Hypothesis

Age‑dependent accumulation of cytoplasmic stress granules traps METTL3/14 away from the nucleus, reducing m6A methylation of transcripts that encode proteasome subunits, autophagy regulators and chaperones. This loss of modification diminishes their translation and stability, weakening proteostatic capacity and creating a feed‑forward loop where proteotoxic stress further seeds stress granules.

Mechanistic rationale

• In Alzheimer’s pyramidal neurons METTL3/14 shifts from a nuclear to a cytosolic‑nuclear distribution [PMC8482683].
• Global m6A levels fall with age across species [PNAS2022] and the age‑associated gain of m6A in 3'UTRs of synaptic genes is lost in AD, accompanied by hypomethylation of ubiquitin‑proteasome transcripts [bioRxiv2025].
• Stress granule nucleators such as G3BP1/2 bind RNA‑binding proteins via low‑complexity domains and can sequester methyltransferases.
• We propose that RS‑rich regions of METTL3/14 interact directly with G3BP1/2, leading to their incorporation into stress granules as neurons age.
• Once sequestered, METTL3/14 cannot access nascent nuclear transcripts, causing hypomethylation of key proteostasis mRNAs.
• Reduced m6A decreases ribosome recruitment and increases decay of these transcripts, lowering proteasome activity and autophagic flux.
• Accumulation of damaged proteins further stimulates stress granule formation, closing the loop.

Testable predictions

1. Co‑immunoprecipitation of METTL3/14 with G3BP1/2 will increase in neuronal lysates from old vs. young mice and correlate with cytoplasmic METTL3/14 signal.
2. Acute induction of stress granules (e.g., arsenite treatment) in young neurons will reproduce the nuclear‑to‑cytosolic shift of METTL3/14 and cause loss of m6A on proteasome subunit mRNAs (measured by MeRIP‑seq).
3. Forced nuclear localization of METTL3/14 (NLS‑METTL3 fusion) in aged neurons will restore m6A on autophagy genes (e.g., Atg5, Becn1) and rescue autophagic flux and proteasome activity without altering total METTL3/14 levels.
4. Pharmacological inhibition of stress granule assembly (ISRIB or 2BAct) in aged animals will prevent METTL3/14 mislocalization, maintain m6A on proteostatic transcripts and improve cognitive performance in age‑related models.

Experimental approach

• Use subcellular fractionation followed by western blot to quantify nuclear vs. cytoplasmic METTL3/14 in mouse cortex at 3, 12, 24 months.
• Perform RNA immunoprecipitation (RIP) for G3BP1/2 and probe for METTL3/14 by western.
• Conduct MeRIP‑seq on nuclear RNA from fractions to map m6A on proteasome and autophagy transcripts across ages.
• Express NLS‑METTL3 via AAV in aged mice; assess LC3‑II/I ratio, p62 degradation, proteasome activity (Suc‑LLVY‑AMC), and behavioral readouts (Morris water maze).
• Treat cohorts with ISRIB (stress granule inhibitor) and compare METTL3/14 localization, m6A levels and proteostatic readouts.

If predictions hold, the data will support a localization‑centric mechanism linking stress granule‑mediated sequestration of METTL3/14 to age‑dependent proteostatic collapse. Failure to observe increased METTL3/14‑G3BP interaction or lack of rescue by nuclear restoration would falsify the hypothesis.