Fig. 2

Methylazoxymethanol (MAM) exposure induces transcriptional mutagenesis in neural stem cells. A Overview of the experiment. Mouse primary hippocampal neural stem cells (NSCs) were cultured in quiescence medium for 3 days. Cell cycle arrest was validated by Ki-67 staining. Next, quiescent NSCs were treated with 1 mM methylazoxymethanol (MAM) acetate or vehicle (PBS) for 1 h, after which cells were rinsed with PBS and cultured for 16 more hours in quiescence medium. Cells were then collected and used for single-cell RNA-seq experiments. B The error spectrum of RNAPII in MAM-treated NSCs shows an increased C → U error rate as compared to vehicle-treated cells. *P < 0.01, unpaired two-tailed t-test. C MAM treatment results in transcripts containing identical errors, termed pseudo-alleles for their ability to generate both WT and mutant transcripts. The graph depicts the ratio of mutant:WT mRNAs detected. Only alleles with more than 10% mutant mRNAs are included (MAM-treated replicate #1). D It is proposed that (1) mutant RNAs, which are the result of transcriptional mutagenesis on unrepaired O⁶-methylguanine (O⁶-mG) DNA lesions, could initiate disease by generating toxic molecules, e.g., misfolded proteins that act as proteopathic seeds. Additionally, (2) an overall increase in the number of erroneous RNAs could overwhelm the cellular protein quality control machinery, potentiating proteotoxic stress phenotypes by impairing the clearance of toxic proteins. Lastly, (3) transcription errors could promote the transition of stressed cells to a dysfunctional state through various mechanisms