Höfundar:
Daníel Heiðar Magnússon, Karen Kristjánsdóttir, Linda Viðarsdóttir, Stefán Sigurðsson
In recent years, mRNA methylations have been shown to play an important role in post-transcriptional level of regulations of gene expression through the addition of methyl groups to RNA. These modifications are dynamic and reversible through the activities of methyltransferases, demethylases and methylation readers. Here we present a novel form of epitranscriptomic regulation of RNF168 mRNA export where we demonstrate that known demethylases FTO, and ALKBH3 regulate mRNA export of RNF168, a key regulator of DNA-DSB repair. These demethylases impact RNF168 mRNA export by removing methylation marks from the RNF168-transcript, FTO removes N6-methyladeonosine (m6A) and ALKBH3 N1-methyladenosine (m1A).
While qPCR experiments showed no change in mRNA expression following silencing of ALKBH3 or FTO gene, western blot and RNA-scope experiments showed significant decrease in protein expression, concluding that ALKBH3 and FTO play a role in regulating mRNA export.
Furthermore, m6A and m1A methylation-specific RNA-immunoprecipitation and qPCR demonstrated epitranscriptomic regulation of RNF168 by ALKBH3 and FTO by confirming the presence of m6A and m1A methylations on the RNF168 mRNA transcript.
Our findings point to a novel epitranscriptomic regulation of RNF168 mRNA export by demethylases ALKBH3 and FTO and, in turn, a novel regulatory mechanism for the DNA-DSB repair pathway. We anticipate there might be other ALKBH3/FTO-affected pathways, therefore, further research could prove beneficial. Misregulation of mRNA methylations can result in physiological defects like cancer and other diseases. For that reason it is vital to understand their function and regulation.