Optogenetic Evidence for the Intrinsic Phase Separation Propensity of the Sgs1 N-Terminal Region: Implications for Assemblysome Formation

Gombás, Bence György and Gábor, Erika and Honti, Viktor and Németh-Szatmári, Orsolya and Jankovics, Ferenc and Villányi, Zoltán (2026) Optogenetic Evidence for the Intrinsic Phase Separation Propensity of the Sgs1 N-Terminal Region: Implications for Assemblysome Formation. BIOMOLECULES, 16 (9). No. 1240. ISSN 2218-273X

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Abstract

Assemblysomes are ribosome-nascent chain condensates that regulate co-translational processes through liquid–liquid phase separation, yet the sequence determinants underlying their formation remain incompletely understood. Previous studies identified the DNA helicase Sgs1 as an assemblysome-associated protein; however, whether its N-terminal region possesses intrinsic phase separation propensity has not been experimentally examined. Here, we investigated the first 135 amino acids of Sgs1 using a light-inducible optoDroplet assay. A mCherry–Cry2–Sgs11–135 fusion construct was compared with the established positive control FUS–mCherry–Cry2 and the negative control mCherry–Cry2 in live HEK293T cells. Following blue-light activation, Sgs11–135 reproducibly formed reversible condensates, indicating intrinsic phase separation propensity. Quantitative image analysis revealed light-dependent increases in condensate number, average condensate area, and integrated condensate fluorescence intensity. Compared with FUS, Sgs11–135 formed slightly fewer and smaller condensates but displayed reproducible light-dependent condensate formation. These findings indicate that the Sgs1 N-terminal region exhibits intrinsic phase separation propensity in a validated optogenetic assay. Although this proof-of-principle study does not establish the molecular mechanism of assemblysome formation, the results are consistent with the hypothesis that the Sgs1 N-terminus may contribute to the multivalent interactions underlying assemblysome organization.

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