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

View Article: PubMed Central - PubMed

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.

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ALKBH3 and ALKBH5 have distinct roles in RNA demethylation.The larger RNA fraction, smaller RNA fraction, tRNA, and mRNA were incubated in the absence (Mock) or presence of ALKBH3 or ALKBH5, enzymatically degraded to nucleosides, and then subjected to LC-ESI-MS/MS. The peak areas of methylated nucleosides were normalized to that of cytidine. (A) ALKBH5, but not ALKBH3, demethylated N6-meA in mRNA. (B) ALKBH3, but not ALKBH5, demethylated N6-meA in tRNA. (C) In the larger RNA fraction, ALKBH3 demethylated 3-meC and 1-meA. On the other hand, ALKBH5 demethylated only N6-meA. (D) In the smaller RNA fraction, ALKBH3 demethylated 3-meC, 1-meA, and N6-meA. On the other hand, ALKBH5 did not demethylate any of the methylated nucleosides. Data show means ± S.D. (n = 3). N.S.: p > 0.05, *p < 0.05, **p < 0.01.
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f3: ALKBH3 and ALKBH5 have distinct roles in RNA demethylation.The larger RNA fraction, smaller RNA fraction, tRNA, and mRNA were incubated in the absence (Mock) or presence of ALKBH3 or ALKBH5, enzymatically degraded to nucleosides, and then subjected to LC-ESI-MS/MS. The peak areas of methylated nucleosides were normalized to that of cytidine. (A) ALKBH5, but not ALKBH3, demethylated N6-meA in mRNA. (B) ALKBH3, but not ALKBH5, demethylated N6-meA in tRNA. (C) In the larger RNA fraction, ALKBH3 demethylated 3-meC and 1-meA. On the other hand, ALKBH5 demethylated only N6-meA. (D) In the smaller RNA fraction, ALKBH3 demethylated 3-meC, 1-meA, and N6-meA. On the other hand, ALKBH5 did not demethylate any of the methylated nucleosides. Data show means ± S.D. (n = 3). N.S.: p > 0.05, *p < 0.05, **p < 0.01.

Mentions: Since ALKBH5 was recently reported to exhibit demethylation activity for N6-meA in mRNA, we compared the demethylation activities of recombinant ALKBH3 and ALKBH5 proteins purified from silkworm pupae (Supplementary Fig. S2), using commercially available bovine mRNA and tRNA as substrates. As shown in Fig. 3A, the demethylation activity of ALKBH5 for N6-meA in mRNA was confirmed, whereas ALKBH3 had no such activity. On the other hand, ALKBH3 showed marked demethylation activity for N6-meA in tRNA, but ALKBH5 did not (Fig. 3B).


AlkB homolog 3-mediated tRNA demethylation promotes protein synthesis in cancer cells
ALKBH3 and ALKBH5 have distinct roles in RNA demethylation.The larger RNA fraction, smaller RNA fraction, tRNA, and mRNA were incubated in the absence (Mock) or presence of ALKBH3 or ALKBH5, enzymatically degraded to nucleosides, and then subjected to LC-ESI-MS/MS. The peak areas of methylated nucleosides were normalized to that of cytidine. (A) ALKBH5, but not ALKBH3, demethylated N6-meA in mRNA. (B) ALKBH3, but not ALKBH5, demethylated N6-meA in tRNA. (C) In the larger RNA fraction, ALKBH3 demethylated 3-meC and 1-meA. On the other hand, ALKBH5 demethylated only N6-meA. (D) In the smaller RNA fraction, ALKBH3 demethylated 3-meC, 1-meA, and N6-meA. On the other hand, ALKBH5 did not demethylate any of the methylated nucleosides. Data show means ± S.D. (n = 3). N.S.: p > 0.05, *p < 0.05, **p < 0.01.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC5304225&req=5

f3: ALKBH3 and ALKBH5 have distinct roles in RNA demethylation.The larger RNA fraction, smaller RNA fraction, tRNA, and mRNA were incubated in the absence (Mock) or presence of ALKBH3 or ALKBH5, enzymatically degraded to nucleosides, and then subjected to LC-ESI-MS/MS. The peak areas of methylated nucleosides were normalized to that of cytidine. (A) ALKBH5, but not ALKBH3, demethylated N6-meA in mRNA. (B) ALKBH3, but not ALKBH5, demethylated N6-meA in tRNA. (C) In the larger RNA fraction, ALKBH3 demethylated 3-meC and 1-meA. On the other hand, ALKBH5 demethylated only N6-meA. (D) In the smaller RNA fraction, ALKBH3 demethylated 3-meC, 1-meA, and N6-meA. On the other hand, ALKBH5 did not demethylate any of the methylated nucleosides. Data show means ± S.D. (n = 3). N.S.: p > 0.05, *p < 0.05, **p < 0.01.
Mentions: Since ALKBH5 was recently reported to exhibit demethylation activity for N6-meA in mRNA, we compared the demethylation activities of recombinant ALKBH3 and ALKBH5 proteins purified from silkworm pupae (Supplementary Fig. S2), using commercially available bovine mRNA and tRNA as substrates. As shown in Fig. 3A, the demethylation activity of ALKBH5 for N6-meA in mRNA was confirmed, whereas ALKBH3 had no such activity. On the other hand, ALKBH3 showed marked demethylation activity for N6-meA in tRNA, but ALKBH5 did not (Fig. 3B).

View Article: PubMed Central - PubMed

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.

No MeSH data available.


Related in: MedlinePlus