Fig. 6: Diminished mTORC1 signalling in differentiated cells triggers MAF1-dependent selective tRNA gene repression.
From: Selective gene expression maintains human tRNA anticodon pools during differentiation
a, Immunoblots of MAF1, phospho-S6K1 (S6K1-P) and phospho-4E-BP1 (4E-BP1-P) in hiPSC, NPC, neurons and CM (n = 3 biological replicates). Samples from both untreated and Torin 1-treated HEK293T cells (250 nM; 1 h) served as controls for mTOR inhibition. b, Immunoblot of a Phos-tag gel for MAF1 in hiPSC, NPC and hiPSC treated with 10 nM rapamycin for 8 h (n = 2 biological replicates). Vinculin (VCL) served as a loading control. c, Immunoblots of MAF1 in CRISPRi lines carrying an sgRNA targeting MAF1. Gene knockdown was induced by the addition of 2 µM doxycycline (Dox) for 3 (hiPSC; top) or 6 d (NPC; bottom). For the hiPSC>NPC samples (middle), 2 µM Dox was added to MAF1 sgRNA-containing hiPSC for 3 d, followed by NPC derivation under continuous Dox treatment (n = 2 biological replicates). d, MA plots (generated by DiffBind) of spike-in-normalized RPC1 counts over tRNA features (±125 bp) versus the log2-transformed fold change for Dox-induced hiPSC (top), hiPSC>NPC (middle) and NPC (bottom) samples carrying an sgRNA targeting MAF1 relative to uninduced controls (n = 2 biological replicates for each cell type). Significantly higher and lower occupancies (FDR ≤ 0.05) are shown in green and purple, respectively. e, Heatmap of RPC1 ChIP–Seq occupancy changes following MAF1 depletion by inducible CRISPRi (+Dox) relative to uninduced controls (−Dox). RPC1 ChIP–Seq read counts over extended tRNA features (±125 bp; left). Normalized signal, accounting for estimated library sizes, was generated from these counts scaled to RPM. DiffBind differential occupancy analysis using spike-in normalization for induced samples relative to the corresponding uninduced controls reported as the log2-tranformed fold change (right; n = 2 biological replicates for each cell type and condition; FDR-adjusted P ≤ 0.05). f, Model of selective tRNA expression following hiPSC differentiation. Rep, replicate. Source numerical data and unprocessed blots are provided.
