.

<table border="0" cellpadding="0" cellspacing="0" width="772">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">Name of Material/ Equipment</td>
			<td style="width:135px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">Gradient Master 108 automated gradient maker</td>
			<td style="width:135px;">BIOCOMP</td>
			<td style="width:119px;">107-201M</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">Auto Densi-Flow two-chamber gradient maker</td>
			<td style="width:135px;">LABCONCO</td>
			<td align="right" style="width:119px;">4517000</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:351px;">Beckman Ultracentrifuge&#160;</td>
			<td style="width:135px;">Beckman Coulter</td>
			<td style="width:119px;">Model # L-100-XP</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">Density gradient fractionator</td>
			<td style="width:135px;">TELEDYNE ISCO</td>
			<td style="width:119px;">69-3873-246</td>
			<td>Composed of: tube piercing system,&#160; peristaltic pump, UA-6 Detector with 254 and 280nm filters and Foxy R1 Fraction Collector</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:351px;">WinDaq data acquisition software</td>
			<td style="width:135px;">DATAQ INSTRUMENTS</td>
			<td style="width:119px;">WINDAQ/LITE</td>
			<td>http://www.dataq.com/products/software/acquisition.htm</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:351px;">Pollyallomer ultracentrifuge tubes (for SW41, 14x89 mm)</td>
			<td style="width:135px;">Beckman Coulter</td>
			<td style="width:119px;">331372</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">Nuclease-free 2-ml centrifuge tubes (dolphin nose)</td>
			<td style="width:135px;">DiaMed</td>
			<td style="width:119px;">PRE1900-N</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">Nuclease-free 2-ml centrifuge tubes (flat bottom)</td>
			<td style="width:135px;">SafeSeal</td>
			<td style="width:119px;">12000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">Sucrose (nucleases-free)</td>
			<td style="width:135px;">Calbiochem</td>
			<td style="width:119px;">573113</td>
			<td>MW = 342.3 g/mol</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">Cycloheximide</td>
			<td style="width:135px;">SIGMA</td>
			<td style="width:119px;">C7698</td>
			<td>MW = 281.4 g/mol. Resuspend in DMSO and keep at -80C for long term sotrage. Caution: TOXIC! Can cause repiratory tracts and skin irritation. Wear mask when weighing.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">Sodium chloride (nucleases-free)</td>
			<td style="width:135px;">OmniPur</td>
			<td style="width:119px;">7710</td>
			<td>MW = 58.44 g/mol</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">MgCl2.6H2O (nucleases-free)</td>
			<td style="width:135px;">OmniPur</td>
			<td style="width:119px;">5980</td>
			<td>MW = 203.3 g/mol</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">Tris Base (nucleases-free)</td>
			<td style="width:135px;">J.T. Baker</td>
			<td style="width:119px;">4109-01</td>
			<td>MW = 121.14 g/mol</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">Triton X-100</td>
			<td style="width:135px;">OmniPur</td>
			<td align="right" style="width:119px;">9400</td>
			<td>Caution: TOXIC! Can cause repiratory tracts and skin irritation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">Recombinant RNasin Ribonuclease Inhibitor</td>
			<td style="width:135px;">Promega</td>
			<td style="width:119px;">N2515</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:351px;">RNaseZap Rnase inactivation solution</td>
			<td style="width:135px;">Ambion, Life Technologies</td>
			<td style="width:119px;">AM9780</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">GlycoBlue Coprecipitant (15 mg/mL)</td>
			<td style="width:135px;">Invitrogen, Life Technologies</td>
			<td style="width:119px;">AM9516</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Acid-Phenol:Chloroform, pH 4.5 (with IAA, 125:24:1)</td>
			<td style="width:135px;">Ambion, Life Technologies</td>
			<td style="width:119px;">AM9722</td>
			<td>Caution: TOXIC ! Can cause repiratory tracts irritation as well as skin irritation and corrosion</td>
		</tr>
	</tbody>
</table>

assessment of selective mrna translation in mammalian cells by polysome profiling

Protein sentezi Düzenleme strese hücre yanıt olarak önemli bir kontrol noktasını temsil eder. Özellikle, gizli RNA, düzenleyici elemanlar tipik olarak belirli bir stres tepkisi için gerekli olan proteinler için kodlayan spesifik mRNA, seçici çeviri izin vermek için gösterilmiştir. Bu mRNA&#39;lardan yanı sıra seçici çeviri sorumlu düzenleyici mekanizmaların karakterizasyonu Kimlik süredir moleküler biyoloji ön planda olmuştur. Polizom profilleme bu çalışmalarda bir dönüm yöntemdir. Polizom profil amacı, farklı kopyalar üzerinde aktif çeviri ribozomlar immobilize edilmesi ile mRNA dönüşümüyle yakalamak ve bu yüzden de çok tercüme transkript ve kötü tercüme olanlar arasında bir ayrım sağlayan, bir sükroz gradyanı üzerinde ultra-santrifüj ile elde edilen poliribozomların ayırmaktır. Bu daha sonra, klasik biyokimyasal ve moleküler biyoloji yöntemleri ile ayırt edilebilir. Önemli bir şekilde, birleştirme syüksek kapasiteli genomik yaklaşımları ile olysome profil öteleme düzenlemenin bir büyük ölçekli analizi sağlar.

Biz son zamanlarda apoptotik inhibitörleri XIAP ve Bcl-xL 12 kodlayan mRNA&#39;lan IRES-barındıran bir seçici çeviri inhibitörü olarak tümör baskılayıcı PDCD4 için yeni bir rol tarif var. Polizom profil IRES-aracılı çeviri PDCD4 özel katılımını göstermek için önemli bir tekniktir. HEK293 hücreleri geçici PDCD4 (Şekil 1A) endojenik düzeylerini tüketmek için transfekte edilmiş ve daha sonra profil analizleri polisom tabi tutuldu. Biz siCTRL ve siPDCD4 ile işlenen hücrelerde (Şekil 1B) karşılaştıran polisom profilindeki değişikliklerle eksikliği ile karar PDCD4 azaltılması kat, küresel hücresel Çeviri zarar almadığı gözlemlenmiştir. Benzer şekilde, XIAP 19 olmayan IRES varyantının polisom dağılımı, işlenmiş ve işlenmemiş hücreleri (Şekil 1C) arasında aynı idi. Buna karşılık, iki IRES-barındıran mRNA&#39;ların polisom dağılımı, XIAP ve Bcl-xL, önemli ölçüde değişmiş edildi. P yokluğundaDCD4 Bu iki mRNA&#39;ların dağılımı ağır ribopolysomes (Şekil 1D, E) içine kaydırılır. Bu XIAP ve Bcl-xL, mRNA (Şekil 1F), bu sonuçlar kararlı durum seviyelerinde değişikliklere eşlik olmadığından daha ileri olarak Western lekeleme (Şekil ile teyit edilmiştir, indirgenmiş PDCD4 seviyeleri ile hücrelerde XIAP ve Bcl-xL translasyonunu daha zengin oldukları bilinir 1G). <img alt="Şekil 1," src="/files/ftp_upload/52295/52295fig1highres.jpg" /> Şekil 1:. Polisom profil XIAP ve Bcl-xL çeviri seçici önleyicisi olarak PDCD4 tanımlayan (A), HEK293 hücreleri PDCD4 siRNA veya kontrol (CTRL) ile muamele edildi, bir anti-PDCD4 lizlendi ve Western blot analizine tabi tutulmuştur siRNA olmayan hedefleme ve anti-Nucleolin antikorları. PDCD4 knock-down kapsamını doğrulamak (B) PDCD4 (A) gibi yıkıldığında veHücre lizatları, profil polisom tabi tutuldu. Temsilci Polizom profil üst panelde gösterilir; XIAP olmayan IRES (C) dağılımı, GAPDH edilene göre, Bcl-xL (D) ve XIAP IRES (E) mRNAlar, her bir parça halinde toplam mRNA yüzde (% mRNA) olarak gösterilmiştir. (F) Sabit -durum mRNA seviyeleri PDCD4, siRNA veya Kontrol (CTRL) siRNA ile muamele görmüş hücreler RT-qPCR ölçüldü. (G) &#39;den Lizatlar (A)&#39; da, anti-XIAP, anti-Bcl-xL, ve birlikte western blot analizine tabi tutulmuştur Anti-Nucleolin antikorlar. (NB. Veri başlangıçta 12 yayınlandı Şekil 1&#39;de sunulan) <a href="https://www.jove.com/files/ftp_upload/52295/52295fig1large.jpg" target="_blank">bu rakamın büyük halini görmek için lütfen buraya tıklayınız.</a>

Watch this Scientific Journal Video about Assessment of Selective mRNA Translation in Mammalian Cells by Polysome Profiling at JoVE.com

Assessment of Selective mRNA Translation in Mammalian Cells by Polysome Profiling

The ability of cells to adapt to stress is crucial for their survival. Regulation of mRNA translation is one such adaptation strategy, providing for rapid regulation of the proteome. Here, we provide a standardized polysome profiling protocol to identify specific mRNAs that are selectively translated under stress conditions.

Polizom Profil Yöntemi ile memeli hücrelerine selektif mRNA dönüşümüyle Değerlendirilmesi

Regulation of protein synthesis represents a key control point in cellular response to stress. In particular, discreet RNA regulatory elements were shown ...

assessment-selective-mrna-translation-mammalian-cells-polysome

Research

JoVE Journal

Biology

27.9K Görüntüleme.  University of Ottawa. The ability of cells to adapt to stress is crucial for their survival. Regulation of mRNA translation is one such adaptation strategy, providing for rapid regulation of the proteome. Here, we provide a standardized polysome profiling protocol to identify specific mRNAs that are selectively translated under stress conditions. 

Video: Assessment of Selective mRNA Translation in Mammalian Cells by Polysome Profiling

Analysis of mRNA Nuclear Export Kinetics in Mammalian Cells by Microinjection

In eukaryotes, messenger RNA (mRNA) is transcribed in the nucleus and must be exported into the cytoplasm to access the translation machinery. Although the nuclear export of mRNA has been studied extensively in Xenopus oocytes1 and genetically tractable organisms such as yeast2 and the Drosophila derived S2 cell line3, few studies had been conducted in mammalian cells. Furthermore the kinetics of mRNA export in mammalian somatic cells could only be inferred indirectly4,5. In order to measure the nuclear export kinetics of mRNA in mammalian tissue culture cells, we have developed an assay that employs the power of microinjection coupled with fluorescent in situ hybridization (FISH). These assays have been used to demonstrate that in mammalian cells, the majority of mRNAs are exported in a splicing dependent manner6,7, or in manner that requires specific RNA sequences such as the signal sequence coding region (SSCR) 6. In this assay, cells are microinjected with either in vitro synthesized mRNA or plasmid DNA containing the gene of interest. The microinjected cells are incubated for various time points then fixed and the sub-cellular localization of RNA is assessed using FISH. In contrast to transfection, where transcription occurs several hours after the addition of nucleic acids, microinjection of DNA or mRNA allows for rapid expression and allows for the generation of precise kinetic data.

Here we describe an assay that employs the power of microinjection coupled with fluorescent in situ hybridization in order to accurately measure the nuclear export kinetics of mRNA in mammalian somatic cells.

Mikroenjeksiyon tarafından Memeli Hücreleri mRNA Nükleer İhracat Kinetik Analizi

In eukaryotes, messenger RNA (mRNA) is transcribed in the nucleus and must be exported into the cytoplasm to access the translation machinery. Although ...

Biyoloji

Analysis of Cell Cycle Position in Mammalian Cells

The regulation of cell proliferation is central to tissue morphogenesis during the development of multicellular organisms. Furthermore, loss of control of cell proliferation underlies the pathology of diseases like cancer. As such there is great need to be able to investigate cell proliferation and quantitate the proportion of cells in each phase of the cell cycle. It is also of vital importance to indistinguishably identify cells that are replicating their DNA within a larger population. Since a cell&prime;s decision to proliferate is made in the G1 phase immediately before initiating DNA synthesis and progressing through the rest of the cell cycle, detection of DNA synthesis at this stage allows for an unambiguous determination of the status of growth regulation in cell culture experiments.
DNA content in cells can be readily quantitated by flow cytometry of cells stained with propidium iodide, a fluorescent DNA intercalating dye. Similarly, active DNA synthesis can be quantitated by culturing cells in the presence of radioactive thymidine, harvesting the cells, and measuring the incorporation of radioactivity into an acid insoluble fraction. We have considerable expertise with cell cycle analysis and recommend a different approach. We Investigate cell proliferation using bromodeoxyuridine/fluorodeoxyuridine (abbreviated simply as BrdU) staining that detects the incorporation of these thymine analogs into recently synthesized DNA. Labeling and staining cells with BrdU, combined with total DNA staining by propidium iodide and analysis by flow cytometry1 offers the most accurate measure of cells in the various stages of the cell cycle. It is our preferred method because it combines the detection of active DNA synthesis, through antibody based staining of BrdU, with total DNA content from propidium iodide. This allows for the clear separation of cells in G1 from early S phase, or late S phase from G2/M. Furthermore, this approach can be utilized to investigate the effects of many different cell stimuli and pharmacologic agents on the regulation of progression through these different cell cycle phases.
In this report we describe methods for labeling and staining cultured cells, as well as their analysis by flow cytometry. We also include experimental examples of how this method can be used to measure the effects of growth inhibiting signals from cytokines such as TGF-&beta;1, and proliferative inhibitors such as the cyclin dependent kinase inhibitor, p27KIP1. We also include an alternate protocol that allows for the analysis of cell cycle position in a sub-population of cells within a larger culture5. In this case, we demonstrate how to detect a cell cycle arrest in cells transfected with the retinoblastoma gene even when greatly outnumbered by untransfected cells in the same culture. These examples illustrate the many ways that DNA staining and flow cytometry can be utilized and adapted to investigate fundamental questions of mammalian cell cycle control.

Determining the cell cycle position of a population of cells, or understanding how signals affect proliferation, can be readily measured by flow cytometry using this protocol. We report a simple experimental approach to staining cells and quantifying their position in the cell cycle.

Memeli Hücreler, Hücre Döngüsü Pozisyon Analizi

The regulation of cell proliferation is central to tissue morphogenesis during the development of multicellular organisms. Furthermore, loss of control of ...

Monitoring Plasmid Replication in Live Mammalian Cells over Multiple Generations by Fluorescence Microscopy

Few naturally-occurring plasmids are maintained in mammalian cells. Among these are genomes of gamma-herpesviruses, including Epstein-Barr virus (EBV) and Kaposi's Sarcoma-associated herpesvirus (KSHV), which cause multiple human malignancies 1-3. These two genomes are replicated in a licensed manner, each using a single viral protein and cellular replication machinery, and are passed to daughter cells during cell division despite their lacking traditional centromeres 4-8.
Much work has been done to characterize the replications of these plasmid genomes using methods such as Southern blotting and fluorescence in situ hybridization (FISH). These methods are limited, though. Quantitative PCR and Southern blots provide information about the average number of plasmids per cell in a population of cells. FISH is a single-cell assay that reveals both the average number and the distribution of plasmids per cell in the population of cells but is static, allowing no information about the parent or progeny of the examined cell.
Here, we describe a method for visualizing plasmids in live cells. This method is based on the binding of a fluorescently tagged lactose repressor protein to multiple sites in the plasmid of interest 9. The DNA of interest is engineered to include approximately 250 tandem repeats of the lactose operator (LacO) sequence. LacO is specifically bound by the lactose repressor protein (LacI), which can be fused to a fluorescent protein. The fusion protein can either be expressed from the engineered plasmid or introduced by a retroviral vector. In this way, the DNA molecules are fluorescently tagged and therefore become visible via fluorescence microscopy. The fusion protein is blocked from binding the plasmid DNA by culturing cells in the presence of IPTG until the plasmids are ready to be viewed.
This system allows the plasmids to be monitored in living cells through several generations, revealing properties of their synthesis and partitioning to daughter cells. Ideal cells are adherent, easily transfected, and have large nuclei. This technique has been used to determine that 84% of EBV-derived plasmids are synthesized each generation and 88% of the newly synthesized plasmids partition faithfully to daughter cells in HeLa cells. Pairs of these EBV plasmids were seen to be tethered to or associated with sister chromatids after their synthesis in S-phase until they were seen to separate as the sister chromatids separated in Anaphase10. The method is currently being used to study replication of KSHV genomes in HeLa cells and SLK cells. HeLa cells are immortalized human epithelial cells, and SLK cells are immortalized human endothelial cells. Though SLK cells were originally derived from a KSHV lesion, neither the HeLa nor SLK cell line naturally harbors KSHV genomes11. In addition to studying viral replication, this visualization technique can be used to investigate the effects of the addition, removal, or mutation of various DNA sequence elements on synthesis, localization, and partitioning of other recombinant plasmid DNAs.

A method of observing individual DNA molecules in live cells is described. The technique is based on the binding of a fluorescently tagged lac repressor protein to binding sites engineered into the DNA of interest. This method can be adapted to follow many recombinant DNAs in live cells over time.

Floresan Mikroskobu ile Çoklu Nesiller boyunca Canlı Memeli Hücrelerinde Plazmid Çoğaltma İzlenmesi

Few naturally-occurring plasmids are maintained in mammalian cells.  Among these are genomes of gamma-herpesviruses, including Epstein-Barr virus  (EBV) ...

Analysis of Translation Initiation During Stress Conditions by Polysome Profiling

Precise control of mRNA translation is fundamental for eukaryotic cell homeostasis, particularly in response to physiological and pathological stress. Alterations of this program can lead to the growth of damaged cells, a hallmark of cancer development, or to premature cell death such as seen in neurodegenerative diseases. Much of what is known concerning the molecular basis for translational control has been obtained from polysome analysis using a density gradient fractionation system. This technique relies on ultracentrifugation of cytoplasmic extracts on a linear sucrose gradient. Once the spin is completed, the system allows fractionation and quantification of centrifuged zones corresponding to different translating ribosomes populations, thus resulting in a polysome profile. Changes in the polysome profile are indicative of changes or defects in translation initiation that occur in response to various types of stress. This technique also allows to assess the role of specific proteins on translation initiation, and to measure translational activity of specific mRNAs. Here we describe our protocol to perform polysome profiles in order to assess translation initiation of eukaryotic cells and tissues under either normal or stress growth conditions.

Here, we describe a method to analyze changes in the initiation of mRNA translation of eukaryotic cells in response to stress conditions. This method is based on the velocity separation on sucrose gradients of translating ribosomes from non-translating ribosomes.

Polizom profilleme Stres Durumlardaki Çeviri başlatılması Analizi

Precise control of mRNA translation is fundamental for eukaryotic cell homeostasis, particularly in response to physiological and pathological stress. ...

Profiling Individual Human Embryonic Stem Cells by Quantitative RT-PCR

Heterogeneity of stem cell population hampers detailed understanding of stem cell biology, such as their differentiation propensity toward different lineages. A single cell transcriptome assay can be a new approach for dissecting individual variation. We have developed the single cell qRT-PCR method, and confirmed that this method works well in several gene expression profiles. In single cell level, each human embryonic stem cell, sorted by OCT4::EGFP positive cells, has high expression in OCT4, but a different level of NANOG expression. Our single cell gene expression assay should be useful to interrogate population heterogeneities.

Single cell gene expression assay is needed for understanding stem cell heterogeneities.

Kantitatif RT-PCR ile Bireysel İnsan Embriyonik Kök Hücreleri profilleme

Heterogeneity of stem cell population hampers detailed understanding of stem cell biology, such as their differentiation propensity toward different ...

Polysome Fractionation and Analysis of Mammalian Translatomes on a Genome-wide Scale

mRNA translation plays a central role in the regulation of gene expression and represents the most energy consuming process in mammalian cells. Accordingly, dysregulation of mRNA translation is considered to play a major role in a variety of pathological states including cancer. Ribosomes also host chaperones, which facilitate folding of nascent polypeptides, thereby modulating function and stability of newly synthesized polypeptides. In addition, emerging data indicate that ribosomes serve as a platform for a repertoire of signaling molecules, which are implicated in a variety of post-translational modifications of newly synthesized polypeptides as they emerge from the ribosome, and/or components of translational machinery. Herein, a well-established method of ribosome fractionation using sucrose density gradient centrifugation is described. In conjunction with the in-house developed &#8220;anota&#8221; algorithm this method allows direct determination of differential translation of individual mRNAs on a genome-wide scale. Moreover, this versatile protocol can be used for a variety of biochemical studies aiming to dissect the function of ribosome-associated protein complexes, including those that play a central role in folding and degradation of newly synthesized polypeptides.

Ribosomes play a central role in protein synthesis. Polyribosome (polysome) fractionation by sucrose density gradient centrifugation allows direct determination of translation efficiencies of individual mRNAs on a genome-wide scale. In addition, this method can be used for biochemical analysis of ribosome- and polysome-associated factors such as chaperones and signaling molecules.

Bir genom Ölçeği Polizom Fraksiyonu ve Memeli Translatomes Analizi

mRNA translation plays a central role in the regulation of gene expression and represents the most energy consuming process in mammalian cells. ...

Measurement of Heme Synthesis Levels in Mammalian Cells

Heme serves as the prosthetic group for a wide variety of proteins known as hemoproteins, such as hemoglobin, myoglobin and cytochromes. It is involved in various molecular and cellular processes such as gene transcription, translation, cell differentiation and cell proliferation. The biosynthesis levels of heme vary across different tissues and cell types and is altered in diseased conditions such as anemia, neuropathy and cancer. This technique uses [4-14C] 5-aminolevulinic acid ([14C] 5-ALA), one of the early precursors in the heme biosynthesis pathway to measure the levels of heme synthesis in mammalian cells. This assay involves incubation of cells with [14C] 5-ALA followed by extraction of heme and measurement of the radioactivity incorporated into heme. This procedure is accurate and quick. This method measures the relative levels of heme biosynthesis rather than the total heme content. To demonstrate the use of this technique the levels of heme biosynthesis were measured in several mammalian cell lines.

Altered intracellular heme levels are associated with common diseases such as cancer. Thus, there is a need to measure heme biosynthesis levels in diverse cells. The goal of this protocol is to provide a fast and sensitive method to measure and compare the levels of heme synthesis in different cells.

Memeli Hücrelerde Heme Sentezi Düzeylerinin Ölçümü

Heme serves as the prosthetic group for a wide variety of proteins known as hemoproteins, such as hemoglobin, myoglobin and cytochromes. It is involved in ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

Yetişkin Fare Kalvaryal Kemik İliği içinde Hematopoietik kök hücrelerin Vivo 4-Boyutlu Takip

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

An Easy Method for Plant Polysome Profiling

Translation of mRNA to protein is a fundamental and highly regulated biological process. Polysome profiling is considered as a gold standard for the analysis of translational regulation. The method described here is an easy and economical way for fractionating polysomes from various plant tissues. A sucrose gradient is made without the need for a gradient maker by sequentially freezing each layer. Cytosolic extracts are then prepared in a buffer containing cycloheximide and chloramphenicol to immobilize the cytosolic and chloroplastic ribosomes to mRNA and are loaded onto the sucrose gradient. After centrifugation, six fractions are directly collected from the bottom to the top of the gradient, without piercing the ultracentrifugation tube. During collection, the absorbance at 260 nm is read continuously to generate a polysome profile that gives a snapshot of global translational activity. Fractions are then pooled to prepare three different mRNA populations: the polysomes, mRNAs bound to several ribosomes; the monosomes, mRNAs bound to one ribosome; and mRNAs that are not bound to ribosomes. mRNAs are then extracted. This protocol has been validated for different plants and tissues including Arabidopsis thaliana seedlings and adult plants, Nicotiana benthamiana, Solanum lycopersicum, and Oryza sativa leaves.

This protocol describes an easy method to extract and fractionate transcripts from plant tissues on the basis of the number of bound ribosomes. It allows a global estimate of translation activity and the determination of the translational status of specific mRNAs.

Bitki Polizom Profiling için bir Kolay Yöntem

Translation of mRNA to protein is a fundamental and highly regulated biological process. Polysome profiling is considered as a gold standard for the ...

Probing mRNA Kinetics in Space and Time in Escherichia coli using Two-Color Single-Molecule Fluorescence In Situ Hybridization

Single-molecule fluorescence in situ hybridization (smFISH) allows for counting the absolute number of mRNAs in individual cells. Here, we describe an application of smFISH to measure the rates of transcription and mRNA degradation in Escherichia coli. As smFISH is based on fixed cells, we perform smFISH at multiple time points during a time-course experiment, i.e., when cells are undergoing synchronized changes upon induction or repression of gene expression. At each time point, sub-regions of an mRNA are spectrally distinguished to probe transcription elongation and premature termination. The outcome of this protocol also allows for analyzing intracellular localization of mRNAs and heterogeneity in mRNA copy numbers among cells. Using this protocol many samples (~50) can be processed within 8 h, like the amount of time needed for just a few samples. We discuss how to apply this protocol to study the transcription and degradation kinetics of different mRNAs in bacterial cells.

Probing mRNA Kinetics in Space and Time in Escherichia coli using Two-Color Single-Molecule Fluorescence In Situ Hybridization

This protocol describes an application of single-molecule fluorescence in situ hybridization (smFISH) to measure the in vivo kinetics of mRNA synthesis and degradation.

Situ Hibridizasyonda İki Renkli Tek Moleküllü Floresan Kullanarak Escherichia coli'de Uzay ve Zamanda MRNA KinetikLerini Sondalama

Single-molecule fluorescence in situ hybridization (smFISH) allows for counting the absolute number of mRNAs in individual cells. Here, we describe an ...