Quantification of mRNA translation in live cells using single-molecule imaging.

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).

Single-Molecule Imaging Uncovers Rules Governing Nonsense-Mediated mRNA Decay.

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.

Imaging Translation Dynamics of Single mRNA Molecules in Live Cells.

mRNA translation is a key step in decoding the genetic information stored in DNA. Regulation of translation efficiency contributes to gene expression control and is therefore important for cell fate and function. Here, we describe a recently developed microscopy-based method that allows for visualization of translation of single mRNAs in live cells. The ability to measure translation dynamics of single mRNAs will enable a better understanding of spatiotemporal control of translation, and will provide unique insights into translational heterogeneity of different mRNA molecules in single cells.

Correction: Resource Availability Modulates the Cooperative and Competitive Nature of a Microbial Cross-Feeding Mutualism.

[This corrects the article DOI: 10.1371/journal.pbio.1002540.].

Resource Availability Modulates the Cooperative and Competitive Nature of a Microbial Cross-Feeding Mutualism.

Mutualisms between species play an important role in ecosystem function and stability. However, in some environments, the competitive aspects of an interaction may dominate the mutualistic aspects. Although these transitions could have far-reaching implications, it has been difficult to study the causes and consequences of this mutualistic-competitive transition in experimentally tractable systems. Here, we study a microbial cross-feeding mutualism in which each yeast strain supplies an essential amino acid for its partner strain. We find that, depending upon the amount of freely available amino acid in the environment, this pair of strains can exhibit an obligatory mutualism, facultative mutualism, competition, parasitism, competitive exclusion, or failed mutualism leading to extinction of the population. A simple model capturing the essential features of this interaction explains how resource availability modulates the interaction and predicts that changes in the dynamics of the mutualism in deteriorating environments can provide advance warning that collapse of the mutualism is imminent. We confirm this prediction experimentally by showing that, in the high nutrient competitive regime, the strains rapidly reach a common carrying capacity before slowly reaching the equilibrium ratio between the strains. However, in the low nutrient regime, before collapse of the obligate mutualism, we find that the ratio rapidly reaches its equilibrium and it is the total abundance that is slow to reach equilibrium. Our results provide a general framework for how mutualisms may transition between qualitatively different regimes of interaction in response to changes in nutrient availability in the environment.

Dynamics of Translation of Single mRNA Molecules In Vivo.

Regulation of mRNA translation, the process by which ribosomes decode mRNAs into polypeptides, is used to tune cellular protein levels. Currently, methods for observing the complete process of translation from single mRNAs in vivo are unavailable. Here, we report the long-term (>1 hr) imaging of single mRNAs undergoing hundreds of rounds of translation in live cells, enabling quantitative measurements of ribosome initiation, elongation, and stalling. This approach reveals a surprising heterogeneity in the translation of individual mRNAs within the same cell, including rapid and reversible transitions between a translating and non-translating state. Applying this method to the cell-cycle gene Emi1, we find strong overall repression of translation initiation by specific 5' UTR sequences, but individual mRNA molecules in the same cell can exhibit dramatically different translational efficiencies. The ability to observe translation of single mRNA molecules in live cells provides a powerful tool to study translation regulation.

Dissecting the roles of human BUB1 in the spindle assembly checkpoint.

Mitotic chromosome segregation is initiated by the anaphase promoting complex/cyclosome (APC/C) and its co-activator CDC20 (forming APC/C(CDC20)). APC/C(CDC20) is inhibited by the spindle assembly checkpoint (SAC) when chromosomes have not attached to spindle microtubules. Unattached kinetochores catalyze the formation of a diffusible APC/C(CDC20) inhibitor that comprises BUBR1 (also known as BUB1B), BUB3, MAD2 (also known as MAD2L1) and a second molecule of CDC20. Recruitment of these proteins to the kinetochore, as well as SAC activation, rely on the mitotic kinase BUB1, but the molecular mechanism by which BUB1 accomplishes this in human cells is unknown. We show that kinetochore recruitment of BUBR1 and BUB3 by BUB1 is dispensable for SAC activation. Unlike its yeast and nematode orthologs, human BUB1 does not associate stably with the MAD2 activator MAD1 (also known as MAD1L1) and, although required for accelerating the loading of MAD1 onto kinetochores, BUB1 is dispensable for the maintenance of steady-state levels of MAD1 there. Instead, we identify a 50-amino-acid segment that harbors the recently reported ABBA motif close to a KEN box as being crucial for the role of BUB1 in SAC signaling. The presence of this segment correlates with SAC activity and efficient binding of CDC20 but not of MAD1 to kinetochores.