Lai, E. C. | bioRxiv Science

bioRxiv2020

miRNAs and neural alternative polyadenylation specify the virgin behavioral state

How are diverse regulatory strategies integrated to impose appropriately patterned gene expression that underlie in vivo phenotypes? Here, we reveal how coordinated miRNA regulation and neural-specific alternative polyadenylation (APA) of a single locus controls complex behaviors. Our entry was the unexpected observation that deletion of Bithorax-Complex (BX-C) miRNAs converts virgin female flies into a subjective post-mated behavioral state, normally induced by seminal proteins following copulation. Strikingly, this behavioral switch is directly attributable to misregulation of homothorax (hth). We localize specific CNS abdominal neurons where de-repressed Hth compromises virgin behavior in BX-C miRNA mutants. Moreover, we use genome engineering to demonstrate that precise mutation of hth 3UTR sites for BX-C miRNAs, or deletion of its neural 3 UTR extension containing most of these sites, both induce post-mated behaviors in virgins. Thus, facilitation of miRNA-mediated repression by neural APA is required for virgin females to execute behaviors appropriate to their internal state.

neuroscience↗

bioRxiv2020

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

The roles of epitranscriptomic modifications in mRNA regulation have recently received substantial attention, with appreciation growing for their phenotypically selective impacts within the animal. We adopted Drosophila melanogaster as a model system to study m6A, the most abundant internal modification of mRNA. Here, we report proteomic and functional analyses of fly m6A-binding proteins, confirming nuclear (YTHDC) and cytoplasmic (YTHDF) YTH domain proteins as the major m6A binders. Since all core m6A pathway mutants are viable, we assessed in vivo requirements of the m6A pathway in cognitive processes. Assays of short term memory revealed an age-dependent requirement of m6A writers working via YTHDF, but not YTHDC, comprising the first phenotypes assigned to Drosophila mutants of the cytoplasmic m6A reader. These factors promote memory via neural-autonomous activities, and are required in the mushroom body, the center for associative learning. To inform their basis, we mapped m6A from wild-type and mettl3 null mutant heads, allowing robust discrimination of Mettl3-dependent m6A sites. In contrast to mammalian m6A, which is predominant in 3 UTRs, Drosophila m6A is highly enriched in 5 UTRs and occurs in an adenosine-rich context. Genomic analyses demonstrate that Drosophila m6A does not directionally affect RNA stability, but is preferentially deposited on genes with low translational efficiency. However, functional tests indicate a role for m6A in translational activation, since we observe reduced nascent protein synthesis in mettl3-KO cells. Finally, we show that ectopic YTHDF can increase m6A target reporter output in an m6A-binding dependent manner, and that this activity is required for in vivo neural function of YTHDF in memory. Altogether, we provide the first tissue-specific m6A maps in this model organism and reveal selective behavioral and translational defects for m6A/YTHDF mutants.

animal behavior and cognition↗