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TDP-43 is autoregulated by multiple excisions of introns in exon6 and reservation of mRNA in nucleus by TDP-43

  • P1-312
  • 小山 哲秀 / Akihide Koyama:1 須貝 章弘 / Akihiro Sugai:2 加藤 泰介 / Taisuke Kato:2 今野 卓哉 / Takuya Konno:2 石原 智彦 / Tomohiko Ishihara:3 西澤 正豊 / Masatoyo Nishizawa:2 小野寺 理 / Osamu Onodera:3 
  • 1:新潟大学研究機構超域学術院 / Center for Transdisciplinary Research, Niigata Univ, Niigata, Japan 2:新潟大学脳研究所神経内科 / Dept. of Neurology, Brain Research Institute, Niigata Univ, Niigata, Japan 3:新潟大学脳研究所分子神経疾患資源解析学分野 / Dept. of Molecular Neuroscience, Brain Research Institute, Niigata Univ, Niigata, Japan 

Background:TAR DNA-binding protein 43 (TDP-43) is a major component of neuronal cytoplasmic inclusions in patients with amyotrophic lateral sclerosis (ALS). One of the significant functions of TDP-43 is that TDP-43 induces its own splicing and regulates its own amounts. The level of TDP-43 should be maintained within adequate level for maintaining cell function and survival. However, these mechanisms is still unclear.
Objective:To investigate the mechanism of TDP-43 auto-regulation.
Materials and Methods:We used Flp-In 293 cell lines in which myc-tagged TDP-43 was induced by doxycycline (Dox). Minigene containing exon6 of TDP-43 was used to investigate depleted TDP-43 effects on the transcripts. Northern blotting of mRNA in cytoplasmic and nuclear extraction and 3'-end qRT-PCR were performed to evaluate the distribution and expression levels of each isoform of the transcripts.
Results:According to northern blotting analysis using polyadenylation (polyA) (+) mRNA extracted from nuclear and cytoplasmic fractions, after induction of ectopic TDP-43-myc by Dox, the endogenous TDP-43 mRNA using proximal polyA site was disappeared from the cytoplasm. In the nucleus, the isoform using distal polyA site showed no obvious change. Upon cycloheximide treatment, the isoforms lacking two or three intra-exonic introns in exon6 were markedly increased in the cytoplasm. These spliced isoforms fulfilled criteria for nonsense-mediated mRNA decay (NMD). Next, to analyze up-regulatory mechanism of TDP-43, we used the minigene under depletion of the endogenous TDP-43 by siRNA. Northern blotting analysis and 3'-end qRT-PCR elucidated that the spliced isoforms were decreased in the cytoplasm, and the unspliced isoform were increased in the cytoplasm. Meanwhile, the unspliced isoform using distal polyA site was decreased in the nucleus.
Conclusion:Increasing TDP-43 induces intra-exonic splicing in exon6 and NMD. In addition, the amounts of TDP-43 altered the use of polyA sites, resulting in regulating intracellular distribution of the mRNA.

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