Cap-dependent translation initiation monitored in living cells

Valentina Gandin (), Brian P. English, Melanie Freeman, Louis-Philippe Leroux, Stephan Preibisch, Deepika Walpita, Maritza Jaramillo and Robert H. Singer ()
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Valentina Gandin: Howard Hughes Medical Institute
Brian P. English: Howard Hughes Medical Institute
Melanie Freeman: Howard Hughes Medical Institute
Louis-Philippe Leroux: Institut National de la Recherche Scientifique (INRS)-Centre Armand-Frappier Santé Biotechnologie (CAFSB)
Stephan Preibisch: Howard Hughes Medical Institute
Deepika Walpita: Howard Hughes Medical Institute
Maritza Jaramillo: Institut National de la Recherche Scientifique (INRS)-Centre Armand-Frappier Santé Biotechnologie (CAFSB)
Robert H. Singer: Howard Hughes Medical Institute

Nature Communications, 2022, vol. 13, issue 1, 1-15

Abstract: Abstract mRNA translation is tightly regulated to preserve cellular homeostasis. Despite extensive biochemical, genetic, and structural studies, a detailed understanding of mRNA translation regulation is lacking. Imaging methodologies able to resolve the binding dynamics of translation factors at single-cell and single-mRNA resolution were necessary to fully elucidate regulation of this paramount process. Here live-cell spectroscopy and single-particle tracking were combined to interrogate the binding dynamics of endogenous initiation factors to the 5’cap. The diffusion of initiation factors (IFs) changed markedly upon their association with mRNA. Quantifying their diffusion characteristics revealed the sequence of IFs assembly and disassembly in cell lines and the clustering of translation in neurons. This approach revealed translation regulation at high spatial and temporal resolution that can be applied to the formation of any endogenous complex that results in a measurable shift in diffusion.

Date: 2022
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DOI: 10.1038/s41467-022-34052-8

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