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.
Highlights
MicroRNAs are ~22 nucleotide regulatory RNAs derived from hairpin precursors [1], and there are 100s ~ 1000 miRNA loci in well-studied animal genomes [2]
We use precise genetic engineering to show that a single endogenous copy of either mir-279 or mir-996 can fully rescue viability, olfactory neuron, and circadian rhythm defects of double deletion animals
These data and genetic reagents set a new foundation for developmental and behavioral studies of this critical miRNA locus. These data demonstrate that multiple loss-of-function phenotypes can be rescued by endogenous expression of divergent seed family members, highlighting the importance and potentially sufficiency of this region for in vivo function
Summary
MicroRNAs (miRNAs) are ~22 nucleotide (nt) regulatory RNAs derived from hairpin precursors [1], and there are 100s ~ 1000 miRNA loci in well-studied animal genomes [2]. It is well-appreciated that knockouts of individual miRNA genes frequently lack substantial phenotypes [12,13], and that the typical range of miRNA-mediated repression is modest [14,15]. Such findings have been interpreted to reflect that miRNAs are usually for "fine-tuning" or "robustness" of gene expression [5,16], but perhaps dispensable for major aspects of development, metabolism and behavior
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