Role of tRNAs in human keratinocytes: an insight on the modifying enzymes NSUN2, CTU2 and ELP3

Cultures of human epidermal stem cells have been widely used to treat skin burns and genetic skin diseases. For successful burn treatment and ex vivo gene therapy of genetic skin conditions, it is essential that the graft contains an adequate proportion of holoclones, the clonal type generated by epidermal stem cells. During cultivation holoclones give rise to transient amplifying progenitors, which generate meroclones and paraclones. A complete understanding of the molecular and biological mechanism driving self-renewal in holoclones as compared to meroclones and paraclones is still missing. Over the past decades, the field of epitranscriptomics has emerged, focusing on specific RNA modifications that endow transcripts with novel features and functions, ultimately influencing protein translation. These processes have been recognized as key regulators in tumorigenesis and in the maintenance of pluripotency. In murine hair follicle stem cells, methylation of a defined subset of tRNAs promotes differentiation by enhancing translational efficiency during lineage commitment, whereas mRNA methylation in progenitor cells is essential for the translation of RNA processing factors and for supporting skin morphogenesis. Our recent single cell RNA profiling of human keratinocyte cultures revealed strong enrichment of tRNA modifications and translational pathways in human holoclones and meroclones. In this project we investigated whether specific translational dynamics are different in stem cells and transient amplifying progenitors. Based on preliminary data, we focused on the role of three writer enzymes, NSUN2, CTU2 and ELP3. We assessed their expression during keratinocytes serial cultivation and their role after specific gene-knockout using CRISPR-Cas9 technology. We obtained a successful gene knock-out after editing and performed clonogenic and biochemical assays on control and edited cells. The data suggest that the loss of NSUN2 and CTU2 in interfollicular epidermis does not significantly alter the clonogenic potential of human primary keratinocytes, while loss of ELP3 seems to be required to sustain keratinocytes clonogenic potential. Further investigations are required to confirm these findings.