NSUN2 integrates glucose and one‑carbon metabolism upstream of Rag GTPase-dependent mTORC1 signaling.

How glucose and one‑carbon metabolism converge on Rag GTPase-dependent mechanistic target of rapamycin complex 1 (mTORC1) remains poorly defined, particularly in the absence of AMP-activated protein kinase (AMPK). This study mapped an AMPK-independent glucose-response pathway in human cells using CRISPR editing, rescue assays, proteomics, thermal stability analysis, and RNA sequencing. In AMPK-deficient human cells, glucose refeeding rapidly reactivated mTORC1, and proteomics of mTORC1-associated complexes identified NSUN2 as a glucose-associated factor. NSUN2 loss markedly reduced glucose-induced mTORC1 activation, whereas re-expression restored it. Genetic analyses placed NSUN2 upstream of the lysosomal Rag module because constitutively active Rag GTPases bypassed NSUN2 deficiency. Structure-function studies showed that the acute signaling role of NSUN2 was largely independent of its catalytic cysteines, but required an N-terminal nutrient-responsive motif and a predicted S-adenosylmethionine (SAM)-responsive segment. SAM increased the thermal stability of wild-type NSUN2, and proteomics and Immunoprecipitation identified methionine adenosyltransferase 2 A (MAT2A), a SAM-producing enzyme, as a glucose-responsive NSUN2 partner. MAT2A knockout reproduced the signaling defect. Transcriptomics further linked NSUN2 to glucose-responsive programs in proteostasis, secretion, and stress adaptation. These results identify NSUN2 as a noncanonical signaling factor that couples glucose and methyl-donor availability to Rag-dependent mTORC1 control and transcriptional adaptation.