The m⁶A Readers YTHDF1 and YTHDF2 Synergistically Control Cerebellar Parallel Fiber Growth by Regulating Local Translation of the Key Wnt5a Signaling Components in Axons

Abstract

Messenger RNA m 6 A modification is shown to regulate local translation in axons. However, how the m 6 A codes in axonal mRNAs are read and decoded by the m 6 A reader proteins is still unknown. Here, it is found that the m 6 A readers YTHDF1 and YTHDF2 are both expressed in cerebellar granule cells (GCs) and their axons. Knockdown (KD) of YTHDF1 or YTHDF2 significantly increases GC axon growth rates in vitro. By integrating anti-YTHDF1&2 RIP-Seq with the quantitative proteomic analysis or RNA-seq after KD of YTHDF1 or YTHDF2, a group of transcripts which may mediate the regulation of GC axon growth by YTHDFs is identified. Among them, Dvl1 and Wnt5a, encoding the key components of Wnt pathway, are further found to be locally translated in axons, which are controlled by YTHDF1 and YTHDF2, respectively. Specific ablation of Ythdf1 or Ythdf2 in GCs increases parallel fiber growth, promotes synapse formation in cerebellum in vivo, and improves motor coordination ability. Together, this study identifies a mechanism by which the m 6 A readers YTHDF1 and YTHDF2 work synergistically on the Wnt5a pathway through regulating local translation in GC axons to control cerebellar parallel fiber development.

The m6A reader YTHDF2 is a negative regulator for dendrite development and maintenance of retinal ganglion cells

“…Ythdf2 fl/fl mice were reported previously ( Yu et al, 2021b ). Six3-cre ( Furuta et al, 2000 ), Thy1-GFP ( Feng et al, 2000 ), and Rosa26-eYFP ( Srinivas et al, 2001 ) mice were from Jackson Laboratory.…”

“…E15.5 retinal neurons were cultured and infected with lentiviral sh Ythdf2 or shCtrl. Sample collection and lysis, protein and peptide preparation were performed following procedures reported previously ( Yu et al, 2021b ). Proteins with fold changes greater than 1.3 and p values less than 0.05 were considered to be regulated by YTHDF2 KD with statistical significance.…”

“…N 6 -methyladenosine (m 6 A) is the most widely distributed and extensively studied internal modification in mRNA (Dominissini et al 2012;Meyer et al 2012;Nachtergaele and He 2018). m 6 A modification has been shown to regulate brain development and functions in the nervous system (Livneh et al 2020;Yu et al 2021). By effectors, most of these studies have focused on its demethylases ("m 6 A erasers") and methyltransferases ("m 6 A writers").…”

Insight into the structure, physiological function, and role in cancer of m6A readers—YTH domain-containing proteins

“…DF1 enhances the translation efficiency of target transcripts in two ways: first, it accelerates the occupancy of the ribosome to methylated mRNA; second, it interacts with initiation factors to promote expression of target mRNA. DF2 was found to regulate the translation of m6A-modified mRNA by cooperating with DF1 or by individually binding to its m6A sites on its 3′UTR [ 31 , 32 ]. DF3 has been reported to primarily regulated the initiation of m6A-methylated mRNA translation by interacting with DF1 in cytoplasmic [ 33 ].…”

“…Sheng et al found that DF2 individually acts as a tumor promoter to enhance tumor growth via facilitating 6PGD mRNA translation [ 31 ]. DF2 also plays its translation regulation function through synthetizing with DF1, and regulates the Wnt5a pathway by promoting the local translation of GC axons, so as to control the development of cerebellar parallel fibers [ 32 ]. Shi et al demonstrated for the first time that DF3 promoted mRNA translation by interacting with ribosomal 40S/60S subunits.…”