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2021 年度 実績報告書

軸索とシナプス形成時において細胞骨格を動的に制御するRNAメチル化修飾の役割

研究課題

研究課題/領域番号 21F51085
配分区分補助金
研究機関国立研究開発法人理化学研究所

研究代表者

王 丹  国立研究開発法人理化学研究所, 生命機能科学研究センター, チームリーダー (50615482)

研究分担者 BROIX LOIC  国立研究開発法人理化学研究所, 生命機能科学研究センター, 外国人特別研究員
研究期間 (年度) 2021-11-18 – 2024-03-31
キーワードm6A signals
研究実績の概要

RNA modification N6-methyl-adenosine (m6A) has been found in axonal and synaptically localized mRNAs whose local translation is required for axon growth, synaptogenesis, and synaptic plasticity. However, the molecular mechanisms responsible for m6a-mediated regulation of postmitotic neuronal development are not completely understood. During the JSPS fellowship, we aim to uncover the role of m6A epitranscriptomic modification in the regulation of microtubule and actin dynamics in the axonal growth cone through APC translation regulation. So far, we have confirmed the down-regulation of APC protein in developing neurons upon m6A reader YTHDF1 protein knock-down. We conducted live-imaging of EB3-GFP comets in the axonal growth cone to evaluate the consequences of YTHDF1 knock-down on microtubule dynamics. We observed an increase in the MT growth speed as well as an increase in the cumulative distance of EB3 comets in the growth cone of YTHDF1-KD neurons in comparison to control. In parallel, we performed immunofluorescence stainings of tyrosinated microtubules and F-actin in cultured neurons transfected with either shControl or shYTHDF1 to determine how the deregulation of YTHDF1 affects the typical growth cone cytoskeleton architecture. We observed that the downregulation of YTHDF1 leads to an increase of the invasion of the growth cone by the dynamic tyrosinated MTs and a decrease of the F-actin staining area in the periphery of the growth cone.

現在までの達成度 (区分)
現在までの達成度 (区分)

2: おおむね順調に進展している

理由

The project is following the planned schedule as in the proposal.

今後の研究の推進方策

Next we will first try to rescue developmental phenotype by reexpressing APC protein. We will transfect primary neurons with an APC-expressing plasmid together with the shRNA directed against YTHDF1 and determine the axonal length and branching. Next, to determine whether the decrease of APC protein level upon YTHDF1 knockdown is due to protein translation defects, we will use a puromycin-proximity ligation assay (Puro-PLA). Neurons are treated with puromycin, a t-RNA analog that incorporates into the nascent polypeptide chain and leads to the release of the newly synthesized peptides, and submitted to a PLA assay with antibodies against puromycin and APC in order to detect the newly synthesized APC proteins. In parallel, we will perform a cycloheximide pulse-chase assay in Neuro-2a cells to measure whether YTHDF1 downregulation is affecting APC protein stability. Finally, in order to determine whether YTHDF1 is binding to APC mRNA to regulate its translation or stability, we plan to use a combination of fluorescence in situ hybridization (FISH) and PLA techniques. We will design FISH Locked-nucleic-acid (LNA) probes conjugated to biotin that will hybridize to APC mRNA and then perform PLA with two antibodies recognizing biotin modification present in the probes and YTHDF1. This approach will allow us to observe and quantify by confocal microscopy the interaction between YTHDF1 and APC in neurons. We will quantify the level of interaction between YTHDF1 and APC after downregulation of the m6a writer METTL14 to assess whether the interaction is m6a dependent.

  • 研究成果

    (1件)

すべて 2022

すべて 学会発表 (1件) (うち国際学会 1件)

  • [学会発表] The m6a reader YTHDF1 modulates APC and controls dendrite and axon development.2022

    • 著者名/発表者名
      Loic Broix
    • 学会等名
      RIKEN BDR Symposium 2022 EMERGENCE in Biological Systems: Challenges to Bridging Hierarchies
    • 国際学会

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公開日: 2022-12-28   更新日: 2023-08-01  

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