Name: ribosome profiling for translation snapshot
Uploaded: 2021-04-07T12:12:00.000Z
Duration: 2 min 24 s
Channel: JoVE

K12 Standards

north-carolina-k-12-science-standard-course-of-study

NC K-12 Science Standard Course of Study

9-12

Biology

heredity-inheritance-and-variation-of-traits

Heredity- Inheritance and Variation of Traits

LS.Bio.8.2

Ribosome Profiling

L’identification des régions d’un génome qui sont transcrites et traduites en protéines est une étape essentielle dans la compréhension d’un génome et de son expression.
Ceci est réalisé à l’aide d’une technique appelée profilage des ribosomes, également appelée ribo-seq. Il cartographie les positions des ribosomes sur l’ARNm et identifie les ARNm qui sont activement traduits en protéines.
Pour isoler l’ARN, les cellules doivent d’abord être lysées pour accéder aux molécules qu’elles contiennent. Le lysat est ensuite traité avec des RNases. Ces enzymes clivent les ARNm qui ne sont pas recouverts de ribosomes, ne laissant que les fragments d’ARNm protégés.
Les fragments protégés par le ribosome sont ensuite séparés des fragments non protégés et clivés à l’aide d’un gradient de saccharose.
Les ribosomes sont ensuite retirés des fragments d’ARNm, et l’ARN est converti en ADN par RT-PCR, en utilisant l’enzyme transcriptase inverse.
Ensuite, l’ADN est séquencé et cartographié sur le génome de référence. Cela détermine l’emplacement exact du ribosome le long de chaque ARNm.
Le profilage des ribosomes peut également aider à identifier les cadres de lecture ouverts ou ORF non reconnus. Un ORF est une région de l’ADN située entre un codon de départ et un codon d’arrêt qui peut être traduite en protéine. La découverte d’ORF peut être utile à l’identification de nouveaux gènes.
Prenons l’étude des modèles d’expression génique dans une lignée cellulaire de mammifère. La procédure expérimentale consiste à exposer les cellules à des stimuli qui peuvent activer l’expression des gènes et initier la synthèse de l’ARNm.
Les ARNm qui sont activement traduits sont identifiés à l’aide du profilage des ribosomes.
Cette expérience révèle que le gène B est traduit alors que les gènes A et C ne le sont pas.
Il montre également qu’une petite région supplémentaire du génome, d’une centaine de nucléotides de long, est activement traduite.
Cette région non reconnue est un cadre de lecture ouvert qui peut coder pour une nouvelle protéine qui est régulée à la hausse par les stimuli expérimentaux.

<ol>
	<li>Brar, G., Weissman, J. Le profilage des ribosomes révèle le quoi, le quand, le où et le comment de la synthèse des protéines. Nat Rev Mol Cell Biol 16, 651 à 664 (2015). https://doi.org/10.1038/nrm4069</li>
	<li>Chung BY, Hardcastle TJ, Jones JD, et al. L’utilisation de nucléases spécifiques au duplex dans le profilage des ribosomes et un progiciel convivial pour l’analyse des données Ribo-seq. ARN. 2015; 21(10):1731-1745. doi :10.1261/rna.052548.115</li>
</ol>

profilage des ribosomes

Le profilage des ribosomes ou ribo-séquençage est une technique de séquençage en profondeur qui produit un instantané de la traduction active dans une cellule. Il séquence sélectivement les ARNm protégés par les ribosomes pour avoir un aperçu du paysage de traduction d’une cellule à un moment donné.

Applications du profilage des ribosomes

Le profilage des ribosomes a de nombreuses applications, notamment la surveillance in vivo de la traduction à l'intérieur d'un type d'organe ou de tissu particulier et la quantification de nouveaux niveaux de synthèse protéique.

La technique permet d'identifier les cadres de lecture ouverts traduits et de découvrir de nouveaux produits traduits, notamment des peptides courts et des isoformes de protéines caractérisées aux fonctions inconnues. Le profilage des ribosomes aide également à identifier plusieurs ARNm dans la cellule qui ne sont pas traduits jusqu'à ce qu'ils reçoivent un signal externe.

Défis techniques du profilage des ribosomes

Le profilage des ribosomes est confronté à de nombreux défis techniques, tels que la nécessité de disposer de grandes quantités d'échantillons, la fiabilité de l'inhibition rapide de la traduction et la contamination par l'ARN.

La technique de congélation flash peut surmonter le défi de l’inhibition rapide de la traduction. Il aide à capturer efficacement la distribution des ribosomes dans les cellules par rapport aux inhibiteurs d'élongation par traduction, tels que le cycloheximide.

L'ARN ribosomal (ARNr) qui se lie à l'ARNm est généralement éliminé lors du profilage des ribosomes. Cependant, quelques contaminants d’ARNr sont parfois encore observés. Les chercheurs utilisent l'enzyme duplex-spécifique nucléase (DSN) pour réduire la contamination par l'ARNr, isolée de l'hépatopancréas du crabe du Kamtchatka, qui clive les hybrides ADNdb et ADN-ARN. Cette méthode est souvent également utilisée pour normaliser les bibliothèques d’ADNc avant le séquençage de nouvelle génération et l’épuisement de l’ARNr des bibliothèques de séquençage d’ARN.

Une autre limite du profilage des ribosomes réside dans l’analyse des données, qui nécessite une expertise adéquate en bioinformatique. Un package R riboSeqR est utilisé pour surmonter ce problème, qui fournit des méthodes pour résoudre les données de profilage ribosomal pour plusieurs échantillons.

Profilage des ribosomes

Le profilage des ribosomes ou ribo-séquençage est une technique de séquençage profond qui produit un instantané de la traduction active dans une cellule. Il séquence sélectivement ...

Education

Enseignement

JoVE Core

Molecular Biology

Biologie moléculaire

Unit 2

Unité 2

Analyzing Gene Expression and Function

Analyse de l'expression et de la fonction géniques

KEY TERMS AND CONCEPTS

TERMES ET CONCEPTS CLÉS

ribosome profiling for translation snapshot

Ribosome profiling is a deep sequencing method that gives a snapshot of active translation in a cell. It sequences the mRNAs protected by ribosomes. This shows which messages are being read to make proteins at a specific moment.

The method has several uses in biology. It can track translation in a living organ or tissue type. It also helps measure new protein synthesis levels. Researchers use it to find translated open reading frames, which are stretches of RNA that can make protein. It can also reveal short peptides, protein isoforms with unknown functions, and mRNAs that stay untranslated until a cell receives an external signal.

Ribosome profiling also has technical limits. It often needs a large sample amount. It depends on stopping translation at the right time, and RNA contamination can be a problem. Flash-freezing can help block translation quickly and capture ribosome positions more accurately than elongation inhibitors such as cycloheximide.

Another challenge is ribosomal RNA, or rRNA, contamination. rRNA is usually removed during the procedure, but some contaminants can remain. Researchers may use duplex-specific nuclease, or DSN, from the hepatopancreas of the Kamchatka crab to reduce this problem. DSN cleaves dsDNA and DNA-RNA hybrids. It is also used to normalize cDNA libraries before next-generation sequencing and to remove rRNA from RNA-seq libraries.

Data analysis can also be difficult because it requires bioinformatics expertise. The R package riboSeqR helps address this issue. It provides methods for analyzing ribosome profiling data from multiple samples.

Ribosome Profiling for Translation Snapshot

ribosome-profiling-for-translation-snapshot

in vitro mutagenesis

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ribosome profiling

Ribosome profiling is a deep sequencing method that gives a snapshot of active translation in a cell. It sequences the mRNAs protected by ribosomes. This shows which messages are being read to make proteins at a specific moment. The method has several uses in biology. It can track translation in a living organ or tissue type. It also helps measure new protein synthesis levels. Researchers use it to find translated open reading frames, which are stretches of RNA that can make protein. It can...

transgenic plants

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key terms and concepts

Learn how ribosome profiling captures active translation, finds translated RNA regions, and handles challenges like rRNA contamination and data analysis.

Ribosome Profiling for Translation Snapshot | JoVE K12

molecular biology

JoVE Core Biologie Moléculaire illustre les processus moléculaires au sein de la cellule grâce à des leçons vidéos claires et concises. Les applications de ces concepts sont ensuite démontrées grâce à des expériences concrètes en laboratoire.

Tennessee Academic Standards

High School

links-among-engineering-technology-science-and-society

Links Among Engineering, Technology, Science, and Society

BIO1.ETS2.1

BIO1.ETS2.3

Virginia  Standards of Learning

HIgh School

BIO.5

BIO.5.e

Alabama Course of Study Science

from-molecules-to-organisms-structures-and-processes

From Molecules to Organisms: Structures and Processes

9-12.B.1.2.c

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Meiosis II

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Oogenesis

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Use models to explain how DNA is passed from parents to offspring through the processes of meiosis and fertilization in sexual reproduction.

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Fixing Double-strand Breaks

Homologous Recombination

Gene Conversion

Restarting Stalled Replication Forks

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LS.Bio.6.2

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Genetic Crosses

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Punnett Squares

Monohybrid Crosses

Dihybrid Crosses

Trihybrid Crosses

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Law of Independent Assortment

Chi-square Analysis

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Analyze and interpret data to compare DNA samples.

LS.Bio.8.1

Understand genetic mechanisms for variation.

Ribosome Profiling for Translation Snapshot

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