The non-canonical Smoothened-AMPK axis regulates Smaug1 biomolecular condensates

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.