Hypothesis: mTOR Hyperactivation Induces Splicing Factor Stoichiometric Imbalance, Driving Transcriptomic Entropy and Metabolic Dysregulation

Core Idea

When mTORC1 activity exceeds a physiologic threshold, it not only promotes growth but also dysregulates the expression and splicing of splicing factor genes (e.g., SRSF1/3/7), altering their relative abundances. This perturbs the spliceosome's composition, leading to widespread isoform switching that decouples transcriptome from metabolome—what we term splicing entropy.

Testable Predictions

• Prediction 1: Graded overexpression of DEPTOR will create a biphasic response: low DEPTOR (high mTOR) increases splicing noise, measured as increased intron retention and exon skipping variance across biological replicates; moderate DEPTOR restores fidelity; high DEPTOR (low mTOR) reduces noise but also lowers biosynthetic output.
• Prediction 2: Genes whose splicing is mTOR‑dependent (the 409 nutrient‑responsive set) will show the greatest increase in isoform variance under mTOR hyperactivation, and their corresponding metabolite levels will become discordant with isoform abundance.
• Prediction 3: Pharmacologic rescue of splicing factor stoichiometry (e.g., inducible expression of a balanced SRSF1/3/7 cocktail) will mitigate the metabolic dysregulation caused by mTOR hyperactivation without altering mTOR signaling per se.

Experimental Approach

1. Generate a doxycycline‑inducible DEPTOR titration series in human fibroblasts.
2. Treat cells with a range of doxycycline concentrations to achieve low, medium, high DEPTOR levels, confirming mTORC1 activity via p‑S6K and p‑4EBP1 westerns.
3. Perform deep RNA‑seq (>=150M reads) and quantify splicing entropy using Shannon entropy of isoform distributions per gene and genome‑wide intron retention variance.
4. Parallel targeted metabolomics (LC‑MS) for glycolysis, PPP, purine/pyrimidine pathways.
5. In a second arm, co‑express a stoichiometric SRSF1/3/7 bundle under a constitutive promoter in the high‑mTOR condition and assess whether splicing entropy and metabolite‑isoform coupling are restored.

Expected Outcomes

If the hypothesis holds, we will observe a clear inflection point where splicing entropy rises sharply as DEPTOR falls below a threshold, correlating with loss of isoform‑metabolite concordance. Restoring splicing factor balance should decouple mTOR activity from splicing noise, demonstrating that mTOR’s role in transcriptomic fidelity is separable from its canonical translational control.

Falsification

If splicing entropy does not change with mTOR activity, or if correcting splicing factor levels fails to rescue metabolite‑isoform coupling despite mTOR hyperactivation, the hypothesis would be refuted, suggesting mTOR’s influence on splicing is indirect or negligible for metabolic output.