ABSTRACT
Biomolecular condensates (BMCs) emerge as important players in RNA regulation. The RNA-binding protein Smaug forms cytosolic BMCs in mammals, insects and yeasts and affects mitochondrial function and/or responses to nutrient deprivation. Here, we found that the non-canonical activation of the Smoothened (SMO)-AMPK pathway, which is known to affect energy metabolism, triggers the immediate disassembly of BMCs formed by a number of human and rodent Smaug orthologs, whereas processing bodies remain rather unaltered. A non-phosphorylatable SMO mutant abrogated the effect, involving SMO phosphorylation in human (h)Smaug1 (also known as SAMD4A) BMCs regulation. Three mechanistically different SMO ligands, namely SAG, GSA-10 and cyclopamine, elicited a similar response, which was blocked upon AMPK pharmacological inhibition. Polysome disassembly by puromycin halted Smaug1 BMC dissolution, thus suggesting that unbound transcripts became translationally active. Single-molecule fluorescent in situ hybridization illustrated the release of UQCRC1 mRNA. Finally, Smaug1 is a phosphoprotein bound by 14-3-3 proteins, and the competitive inhibitor difopein blocked the response to non-canonical SMO stimulation. We propose that the regulated condensation and dispersion of Smaug1 BMCs generate translational changes that contribute to metabolic regulation downstream of the non-canonical SMO-AMPK axis.
Footnotes
Author contributions
Conceptualization: G.L.B.; Data curation: M.G.T., A.J.F.-A., L.H.C., M.H., A.C., G.L.B., H.E.G.; Formal analysis: M.G.T., A.J.F.-A., M.G., F.C.A., L.H.C., J. Pimentel, M.L.P., L.B., P.E.L.S., T.P., H.E.G., G.L.B.; Funding acquisition: M.G.T., A.J.F.-A., M.C.-F., H.E.G., G.L.B.; Investigation: M.G.T., A.J.F.-A., M.G., F.C.A., L.H.C., J. Pimentel, J. Pessoa, M.L.P., L.B., M.H., A.C., P.E.L.S., T.P., H.E.G.; Methodology: M.G.T., A.J.F.-A., M.G., F.C.A., L.H.C., J. Pimentel, J. Pessoa, M.L.P., M.H., A.C., P.E.L.S., H.E.G., G.L.B.; Project administration: G.L.B., M.C.-F., H.E.G.; Resources: D.M.B., M.C.-F., H.E.G., G.L.B.; Supervision: M.G.T., A.J.F.-A., M.C.-F., H.E.G., G.L.B.; Validation: M.G.T., A.J.F.-A., M.G., F.C.A., L.H.C.; Visualization: M.G.T., A.J.F.-A., G.L.B.; Writing – original draft: G.L.B.; Writing – review & editing: M.G.T., A.J.F.-A., J. Pessoa, D.M.B., H.E.G., G.L.B.
Funding
This work was supported by the Agencia Nacional de Promoción Científica y Tecnológica (ANPCyT, Argentina; PICT 2014-3658 to G.L.B. and H.E.G.; PICT 2018-03190 and PICT 2020-2195 to G.L.B..; PICT 2018-01790 to M.G.T. and A.J.F.-A.; PICT 2018-01516 to H.E.G.), the Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET, Argentina; PIP 11220200103180CO to A.J.F.-A.); the Fundação para a Ciência e a Tecnologia (Portugal) and FEDER (grant 2022.01199.PTDC to J. Pessoa) and H2020-Marie Sklodowska-Curie Research and Innovation Staff Exchanges (734825-LysoMod) to M.C.-F.
Data availability
All relevant data can be found within the article and its supplementary information.
