Transcriptional interference by antisense RNA is required for circadian clock function

Eukaryotic circadian oscillators consist of negative feedback loops that generate endogenous rhythmicities(1). Natural antisense RNAs are found in a wide range of eukaryotic organisms(2-5). Nevertheless, the physiological importance and mode of action of most antisense RNAs is not clear(6-9). frequency (frq) encodes a component of the Neurospora core circadian negative feedback loop which was thought to generate sustained rhythmicity(10). Transcription of qrf, the long non-coding frq antisense RNA, is light induced, and its level oscillates in antiphase to frq sense RNA(3). Here we show that qrf transcription is regulated by both light-dependent and -independent mechanisms. Light-dependent qrf transcription represses frq expression and regulates clock resetting. qrf expression in the dark, on the other hand, is required for circadian rhythmicity. frq transcription also inhibits qrf expression and surprisingly, drives the antiphasic rhythm of qrf transcripts. The mutual inhibition of frq and qrf transcription thus forms a double negative feedback loop that is interlocked with the core feedback loop. Genetic and mathematical modeling analyses indicate that such an arrangement is required for robust and sustained circadian rhythmicity. Moreover, our results suggest that antisense transcription inhibits sense expression by mediating chromatin modifications and premature transcription termination. Together, our results established antisense transcription as an essential feature in a circadian system and shed light on the importance and mechanism of antisense action.