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

Identifying regions in a genome that are transcribed and translated into proteins is a vital step in understanding a genome and its expression.
This is achieved with a technique called ribosome profiling, also referred to as ribo-seq. It maps the positions of ribosomes on mRNA and identifies mRNAs that are being actively translated into proteins.
To isolate the RNA, cells must first be lysed to access the molecules inside them. The lysate is then treated with RNases. These enzymes cleave the mRNAs that are not covered with ribosomes, leaving only the protected mRNA fragments.
The ribosome protected fragments are then separated from the unprotected, cleaved fragments using a sucrose gradient.
The ribosomes are then removed from the mRNA fragments, and the RNA are converted into DNA by RT-PCR, using the enzyme reverse transcriptase.
Next, the DNA is sequenced and mapped on the reference genome. This determines the exact location of the ribosome along each mRNA.
Ribosome profiling can also help to identify unrecognized open reading frames or ORFs. An ORF is a region of DNA between a start codon and a stop codon that can be translated into protein. Finding ORFs can be helpful in the identification of new genes.
Consider the study of gene expression patterns in a mammalian cell line. The experimental procedure involves exposing the cells to stimuli that may turn on gene expression and initiate mRNA synthesis.
The mRNAs that are being actively translated are identified using ribosome profiling.
This experiment reveals that gene B is being translated while genes A and C are not.
It also shows an additional small region of the genome around a hundred nucleotides long is being actively translated.
This unrecognized region is an open reading frame that may code for a novel protein that is upregulated by the experimental stimuli.

ribosoomprofilering

Ribosoomprofilering of ribo-sequencing is een diepe sequencingtechniek die een momentopname produceert van actieve vertaling in een cel. Het sequenteert selectief de door ribosomen beschermde mRNA’s om inzicht te krijgen in het vertaallandschap van een cel op een bepaald moment.

Toepassingen van ribosoomprofilering

Ribosoomprofilering kent vele toepassingen, waaronder in vivo monitoring van de vertaling binnen een bepaald orgaan- of weefseltype en het kwantificeren van nieuwe eiwitsyntheseniveaus.

De techniek helpt vertaalde open leesframes te identificeren en nieuwe vertaalde producten te ontdekken, waaronder korte peptiden en isovormen van gekarakteriseerde eiwitten met onbekende functies. Ribosoomprofilering helpt ook bij het identificeren van verschillende mRNA's in de cel die pas worden vertaald als ze een extern signaal ontvangen.

Technische uitdagingen van ribosoomprofilering

Ribosoomprofilering wordt geconfronteerd met veel technische uitdagingen, zoals de behoefte aan grote hoeveelheden monsters, de betrouwbaarheid van het tijdig remmen van translatie en RNA-besmetting.

De flash-freezingtechniek kan de uitdaging van snelle translatieremming overwinnen. Het helpt de ribosoomverdeling in cellen efficiënt vast te leggen in vergelijking met remmers van de translatie-verlenging, zoals cycloheximide.

Ribosomaal RNA (rRNA) dat aan het mRNA bindt, wordt doorgaans verwijderd tijdens ribosoomprofilering. Soms worden echter nog steeds enkele rRNA-verontreinigingen waargenomen. Onderzoekers gebruiken het duplex-specifieke nuclease (DSN)-enzym om rRNA-besmetting te verminderen, geïsoleerd uit de hepatopancreas van de Kamtsjatka-krab, die dsDNA- en DNA-RNA-hybriden splitst. Deze methode wordt vaak ook gebruikt om cDNA-bibliotheken te normaliseren vóór sequencing van de volgende generatie en de uitputting van rRNA uit RNA-seq-bibliotheken.

Een andere beperking van ribosoomprofilering is het analyseren van gegevens, waarvoor de juiste expertise op het gebied van bio-informatica vereist is. Om dit probleem op te lossen wordt een R-pakket riboSeqR gebruikt, dat methoden biedt voor het oplossen van ribosomale profileringsgegevens voor meerdere monsters.

Ribosoomprofilering

Ribosome profiling or ribo-sequencing is a deep sequencing technique that produces a snapshot of active translation in a cell. It selectively sequences ...

Education

JoVE Core

Molecular Biology

Moleculaire biologie

Unit 2

Onderdeel 2

Analyzing Gene Expression and Function

Analyse van genexpressie en functie

KEY TERMS AND CONCEPTS

BELANGRIJKE TERMEN EN CONCEPTEN

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

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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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LS.Bio.8.1

Understand genetic mechanisms for variation.

Ribosome Profiling for Translation Snapshot

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