Bu eser sağlık hibe AG040191 ve AG054566 için VML Ulusal Enstitüleri tarafından desteklenmiştir. VML yaşlanma araştırma Amerikan Federasyonu AFAR araştırma bursu alıcıdan iken bu araştırma yapılmıştır.

<ol>
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Yazarlar onlar rakip hiçbir mali çıkarları var bildirin.

Ribo Seq yaklaşım mRNA çeviri vivo içinde genom çapında düzey 3analizi için güçlü bir teknoloji olarak ortaya çıkmıştır. Çeviri ile tek-kodonu kararlılık izleme sağlar, bu yaklaşımı kullanarak çalışmalar translasyonel yönetmelik anlayışımızı katkıda bulunmuştur. Avantajları rağmen Ribo Seq bazı sınırlamaları vardır. Ribozomal RNA (rRNA) her zaman sırasında yalıtım Ribo Seq deneyler 9,25...

Hücredeki protein ifade yönetmelikte önemli bir rol oynayan temel işlemlerden biri mRNA çevrilmiştir. Bu nedenle, mRNA çeviri sıkı farklı iç ve dış fizyolojik uyaranlar 1,2yanıt olarak kontrol edilir. Ancak, translasyonel düzenleme mekanizmaları çöküşünde kalır. Burada, biz genom çapında miktar mayası çeviri için protokol ribozom profil oluşturma açıklanmaktadır. Ribozom profil çıkarma tekniği genel amacı çalışma ve çeşitli hücresel koşullarda belirli mRNA'ların çeviri ölçmek etmektir. Bu teknik ribozom doluluk genom boyunca kantitatif analiz için yeni nesil sıralama kullanır ve protein sentezi vivo içinde tek kodon çözünürlük 3,4oranı izleme sağlar. Şu anda, bu yöntem protein çeviri düzeyde ölçmenin en gelişmiş araçlar sağlar ve mevcut diğer tekniklerle, örneğin microarrays ortaya bilgi veren yararlı keşif aracı olduğu ispatlanmıştır veya Çeviri Durumu dizi analizi (TSAA) 5. Transkript düzeyleri ve çevirim çıkış birleştirilmiş değişiklikleri ile ilgili raporlar profil oluşturma ribozom da diğer yöntemlerine göre çok daha fazla hassasiyet sağlar.
Bu yaklaşım mRNA parçaları 3ribozom korumalı derin sıralama üzerinde temel alır. Ribozomlarda protein çeviri sırasında korumak ~ 28 nt bölümlerini (ayak izleri olarak adlandırılır) mRNA 6. Ribozom tarafından korunan parçaları dizisi belirleyerek, Ribo Seq ribozomlara çevrilmiş mRNA üzerinde konumunu harita ve mRNA hangi bölgelerinde aktif protein 3,7olarak tercüme edilebilir muhtemeldir tanımlamak. Buna ek olarak, biz kantitatif mRNA tercümesi verilen bir mRNA transkript hizalamak ayak izleri sayısını sayarak ölçebilirsiniz.
Ribozom tarafından korunan parçaları ayırmak için hücre lysates başlangıçta ribonükleaz sindirim tarafından takip ribozomlara oyalamak için bir çeviri önleyici ile tedavi edilir. Ücretsiz mRNA ve ribozomlar tarafından korunmamasına çevrilmiş mRNA'ların bölümlerini ribonükleaz tarafından bozulmuş ise, ribozom korumalı mRNA parçaları sağlam ribozom-ayak izi kompleksleri arındırıcı tarafından elde edilebilir. Bu mRNA ayak sonra cDNA kitaplığa dönüştürülür ve derin sıralama (şekil 1) tarafından analiz. Ribozom profil oluşturma için paralel olarak sağlam mRNA aynı örneğinden ayıklanmış ve sıralı. MRNA bereket ölçümleri ile Ribo-Seq tarafından tanımlanan çeviri düzeyini karşılaştırarak, biz özellikle up - veya aşağı-düzenlenmiş çeviri düzeyde olan genlerin tanımlamak ve mRNA genom geneli düzeyinde verimliliğini çeviri hesaplamak. Bu makalede açıklanan protokol için Maya belirli olmakla birlikte, aynı zamanda diğer sistemlerde Ribo Seq Protokolü kurmaya çalışacağız araştırmacılar için yararlı olmalıdır.

<table><tbody><tr><td>0.45 &amp;mu;M membrane filters</td><td>Millipore</td><td>HVLP04700</td><td></td></tr><tr><td>0.5 M EDTA</td><td>Invitrogen</td><td>AM9261</td><td></td></tr><tr><td>0.5 mL centrifugal filters (100 kDa MWCO)</td><td>Millipore</td><td>UFC510024</td><td></td></tr><tr><td>1 M Tris-HCl, pH 7.0</td><td>Invitrogen</td><td>AM9850G</td><td></td></tr><tr><td>1 M Tris-HCl, pH 7.5 (pH 8.0 at 4&amp;deg;C)</td><td>Invitrogen</td><td>15567-027</td><td></td></tr><tr><td>10X TBE buffer</td><td>Invitrogen</td><td>AM9863</td><td></td></tr><tr><td>10% TBE-urea gel</td><td>Invitrogen</td><td>EC6875BOX</td><td></td></tr><tr><td>15% TBE-urea gel</td><td>Invitrogen</td><td>EC6885BOX</td><td></td></tr><tr><td>2 M MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>RPI</td><td>M24500-10.0</td><td></td></tr><tr><td>2X TBE-urea sample buffer</td><td>Invitrogen</td><td>LC6876</td><td></td></tr><tr><td>3M NaOAc, pH 5.5</td><td>Invitrogen</td><td>AM9740</td><td></td></tr><tr><td>5&#039; Deadenylase (10 U/&amp;mu;L)</td><td>Epicentre</td><td>DA11101K</td><td></td></tr><tr><td>5X Nucleic acid sample loading buffer</td><td>Bio-Rad</td><td>161-0767</td><td></td></tr><tr><td>8% TBE gel</td><td>Invitrogen</td><td>EC6215BOX</td><td></td></tr><tr><td>Acid-Phenol:Chloroform, pH 4.5 (with IAA, 125:24:1)</td><td>Invitrogen</td><td>AM9722</td><td></td></tr><tr><td>Blue light transilluminator</td><td>Clare Chemical Research</td><td>DR-46B</td><td></td></tr><tr><td>Chrome-steel beads, 3.2 mm</td><td>BioSpec Products</td><td>11079132c</td><td></td></tr><tr><td>Cryogrinder</td><td>Biospec product</td><td>3110BX</td><td></td></tr><tr><td>Cycloheximide</td><td>RPI</td><td>C81040-5.0</td><td></td></tr><tr><td>Data Acquisition System</td><td>DATAQ Instruments</td><td>DI-245</td><td></td></tr><tr><td>Deoxynucleotide (dNTP) solution mix (10 mM)</td><td>NEB</td><td>N0447L</td><td></td></tr><tr><td>Glycogen</td><td>Invitrogen</td><td>AM9510</td><td></td></tr><tr><td>Gradient fractionation system</td><td>Brandel</td><td>BR-184X</td><td></td></tr><tr><td>High-fidelity DNA polymerase (2,000 U/mL)</td><td>NEB</td><td>M0530S</td><td>Supplied with 5X Phusion HF Buffer</td></tr><tr><td>Next-generation sequencing library quantification kit</td><td>Kapa Biosystems</td><td>KK4824</td><td></td></tr><tr><td>Nucleic acid gel stain</td><td>Invitrogen</td><td>S11494</td><td></td></tr><tr><td>Optima XE-90 ultracentrifuge</td><td>Beckman Coulter</td><td>A94471</td><td></td></tr><tr><td>Poly(A) mRNA isolation kit</td><td>Invitrogen</td><td>61011</td><td></td></tr><tr><td>Rec J exonuclease (10 U/&amp;mu;L)</td><td>Epicentre</td><td>RJ411250</td><td></td></tr><tr><td>Reverse transcriptase (200 U/&amp;mu;L)</td><td>Invitrogen</td><td>18080093</td><td>Supplied with 5X first-strand buffer and 0.1 M DTT</td></tr><tr><td>RNA fragmentation buffer</td><td>NEB</td><td>E6186A</td><td></td></tr><tr><td>RNase I (100 U/&amp;mu;L)</td><td>Invitrogen</td><td>AM2295</td><td></td></tr><tr><td>RNase inhibitor (20 U/&amp;mu;L)</td><td>Invitrogen</td><td>AM2696</td><td></td></tr><tr><td>Silicone rubber caps</td><td>BioSpec Products</td><td>2008</td><td></td></tr><tr><td>ssDNA ligase (100 U/&amp;mu;L)</td><td>Epicentre</td><td>CL9021K</td><td>Supplied with 10X CircLigase II buffer and 50 mM MnCl&lt;sub&gt;2&lt;/sub&gt;</td></tr><tr><td>Stainless steel microvials, 1.8 mL</td><td>BioSpec Products</td><td>2007</td><td></td></tr><tr><td>Sucrose</td><td>RPI</td><td>S24060-5000.0</td><td></td></tr><tr><td>SW-41 Ti rotor</td><td>Beckman Coulter</td><td>331362</td><td></td></tr><tr><td>Syringe pump</td><td>New Era Pump Systems</td><td>NE-300</td><td></td></tr><tr><td>T4 polynucleotide kinase (10,000 U/mL)</td><td>NEB</td><td>M0201S</td><td>Supplied with 10X T4 polynucleotide kinase buffer</td></tr><tr><td>T4 RNA ligase 2 truncated KQ (200,000 U/mL)</td><td>NEB</td><td>M0373S</td><td>Supplied with 10X T4 RNA ligase buffer and 50% PEG8000</td></tr><tr><td>Thermal cycler</td><td>Bio-Rad</td><td>1851148</td><td></td></tr><tr><td>Thinwall polyallomer tubes, 13.2 mL</td><td>Beckman Coulter</td><td>331372</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma Aldrich</td><td>X100-100ML</td><td></td></tr><tr><td>UV monitor</td><td>Bio-Rad</td><td>7318160</td><td></td></tr><tr><td>Saccharomyces cerevisiae strain BY4741</td><td>Open Biosystems</td><td>YSC1048</td><td></td></tr></tbody></table>

genome-wide quantification of translation in budding yeast by ribosome profiling

MRNA tercüme proteinler düzenleme birkaç kat içeren karmaşık bir işlemdir. Bu kez değişiklikleri protein sentezi mRNA transkripsiyon değişiklikleri yansıtmak, ama pek çok özel durumlar gözlenmiştir varsayılır. Son zamanlarda, ribozom profil oluşturma (Ribo Seq) adında bir teknik veya kimlik, mRNA hangi bölgelerinde protein ve miktar çeviri genom geneli düzeyinde çevriliyor yüksek doğrulukla sağlar güçlü bir yöntem olarak ortaya çıkmıştır. Burada, genom çapında miktar Ribo Seq mayası içinde kullanarak çeviri için Genelleştirilmiş bir iletişim kuralı mevcut. Buna ek olarak, mRNA bereket ölçümleri ile Ribo Seq veri birleştirerek aynı anda mRNA transkript aynı örnek içinde binlerce çeviri verimliliğini ölçmek ve bu parametreler deneysel cevaben değişiklikleri karşılaştırmak için bize izin verir işlemler veya farklı fizyolojik devletler. Biz üretimi ile birlikte uygun derin sıralama için nükleaz sindirim, bozulmamış ribozom-ayak izi kompleksleri Sükroz degrade ayırma ile yalıtım ve hazırlık DNA kütüphanelerinin kullanarak ribozom ayak izi için detaylı bir protokol tarif Kalite denetimleri doğru analiz vivo içinde çeviri sağlamak için gerekli.

Veri profil oluşturma ribozom bioinformatic çözümlenmesi için detaylı boru hatları-si olmak be daha önce açıklanan 8,9. Buna ek olarak, birçok araştırma grupları farklı gen ifade analizi ve ribozom yöntemi 10,11,12 profil oluşturma için özel sıralama veri işleme için Biyoinformatik araçlarını geliştirdik ,<...

Watch this Scientific Journal Video about Genome-wide Quantification of Translation in Budding Yeast by Ribosome Profiling at JoVE.com

Genome-wide Quantification of Translation in Budding Yeast by Ribosome Profiling

Translasyonel yönetmelik protein bereket kontrolünde önemli bir rol oynar. Burada, kantitatif analiz tomurcuklanma Maya Saccharomyces cerevisiaeçeviri için bir yüksek-den geçerek yöntemi açıklanmaktadır.

Genom çapında Quantification ribozom profil oluşturma tarafından mayası çeviri

Translation of mRNA into proteins is a complex process involving several layers of regulation. It is often assumed that changes in mRNA transcription ...

genome-wide-quantification-translation-budding-yeast-ribosome

Universidad de Cartagena UDEC

Research

JoVE Journal

Biochemistry

11.4K Görüntüleme.  Boston University School of Medicine. Translasyonel yönetmelik protein bereket kontrolünde önemli bir rol oynar. Burada, kantitatif analiz tomurcuklanma Maya Saccharomyces cerevisiaeçeviri için bir yüksek-den geçerek yöntemi açıklanmaktadır.

Video: Genome-wide Quantification of Translation in Budding Yeast by Ribosome Profiling

Quantitative Immunofluorescence to Measure Global Localized Translation

The mechanisms regulating mRNA translation are involved in various biological processes, such as germ line development, cell differentiation, and organogenesis, as well as in multiple diseases. Numerous publications have convincingly shown that specific mechanisms tightly regulate mRNA translation. Increased interest in the translation-induced regulation of protein expression has led to the development of novel methods to study and follow de novo protein synthesis in cellulo. However, most of these methods are complex, making them costly and often limiting the number of mRNA targets that can be studied. This manuscript proposes a method that requires only basic reagents and a confocal fluorescence imaging system to measure and visualize the changes in mRNA translation that occur in any cell line under various conditions. This method was recently used to show localized translation in the subcellular structures of adherent cells over a short period of time, thus offering the possibility of visualizing de novo translation for a short period during a variety of biological processes or of validating changes in translational activity in response to specific stimuli.

This manuscript describes a method to visualize and quantify localized translation events in subcellular compartments. The approach proposed in this manuscript requires a basic confocal imaging system and reagents and is rapid and cost-effective.

Nicel ayirt ölçmek küresel çeviri lokalize

The mechanisms regulating mRNA translation are involved in various biological processes, such as germ line development, cell differentiation, and ...

Biyokimya

Saccharomyces cerevisiae Metabolic Labeling with 4-thiouracil and the Quantification of Newly Synthesized mRNA As a Proxy for RNA Polymerase II Activity

Global defects in RNA polymerase II transcription might be overlooked by transcriptomic studies analyzing steady-state RNA. Indeed, the global decrease in mRNA synthesis has been shown to be compensated by a simultaneous decrease in mRNA degradation to restore normal steady-state levels. Hence, the genome-wide quantification of mRNA synthesis, independently from mRNA decay, is the best direct reflection of RNA polymerase II transcriptional activity. Here, we discuss a method using non-perturbing metabolic labeling of nascent RNAs in Saccharomyces cerevisiae (S. cerevisiae). Specifically, the cells are cultured for 6 min with a uracil analog, 4-thiouracil, and the labeled newly transcribed RNAs are purified and quantified to determine the synthesis rates of all individual mRNA. Moreover, using labeled Schizosaccharomyces pombe cells as internal standard allows comparing mRNA synthesis in different S. cerevisiae strains. Using this protocol and fitting the data with a dynamic kinetic model, the corresponding mRNA decay rates can be determined.

Saccharomyces cerevisiae Metabolic Labeling with 4-thiouracil and the Quantification of Newly Synthesized mRNA As a Proxy for RNA Polymerase II Activity

The protocol described here is based on the genome-wide quantification of newly synthesized mRNA purified from yeast cells labeled with 4-thiouracil. This method allows to measure mRNA synthesis uncoupled from mRNA decay and, thus, provides an accurate measurement of RNA polymerase II transcription.

Saccharomyces cerevisiae Metabolik 4-thiouracil ve bir miktar ile etiketleme yeni mRNA RNA polimeraz II etkinlik için bir Proxy olarak sentezlenmiş

Global defects in RNA polymerase II transcription might be overlooked by transcriptomic studies analyzing steady-state RNA. Indeed, the global decrease in ...

Genetik

RIBO-seq in Bacteria: a Sample Collection and Library Preparation Protocol for NGS Sequencing

The ribosome profiling technique (RIBO-seq) is currently the most effective tool for studying the process of protein synthesis in vivo. The advantage of this method, in comparison to other approaches, is its ability to monitor translation by precisely mapping the position and number of ribosomes on a mRNA transcript.
In this article, we describe the consecutive stages of sample collection and preparation for RIBO-seq method in bacteria, highlighting the details relevant to the planning and execution of the experiment.
Since the RIBO-seq relies on intact ribosomes and related mRNAs, the key step is rapid inhibition of translation and adequate disintegration of cells. Thus, we suggest filtration and flash-freezing in liquid nitrogen for cell harvesting with an optional pretreatment with chloramphenicol to arrest translation in bacteria. For the disintegration, we propose grinding frozen cells with mortar and pestle in the presence of aluminum oxide to mechanically disrupt the cell wall. In this protocol, sucrose cushion or a sucrose gradient ultracentrifugation for monosome purification is not required. Instead, mRNA separation using polyacrylamide gel electrophoresis (PAGE) followed by the ribosomal footprint excision (28-30 nt band) is applied and provides satisfactory results. This largely simplifies the method as well as reduces the time and equipment requirements for the procedure. For library preparation, we recommend using the commercially available small RNA kit for Illumina sequencing from New England Biolabs, following manufacturer&#39;s guidelines with some degree of optimization.
The resulting cDNA libraries present appropriate quantity and quality required for next generation sequencing (NGS). Sequencing of the libraries prepared according to the described protocol results in 2 to 10 mln uniquely mapped reads per sample providing sufficient data for comprehensive bioinformatic analysis. The protocol we present is quick and relatively easy and can be performed with standard laboratory equipment.

Here we describe the stages of sample collection and preparation for RIBO-seq in bacteria. Sequencing of the libraries prepared according to these guidelines results in sufficient data for comprehensive bioinformatic analysis. The protocol we present is simple, uses standard laboratory equipment and takes seven days from lysis to obtaining libraries.

Bakterilerde RIBO-seq: NGS Dizilimi için Örnek Toplama ve Kütüphane Hazırlama Protokolü

The ribosome profiling technique (RIBO-seq) is currently the most effective tool for studying the process of protein synthesis in vivo. The advantage of ...

Polysome Profiling without Gradient Makers or Fractionation Systems

Polysome fractionation by sucrose density gradient centrifugation is a powerful tool that can be used to create ribosome profiles, identify specific mRNAs being translated by ribosomes, and analyze polysome associated factors. While automated gradient makers and gradient fractionation systems are commonly used with this technique, these systems are generally expensive and can be cost-prohibitive for laboratories that have limited resources or cannot justify the expense due to their infrequent or occasional need to perform this method for their research. Here, a protocol is presented to reproducibly generate polysome profiles using standard equipment available in most molecular biology laboratories without specialized fractionation instruments. Moreover, a comparison of polysome profiles generated with and without a gradient fractionation system is provided. Strategies to optimize and produce reproducible polysome profiles are discussed. Saccharomyces cerevisiae is utilized as a model organism in this protocol. However, this protocol can be easily modified and adapted to generate ribosome profiles for many different organisms and cell types.

This protocol describes how to generate a polysome profile without using automated gradient makers or gradient fractionation systems.

Degrade Yapıcılar veya Fraksiyonasyon Sistemleri Olmadan Polizom Profilleme

Polysome fractionation by sucrose density gradient centrifugation is a powerful tool that can be used to create ribosome profiles, identify specific mRNAs ...

Eukaryotic Polyribosome Profile Analysis

Protein synthesis is a complex cellular process that is regulated at many levels. For example, global translation can be inhibited at the initiation phase or the elongation phase by a variety of cellular stresses such as amino acid starvation or growth factor withdrawal. Alternatively, translation of individual mRNAs can be regulated by mRNA localization or the presence of cognate microRNAs. Studies of protein synthesis frequently utilize polyribosome analysis to shed light on the mechanisms of translation regulation or defects in protein synthesis. In this assay, mRNA/ribosome complexes are isolated from eukaryotic cells.  A sucrose density gradient separates mRNAs bound to multiple ribosomes known as polyribosomes from mRNAs bound to a single ribosome or monosome. Fractionation of the gradients allows isolation and quantification of the different ribosomal populations and their associated mRNAs or proteins. Differences in the ratio of polyribosomes to monosomes under defined conditions can be indicative of defects in either translation initiation or elongation/termination. Examination of the mRNAs present in the polyribosome fractions can reveal whether the cohort of individual mRNAs being translated changes with experimental conditions. In addition, ribosome assembly can be monitored by analysis of the small and large ribosomal subunit peaks which are also separated by the gradient. In this video, we present a method for the preparation of crude ribosomal extracts from yeast cells, separation of the extract by sucrose gradient and interpretation of the results.  This procedure is readily adaptable to mammalian cells.

This article describes a protocol for the extraction of translating ribosomes from eukaryotic cells. Once extracted, ribosomes are separated into monosomes and polyribosomes by sucrose gradient fractionation to allow different ribosomal populations to be analyzed. As such, this method is the gold standard for examining the regulation of translation.

Ökaryotik Polyribosome Profil Analizi

Protein synthesis is a complex cellular process that is regulated at many levels. For example, global translation can be inhibited at the initiation phase ...

Biyoloji

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. ...

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. ...

Assessment of Selective mRNA Translation in Mammalian Cells by Polysome Profiling

Regulation of protein synthesis represents a key control point in cellular response to stress. In particular, discreet RNA regulatory elements were shown to allow to selective translation of specific mRNAs, which typically encode for proteins required for a particular stress response. Identification of these mRNAs, as well as the characterization of regulatory mechanisms responsible for selective translation has been at the forefront of molecular biology for some time. Polysome profiling is a cornerstone method in these studies. The goal of polysome profiling is to capture mRNA translation by immobilizing actively translating ribosomes on different transcripts and separate the resulting polyribosomes by ultracentrifugation on a sucrose gradient, thus allowing for a distinction between highly translated transcripts and poorly translated ones. These can then be further characterized by traditional biochemical and molecular biology methods. Importantly, combining polysome profiling with high throughput genomic approaches allows for a large scale analysis of translational regulation.

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 ...

Rapid In Vivo Fixation and Isolation of Translational Complexes from Eukaryotic Cells

Rapid responses involving fast redistribution of messenger(m)RNA and alterations of mRNA translation are pertinent to ongoing homeostatic adjustments of the cells. These adjustments are critical to eukaryotic cell survivability and &#39;damage control&#39; during fluctuating nutrient and salinity levels, temperature, and various chemical and radiation stresses. Due to the highly dynamic nature of the RNA-level responses, and the instability of many of the RNA:RNA and RNA:protein intermediates, obtaining a meaningful snapshot of the cytoplasmic RNA state is only possible with a limited number of methods. Transcriptome-wide, RNA-seq-based ribosome profiling-type experiments are among the most informative sources of data for the control of translation. However, absence of a uniform RNA and RNA:protein intermediate stabilization can lead to different biases, particularly in the fast-paced cellular response pathways. In this article, we provide a detailed protocol of rapid fixation applicable to eukaryotic cells of different permeability, to aid in RNA and RNA:protein intermediate stabilization. We further provide examples of isolation of the stabilized RNA:protein complexes based on their co-sedimentation with ribosomal and poly(ribo)somal fractions. The separated stabilized material can be subsequently used as part of ribosome profiling-type experiments, such as in Translation Complex Profile sequencing (TCP-seq) approach and its derivatives. Versatility of the TCP-seq-style methods has now been demonstrated by the applications in a variety of organisms and cell types. The stabilized complexes can also be additionally affinity-purified and imaged using electron microscopy, separated into different poly(ribo)somal fractions and subjected to RNA sequencing, owing to the ease of the crosslink reversal. Therefore, methods based on snap-chilling and formaldehyde fixation, followed by the sedimentation-based or other type of RNA:protein complex enrichment, can be of particular interest in investigating finer details of rapid RNA:protein complex dynamics in live cells.

Rapid In Vivo Fixation and Isolation of Translational Complexes from Eukaryotic Cells

We present a technique to rapidly stabilize translational (protein biosynthesis) complexes with formaldehyde crosslinking in live yeast and mammalian cells. The approach enables dissecting transient intermediates and dynamic RNA:protein interactions. The crosslinked complexes can be used in multiple downstream applications such as in deep sequencing-based profiling methods, microscopy, and mass-spectrometry.

Ökaryotik Hücrelerden Çeviri Komplekslerinin Hızlı In Vivo Fiksasyonu ve İzolasyonu

Rapid responses involving fast redistribution of messenger(m)RNA and alterations of mRNA translation are pertinent to ongoing homeostatic adjustments of ...

Global Identification of Co-Translational Interaction Networks by Selective Ribosome Profiling

In recent years, it has become evident that ribosomes not only decode our mRNA but also guide the emergence of the polypeptide chain into the crowded cellular environment. Ribosomes provide the platform for spatially and kinetically controlled binding of membrane-targeting factors, modifying enzymes, and folding chaperones. Even the assembly into high-order oligomeric complexes, as well as protein-protein network formation steps, were recently discovered to be coordinated with synthesis.
Here, we describe Selective Ribosome Profiling, a method developed to capture co-translational interactions in vivo. We will detail the various affinity purification steps required for capturing ribosome-nascent-chain complexes together with co-translational interactors, as well as the mRNA extraction, size exclusion, reverse transcription, deep-sequencing, and big-data analysis steps, required to decipher co-translational interactions in near-codon resolution.

Co-translational interactions play a crucial role in nascent-chain modifications, targeting, folding, and assembly pathways. Here, we describe Selective Ribosome Profiling, a method for in vivo, direct analysis of these interactions in the model eukaryote Saccharomyces cerevisiae.

Ko-Translasyonel Etkileşim Ağlarının Seçici Ribozom Profilleme ile Küresel Tanımlanması

In recent years, it has become evident that ribosomes not only decode our mRNA but also guide the emergence of the polypeptide chain into the crowded ...