RESUMEN
mRNA translation is a key step in decoding genetic information. Genetic decoding is surprisingly heterogeneous because multiple distinct polypeptides can be synthesized from a single mRNA sequence. To study translational heterogeneity, we developed the MoonTag, a fluorescence labeling system to visualize translation of single mRNAs. When combined with the orthogonal SunTag system, the MoonTag enables dual readouts of translation, greatly expanding the possibilities to interrogate complex translational heterogeneity. By placing MoonTag and SunTag sequences in different translation reading frames, each driven by distinct translation start sites, start site selection of individual ribosomes can be visualized in real time. We find that start site selection is largely stochastic but that the probability of using a particular start site differs among mRNA molecules and can be dynamically regulated over time. This study provides key insights into translation start site selection heterogeneity and provides a powerful toolbox to visualize complex translation dynamics.
Asunto(s)
Colorantes Fluorescentes/química , ARN Mensajero/metabolismo , Imagen Individual de Molécula/métodos , Regiones no Traducidas 3' , Regiones no Traducidas 5' , Línea Celular Tumoral , Genes Reporteros , Células HEK293 , Humanos , Iniciación de la Cadena Peptídica Traduccional , ARN Mensajero/química , Ribosomas/metabolismo , Anticuerpos de Dominio Único/química , Anticuerpos de Dominio Único/inmunologíaRESUMEN
Nonsense-mediated decay (NMD) is a surveillance system that degrades mRNAs containing a premature termination codon (PTC) and plays important roles in protein homeostasis and disease. The efficiency of NMD is variable, impacting the clinical outcome of genetic mutations. However, limited resolution of bulk analyses has hampered the study of NMD efficiency. Here, we develop an assay to visualize NMD of individual mRNA molecules in real time. We find that NMD occurs with equal probability during each round of translation of an mRNA molecule. However, this probability is variable and depends on the exon sequence downstream of the PTC, the PTC-to-intron distance, and the number of introns both upstream and downstream of the PTC. Additionally, a subpopulation of mRNAs can escape NMD, further contributing to variation in NMD efficiency. Our study uncovers real-time dynamics of NMD, reveals key mechanisms that influence NMD efficiency, and provides a powerful method to study NMD.
Asunto(s)
Codón sin Sentido/genética , Degradación de ARNm Mediada por Codón sin Sentido/genética , ARN Mensajero/genética , Codón sin Sentido/química , Exones/genética , Humanos , Intrones/genética , Mutación/genética , Estabilidad del ARN/genética , ARN Mensajero/química , Imagen Individual de MoléculaRESUMEN
During mRNA translation, the genetic information stored in mRNA is translated into a protein sequence. It is imperative that the genetic information is translated with high precision. Surprisingly, however, recent experimental evidence has demonstrated that translation can be highly heterogeneous, even among different mRNA molecules derived from a single gene in an individual cell; multiple different polypeptides can be produced from a single mRNA molecule and the rate of translation can vary in both space and time. However, whether translational heterogeneity serves an important cellular function, or rather predominantly represents gene expression 'noise' remains an open question. In this review, we discuss the molecular basis and potential functions of such translational heterogeneity.
Asunto(s)
Biosíntesis de Proteínas/genética , ARN Mensajero/genética , Secuencia de Aminoácidos , Animales , Humanos , Modelos Biológicos , ARN Mensajero/química , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/metabolismo , Ribosomas/metabolismoRESUMEN
mRNA translation is a key step in gene expression. Proper regulation of translation efficiency ensures correct protein expression levels in the cell, which is essential to cell function. Different methods used to study translational control in the cell rely on population-based assays that do not provide information about translational heterogeneity between cells or between mRNAs of the same gene within a cell, and generally provide only a snapshot of translation. To study translational heterogeneity and measure translation dynamics, we have developed microscopy-based methods that enable visualization of translation of single mRNAs in live cells. These methods consist of a set of genetic tools, an imaging-based approach and sophisticated computational tools. Using the translation imaging method, one can investigate many new aspects of translation in single living cells, such as translation start-site selection, 3'-UTR (untranslated region) translation and translation-coupled mRNA decay. Here, we describe in detail how to perform such experiments, including reporter design, cell line generation, image acquisition and analysis. This protocol also provides a detailed description of the image analysis pipeline and computational modeling that will enable non-experts to correctly interpret fluorescence measurements. The protocol takes 2-4 d to complete (after cell lines expressing all required transgenes have been generated).