Post-transcriptional regulation of Ribosome Biogenesis upon treatment with MHY1485

Ribosome biogenesis is a critical aspect of cell differentiation. Ribosome synthesis has been previously reported to be regulated at the transcriptional and post-transcriptional levels. Poly(A) tail-length processing is a hallmark of post-transcriptional regulation associated with different steps of transcript metabolism. Here we monitor the contribution of mTOR pathway activation upon treatment with the agonist MHY1485 in shaping the poly(A) tail profile of undifferentiated P19 cells. We found that transcripts with a 5' terminal oligopyrimidine (TOP) motif, including those encoding for ribosomal proteins, specifically accumulate with poly(A) tails ~60 nucleotides long upon mTOR activation. The day after seeding, undifferentiated P19 cells were treated with 10 µM final concentration of MHY1485 (Selleckchem) for 24 hours, or 48 hours with media changed every 24 hours containing fresh MHY1485 solution. Cells exposed to the same amount of DMSO were used as control. RNA was collected, and Direct RNA seq libraries were prepared in duplicates for each timepoint and run on a Nanopore GridIon device. Reads were mapped to the mouse transcriptome, and their poly(A) tail lengths were determined. This information was then used to monitor the impact of mTOR activation on mRNA poly(A) tail lengths.