A neural m⁶A/Ythdf pathway is required for learning and memory in Drosophila

Epitranscriptomic modifications can impact behavior. Here, we used Drosophila melanogaster to study N⁶-methyladenosine (m⁶A), the most abundant modification of mRNA. Proteomic and functional analyses confirm its nuclear (Ythdc1) and cytoplasmic (Ythdf) YTH domain proteins as major m⁶A binders. Assays of short term memory in m⁶A mutants reveal neural-autonomous requirements of m⁶A writers working via Ythdf, but not Ythdc1. Furthermore, m⁶A/Ythdf operate specifically via the mushroom body, the center for associative learning. We map m⁶A from wild-type and Mettl3 mutant heads, allowing robust discrimination of Mettl3-dependent m⁶A sites that are highly enriched in 5' UTRs. Genomic analyses indicate that Drosophila m⁶A is preferentially deposited on genes with low translational efficiency and that m⁶A does not affect RNA stability. Nevertheless, functional tests indicate a role for m⁶A/Ythdf in translational activation. Altogether, our molecular genetic analyses and tissue-specific m⁶A maps reveal selective behavioral and regulatory defects for the Drosophila Mettl3/Ythdf pathway.