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

Lijuan Kan,Eric C Lai,Wei Dai,Adam Claridge-Chang,Eun Sil Park,Ralph E Kleiner,Stanislav Ott,Brian Joseph

doi:10.1038/s41467-021-21537-1

Abstract

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.

Highlights

In our effort to identify additional factors that regulate memory, we were enticed by the “epitranscriptome”, the multitude of modified bases that exist beyond the standard RNA nucleotides
Evidence has been shown for preferential association to m6A vs. A for non-YTH proteins, the YTH domain is the only module for which the structural basis of selective m6A binding is known
We examined C-to-T crosslinking-induced mutations following adenosine residues (CIMs), which have been taken to represent individual m6A site in m6A individual-nucleotide-resolution cross-linking and immunoprecipitation (miCLIP) data[61,66]

Summary

Results

Drosophila Ythdc[1] and Ythdf bind m6A in A-rich contexts. In mammals, two general classes of m6A-binding proteins (“readers”) are recognized, based on whether they contain or lack a YTH domain[22]. As our previous mapping suggested that Drosophila m6A modifications are biased to have upstream adenosines[55], we compared AAm6ACU/AAACU probes Both Ythdc[1] and Ythdf exhibited clearly preferential binding to methylated adenosine in this context (Fig. 1B, C). All nonneuronal cells of the intact animal lack Mettl[3] These flies exhibited normal STM (Fig. 2G), providing stringent evidence that the odor avoidance behavioral defect of m6A knockouts is strictly due to a cell-autonomous function of Mettl[3] in neurons. 20-day-old elav-Gal4 > UAS-Ythdf[RNAi] flies exhibited impaired STM (Fig. 3G) These data indicate that cytoplasmic readout of m6A by Ythdf is required for normal function of memory-storing neurons in older flies.

F WT Head miCLIP peaks

Discussion

Methods