Multiple In Vivo Biological Processes Are Mediated by Functionally Redundant Activities of Drosophila mir-279 and mir-996

Abstract

While most miRNA knockouts exhibit only subtle defects, a handful of miRNAs are profoundly required for development or physiology. A particularly compelling locus is Drosophila mir-279, which was reported as essential to restrict the emergence of CO2-sensing neurons, to maintain circadian rhythm, and to regulate ovarian border cells. The mir-996 locus is located near mir-279 and bears a similar seed, but they otherwise have distinct, conserved, non-seed sequences, suggesting their evolutionary maintenance for separate functions. We generated single and double deletion mutants of the mir-279 and mir-996 hairpins, and cursory analysis suggested that miR-996 was dispensable. However, discrepancies in the strength of individual mir-279 deletion alleles led us to uncover that all extant mir-279 mutants are deficient for mature miR-996, even though they retain its genomic locus. We therefore engineered a panel of genomic rescue transgenes into the double deletion background, allowing a pure assessment of miR-279 and miR-996 requirements. Surprisingly, detailed analyses of viability, olfactory neuron specification, and circadian rhythm indicate that miR-279 is completely dispensable. Instead, an endogenous supply of either mir-279 or mir-996 suffices for normal development and behavior. Sensor tests of nine key miR-279/996 targets showed their similar regulatory capacities, although transgenic gain-of-function experiments indicate partially distinct activities of these miRNAs that may underlie that co-maintenance in genomes. Altogether, we elucidate the unexpected genetics of this critical miRNA operon, and provide a foundation for their further study. More importantly, these studies demonstrate that multiple, vital, loss-of-function phenotypes can be rescued by endogenous expression of divergent seed family members, highlighting the importance of this miRNA region for in vivo function.

Neural specificity of the RNA-binding protein Elav is achieved by post-transcriptional repression in non-neural tissues

“…This was coupled to bioinformatic evidence that the miR-279/996 seed is enriched for conserved targets that are neurally expressed (Stark et al, 2005). Finally, deletion mutants of mir-279/996 reveal defects in olfactory sensory organs, causing inappropriate specification of ectopic CO 2 -sensing, Elav + neurons within the maxillary palp (Cayirlioglu et al, 2008;Sun et al, 2015). The developmental basis of this phenotype remains unknown, but a plausible hypothesis based on their expression and computational patterns as 'anti-neuronal' determinants might be that they stem from the transformation of a non-neuronal sensory lineage cell into an ectopic neuron.…”

“…We knocked GFP into a 16.6 kb mir-279/996 genomic backbone (Fig. 4A), which we showed fully rescues mir-279/996 null animals (Sun et al, 2015). This reporter generated ubiquitous GFP in imaginal discs (Fig.…”

“…In particular, our previous studies indicated that miR-279/996 are specifically deployed in sensory organs and regulate neural specification (Cayirlioglu et al, 2008;Sun et al, 2015), and thus did not hint at their ubiquitous expression. …”

“…We also identified a less-conserved 7mer-1A site for the miR-279/996/286 family. Expression of miR-286 is restricted to the early embryo, whereas miR-279 and miR-996 are co-expressed from a genomic cluster and are detected throughout development (Mohammed et al, 2014;Sun et al, 2015). We recently characterized a double-deletion allele [15C] that specifically removes mir-279/mir-996 (Sun et al, 2015), and showed that both miRNAs contribute to a variety of neural phenotypes previously ascribed to the sole loss of miR-279 (Cayirlioglu et al, 2008;Luo and Sehgal, 2012).…”

“…RNAi lines were from the Vienna Drosophila RNAi Center: UAS-GW182-RNAi (VDRC 103581), UAS-elav-RNAi (VDRC 37915) and UASmei-P26-RNAi (VDRC 101060). Other published mutants and transgenes included elav[4] (Campos et al, 1985), elav-lacZ (Yao and White, 1994), mir-7[delta1] (Li and Carthew, 2005), mir-8[delta1] (Shcherbata et al, 2007), mir-279/996[15C] (Sun et al, 2015), 16.6 kb mir-279/996-GFP (Sun et al, 2015), UAS-elav[2e2], UAS-elav[3e3], ubi-GFP-UgGH (Toba et al, 2002) and UAS-mei-P26 (Page et al, 2000). A detailed summary of the genotypes analyzed is provided in the supplementary Materials and Methods.…”