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

K12 Standards

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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

Identyfikacja regionów w genomie, które są transkrybowane i tłumaczone na białka, jest istotnym krokiem w zrozumieniu genomu i jego ekspresji.
Osiąga się to za pomocą techniki zwanej profilowaniem rybosomów, określanej również jako rybosekwencjonowanie. Mapuje pozycje rybosomów na mRNA i identyfikuje mRNA, które są aktywnie przekształcane w białka.
Aby wyizolować RNA, komórki muszą najpierw zostać poddane lizie, aby uzyskać dostęp do znajdujących się w nich cząsteczek. Lizat jest następnie traktowany RNazami. Enzymy te rozszczepiają mRNA, które nie są pokryte rybosomami, pozostawiając tylko chronione fragmenty mRNA.
Fragmenty chronione rybosomem są następnie oddzielane od niezabezpieczonych, rozszczepionych fragmentów za pomocą gradientu sacharozy.
Rybosomy są następnie usuwane z fragmentów mRNA, a RNA jest przekształcane w DNA za pomocą RT-PCR, przy użyciu enzymu odwrotnej transkryptazy.
Następnie DNA jest sekwencjonowane i mapowane w genomie referencyjnym. Określa to dokładną lokalizację rybosomu wzdłuż każdego mRNA.
Profilowanie rybosomów może również pomóc w identyfikacji nierozpoznanych otwartych ramek odczytu lub ORF. ORF to region DNA między kodonem start a kodonem stop, który może ulec translacji na białko. Znalezienie ORF może być pomocne w identyfikacji nowych genów.
Weźmy pod uwagę badanie wzorców ekspresji genów w linii komórkowej ssaków. Procedura eksperymentalna polega na wystawieniu komórek na bodźce, które mogą włączyć ekspresję genów i zainicjować syntezę mRNA.
MRNA, które są aktywnie ulegane translacji, są identyfikowane za pomocą profilowania rybosomów.
Ten eksperyment ujawnia, że gen B ulega translacji, podczas gdy geny A i C nie.
Pokazuje również, że dodatkowy mały obszar genomu o długości około stu nukleotydów jest aktywnie translowany.
Ten nierozpoznany region jest otwartą ramką odczytu, która może kodować nowe białko, które jest regulowane w górę przez bodźce eksperymentalne.

<ol>
	<li>Brar, G., Weissman, J. Profilowanie rybosomów ujawnia co, kiedy, gdzie i jak syntezy białek. Nat Rev Mol Cell Biol 16, 651–664 (2015). https://doi.org/10.1038/nrm4069</li>
	<li>Chung BY, Hardcastle TJ, Jones JD i wsp. Zastosowanie nukleazy specyficznej dla dupleksu w profilowaniu rybosomów oraz przyjazny dla użytkownika pakiet oprogramowania do analizy danych Ribo-seq. RNA. 2015; 21(10):1731-1745. doi:10.1261/rna.052548.115</li>
</ol>

profilowanie rybosomów

Profilowanie rybosomów lub sekwencjonowanie ryb to technika głębokiego sekwencjonowania, która pozwala uzyskać migawkę aktywnej translacji w komórce. Selektywnie sekwencjonuje mRNA chronione przez rybosomy, aby uzyskać wgląd w krajobraz translacji komórki w dowolnym momencie. <h4>Zastosowania profilowania rybosomów</h4> Profilowanie rybosomów ma wiele zastosowań, w tym monitorowanie translacji in vivo wewnątrz określonego typu narządu lub tkanki oraz ilościowe określanie poziomów syntezy nowych białek. Technika ta pomaga zidentyfikować otwarte ramki odczytu i odkryć nowe produkty, w tym krótkie peptydy i izoformy scharakteryzowanych białek o nieznanych funkcjach. Profilowanie rybosomów pomaga również w identyfikacji kilku mRNA w komórce, które nie ulegają translacji, dopóki nie otrzymają sygnału zewnętrznego. <h4>Wyzwania techniczne związane z profilowaniem rybosomów</h4> Profilowanie rybosomów wiąże się z wieloma wyzwaniami technicznymi, takimi jak wymóg dotyczący dużych ilości próbek, niezawodność hamowania translacji w odpowiednim czasie i zanieczyszczenie RNA. Technika błyskawicznego zamrażania może przezwyciężyć wyzwanie polegające na szybkim hamowaniu translacji. Pomaga skutecznie uchwycić rozkład rybosomów w komórkach w porównaniu z inhibitorami wydłużania translacji, takimi jak cykloheksymid. Rybosomowy RNA (rRNA), które wiążą się z mRNA, są na ogół usuwane podczas profilowania rybosomów. Czasami jednak nadal obserwuje się kilka zanieczyszczeń rRNA. Naukowcy wykorzystują enzym nukleazę specyficzną dla dupleksu (DSN), aby zmniejszyć zanieczyszczenie rRNA wyizolowanym z wątrobowotrzustki kraba kamczackiego, które rozszczepia hybrydy dsDNA i DNA-RNA. Metodę tę często stosuje się również do normalizacji bibliotek cDNA przed sekwencjonowaniem nowej generacji i wyczerpaniem rRNA z bibliotek seq RNA. Kolejnym ograniczeniem profilowania rybosomów jest analiza danych, która wymaga odpowiedniej wiedzy z zakresu bioinformatyki. Aby rozwiązać ten problem, stosuje się pakiet R riboSeqR, który zapewnia metody rozwiązywania danych profilowania rybosomów dla wielu próbek.

Profilowanie rybosomów

Profilowanie rybosomów lub sekwencjonowanie rybosomów to technika głębokiego sekwencjonowania, która tworzy migawkę aktywnej translacji w komórce. Selektywnie sekwencjonuje ...

Education

JoVE Core

Molecular Biology

Biologia molekularna

Unit 2

Jednostka 2

Analyzing Gene Expression and Function

Analiza ekspresji i funkcji genów

KEY TERMS AND CONCEPTS

Kluczowe terminy i pojęcia

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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epistasis analysis

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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 Molecular Biology showcases the molecular processes of the cell through clear and concise animated video lessons. Additionally, in scientist-in-action videos, these concepts' application is demonstrated through real experiments in modern laboratories.

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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Meiosis I

Meiosis II

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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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