Circadian rhythmicity is a fundamental aspect of the temporal organization of cells in the body, and it modulates much of biochemistry, physiology, behavior, and pathological state. Essentially every cell in the body is capable of generating circadian rhythmicity in mammals, and within each cell, a set of clock genes (which are highly conserved among animals) form transcription?translation feedback loops that drive circadian oscillation. Even though the molecular basis of circadian rhythmicity is thought to be well-characterized, recent transcriptome analyses identified a novel transcript that appears to play an interesting role in these core clock loops. This transcript, Per2AS, is a natural antisense transcript, a class of non-coding RNAs (ncRNAs), of Period2 (Per2), one of the core clock genes. Per2AS is transcribed from the strand opposite to Per2 and its expression is rhythmic and antiphasic to Per2. Given that Per2AS expression is rhythmic and antiphasic to its sense partner and that rhythmic transcription is more energy-consuming than non-rhythmic transcription, it is hypothesized that Per2AS is a functional molecule and plays an important role in the mammalian circadian clock system. Even though ncRNAs were originally considered to be mere transcriptional noise and lack defined functions, a few dozen examples have expanded the scope of ncRNAs from mere ?junk? to functional molecules having a wide spectrum of regulatory roles. In fact, antisense transcripts of a core clock gene have been reported in Neurospora crassa and Antheraea pernyi and been shown to confer robust and sustained rhythmicity, implying that sense-antisense interactions of a core clock gene constitute a common mechanism for circadian clock regulation across kingdoms.
Three specific aims have been designed to test our central hypothesis and define the biological function of Per2AS in the mammalian circadian clock system.
The first aim addresses whether the presence, rhythmicity, and phase of Per2AS expression relative to Per2 are biologically significant.
The second aim takes advantage of traditional strategies and directly asks whether perturbations of Per2AS expression result in changes in the circadian clock machinery.
The third aim, based on our preliminary data, focuses on specific molecules that Per2AS may regulate in order to shed light on the molecular function(s) of Per2AS. Successful completion of this study not only advances our knowledge of circadian biology but also of regulatory ncRNAs. Only in relatively few cases have interactions between sense and antisense RNA pairs been explored and the physiological importance and mode-of-action of these pairs remain poorly understood. Outcomes from the proposed project will have significant impact in understanding the role of antisense transcripts and shed light on the molecular mechanisms by which antisense transcripts elicit physiological functions without producing a protein. Deepening the understanding of the clock oscillatory mechanism and how a non-coding gene (i.e., Per2AS) contributes to the clock output will help us to, ultimately, develop therapeutics for people who suffer from disrupted internal clocks.
Statement Disruptionofourinternaltimingsystemcanleadtomanypathologicalstatesinhumans,andyet,inthis modernworld,ourinternalclockisconstantlyabusedbylifestylefactorssuchasjetlag,artificiallight,andshift work.Thisproposalfocusesondeepeningourunderstandingofhowdailyrhythmsaregeneratedbyexploring thefunctionofarecentlydiscoverednon-codinggeneinthemammaliancircadianclocksystemusingboth experimentalandmathematicalapproachestoprovideanaccurate,quantitativeanalysisofcircadiangene regulatorynetworks.Outcomesfromthisstudywillhelpusdeveloptherapeuticsforpeoplewhosufferfrom disruptedinternalclocks.