Rosenblum Lab Molecular and Cellular Mechanisms Underlying Learning and Memory

    Measuring mRNA translation in live neurons

    In order to measure mRNA translation in live neurons we adopted and modified a cutting edge method developed by Anima Biotech and termed Protein Synthesis Monitoring (PSM). The technology enables viewing global translation using fluorescently labeled tRNAs (Figure 1). Fluorescently labeled tRNA molecules provide insight about the dynamic localization of translation machinery and allow quantitative measurement of protein synthesis in live cells.

    This technology monitors the actual process of translation by providing cells with tRNAs labeled as fluorescence resonance energy transfer (FRET) pairs, and exploiting their proximity when in the A and P sites of translating ribosomes. An in situ FRET signal is generated only when a donor-labeled tRNA and an acceptor-labeled tRNA come in close contact (< 7 nm). This occurs when the tRNAs are utilized within the A and P sites of ribosomes during the elongation cycle. The intensity of the FRET signal correlates with the number of ribosomes engaged in protein synthesis, providing a real-time, live-cell assay for visualizing the subcellular loci and rates of protein synthesis.



     Figure1. mRNA translation is evenly distributed throughout the neuron. Mouse cortical neurons were transfected with fluorescently labeled tRNA (donor (red tRNA) or acceptor (magenta tRNA) or with both (raw FRET)).  cFRET was calculated by FRET signal after subtraction of bleed through from Donor only and acceptor only in the Donor-acceptor channel.


    Measuring mRNA translation in neuronal processes and somata by tRNA-FRET. Koltun B, Ironi S, Gershoni-Emek N, Barrera I, Hleihil M, Nanguneri S, Sasmal R, Agasti SS, Nair D, Rosenblum K. Nucleic acid research 2020


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