Splicing The Clock Is Timeless: From Fruit Fly Behavior To Mammalian Retinal Degeneration

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Doctor of Philosophy (PhD)
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Cell & Molecular Biology
alternative splicing
circadian clock
Molecular Biology
Neuroscience and Neurobiology
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Circadian rhythms synchronize intrinsic cycles of physiology and behavior with diurnal environmental oscillations of light and temperature. Circadian transcription-translation feedback loops (TTFLs), in which the rhythmic expression of core clock proteins negatively regulates their own expression, are a central mechanism of all eukaryotic circadian clocks. How TTFLs maintain separate phases of transcription and negative feedback and generate a ~24 hour period is a key unresolved question in the field. In this thesis, we uncover a novel splicing factor-mediated mechanism that sets the pace of the clock in both fruit flies and mice. In a Drosophila screen for novel clock regulators, we identify pre-mRNA splicing kinase 4 (prp4) and show its requirement for establishing a delay between transcriptional activation and repression in the TTFL. One of the clock-relevant targets of PRP4 is a retained intron in tim (that we call tim-tiny). We characterize a mechanism by which a splice choice at tim-tiny affects TIM accumulation to set the timing of the clock in constant dark and under temperature cycles. In addition to PRP4, we identify a circadian function for multiple components of U4/U5.U6 triple small nuclear ribonucleoprotein (tri-snRNP), and propose a conserved circadian role for these splicing factors. Our studies in the mouse model of retinitis pigmentosa (RP) support this hypothesis and demonstrate that a Prpf8 knockin mutation lengthens circadian wheel-running behavior and dampens the diurnal rhythm in select transcripts of the mouse retina. We explore the relevance of these findings in the context of RP pathology, in particular asking whether clock disruption could contribute to retinal degeneration.

Amita Sehgal
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