Purinosomes and lysosomes interact to maintain the purine pools

Purines are the building blocks of DNA/RNA and hence essential metabolites. While the contributions of external purine salvage as well as the de novo purine biosynthesis (DNPB) have been widely studied, the contribution of lysosome mediated DNA/RNA digestion and external reabsorption into the cytosol remains unknown. Here, we address that question as well as the role of lysosome-mediated purine recycling and its coordination with DNPB in maintaining total purine pools in human cancer cell lines. By combining in-cell stable isotope incorporation assay with quantitative metabolomics we show: cellular uptake of external purines and their internal generation are equivalent; an upregulation in lysosome biogenesis that functions in response to purine deficiency caused by methotrexate (MTX) and lometrexol (LTX) treatment. This leads to increased RNA digestion as visualized by a newly developed intracellular RNA-FRET oligo assay. Interestingly, downregulation of lysosomal RNase activity through knockdown of RNAseT2 increased RNA accumulation and a compensating increase in DNPB.