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

La identificación de regiones en un genoma que se transcriben y traducen en proteínas es un paso vital para comprender un genoma y su expresión.
Esto se logra con una técnica llamada perfil de ribosomas, también conocida como ribo-seq. Mapea las posiciones de los ribosomas en el ARNm e identifica los ARNm que se traducen activamente en proteínas.
Para aislar el ARN, primero se deben lisar las células para acceder a las moléculas que contienen. A continuación, el lisado se trata con RNasas. Estas enzimas escinden los ARNm que no están cubiertos con ribosomas, dejando solo los fragmentos de ARNm protegidos.
A continuación, los fragmentos protegidos por el ribosoma se separan de los fragmentos escindidos no protegidos mediante un gradiente de sacarosa.
A continuación, los ribosomas se eliminan de los fragmentos de ARNm y el ARN se convierte en ADN mediante RT-PCR, utilizando la enzima transcriptasa inversa.
A continuación, se secuencia el ADN y se mapea en el genoma de referencia. Esto determina la ubicación exacta del ribosoma a lo largo de cada ARNm.
El perfil de ribosomas también puede ayudar a identificar marcos de lectura abiertos o ORF no reconocidos. Un ORF es una región del ADN entre un codón de inicio y un codón de parada que se puede traducir en proteína. Encontrar ORFs puede ser útil en la identificación de nuevos genes.
Consideremos el estudio de los patrones de expresión génica en una línea celular de mamíferos. El procedimiento experimental consiste en exponer las células a estímulos que pueden activar la expresión génica e iniciar la síntesis de ARNm.
Los ARNm que se traducen activamente se identifican mediante el perfil de ribosomas.
Este experimento revela que el gen B se está traduciendo mientras que los genes A y C no.
También muestra una pequeña región adicional del genoma, de alrededor de cien nucleótidos de largo, que se está traduciendo activamente.
Esta región no reconocida es un marco de lectura abierto que puede codificar una nueva proteína que está regulada al alza por los estímulos experimentales.

<ol>
	<li>Brar, G., Weissman, J. El perfil de ribosomas revela el qué, cuándo, dónde y cómo de la síntesis 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. El uso de nucleasa específica dúplex en el perfil de ribosomas y un paquete de software fácil de usar para el análisis de datos Ribo-seq. ARN. 2015; 21(10):1731-1745. doi:10.1261/rna.052548.115</li>
</ol>

perfilado de ribosomas

La elaboración de perfiles de ribosomas o ribosecuenciación es una técnica de secuenciación profunda que produce una instantánea de la traducción activa en una célula. Secuencia selectivamente los ARNm protegidos por ribosomas para obtener información sobre el panorama de traducción de una célula en un momento dado.

Aplicaciones de la elaboración de perfiles de ribosomas.

La elaboración de perfiles de ribosomas tiene muchas aplicaciones, incluida la monitorización in vivo de la traducción dentro de un órgano o tipo de tejido en particular y la cuantificación de nuevos niveles de síntesis de proteínas.

La técnica ayuda a identificar marcos de lectura abiertos traducidos y a descubrir nuevos productos traducidos, incluidos péptidos cortos e isoformas de proteínas caracterizadas con funciones desconocidas. El perfilado de ribosomas también ayuda a identificar varios ARNm en la célula que no se traducen hasta que reciben una señal externa.

Desafíos técnicos del perfilado de ribosomas

La elaboración de perfiles de ribosomas enfrenta muchos desafíos técnicos, como el requisito de grandes cantidades de muestras, la confiabilidad en la inhibición oportuna de la traducción y la contaminación del ARN.

La técnica de congelación instantánea puede superar el desafío de la inhibición de la traducción rápida. Ayuda a capturar eficientemente la distribución de los ribosomas dentro de las células en comparación con los inhibidores del alargamiento de la traducción, como la cicloheximida.

El ARN ribosómico (ARNr) que se une al ARNm generalmente se elimina durante la elaboración del perfil de ribosomas. Sin embargo, a veces todavía se observan algunos contaminantes de ARNr. Los investigadores utilizan la enzima nucleasa específica dúplex (DSN) para reducir la contaminación por ARNr, aislada del hepatopáncreas del cangrejo de Kamchatka, que escinde híbridos de ADNds y ADN-ARN. Este método también se utiliza a menudo para normalizar bibliotecas de ADNc antes de la secuenciación de próxima generación y el agotamiento del ARNr de las bibliotecas de RNA-seq.

Otra limitación de la elaboración de perfiles de ribosomas es el análisis de datos, lo que requiere la experiencia adecuada en bioinformática. Se utiliza un paquete R riboSeqR para superar este problema, que proporciona métodos para resolver datos de perfiles ribosomales para múltiples muestras.

Perfilado de ribosomas

El perfil de ribosomas o ribosecuenciación es una técnica de secuenciación profunda que produce una instantánea de la traducción activa en una célula. Secuencias selectivamente ...

Education

Educación

JoVE Core

Molecular Biology

Biología Molecular

Unit 2

Unidad 2

Analyzing Gene Expression and Function

Análisis de la expresión y función génica

KEY TERMS AND CONCEPTS

TÉRMINOS Y CONCEPTOS CLAVE

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

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test cross

Test Cross in Genetic Inheritance

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complementation tests

Using Crosses to Group Mutations

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single nucleotide polymorphisms-snps

SNPs as Genetic Markers and Variants

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reproductive cloning

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crispr

CRISPR Gene Editing Basics

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experimental rnai

RNAi for Gene Silencing and Discovery

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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

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gene therapy

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epistasis analysis

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genome-wide association studies-gwas

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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 Biología Molecular presenta los procesos moleculares de la célula a través de lecciones claras y concisas de animaciones audiovisuales. Además, en los vídeos de científicos en acción, se muestran aplicaciones de estos conceptos mediante experimentos reales en laboratorios especializados.

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

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Meiosis I

Meiosis II

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Crossing Over

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Oogenesis

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

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Proofreading

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Replication in Prokaryotes

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Replication in Eukaryotes

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Overview of DNA Repair

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Nucleotide Excision Repair

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Fixing Double-strand Breaks

Homologous Recombination

Gene Conversion

Restarting Stalled Replication Forks

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LS.Bio.6.2

An Overview of Genetic Analysis

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Genetic Crosses

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Punnett Squares

Monohybrid Crosses

Dihybrid Crosses

Trihybrid Crosses

Trihybrid Crosses
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Law of Independent Assortment

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Pedigree Analysis

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Background and Environment Affect Phenotype

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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.

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Probability Laws

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The Concept of Multiple Allelism

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

LS.Bio.7.2

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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

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DNA Isolation

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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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