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bioRxiv · 10.64898/2026.05.13.724860

Mapping the diffusional landscape of short NEAT1 in living cells

Zappone, S.·Perego, E.·Slenders, E.·Diaspro, A.·Oneto, M.·Sunbul, M.·Vicidomini, G.
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Abstract

The long non-coding RNA NEAT1 is a fundamental architect of nuclear condensates, specifically paraspeckles. While the scaffold-essential isoform NEAT1-2 has been extensively characterized, the function and dynamics of its shorter isoform, NEAT1-1, remain poorly understood. Investigating NEAT1-1 in live cells has been historically hindered by its genomic overlap with NEAT1-2. Traditional visualization study designs require either the genetic ablation of NEAT1-2, which disrupts paraspeckle integrity, or the use of bulky tandem tagging arrays, which can sterically hinder RNA folding and partitioning. Here, we implemented a non-invasive imaging strategy and performed diffusivity analysis of NEAT1-1 using the fluorescence light-up aptamer biRhoBAST. This small, high-affinity RNA tag enables high-contrast visualization of NEAT1-1 while preserving the structural integrity of both isoforms and their associated nuclear bodies. By combining imaging and fluorescence fluctuation spectroscopy, we provide characterization of NEAT1-1 within intact micro-and para-speckles. Our results reveal that NEAT1-1 is not purely sequestered within visible condensates; rather, a fraction exists in a distinct diffusive state within the nucleoplasm, likely as nanoscale complexes. These findings suggest that NEAT1-1 possesses a previously unrecognized regulatory role independent of the primary paraspeckle scaffold, offering new insights into the functional diversity of the lncRNA isoforms.

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Zappone, S., Perego, E., Slenders, E., Diaspro, A., Oneto, M., Sunbul, M., Vicidomini, G.. 2026-05-16. Mapping the diffusional landscape of short NEAT1 in living cells. https://doi.org/10.64898/2026.05.13.724860

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