Fluorescence Imaging Methods to Investigate Translation in Single Cells

Abstract

Translation is the fundamental biological process that converts the genetic information in messenger RNAs (mRNAs) into functional proteins. Translation regulation allows cells to control when, where, and how many proteins are synthesized. Much of what we know about translation comes from ensemble approaches that measure the average of many cells. The cellular and molecular heterogeneity in the regulation of translation remains largely elusive. Fluorescence microscopy allows interrogation of biological problems with single-molecule, single-cell sensitivity. In recent years, improved design of reagents and microscopy tools has led to improved spatial and temporal resolution of translation imaging. It is now possible to track global translation in specific subcellular compartments and follow the translation dynamics of single transcripts. Highlighted here is the recent progress in translation imaging with emphasis on in vivo translation dynamics. These tools will be invaluable to the study of translation regulation.

The structural basis for RNA selectivity by the IMP family of RNA-binding proteins

“…With over 1000 RBPs expressed in humans 1 , their ability to regulate RNA is extensive. RBPs participate in all aspects of an RNA’s life 2 , many are deposited as soon as the RNA is transcribed and they contribute to control over export, localization 3 , translation 4 , and decay 5 . Progress has been made in studying RBPs on the atomic level 6 , with single cell resolution 7 , as well as on a genome wide level by profiling the RNAs that interact with individual RBPs 8 .…”

Principles of Translational Control

“…mRNA Levels mRNA levels obviously play a role in determining the amounts of protein produced by the translational machinery. As mentioned earlier, mRNA levels are determined by their rates of transcription and processing, but also by their rates of transport into the cytoplasm (Biswas et al 2018) and by their rates of degradation that may be coupled to or influenced by their translation (Heck and Wilusz 2018), by their sequences (Karousis and Mühlemann 2018), as well as by microRNAs (Duchaine and Fabian 2018). mRNAs also can be sequestered in PBs and SGs (Ivanov et al 2018) where they are not being translated.…”

“…But mRNA levels alone do not determine the amounts of proteins in cells, as some mRNAs are not actively translated, or are translated poorly, being bound to proteins, called messenger ribonucleoproteins (mRNPs), or sequestered in stress granules (SGs) or processing bodies (PBs) (Ivanov et al 2018). In addition, translation rates are not always uniform among different mRNA species or along individual mRNAs (Biswas et al 2018;Ingolia et al 2018).…”

The travels of mRNAs in neurons: do they know where they are going?

“…There are also technical limitations to observing these events in living cells. To visualize the RNA localization machinery such as mRNAs, adaptor proteins, molecular motors and translated proteins simultaneously at high resolution, novel orthogonal tagging systems beyond fluorescent proteins are needed for multiplex imaging of many different components [21,23,54-56]. Such advances will aid in the development of strategies to see how a neuron handles the constant delivery of multiple mRNPs to its processes.…”