Improving Human Epidermal Stem Cell in Vitro Culture Conditions by Uncovering How RNA Methylation Modulate Non-Coding RNAs

Abstract

Post-transcriptional RNA modifications are increasingly implicated in regulating stem cell (SC) fate and homeostasis, offering new insight in understanding stem cell biology and novel methods for in vitro culturing. 5-methylcytosine (m5C) is well-characterized for its functional significance in the modification of non-coding RNAs, such as tRNAs, rRNAs, and vault RNAs (vtRNAs). In particular, NSUN2mediated m5C methylation of vtRNA1.1 plays a critical role in regulating its cleavage into small miRNA-like molecules known as small vault RNAs (svRNAs), with emerging evidence indicating a potential involvement of Drosha and Dicer in this process. Some of the processed svRNAs (1-4) can form RNA-induced silencing complex (RISC) with Argonaute protein and regulates gene expression by binding to their target mRNAs. svRNA4 levels are compromised in contrast to svRNA1 in the absence of NSUN2 mediated m5c, indicating that vtRNA1.1 methylation acts as molecular switch to differentially produce specific svRNAs.

In this work, we confirm the role of Drosha in processing vaultRNA1.1 to svRNA1 by binding to the unmethylated form with higher affinity. On the other hand, our findings suggest that Dicer may not be involved in the processing of vtRNA1.1 and could potentially inhibit its cleavage instead. We further demonstrate the important roles of Drosha and Dicer as RNAse III enzymes in maintaining epidermal SC homeostasis and self-renewal. Knock down of both Drosha and Dicer endonuclease severely affected the stem cell phenotype and promoted their differentiation.

Additionally, we uncover that the selective processing of vtRNA1.1 to svRNA4 maintains the self-renewal state of Epidermal CSs by targeting the master differentiation regulator of skin, OVOL1, via a miRNA-mRNA mechanism. In contrast, svRNA1 promotes cell differentiation and indirectly inhibits EMT pathways, highlighting the opposing regulatory roles of svRNA1 and svRNA4 in stem cell fate determination.

Finally, we evaluated the viability of svRNA4 treatment in the long-term culture and maintenance of epidermal SCs. Our results indicate that svRNA4 treatment facilitates extended cell expansion and supports functional differentiation. Based on these findings, we explored and proposed alternative methods for the efficient incorporation of svRNA4 into cell culture media, aiming to optimize its application for sustained stem cell maintenance and expansion. Our findings provide novel insights into small non-coding RNA processing mechanisms that regulate stem cell fate and offer potential avenues for enhancing in vitro stem cell culture techniques for therapeutic purposes.

Date of Award	8 Jan 2025
Original language	American English
Supervisor	Abdulrahim Sajini (Supervisor)