m6A Modifications of mRNA in Biotechnology

Beatriz Carvalho Sobreira

Key information

Authors:

Beatriz Carvalho Sobreira (Beatriz Carvalho Sobreira)

Supervisors:

Markus Schosserer; Cláudia Alexandra Martins Lobato da Silva (Cláudia Lobato da Silva)

Published in

July 1, 2022

Abstract

RNA modifications represent a novel layer of the regulatory mechanisms of gene expression that hasn't been yet covered by the field of epigenetics. Particularly N6-methyladenoside (m6A), the most abundant modification in mammalian mRNA, was proved to play crucial roles in almost all aspects of mRNA processing including splicing, translation, stability, and decay. Although its role in these processes is assured to be crucial, the precise mechanisms remain unknown. In an attempt to get a deeper understanding of m6A mechanisms and their repercussions on the cells, m6A modifications were quantified by a dot blot in different stages of cell growth during a batch experiment, in three cells lines (CHO-K1, CHO-K1-Hy and CHO-K1-HyHer), growing at 37ºC and 42ºC. After testing several primary and secondary antibodies, as well as positive and negative controls, it was not possible to compare the levels of m6A throughout the batch experiment at 37ºC as the dot blot signals were not specific, neither at 42ºC as the cells immediately died at such high temperature. Furthermore, m1A modifications in CHO-K1 mRNA were assessed and its presence was confirmed by loading its cDNA originated from reverse transcription in 2% agarose and 10% TBE gels.