miRNAs and Neural Alternative Polyadenylation Specify the Virgin Behavioral State

Abstract

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 3'UTR 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.

A double negative post-transcriptional regulatory circuit underlies the virgin behavioral state

“…We employed SAG-1 neurons for quantitative analysis of Hth and Dsx within identified VNC nuclei, taking great care to stain wild-type and mutant VNC in the same wells and to image them in parallel using identical settings (see Methods). Although SAG-1 neurons did not necessarily accumulate the highest levels of Hth seen amongst total VT-7068 or VT-50405 neurons (Garaulet et al, 2020), we could reliably observe elevated Hth in identified SAG-1 neurons, in both miRNA deletion and hth [BSmut] mutants, concomitant with downregulation of Dsx ( Figure 2G). In these same SAG-1 neurons, wildtype Dsx exhibits a somewhat bimodal distribution ( Figure 2G).…”

“…To be certain of this effect, we quantified all Dsx+ neurons throughout the volume of the VNC, and observed ~150 fewer Dsx+ neurons in both mutant conditions (Figure 2E), but found that the difference was larger when restricting the analysis to the dorsal half of the VNC (Figure 2E). We recently showed that repression of hth by BX-C miRNAs that is relevant for female PMRs occurs in several populations of abdominal VNC neurons, marked by the VT-7068, VT-454 and VT-50405 Gal4 drivers (i.e., VT-switch lines) (Feng et al, 2014;Garaulet et al, 2020). However, the numbers of neurons labeled by these drivers (~80-300) were not substantially affected in miRNA mutants ( Figure 2F).…”

“…mutants, ectopic Hth proteins accumulate in both miR-iab-4 and miR-iab-8 domains from larval stages ( Figure 1C) to adult (Garaulet et al, 2014;Garaulet et al, 2020). Specific mutations of all miR-iab-4/8 binding sites in the 3' UTR of the homeodomain-encoding isoform of homothorax (hth[BSmut]) also derepress Hth protein ( Figure 1C).…”

“…phenotype in female virgins, ectopic Hth in hth [BSmut] is most overt within the miR-iab-8 domain ( Figure 1C), and is sufficient to induce PMRs in virgin females (Garaulet et al, 2020).…”

“…Recently, we made surprising genetic observations regarding implementation of the female virgin behavioral state. In particular, post-transcriptional suppression of the homeobox gene homothorax (hth) within the VNC is critical to implement the virgin behavioral state (Garaulet et al, 2020). Of note, deletion of the Bithorax Complex (BX-C) miRNA locus (mir-iab-4/8), point mutations of BX-C miRNA binding sites in hth, or deletion of the hth neural-specific 3' UTR extension bearing many of these miRNA sites, all cause mutant female virgins to perform a host of behaviors characteristic of subjectively mated flies.…”