Abstract
Circadian rhythms are endogenous, self-perpetuating patterns of gene expression which oscillate with a period of 24 hours that matches the rotation of the earth. The transcriptomic state of the organism within this pattern allows it to anticipate and adapt to environmental changes by driving physiological and behavioral changes. The driver of circadian rhythms is the molecular clock, a transcription-translation feedback loop that makes each cell an autonomous oscillator and, in organisms with neural tissue, drive patterns of neuronal activation that can set a master pacemaker for the entire organism. The molecular clock is robust, resisting stochastic perturbations in intercellular signaling or gene expression, and also plastic, taking certain environmental inputs, such as light pulses, to re-entrain the timing of the circadian rhythms. The source of this robustness is found in the complex regulation of molecular clock components. In this dissertation we aimed to understand how the mechanisms of post-transcriptional regulation (PTR) acting on the molecular clock which contribute to its timing and robustness. We used luciferase reporters with circadian 3′UTRs to measure the effect of PTR on gene expression of steady state mRNA. The reporters were expressed inducibly from Schneider 2 cell culture, and, for the first time, from Drosophila melanogaster in vivo allowing measurement from live flies. In combination with miRNA sponges and overexpression, we found that reporters with the 3′UTR of the cold expressed isoform of timeless (tim), tim-cold and tim-sc, are regulated by micro RNAs (miRNAs). The tim-cold reporter was sensitive to knockdown (KD) and overexpression (OE) of miR-bantam. The tim-sc reporter was sensitive to KD and OE of miR-313. This agrees with previous research from the lab that indicated that tim-cold is likely bound by Argonaute 1.We studied PTR affecting the clock gene Clock (Clk), using reporters and biochemical methods to identify proteins associated with the Clk 3′UTR. Using in vitro RAP-MS methods we found that the splicing factor U1 is associated with the Clk 3′UTR. We then adapted Oligonucleotide Proximity Interactome Mapping (O-MAP), recently published in mammalian systems, for using in Schneider 2 cell culture, and demonstrated that it can be used in vivo in fixed fly brains. O-MAP identified further spliceosomal proteins, in particular U1 and U2 snRNPs and their protein partners, as being associated with the Clk 3′UTR. KD of certain splicing proteins reduced the rhythmic activity of flies in constant dark conditions, an output of the circadian clock. Future work will seek to establish a mechanistic connection between these proteins and circadian regulation. Mutation of a strongly predicted U1 binding site in the Clk 3′UTR increased reporter activity by 2 fold, indicating that U1 binding is important for PTR of Clk and that Clk mRNA might be targeted by the nuclear exosome. Finally, we developed an allele of endogenous Clk edited with CRISPR-Cas9 which can inducibly switch its 3′UTR for a control. This allele will be critical for studying the important of PTR in the molecular clock without any confounding developmental effects.