N6–methyl adenosine (m6A) is the most abundant internal modification on eukaryotic mRNA and has been implicated in a wide range of fundamental cellular processes. This modification is regulated and interpreted by a set of writer, eraser, and reader proteins. To date, there have been no reports on the potential of mRNA epigenetic regulators to influence recombinant protein expression in mammalian cells. In this study we evaluated the potential of manipulating the expression of the m6A YTH domain-containing readers, YTHDF1, 2, and 3 to improve recombinant protein yield based on their role in regulating mRNA stability and promoting translation. Using siRNA-mediated gene depletion, cDNA over-expression and methylation-specific RNA immunoprecipitation, we demonstrate that (i) knock-down of YTHDF2 enhances (~2-fold) the levels of recombinant protein derived from GFP and EPO transgenes in CHO cells; (ii) the effects of YTHDF2 depletion on transgene expression is m6A-mediated and (iii) YTHDF2 depletion or over-expression of YTHDF1 increases viral protein expression and yield of infectious lentiviral particles (~2-3 fold) in HEK293 cells. We conclude that various transgenes can be subjected to regulation by m6A regulators in mammalian cell lines and that these findings demonstrate the utility of epi-transcriptomic-based approaches to host cell line engineering for improved recombinant protein and viral vector production.

N6-methylated adenosine (m6A) and N1-methylated adenosine (m1A) are two epi-transcriptomic modifications on eukaryotic mRNA which have recently been rediscovered and are generating considerable interest. M6A methylation impacts on all aspects of cellular RNA metabolism and numerous physiological processes. Although less abundant than the m6A epitranscriptomic mark, m1A methylation has recently also attracted interest due to its dynamic nature in response to physiological changes. We investigated the role of the m6A and m1A methylation regulators on the expression of a transgene in Chinese Hamster Ovary (CHO) cells - the host cell of choice in producing biopharmaceutical proteins commercially. Using siRNA-mediated gene depletion and methylation-specific RNA immunoprecipitation with anti-m6A or m1A-antibodies, we show that (i) knock-down of the m6A ‘reader’ YTHDF2 or the m1A ‘eraser’ ALKBH3 dramatically impacts transgene expression; (ii) the effects of YTHDF2 and ALKBH3 depletion on transgene expression are m6A- and m1A-mediated. We conclude that the expression of transgenes in CHO cells can be subjected to regulation by both m6A and m1A regulators. These findings open up the prospect of previously unexplored epi-transcriptomic-based approaches to CHO cell line engineering for improved recombinant protein production.