Roles of mRNA modification and local translation in neuronal development

Abstract

Local translation is a crucial mechanism that functions throughout the development and adulthood of the nervous system, ranging from axon growth to synaptic plasticity. It has been shown that N6-methyladenosine (m6A) modification regulates local translation in axons. However, how the m6A codes in axonal mRNAs are read and decoded by the m6A reader proteins is still unknown. Besides, the involvement of local translation in presynaptic differentiation remains mostly unknown, especially at the developing neuromuscular junctions (NMJs). In this thesis work, I found that the m6A readers YTHDF1 and YTHDF2 are expressed in cerebellar granule cells (GCs) and their axons. Knockdown (KD) of YTHDF1 or YTHDF2 by using lentivirus significantly increased GC axon growth rates in vitro. By integrating anti-YTHDF1 RNA immunoprecipitation-sequencing (RIP-seq) with the quantitative proteomic analysis after KD of YTHDF1 or combining anti-YTHDF2 RIP-seq with RNA-seq after KD of YTHDF2, several critical transcripts were identified which may potentially mediate GC axon growth under the regulation of YTHDF1 or YTHDF2. Among them, Dvl1 and Wnt5a, encoding the key regulators of the Wnt/planar cell polarity (PCP) signaling pathway, were further found to be locally translated in axons and controlled by YTHDF1 and YTHDF2, respectively. Specific deletion of Ythdf1 or Ythdf2 in GCs increased parallel fiber growth, promoted synapse formation in the cerebellum in vivo, and improved motor coordination ability. Together, this work identifies a mechanism by which the m6A readers YTHDF1 and YTHDF2 work synergistically on the Wnt5a pathway through regulating local translation in GC axons to control cerebellar parallel fiber development. I also found that local translation is required in agrin-induced presynaptic differentiation in cultured Xenopus spinal motor neurons. Newly synthesized proteins, labeled by click-reactive cell-permeable O-propargyl-puromycin (OPP), are prominently located at the agrin bead-axon contacts. The mTOR/4E-BP1 signaling pathway was found to regulate agrin-induced local protein synthesis. Moreover, live-cell time-lapse imaging showed the local capturing and immobilization of ribonucleoprotein particles at agrin bead-axon contacts. MT1-MMP mRNA can be locally translated at bead-contacted axons as revealed by newly synthesized photoconvertible fluorescent protein Kaede linked to the untranslated region of MT1-MMP. Taken together, this work reveals a novel role of MT1-MMP local translation in regulating agrin-induced presynaptic differentiation.