| 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.
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| 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.
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