Measuring mRNA translation in neuronal processes and somata by tRNA-FRET.

Bella Koltun,Deepak Nair,Iliana Barrera,Kobi Rosenblum,Mohammad Hleihil,Noga Gershoni-Emek,Ranjan Sasmal,Sarit S Agasti,Siddharth Nanguneri,Sivan Ironi

doi:10.1093/nar/gkaa042

Abstract

In neurons, the specific spatial and temporal localization of protein synthesis is of great importance for function and survival. Here, we visualized tRNA and protein synthesis events in fixed and live mouse primary cortical culture using fluorescently-labeled tRNAs. We were able to characterize the distribution and transport of tRNAs in different neuronal sub-compartments and to study their association with the ribosome. We found that tRNA mobility in neural processes is lower than in somata and corresponds to patterns of slow transport mechanisms, and that larger tRNA puncta co-localize with translational machinery components and are likely the functional fraction. Furthermore, chemical induction of long-term potentiation (LTP) in culture revealed up-regulation of mRNA translation with a similar effect in dendrites and somata, which appeared to be GluR-dependent 6 h post-activation. Importantly, measurement of protein synthesis in neurons with high resolutions offers new insights into neuronal function in health and disease states.

Highlights

Proteostatic processes, including protein synthesis and/or degradation and the mechanisms regulating them, are key to the cellular ability to respond to environmental changes
We examined whether introduction of fluorescentlylabeled tRNA adversely affects neuronal health or function, as previous work with fluorescently-labeled tRNA has far been limited to somatic cells and cell lines [38,46]
We employed the tRNA-Forster Resonance Energy Transfer (FRET) technique to establish a novel application of a method that enables the visualization and quantification of both tRNA dynamics and mRNA translation at a subcellular resolution [26,38]

Summary

Introduction

Proteostatic processes, including protein synthesis and/or degradation and the mechanisms regulating them, are key to the cellular ability to respond to environmental changes. In neuronal processes, which often traverse distances several orders of magnitude larger than the cell body, the regulation of local translation is fundamental for maintaining their distinct functionalities, including signaling and synaptic plasticity [1,2,3,4]. Dysregulation of these mechanisms underlies various neurodevelopmental and neurodegenerative pathologies Both the initiation and elongation phases of mRNA translation and the respective translation factors that mediate them have been suggested as innovative targets for memory enhancement in health and disease [6,8,9,10,11,12,13]. Labeled and unlabeled samples of bulk yeast tRNA were sent to GenXPro (Frankfurt, Germany). The sequence has been deposited in the NCBI SRA database, BioProject ID: PRJNA592850; BioSample accessions: SAMN13441002, SAMN13441003

Methods

Results

Conclusion