McGhee, E. | Search bioRxiv

bioRxiv2023

High-efficiency digitally scanned light-sheet fluorescence lifetime microscopy (DSLM-FLIM)

Recently-developed kilopixel single-photon avalanche diode (SPAD) arrays with in-pixel timing hold great promise for fluorescence lifetime imaging microscopy of dynamic samples, thanks to their widefield single-photon time-of-arrival imaging capabilities. However digitally-scanned light-sheet microscope (DSLM) and two-photon microscope systems present significant technical barriers which have to date prevented full and efficient use of the capabilities of SPAD arrays. Because the 12.4 kHz frame-rate of our array camera is faster than achievable DSLM scan rates, most pixels would be sitting idle most of the time. We present a new optical design based around astigmatic imaging optics, enabling rapid and efficient acquisition of fluorescence lifetime imaging data. We demonstrate our system with both one- and two-photon excitation sources, validate performance with lifetime reference beads, and demonstrate separation of similar fluorescence emission spectra in biological samples via lifetime contrast.

biophysics↗

bioRxiv2022

Purine-rich RNA sequences in the 5'UTR site-specifically regulate eIF4A1-unwinding through eIF4A1-multimerisation to facilitate translation

Oncogenic translational programmes underpin cancer development and are often driven by dysregulation of oncogenic signalling pathways that converge on the eukaryotic translation initiation (eIF) 4F complex. Altered eIF4F activity promotes translation of oncogene mRNAs that typically contain highly structured 5UTRs rendering their translation strongly dependent on RNA unwinding by the DEAD-box helicase eIF4A1 subunit of the eIF4F complex. While eIF4A1-dependent mRNAs have been widely investigated, it is still unclear how highly structured mRNAs recruit and activate eIF4A1 unwinding specifically to facilitate their preferential translation. Here, we show that RNA sequence motifs regulate eIF4A1 unwinding activity in cells. Our data demonstrate that eIF4A1-dependent mRNAs contain AG-rich motifs within their 5UTR which recruit and stimulate eIF4A1 unwinding of localised RNA structure to facilitate mRNA translation. This mode of eIF4A1 regulation is used by mRNAs encoding components of mTORC-signalling and cell cycle progression and renders these mRNAs particularly sensitive to eIF4A1-inhibition. Mechanistically, we show that binding of eIF4A1 to AG-rich sequences leads to multimerization of eIF4A1 with eIF4A1 subunits performing distinct enzymatic activities. Our structural data suggest that RNA-binding of multimeric eIF4A1 induces conformational changes in the RNA substrate resulting in an optimal positioning of eIF4A1 proximal to the RNA duplex region that supports efficient unwinding. Hence, we conclude a model in which mRNAs utilise AG-rich sequences to specifically recruit eIF4A1, enabling assembly of the helicase-active multimeric eIF4A1 complex, and positioning these complexes proximal to stable localised RNA structure allowing ribosomal subunit scanning.

molecular biology↗