Circadian Expression of Rev-erb? mRNA and its
Correlation with Expression of Variant Thyroid Hormone Receptors

Karolynn G. Pohl
Marquette University
Mentor:  Dr. Stephen Munroe

In the mammalian genome there exists overlapping genes in which part or all of one gene overlaps a second gene on the opposite strand.  When the overlapping genes include overlapping exons, mRNA transcripts produced from these two genes may base pair to one another, creating regions of double-stranded RNA.  One such example of cis-antisense mRNAs in rat is the TR? / Rev-erb? locus. Thyroid hormone receptor isoforms, Tr?1 and Tr?2, are generated by alternative processing of TR? pre-mRNA.  The 3’ exon (exon 10) of TR?2 mRNA, which is absent from TR?1, overlaps Rev-erb? mRNA.  Therefore Rev-erb? mRNA overlaps TR?2 but not TR?1 mRNA. A hypothetical model for the regulation of TR? pre-mRNA splicing is one in which Rev-erb? mRNA, by binding to the TR?2 region of the TR? pre-mRNA, down-regulates the splicing of TR?2 and thereby increases the ratio of TR?1 to TR?2 mRNA in the cell.

The Rev-erb? protein is a transcription factor and a core component of the molecular clock in mammals. The molecular clock is an interacting set of proteins in the individual cell in which levels of the component transcription factors rise and fall with a 24-hr periodicity.  This clock works by way of interconnected positive and a negative feedback loops.  Due to Rev-erb?’s identity as a clock component, levels of Rev-erb? mRNA vary with a regular circadian period (i.e., about 24 hours).  These oscillations of Rev-erb? mRNA provide an endogenous system in which the correlation between rev-erb? levels and TR? splicing can be studied.  The antisense hypothesis predicts that TR?2  mRNA may cycle anti-phasic to Rev-erb? mRNA.

Rat1A fibroblast cells were chosen for my study because the cells had previously been shown to oscillate in synchrony after a serum shock (Balsalobre and Schibler, Cell 1998). RNA was extracted from oscillating cell cultures and the levels of Rev-erb?, TR?1, TR?2, and Per2 (another clock component) were determined using real-time PCR.  About 20 plates of cells were grown to confluence for six days.  On day six each plate was subjected to media containing 50% horse serum in order to induce synchronized circadian expression in the cells.  One plate was collected immediately (time 0) and others at specific intervals: 1hr, 4hr, 8hr, 12hr, 16hr…etc. Three complete experiments were performed during the course of this project.

The results from each experiment confirm that Rev-erb? and Per2 fluctuate as shown before by Balsalobre and Schibler.  This indicates the success of the experimental protocol, including the serum shock and the use of real-time PCR to determine levels of Rev-erb? and Per2 mRNA levels.  Variations in the levels of TR?1 and TR?2 mRNA are less clear.  However, in some experiments TR?2 appears to vary with a circadian periodicity offset from that of Rev-erb? and roughly parallel to that of TR?1.  This apparent cycling of TR?2 partially anti-phasic to Rev-erb? provides an interesting system for further analysis of possible interactions between these complimentary mRNAs. 
 
 

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