Synonymous codon usage regulates translation initiation.

Nonoptimal synonymous codons repress gene expression, but the underlying mechanisms are poorly understood. We and others have previously shown that nonoptimal codons slow translation elongation speeds and thereby trigger messenger RNA (mRNA) degradation. Nevertheless, transcript levels are often insufficient to explain protein levels, suggesting additional mechanisms by which codon usage regulates gene expression. Using reporters in human and Drosophila cells, we find that transcript levels account for less than half of the variation in protein abundance due to codon usage. This discrepancy is explained by translational differences whereby nonoptimal codons repress translation initiation. Nonoptimal transcripts are also less bound by the translation initiation factors eIF4E and eIF4G1, providing a mechanistic explanation for their reduced initiation rates. Importantly, translational repression can occur without mRNA decay and deadenylation, and it does not depend on the known nonoptimality sensor, CNOT3. Our results reveal a potent mechanism of regulation by codon usage where nonoptimal codons repress further rounds of translation.

A Multi-color Bicistronic Biosensor to Compare the Translation Dynamics of Different Open Reading Frames at Single-molecule Resolution in Live Cells.

Here, we describe how to image and quantitate the translation dynamics of a bicistronic biosensor that we recently created to fairly compare cap-dependent and IRES-mediated translation at single-molecule resolution in live human cells. This technique employs a pair of complementary intrabodies loaded into living cells that co-translationally bind complementary epitopes in the two separate ORFs of the bicistronic biosensor. This causes the biosensor to fluoresce in different colors depending on which ORF/epitopes are translated. Using the biosensor together with high-resolution fluorescence microscopy and single-molecule tracking analysis allows for the quantitative comparison of translation dynamics between the two ORFs at a resolution of tens-of-nanometers in space and sub-seconds in time, which is not possible with more traditional GFP or luciferase reporters. Since both ORFs are on the same biosensor, they experience the same microenvironment, allowing a fair comparison of their relative translational activities. In this protocol, we describe how to get this assay up and running in cultured human cells so that translation dynamics can be studied under both normal and stressful cellular conditions. We also provide a number of useful tips and notes to help express components at appropriate levels inside cells for optimal live cell imaging. Graphical abstract: Steps required for 3-color single-molecule translation imaging and analysis.

Lighting up single-mRNA translation dynamics in living cells.

Over the past five years, technological advances have made it possible to image the translation of single mRNA in the natural context of living cells. With these advances, researchers are beginning to shed light on when, where, and to what degree mRNA are translated with single-molecule precision. These works provide insight into the heterogeneity of translation amongst single transcripts, behavior that is averaged out in complementary bulk assays. In this review, we discuss the rapidly maturing field of live-cell, single-mRNA imaging of translation, beginning with a brief overview of recent technological advances. The remainder of the review focuses on the new biological insights gained from these technologies. We conclude with a discussion of the future of this technology.