The role of RNA methylation in cytoskeleton regulation during axon development

RNA甲基化在轴突发育过程中细胞骨架调节中的作用

基本信息

批准号：
22KF0399
负责人：
王丹
金额：
$ 1.54万
依托单位：
Institute of Physical and Chemical Research
依托单位国家：
日本
项目类别：
Grant-in-Aid for JSPS Fellows
财政年份：
2023
资助国家：
日本
起止时间：
2023-03-08 至 2024-03-31
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-22KF0399/
关键词：
m6A signals

项目摘要

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.

RNA修饰N6-甲基腺苷（M6A）已在轴突和突触局部的mRNA中发现，其局部翻译是轴突生长，突触发生和突触可塑性所必需的。然而，尚不完全了解负责M6A介导的有丝分裂后神经元发育调节的分子机制。在JSP奖学金期间，我们的目标是通过APC翻译调节来揭示M6A表演体修饰在轴突生长锥中微管和肌动蛋白动力学调节中的作用。到目前为止，我们已经证实了在M6A读取器YTHDF1蛋白敲低的神经元中APC蛋白的下调。我们在轴突生长锥中对EB3-GFP彗星进行了实影现象，以评估YTHDF1敲低微管动力学的后果。我们观察到与对照相比，我们观察到MT生长速度的提高以及EB3彗星在YTHDF1-KD神经元生长锥中的累积距离增加。同时，我们在用Shcontrol或Shythdf1转染的培养神经元中进行了酪液微管和F-肌动蛋白的免疫荧光染色，以确定YTHDF1的放松管制如何影响典型的生长锥细胞骨架结构。我们观察到，YTHDF1的下调导致通过动态酪乳MTS的侵袭增加生长锥的侵袭，并减少生长锥外围的F-肌动蛋白染色区域。