mir-276a strengthens Drosophila circadian rhythms by regulating timeless expression

Circadian rhythms in metazoan eukaryotes are controlled by an endogenous molecular clock. It functions in many locations, including subsets of brain neurons (clock neurons) within the central nervous system. Although the molecular clock relies on transcription/translation feedback loops, posttranscriptional regulation also plays an important role. Here, we show that the abundant Drosophila melanogaster microRNA mir-276a regulates molecular and behavioral rhythms by inhibiting expression of the important clock gene timeless (tim). Misregulation of mir-276a in clock neurons alters tim expression and increases arrhythmicity under standard constant darkness (DD) conditions. mir-276a expression itself appears to be light-regulated because its levels oscillate under 24-h light-dark (LD) cycles but not in DD. mir-276a is regulated by the transcription activator Chorion factor 2 in flies and in tissue-culture cells. Evidence from flies mutated using the clustered, regularly interspaced, short palindromic repeats (CRISPR) tool shows that mir-276a inhibits tim expression: Deleting the mir-276a-binding site in the tim 3' UTR causes elevated levels of TIM and similar to 50% arrhythmicity. We suggest that this pathway contributes to the more robust rhythms observed under light/dark LD conditions than under DD conditions.