AlkB homolog 3-mediated tRNA demethylation promotes protein synthesis in cancer cells

Yuko Ueda,Yasuyuki Fusamae,Kentaro Jingushi,Hiroaki Hase,Kazutake Tsujikawa,Ikumi Ooshio,Megumi Kawaguchi,Kazumasa Hirata,Kazuo Harada,Kaori Kitae

doi:10.1038/srep42271

Abstract

The mammalian AlkB homolog (ALKBH) family of proteins possess a 2-oxoglutarate- and Fe(II)-dependent oxygenase domain. A similar domain in the Escherichia coli AlkB protein catalyzes the oxidative demethylation of 1-methyladenine (1-meA) and 3-methylcytosine (3-meC) in both DNA and RNA. AlkB homolog 3 (ALKBH3) was also shown to demethylate 1-meA and 3-meC (induced in single-stranded DNA and RNA by a methylating agent) to reverse the methylation damage and retain the integrity of the DNA/RNA. We previously reported the high expression of ALKBH3 in clinical tumor specimens and its involvement in tumor progression. In this study, we found that ALKBH3 effectively demethylated 1-meA and 3-meC within endogenously methylated RNA. Moreover, using highly purified recombinant ALKBH3, we identified N6-methyladenine (N6-meA) in mammalian transfer RNA (tRNA) as a novel ALKBH3 substrate. An in vitro translation assay showed that ALKBH3-demethylated tRNA significantly enhanced protein translation efficiency. In addition, ALKBH3 knockdown in human cancer cells impaired cellular proliferation and suppressed the nascent protein synthesis that is usually accompanied by accumulation of the methylated RNAs. Thus, our data highlight a novel role for ALKBH3 in tumor progression via RNA demethylation and subsequent protein synthesis promotion.

Highlights

Escherichia coli (E. coli) AlkB protein is a 2-oxoglutarate (2-OG)- and Fe(II)-dependent oxygenase that catalyzes the direct oxidative demethylation of 1-meA and 3-meC and plays a central role in DNA and RNA methylation damage repair[1,2,3]
To evaluate the RNA demethylation activity of AlkB homolog 3 (ALKBH3), total RNA purified from the human pancreatic cancer cell line PANC-1 was incubated with the silkworm-derived recombinant ALKBH3 protein[18]
The reason is that the methylating agent-induced 1-meA and 3-meC in DNA/RNA are elucidated to be demethylated by ALKBH3, and N6-meA in messenger RNA (mRNA) was recently clarified to be a substrate for ALKBH5 and FTO (ALKBH9)[15,16]

Summary

Introduction

Escherichia coli (E. coli) AlkB protein is a 2-oxoglutarate (2-OG)- and Fe(II)-dependent oxygenase that catalyzes the direct oxidative demethylation of 1-meA and 3-meC and plays a central role in DNA and RNA methylation damage repair[1,2,3]. The overexpression of ALKBH3 in some cancer cells is reported to repair the methylation damage in ssDNA, with the assistance of the helicase activating signal co-integrator complex 3 (ASCC3)[11]. Since ALKBH3 has a substrate preference for RNA as well as ssDNA, the defective repair of RNA methylation damage induced by endogenous and exogenous methylating agents may contribute to cancer progression. Localized to the nucleus[6] and ALKBH5 is reported to be in nuclear speckles[16] These ALKBH family members may have different spatial and temporal functions in response to DNA/RNA methylation. ALKBH3 knockdown increases the 1-meA level in RNA and decreases nascent protein levels in the PANC-1 cell line These findings contribute to novel progress in the field of RNA epigenetics

Methods

Results

Conclusion