Improving Human Epidermal Stem Cells in vitro Culture Condition by Uncovering How RNA Methylation Modulate Non-Coding RNAs

Abstract

The deposition of post-transcriptional RNA modifications into RNA molecules is implicated in regulating stem cell homeostasis and gene expression. 5-methylcytosine (m5c) is currently best described for its functional roles in non-coding RNAs including tRNAs, rRNAs, and vault (VT) RNAs. NSUN2-mediated methylation targets VTRNA1.1; which accordingly regulates its cleavage into regulatory RNAs called small vault RNAs (svRNAs). The derived svRNAs can act as regulatory RNAs similar to miRNAs in regulating gene expression but are processed by a mechanism different from miRNAs. Of these svRNAs (1-4), svRNA1 and svRNA4 are found to be m5c sensitive and act opposite to each other. When NSUN2 expression is mitigated during epidermal differentiation, svRNA4 levels are compromised compared to svRNA1. This supports an inverse relationship of svRNA4 with OVOL1, a master differentiation regulator of the skin; which increases during differentiation. Therefore, supporting an mRNA-microRNA relationship between svRNA4 and OVOL1. Interestingly, upon experimental silencing of svRNA4, epidermal stem cells differentiate. The exact mechanism behind svRNA1 and svRNA4 processing is not known, however, our VTRNA pull-downs strongly suggested DROSHA, an RNA endonuclease, having an active role. I found that knocking-down DROSHA in epidermal stem cells upregulated differentiation markers. I further confirmed that the biogenesis of svRNA1 was dependent on DROSHA endonuclease. In contrast, the biogenesis of svRNA4 was independent of DROSHA. This might suggest that the RNA endonuclease Dicer plays an active role in processing svRNA4. My results provide the basis for further analysis of DROSHA in svRNA processing. Furthermore, I explored the importance of OVOL1 and svRNA4 in cellular survival and differentiation. By using chemically defined cultures to maintain, and proliferate human epidermal stem cells, I discovered that OVOL1 overexpression initiated premature differentiation and dynamically induced VTRNA1.1 expression. Finally, I showed that svRNA4 targeted OVOL1’s 3’UTR and maintained a progenitor state of epidermal stem cells. Collectively, the results demonstrate how RNA modification could be utilized to successfully grow epidermal stem cells in vitro for regenerative medicine. Moreover, future research should engineer a novel culture conditions to grow skin stem cells in functional assays and entitle them to reconstitute skin when required by using small non-coding RNAs.

Date of Award	Jan 2021
Original language	American English
Supervisor	Abdulrahim Sajini (Supervisor)