The DNA methylation status of TARDBP 3'-UTR affects its alternative splicing Yuka Koike1, Akihiro Sugai1, Akio Yokoseki1, Osamu Onodera1* 1Department of Neurology, Niigata University *Corresponding Author
[Background] Sporadic amyotrophic lateral sclerosis (SALS) is characterized by the accumulation of TDP-43, and an increase in expression level of TDP-43 contributes to the molecular mechanism of SALS. The molecular mechanism of the increase in expression level of TDP-43 is still unknown. The expression level of TDP-43 is auto-regulated by alternative splicing utilizing introns within TARDBP 3'-UTR . We hypothesize that impairment of this autoregulation mechanism contributes to the molecular mechanism of the increased expression level of TDP-43. DNA methylation status has been proposed as a factor influencing alternative splicing. The TARDBP 3'-UTR is rich in CpGs, which are preferentially methylated, suggesting that methylation of TARDBP 3'-UTR may contribute to its alternative splicing. However, the effect of TARDBP 3'-UTR methylation status on its alternative splicing has not been investigated. Here we investigated whether the methylation status of this region affects alternative splicing and expression levels of TARDBP mRNA. [Methods] The CpGs near the alternative splicing site of TARDBP 3'-UTR was specifically de-methylated or methylated using dCas9-TET1-guideRNA-GFP vector (TET1 vector) or Cas9-DNMT3A-guideRNA-GFP vector (DNMT3A vector), respectively [2, 3]. GFP positive cells were isolated by flow cytometer 48 hours after transfection of these vectors into HEK 293T cells. Bisulfite next-generation sequencing was used to determine the methylation status. [Results] The methylation rate of 6 CpGs near the alternative splicing site of TARDBP 3'-UTR in the cells transfected with TET1 vector, DNMT3A vector, or control vector were 54.6 ± 10.7%, 96.2 ± 3.2%, or 94.4 ± 3.1%, respectively. The transfection of TET1 vector decreased the alternative splicing and increased TARDBP mRNA to 1.9 times higher. The transfection of DNMT3A vector didn't change the alternative splicing and the level of TARDBP mRNA. [Conclusion] The demethylation of CpGs near the alternative splicing site of TARDBP 3'-UTR increased the level of TARDBP mRNA via alteration of alternative splicing.
References  Koyama et al. Nucleic Acids Res 2016  Morita et al. Nat Biotechnol 2016  Vojta, et al. Nucleic Acid Res 2016
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