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

Die Identifizierung von Regionen in einem Genom, die transkribiert und in Proteine übersetzt werden, ist ein wichtiger Schritt zum Verständnis eines Genoms und seiner Expression.
Dies wird mit einer Technik namens Ribosomenprofilierung erreicht, die auch als Ribo-Seq bezeichnet wird. Es kartiert die Positionen von Ribosomen auf mRNA und identifiziert mRNAs, die aktiv in Proteine übersetzt werden.
Um die RNA zu isolieren, müssen die Zellen zunächst lysiert werden, um an die darin enthaltenen Moleküle zu gelangen. Das Lysat wird dann mit RNasen behandelt. Diese Enzyme spalten die mRNAs, die nicht mit Ribosomen bedeckt sind, so dass nur die geschützten mRNA-Fragmente übrig bleiben.
Die ribosomengeschützten Fragmente werden dann mit Hilfe eines Saccharosegradienten von den ungeschützten, gespaltenen Fragmenten getrennt.
Anschließend werden die Ribosomen aus den mRNA-Fragmenten entfernt und die RNA mittels RT-PCR mit dem Enzym reverse Transkriptase in DNA umgewandelt.
Als nächstes wird die DNA sequenziert und auf dem Referenzgenom kartiert. Dadurch wird die genaue Position des Ribosoms entlang jeder mRNA bestimmt.
Die Erstellung von Ribosomenprofilen kann auch helfen, unerkannte offene Leserahmen oder ORFs zu identifizieren. Ein ORF ist eine DNA-Region zwischen einem Start- und einem Stoppcodon, die in ein Protein übersetzt werden kann. Das Auffinden von ORFs kann bei der Identifizierung neuer Gene hilfreich sein.
Denken Sie an die Untersuchung von Genexpressionsmustern in einer Säugetierzelllinie. Das experimentelle Verfahren besteht darin, die Zellen Stimuli auszusetzen, die die Genexpression aktivieren und die mRNA-Synthese initiieren können.
Die mRNAs, die aktiv translatiert werden, werden mittels Ribosomen-Profiling identifiziert.
Dieses Experiment zeigt, dass Gen B translatiert wird, während die Gene A und C dies nicht tun.
Es zeigt auch, dass eine weitere kleine Region des Genoms, die etwa hundert Nukleotide lang ist, aktiv translatiert wird.
Diese unerkannte Region ist ein offener Leserahmen, der für ein neuartiges Protein kodieren kann, das durch die experimentellen Stimuli hochreguliert wird.

<ol>
	<li>Brar, G., Weissman, J. Die Erstellung von Ribosomenprofilen zeigt das Was, Wann, Wo und Wie der Proteinsynthese. Nat Rev Mol Cell Biol 16, 651–664 (2015). https://doi.org/10.1038/nrm4069</li>
	<li>Chung BY, Hardcastle TJ, Jones JD, et al. Die Verwendung von Duplex-spezifischer Nuklease bei der Ribosomenprofilierung und ein benutzerfreundliches Softwarepaket für die Ribo-seq-Datenanalyse. RNS. 2015; 21(10):1731-1745. doi:10.1261/rna.052548.115</li>
</ol>

ribosomen-profiling

Das Ribosomen-Profiling oder Ribo-Sequenzierung ist eine Tiefensequenzierungstechnik, die eine Momentaufnahme der aktiven Translation in einer Zelle liefert. Sie sequenziert selektiv die von Ribosomen geschützten mRNAs, um Einblick in die Translationslandschaft einer Zelle zu einem bestimmten Zeitpunkt zu erhalten.

Anwendungen des Ribosomen-Profiling

Das Ribosomen-Profiling hat viele Anwendungen, einschließlich der in vivo Überwachung der Translation in einem bestimmten Organ oder Gewebetyp und der Quantifizierung der Neusynthese von Proteinen.

Die Technik hilft, translatierte offene Leserahmen zu identifizieren und neue translatierte Produkte zu entdecken, einschließlich kurzer Peptide und Isoformen charakterisierter Proteine mit unbekannten Funktionen. Das Ribosomen-Profiling trägt auch dazu bei, mehrere mRNAs in der Zelle zu identifizieren, die nicht übersetzt werden, bis sie ein externes Signal erhalten.

Technische Herausforderungen des Ribosomen-Profiling

Das Ribosomen-Profiling steht vor vielen technischen Herausforderungen, wie der Notwendigkeit großer Probenmengen, der Abhängigkeit von der rechtzeitigen Hemmung der Translation und der RNA-Kontamination.

Die Schockgefriertechnik kann die Herausforderung der schnellen Translationshemmung überwinden. Sie hilft, die Ribosomenverteilung innerhalb der Zellen im Vergleich zu Translationsverlängerungshemmern wie Cycloheximid effizient zu erfassen.

Ribosomale RNA (rRNA), die an die mRNA bindet, wird beim Ribosomen-Profiling normalerweise entfernt. Manchmal werden jedoch immer noch einige rRNA-Kontaminanten beobachtet. Forscher verwenden das Enzym Duplex-spezifische Nuklease (DSN), das aus dem Hepatopankreas der Kamtschatka-Krabbe isoliert wird und sowohl dsDNA als auch DNA-RNA-Hybride spaltet. Diese Methode wird oft auch verwendet, um cDNA-Bibliotheken vor der Hochdurchsatz-Sequenzierung zu normalisieren und rRNA aus RNA-seq-Bibliotheken zu entfernen.

Eine weitere Einschränkung des Ribosomen-Profiling liegt in der Analyse der Daten, die entsprechende bioinformatische Fachkenntnisse erfordern. Um dieses Problem zu überwinden, wird das R-Paket riboSeqR verwendet, das Methoden zur Auswertung von Ribosomen-Profiling-Daten für mehrere Proben bietet.

Ribosomen-Profiling

Ribosomen-Profiling oder Ribo-Sequenzierung ist eine tiefe Sequenzierungstechnik, die eine Momentaufnahme der aktiven Translation in einer Zelle erstellt. Es sequenziert selektiv ...

Education

Lehre

JoVE Core

Molecular Biology

Molekularbiologie

Unit 2

Einheit 2

Analyzing Gene Expression and Function

Analyse von Genexpression und -funktion

KEY TERMS AND CONCEPTS

KERNBEGRIFFE UND KONZEPTE

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

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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...

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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

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Tennessee Academic Standards

High School

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Links Among Engineering, Technology, Science, and Society

BIO1.ETS2.1

BIO1.ETS2.3

Virginia  Standards of Learning

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BIO.5.e

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From Molecules to Organisms: Structures and Processes

9-12.B.1.2.c

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Understand genetic mechanisms for variation.

Ribosome Profiling for Translation Snapshot

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