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

Идентификация областей генома, которые транскрибируются и транслируются в белки, является жизненно важным шагом в понимании генома и его экспрессии.
Это достигается с помощью метода, называемого рибосомным профилированием, также называемого рибо-секвенированием. Он отображает положение рибосом на мРНК и идентифицирует мРНК, которые активно транслируются в белки.
Чтобы изолировать РНК, клетки сначала должны быть лизированы, чтобы получить доступ к молекулам внутри них. Затем лизат обрабатывают РНКазами. Эти ферменты расщепляют мРНК, которые не покрыты рибосомами, оставляя только защищенные фрагменты мРНК.
Затем защищенные рибосомами фрагменты отделяются от незащищенных, расщепленных фрагментов с помощью градиента сахарозы.
Затем рибосомы удаляются из фрагментов мРНК, и РНК преобразуется в ДНК методом ОТ-ПЦР с использованием фермента обратной транскриптазы.
Затем ДНК секвенируется и картируется на референсном геноме. Это определяет точное расположение рибосомы вдоль каждой мРНК.
Профилирование рибосом также может помочь идентифицировать нераспознанные открытые рамки считывания или ORF. ORF — это участок ДНК между стартовым кодоном и стоп-кодоном, который может быть транслирован в белок. Обнаружение ORF может быть полезным для идентификации новых генов.
Рассмотрим изучение паттернов экспрессии генов в клеточной линии млекопитающих. Экспериментальная процедура включает в себя воздействие на клетки стимулов, которые могут включить экспрессию генов и инициировать синтез мРНК.
МРНК, которые активно транслируются, идентифицируются с помощью профилирования рибосом.
Этот эксперимент показывает, что ген В транслируется, а гены А и С — нет.
Это также показывает, что дополнительный небольшой участок генома длиной около ста нуклеотидов активно транслируется.
Эта нераспознанная область представляет собой открытую рамку считывания, которая может кодировать новый белок, который активируется экспериментальными стимулами.

<ol>
	<li>Брар, Г., Вайсман, Д. Профилирование рибосом раскрывает что, когда, где и как происходит синтез белка. Nat Rev Mol Cell Biol 16, 651–664 (2015). https://doi.org/10.1038/nrm4069</li>
	<li>Чунг Б.И., Хардкасл Т.Дж., Джонс Д.Д. и др. Использование дуплекс-специфической нуклеазы в профилировании рибосом и удобный для пользователя программный комплекс для анализа данных Ribo-seq. РНК. 2015; 21(10):1731-1745. DOI:10.1261/rna.052548.115</li>
</ol>

Профилирование рибосом

Профилирование рибосом или рибосеквенирование — это метод глубокого секвенирования, позволяющий получить снимок активной трансляции в клетке. Он выборочно секвенирует мРНК, защищенные рибосомами, чтобы получить представление о трансляционном ландшафте клетки в любой момент времени. <h4>Применение профилирования рибосом</h4> Профилирование рибосом имеет множество применений, включая мониторинг трансляции внутри определенного органа или типа ткани in vivo и количественную оценку уровней синтеза новых белков. Этот метод помогает идентифицировать транслированные открытые рамки считывания и обнаруживать новые транслируемые продукты, включая короткие пептиды и изоформы охарактеризованных белков с неизвестными функциями. Профилирование рибосом также помогает идентифицировать несколько мРНК в клетке, которые не транслируются до тех пор, пока не получат внешний сигнал. <h4>Технические проблемы профилирования рибосом</h4> Профилирование рибосом сталкивается со многими техническими проблемами, такими как потребность в большом количестве образцов, зависимость от своевременного ингибирования трансляции и загрязнение РНК. Метод мгновенного замораживания может решить проблему быстрого ингибирования трансляции. Он помогает эффективно улавливать распределение рибосом внутри клеток по сравнению с ингибиторами элонгации трансляции, такими как циклогексимид. Рибосомальная РНК (рРНК), которая связывается с мРНК, обычно удаляется во время профилирования рибосом. Однако иногда некоторые примеси рРНК все же наблюдаются. Для уменьшения загрязнения рРНК исследователи используют фермент дуплекс-специфической нуклеазы (DSN), выделенный из гепатопанкреаса камчатского краба, который расщепляет дцДНК и гибриды ДНК-РНК. Этот метод также часто используется для нормализации библиотек кДНК перед секвенированием следующего поколения и исчерпанием рРНК из библиотек РНК-секвенирования. Еще одним ограничением профилирования рибосом является анализ данных, который требует соответствующих знаний в области биоинформатики. Для решения этой проблемы используется пакет RriboSeqR, который предоставляет методы для разрешения данных профиля рибосом для нескольких образцов.

Рибосомное профилирование или рибосеквенирование — это метод глубокого секвенирования, который позволяет получить снимок активной трансляции в клетке. Он выборочно секвенирует...

Education

JoVE Core

Molecular Biology

Молекулярная биология

Unit 2

Раздел 2

Analyzing Gene Expression and Function

Анализ экспрессии и функций генов

KEY TERMS AND CONCEPTS

ОСНОВНЫЕ ТЕРМИНЫ И ПОНЯТИЯ

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

Transgenic organisms are living things with DNA added from another organism. A transgene is the inserted gene. Scientists use this method to change an organism’s traits and to study how genes work. To make a transgenic organism, researchers first identify the gene they want to add. They then place that gene into the target organism’s DNA. Once the gene is integrated, the organism can pass it to new cells as it grows. This approach is useful in biology because it helps scientists analyze gene...

reproductive cloning

Cloning an Organism by Reproduction

Reproductive cloning creates a new organism that is genetically the same as the donor. It is different from cloning genes or cells because the goal is to make a whole living individual. This process usually relies on somatic cell nuclear transfer. In this method, the nucleus from a body cell is placed into an egg cell that has had its own nucleus removed. The egg can then develop into an embryo with DNA from the donor cell. Reproductive cloning is used to produce a copy of an organism, such...

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...

reporter genes

Reporter Genes in Cells and Plasmids

Reporter genes are protein-coding genes used to track gene activity in cells. They are often tagged to a gene of interest so scientists can see whether that gene is present or active. When the tagged genes are expressed together, reporter genes can produce visible signals such as fluorescence or luminescence. These signals help show the presence or absence of a gene of interest in an organism. They can also reveal the pattern of gene expression or the location of a DNA segment or protein...

in-situ hybridization

ISH Probes for DNA and RNA Detection

In situ hybridization (ISH) uses labeled probes to detect and localize specific DNA or RNA molecules in cells, tissues, or tissue sections. The probe is a complementary strand of DNA or RNA that binds to a matching nucleotide sequence in the sample. Since its first use in 1969 for nucleic acid studies, ISH has become an important tool in research and in clinical diagnosis. Several probe types can be used in ISH. These include single-stranded DNA probes, double-stranded DNA probes, antisense...

chromatin immunoprecipitation- chip

ChIP Methods for DNA-Protein Binding

Chromatin immunoprecipitation, or ChIP, is an antibody-based method for finding where proteins bind to DNA. It is used to identify DNA sites bound by transcription factors or histone proteins. ChIP also helps show which histone changes are present, including acetylation, phosphorylation, and methylation. ChIP is commonly divided into two forms: X-ChIP and N-ChIP. X-ChIP first cross-links proteins to DNA inside the cell, then breaks the DNA into pieces by sonication. The protein-DNA complexes...

synthetic biology

Synthetic Biology in Crops and Cells

Synthetic biology combines engineering, molecular biology, cell biology, and systems biology to redesign living systems. Scientists use it to remodel organisms from nature or to build new synthetic organisms. These tools can support protein or enzyme production, bioremediation, the production of useful macromolecules, and the addition of helpful traits to crops. One well-known example is golden rice. This genetically modified rice plant makes grains rich in β-carotene, which is a precursor of...

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

Gene therapy is a treatment approach that changes gene activity to help address disease. It focuses on the genetic cause of a problem rather than only easing symptoms. In this way, it is used in disease intervention. One common strategy is to replace a faulty gene with a normal copy. Another strategy is to inactivate a gene that is causing harm. Gene therapy can also introduce a new gene to help cells work more normally.

epistasis analysis

Gene Interaction Maps in Epistasis

Gene interaction maps in epistasis show how different genes work together to shape a phenotype. In epistasis, the interaction of genes or alleles at different locations affects the final trait. This often happens when one gene masks or interferes with the expression of another gene. Epistasis is common when genes are part of the same biochemical pathway. In that case, the expression of one gene can depend on a gene product made earlier in the pathway. Genes that mask or inhibit other genes in...

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 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

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

Mutations are changes in the sequence of DNA. These changes can occur spontaneously or they can be induced by exposure to environmental factors. Mutations ...

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

Genome Size and the Evolution of New Genes

types-of-genetic-transfer-between-organisms

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
Cells are regularly exposed to mutagens&#8212;factors in the environment that can damage DNA and generate mutations. UV ...

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
Lucien Cu&#233;not discovered lethal alleles in 1905 while studying the inheritance of coat color in mice. The agouti gene is ...

Polygenic Traits

background-and-environment-affect-phenotype

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

Many human characteristics, like height, are shaped by both nature&mdash;in other words, by our genes&mdash;and by nurture, or our environment. For ...

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

The polymerase chain reaction, or PCR, is a technique used to amplify DNA through thermocycling &#8211; cyles of temperature changes at fixed time ...

DNA Gel Electrophoresis

DNA gel electrophoresis is a technique used for the detection and separation of DNA molecules. An electric field is applied to a gel matrix comprised of ...

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 ...

Restriction Enzymes

Restriction enzymes are bacterial enzymes used to cut DNA in a sequence-specific manner. To cleave DNA, they bind to specific palindromic sequences called ...

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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