The role of RNA methylation in cytoskeleton regulation during axon development

RNA修饰N6-甲基腺苷(M6A)在轴突和突触定位的mRNAs中被发现，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，m6A调节有丝分裂后神经元发育的分子机制尚不完全清楚。在JSPS研究员期间，我们的目标是揭示m6A表位转录修饰通过APC翻译调节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M6A阅读器YTHDF1蛋白下调APC蛋白在发育中的神经元中的下调。我们在轴突生长锥内对EB3-GFP彗星进行了活体成像，以评估YTHDF1基因敲除对微管动力学的影响。我们观察到，与对照组相比，YTHDF1-KD神经元的MT生长速度加快，EB3彗星在生长锥的累积距离增加。同时，我们对转染shControl或shYTHDF1的培养神经元中酪氨酸化的微管和F-肌动蛋白进行了免疫荧光染色，以确定YTHDF1的解除调控如何影响典型的生长锥细胞骨架结构。我们观察到，YTHDF1的下调导致动态酪氨酸化的MTS对生长锥的侵袭增加，生长锥外围的F-肌动蛋白染色面积减少。

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.