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

Identificar regiões em um genoma que são transcritas e traduzidas em proteínas é um passo vital para entender um genoma e sua expressão.
Isso é conseguido com uma técnica chamada perfil de ribossomo, também conhecida como ribo-seq. Ele mapeia as posições dos ribossomos no mRNA e identifica os mRNAs que estão sendo ativamente traduzidos em proteínas.
Para isolar o RNA, as células devem primeiro ser lisadas para acessar as moléculas dentro delas. O lisado é então tratado com RNases. Essas enzimas clivam os mRNAs que não são cobertos por ribossomos, deixando apenas os fragmentos de mRNA protegidos.
Os fragmentos protegidos por ribossomos são então separados dos fragmentos clivados desprotegidos usando um gradiente de sacarose.
Os ribossomos são então removidos dos fragmentos de mRNA e o RNA é convertido em DNA por RT-PCR, usando a enzima transcriptase reversa.
Em seguida, o DNA é sequenciado e mapeado no genoma de referência. Isso determina a localização exata do ribossomo ao longo de cada mRNA.
O perfil do ribossomo também pode ajudar a identificar quadros de leitura abertos não reconhecidos ou ORFs. Uma ORF é uma região do DNA entre um códon de início e um códon de parada que pode ser traduzido em proteína. Encontrar ORFs pode ser útil na identificação de novos genes.
Considere o estudo dos padrões de expressão gênica em uma linhagem celular de mamíferos. O procedimento experimental envolve a exposição das células a estímulos que podem ativar a expressão gênica e iniciar a síntese de mRNA.
Os mRNAs que estão sendo traduzidos ativamente são identificados usando o perfil do ribossomo.
Este experimento revela que o gene B está sendo traduzido, enquanto os genes A e C não.
Ele também mostra que uma pequena região adicional do genoma com cerca de cem nucleotídeos de comprimento está sendo traduzida ativamente.
Essa região não reconhecida é um quadro de leitura aberto que pode codificar uma nova proteína que é regulada positivamente pelos estímulos experimentais.

<ol>
	<li>Brar, G., Weissman, J. O perfil do ribossomo revela o quê, quando, onde e como da síntese de proteínas. 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. O uso de nuclease duplex-específica no perfil de ribossomos e um pacote de software amigável para análise de dados Ribo-seq. RNA. 2015; 21(10):1731-1745. DOI:10.1261/RNA.052548.115</li>
</ol>

perfil do ribossomo

O perfil do ribossomo ou sequenciamento do ribossomo é uma técnica de sequenciamento profundo que produz um instantâneo da tradução ativa em uma célula. Ele sequencia seletivamente os mRNAs protegidos pelos ribossomos para obter uma visão do cenário de tradução de uma célula em qualquer momento. <h4>Aplicações de perfil de ribossomo</h4> O perfil do ribossomo tem muitas aplicações, incluindo o monitoramento in vivo da tradução dentro de um determinado órgão ou tipo de tecido e a quantificação de novos níveis de síntese protéica. A técnica ajuda a identificar quadros de leitura abertos traduzidos e a descobrir novos produtos traduzidos, incluindo peptídeos curtos e isoformas de proteínas caracterizadas com funções desconhecidas. O perfil do ribossomo também ajuda na identificação de vários mRNAs na célula que não são traduzidos até receberem um sinal externo. <h4>Desafios técnicos do perfil de ribossomos</h4> O perfil do ribossomo enfrenta muitos desafios técnicos, como a necessidade de grandes quantidades de amostras, a confiabilidade da inibição oportuna da tradução e a contaminação por RNA. A técnica de congelamento rápido pode superar o desafio da inibição rápida da tradução. Ajuda a capturar eficientemente a distribuição do ribossomo dentro das células em comparação com inibidores do alongamento da tradução, como a cicloheximida. O RNA ribossômico (rRNA) que se liga ao mRNA é geralmente removido durante o perfil do ribossomo. No entanto, às vezes alguns contaminantes de rRNA ainda são observados. Os pesquisadores usam a enzima nuclease específica de duplex (DSN) para reduzir a contaminação por rRNA, isolada do hepatopâncreas do caranguejo Kamchatka, que cliva híbridos de dsDNA e DNA-RNA. Este método também é frequentemente usado para normalizar bibliotecas de cDNA antes do sequenciamento de próxima geração e do esgotamento do rRNA das bibliotecas de RNA-seq. Outra limitação do perfil do ribossomo está na análise de dados, o que requer o conhecimento adequado em bioinformática. Um pacote R riboSeqR é usado para superar esse problema, que fornece métodos para resolver dados de perfil ribossômico para múltiplas amostras.

Perfil do Ribossomo

O perfil de ribossomo ou sequenciamento de ribo é uma técnica de sequenciamento profundo que produz um instantâneo da tradução ativa em uma célula. Ele sequencia seletivamente ...

Education

JoVE Core

Molecular Biology

Biologia Molecular

Unit 2

Unidade 2

Analyzing Gene Expression and Function

Analisando a Expressão e a Função Gênica

KEY TERMS AND CONCEPTS

TERMOS-CHAVE E CONCEITOS

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

In Vitro Mutagenesis for Gene Analysis

In vitro mutagenesis is a lab method used to change DNA and study gene function. It helps scientists test how specific DNA changes affect a gene’s activity and the traits it controls. A common approach is to make a known change in a DNA sequence and then examine the result. This can be done by deleting, inserting, or altering selected bases in a gene. The method is useful for linking a DNA change to a biological effect. Researchers use in vitro mutagenesis in molecular biology, genetics, and...

genetic screens

Using Genetic Screens to Find Disease Genes

Genetic screens are tools used to find genes and mutations linked to specific traits. They are also used to identify people or groups at risk for genetic diseases. That can support early intervention, targeted therapy, and reproductive choices. Forward genetic screens, also called classical screens, start with random DNA changes in an organism. Researchers may use radiation, mutagens, or added bases to create mutations that lead to visible phenotype changes. The mutant is then inbred so...

test cross

Test Cross in Genetic Inheritance

A test cross is a genetics tool used to determine the genotype of an organism with a dominant trait. It helps reveal whether the dominant individual is homozygous or heterozygous by crossing it with a recessive individual. The outcome of the cross shows the hidden genotype. If any offspring show the recessive trait, the dominant parent must carry one recessive allele. If all offspring show the dominant trait, the dominant parent is likely homozygous dominant. Test crosses are common in basic...

complementation tests

Using Crosses to Group Mutations

A complementation test uses a simple cross to find out whether two mutations are in the same gene or in different genes. It helps show how mutations are arranged on chromosomes. Edward Lewis first performed this test in the 1940s while studying fruit flies. To do the test, organisms heterozygous for different mutations are crossed pairwise in all combinations. If the mutations are in different genes, they can complement each other. In that case, each mutation provides the missing genetic...

single nucleotide polymorphisms-snps

SNPs as Genetic Markers and Variants

Single nucleotide polymorphisms, or SNPs, are small DNA changes at one specific genomic position. They are the most common type of sequence variation in the human genome. A point mutation counts as an SNP when it appears in more than 1% of the population.

bacterial transformation

Bacterial Transformation in the Lab

Bacterial transformation is a lab method used to move new DNA into bacteria. Scientists use it to change how bacteria behave or to help them carry a gene of interest. In high school biology, this process is often shown with plasmids, which are small circular DNA molecules. During transformation, bacteria take up DNA from their surroundings. The new DNA can then be used by the cell, depending on the experiment. This technique is an important part of genetic engineering and is widely used in...

transgenic organisms

Making Transgenic Organisms

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reproductive cloning

Cloning an Organism by Reproduction

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crispr

CRISPR Gene Editing Basics

CRISPR is a gene editing tool that helps scientists make targeted changes to DNA. It uses a guide system to find a chosen sequence and a cutting protein to act at that spot. This method is based on a natural defense system found in bacteria. In high school biology, it is often studied as a way to see how genes can be edited with precision. CRISPR is widely used in molecular biology research. It helps scientists explore how genes function and how DNA changes can affect living things.

experimental rnai

RNAi for Gene Silencing and Discovery

RNA interference (RNAi) is a gene-silencing process used to reduce gene expression. It can block transcription or trigger RNA degradation. Scientists first discovered this mechanism in plants in 1998 through Andrew Fire and Craig Mello. RNAi has since been found in many eukaryotes, including protozoa, flies, nematodes, insects, parasites, and mammals. Researchers use it as a practical tool to study gene function. It can show what a gene does without permanently changing the organism’s DNA. <br...

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Reporter Genes in Cells and Plasmids

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in-situ hybridization

ISH Probes for DNA and RNA Detection

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chromatin immunoprecipitation- chip

ChIP Methods for DNA-Protein Binding

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synthetic biology

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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

Transgenic Plants for Better Crops

Transgenic plants are crops that have been changed with recombinant DNA technology, also called transgenesis. This method adds a foreign gene or removes a harmful one from an organism. The result is a genetically modified organism. The first transgenic plant was a tobacco plant made in 1983, and it was resistant to tobacco mosaic virus. Since then, transgenic plants have been developed for many uses in agriculture, horticulture, and ornamental plants. Some are designed to resist pests or...

gene therapy

Gene Therapy for Disease Treatment

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epistasis analysis

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genome-wide association studies-gwas

Using SNP Patterns to Link Disease Risk

Genome-wide association studies, or GWAS, look for common SNPs, or single nucleotide polymorphisms, that may be linked to disease. Researchers compare how often a SNP appears in people with a disease and in people without it. When a SNP is seen more often in the disease group, it is considered associated with that disease. GWAS uses chi-square analysis to test the chance that an allele is linked to the disease. This approach does not require scientists to first find the target gene behind the...

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 Biologia Molecular mostra os processos moleculares da célula através de vídeos de animação claros e concisos. Além disso, nos vídeos de Cientistas em ação, a aplicação desses conceitos é demonstrada por meio de experimentos reais em laboratórios modernos.

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

What is Meiosis?

Meiosis is the process by which diploid cells divide to produce haploid daughter cells. In humans, each diploid cell contains 46 chromosomes, half from ...

Meiosis I

Meiosis II

Meiosis vs. Mitosis

Crossing Over

Crossing over is the exchange of genetic information between homologous chromosomes during prophase I of meiosis I. Genetic recombination gives rise to ...

Nondisjunction

Nondisjunction is the failure of homologous chromosomes or sister chromatids to separate correctly and move to the opposite poles of the cells. This ...

Spermatogenesis

Spermatogenesis is the process by which haploid sperm cells are produced in the male testes. It starts with stem cells located close to the outer rim of ...

Oogenesis

In human women, oogenesis produces one mature egg cell or ovum for every precursor cell that enters meiosis. This process differs in two unique ways from ...

Fertilization

During fertilization, an egg and sperm cell fuse to create a new diploid structure. In humans, the process occurs once the egg has been released from the ...

Cleavage and Blastulation

After a large-single-celled zygote is produced via fertilization, the process of cleavage occurs while zygotes travel through the uterine tube. Cleavage ...

Gastrulation

Gastrulation establishes the three primary tissues of an embryo: the ectoderm, mesoderm, and endoderm. This developmental process relies on a series of ...

Neurulation

Neurulation is the embryological process which forms the precursors of the central nervous system and occurs after gastrulation has established the three ...

Cell Migration

Cell migration, the process by which cells move from one location to another, is essential for the proper development and viability of organisms ...

Determination

During embryogenesis, cells become progressively committed to different fates through a two-step process: specification followed by determination. ...

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

LS.Bio.6.1

Mutations

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genome-size-and-the-evolution-of-new-genes

Genome Size and the Evolution of New Genes

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Types of Genetic Transfer Between Organisms

Base-pairing and DNA Repair

The DNA Replication Fork

Lagging Strand Synthesis

The Replisome

Proofreading

Synthesis of new DNA molecules is carried out by the enzyme DNA polymerase, which adds nucleotides on the daughter strand complementary to the template ...

Replication in Prokaryotes

DNA replication has three main steps: initiation, elongation, and termination. Replication in prokaryotes begins when initiator proteins bind to the ...

Replication in Eukaryotes

In eukaryotic cells, DNA replication is highly conserved and tightly regulated. Multiple linear chromosomes must be duplicated with high fidelity before ...

Overview of DNA Repair

Base Excision Repair

Nucleotide Excision Repair

DNA Distortion and Damage
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Mismatch Repair

Organisms are capable of detecting and fixing nucleotide mismatches that occur during DNA replication. This sophisticated process requires identifying the ...

Fixing Double-strand Breaks

Homologous Recombination

Gene Conversion

Restarting Stalled Replication Forks

Construct an explanation to summarize how inheritable genetic variations may result from: new genetic combinations in meiosis, mutations during replication, or mutations caused by environmental factors.

LS.Bio.6.2

An Overview of Genetic Analysis

An organism&#8217;s physical traits, or phenotype, are a product of its genotype, which is the combination of alleles (gene variants) inherited from its ...

Genetic Crosses

To dissect genetic processes or create organisms with novel suites of traits, scientists can perform genetic crosses, or the purposeful mating of two ...

Punnett Squares

Monohybrid Crosses

Dihybrid Crosses

Trihybrid Crosses

Trihybrid Crosses
Some of Mendel&#8217;s crosses examined three pairs of contrasting characteristics. Such a cross is called a trihybrid cross. A ...

Law of Independent Assortment

Chi-square Analysis

The chi-square test is a statistical hypothesis test. It is used to check whether there is a significant difference between an expected value and an ...

Pedigree Analysis

Multiple Allele Traits

Incomplete Dominance

Gregor Mendel&#39;s work (1822 - 1884) was primarily focused on pea plants. Through his initial experiments, he determined that every gene in a diploid ...

Lethal Alleles

Agouti: A Lethal Allele
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Polygenic Traits

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Background and Environment Affect Phenotype

Although the genetic makeup of an organism plays a major role in determining the phenotype, there are also several environmental factors, such as ...

Use mathematics and computational thinking to predict the variation and distribution of expressed traits based on: Mendelian inheritance, co-dominance, incomplete dominance, multiple alleles, and sex-linked inheritance.

LS.Bio.7.1

Overview

A pedigree is a diagram displaying a family&#x2019;s history of a trait. Analyzing pedigrees can reveal (1) whether a trait is dominant or ...

Probability Laws

Overview

The probability of inheriting a trait can be calculated using the sum and product rules. The sum rule is used to calculate the probability of ...

The Concept of Multiple Allelism

Multiple allelism describes genes that exist in three or more allelic forms. Although diploid organisms, like humans, ...

Analyze and interpret data to explain how polygenic traits result in a wide range of phenotypes.

LS.Bio.7.2

Pleiotropy

Pleiotropy is the phenomenon in which a single gene impacts multiple, seemingly unrelated phenotypic traits. For example, defects in the SOX10 gene cause ...

Nature and Nurture

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Construct an explanation to summarize how traits result from interactions of genetic factors (multiple genes and/or alleles) and environmental factors.

LS.Bio.7.3

PCR: The Polymerase Chain Reaction

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DNA Gel Electrophoresis

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DNA Isolation

DNA isolation protocols can be fast and straightforward or complex and time-consuming depending on the type and quality of DNA required for further ...

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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