Solving the Mysteries of Reversible Messenger RNA Methylation

Overview

Objectives/Goals The objective was to determine whether ALKBH5 is a new m6A demethylase in vitro and in vivo and to determine whether its demethylation effects had impacts on cellular processes. Methods/Materials siRNA was used to selectively knock out ALKBH5 expression in HeLa cells. Western Blot analysis was performed to confirm the change in ALKBH5 expression levels compared to control cells. Antibodies were used to separate mRNA into two fractions, one containing m6A and one absent of m6A. qRT-PCR was used to analyze the abundance of genes believed to be substrates of ALKBH5 in each of the fractions. Different oligonucleoutides were synthesized to determine the substrate-specificity of ALKBH5, QQQ-LC/MS was used to analyze demethylation activity. Finally, flow cytometry coupled with the knockout of ALKBH5 in HeLa cells was used to analyze if ALKBH5's demethylation activity had impacts on cellular processes such as nuclear export. Results qRT-PCR analysis showed that the knockout of ALKBH5 in HeLa cells increased m6A enrichment in all experimental genes while the control gene remained relatively constant. QQQ-LC/MS analysis of the synthesized oligonucleotides showed that ALKBH5 has strong preference for ssDNA however, as DNA is located mainly in the nucleus, and ssDNA rarely exists in vivo, ssRNA such as mRNA is the most likely substrate of ALKBH5. Furthermore ALKBH5 demonstrates sequence specificity as it prefers the known consensus sequence for m6A as opposed to a randomly generated sequence. Finally, in the flow cytometry experiment, knocking out ALKBH5 significantly increased RNA export. Conclusions/Discussion My experiment further validates the significance of the mRNA epigenetic regulatory system. I demonstrated ALKBH5's demethylation capabilities in vivo. Furthermore I discovered that single stranded RNA is the most likely substrate of ALKBH5. Finally I showed that these effects impact cellular processes such as nuclear export. These findings demonstrate the significance of the mRNA epigenetic regulatory system and may prove significant in studying heritable diseases.

Awards (1)

• First Place

Competition history

• CSEF 2013 Biochemistry/ Molecular Biology · Entry S0512

Resources

• CSEF project summary ↗

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