Abstract
The circadian clock is encoded by a negative transcriptional feedback loop that coordinates physiology and behavior through molecular programs that remain incompletely understood. Here, we reveal rhythmic genome-wide alternative splicing (AS) of pre-mRNAs encoding regulators of peptidergic secretion within pancreatic β cells that are perturbed in Clock-/- and Bmal1-/- β-cell lines. We show that the RNA-binding protein THRAP3 (thyroid hormone receptor-associated protein 3) regulates circadian clock-dependent AS by binding to exons at coding sequences flanking exons that are more frequently skipped in clock mutant β cells, including transcripts encoding Cask (calcium/calmodulin-dependent serine protein kinase) and Madd (MAP kinase-activating death domain). Depletion of THRAP3 restores expression of the long isoforms of Cask and Madd, and mimicking exon skipping in these transcripts through antisense oligonucleotide delivery in wild-type islets reduces glucose-stimulated insulin secretion. Finally, we identify shared networks of alternatively spliced exocytic genes from islets of rodent models of diet-induced obesity that significantly overlap with clock mutants. Our results establish a role for pre-mRNA alternative splicing in β-cell function across the sleep/wake cycle.
Highlights
The circadian clock is an endogenous timing system that coordinates organismal physiology in anticipation of the sleep–wake cycle (Bass and Takahashi 2010; Asher and Schibler 2011; Bass 2012)
To establish the trajectories of alternative splicing (AS) events throughout the time series to assess rhythmic AS across the day, we calculated ψ for all common AS events from each of the three replicate samples for each time point and identified statistically significant oscillations in ψ using JTK_cycle (Hughes et al 2010), allowing for period lengths of 20, 24, or 28 h. This analysis revealed a total of 257 oscillatory AS events, consisting of 172 skipped exons (SE), 44 alternative 3′ splice sites (A3SS), alternative 5′ splice sites (A5SS), and mutually exclusive exons (MXE) events across 241 unique genes (Fig. 1A; Supplemental Table S1), which collectively displayed a bimodal distribution in peak phases occurring ∼44 and 52 h after shock (Fig. 1B)
Most studies have focused on using mRNA- and nascent RNA sequencing in tandem with assays for measuring transcription factors (TFs) binding to understand the role of the molecular clock in initiating RNA synthesis (Fang et al 2014; Zhang et al 2014)
Summary
The circadian clock is an endogenous timing system that coordinates organismal physiology in anticipation of the sleep–wake cycle (Bass and Takahashi 2010; Asher and Schibler 2011; Bass 2012). Oscillating AS events have been shown to be driven by both the internal core circadian clock and environmental timing cues such as temperature, which drives the rhythmic phosphorylation and function of serine–arginine (SR)-containing spliceosomal proteins as well as the splicing efficiency and stabilization of cold-inducible RNA-binding protein (Cirp) (Morf et al 2012; Gotic et al 2016; Preußner et al 2017). We used genomewide transcriptomics to quantify changes in AS events and expression in circadian controlled RNAs. We found that wild-type (WT) islets display rhythmic expression of RBPs and AS of mRNAs encoding protein regulators of peptidergic secretion, and that clock nullizygous β cells display significantly increased exon skipping events in genes that control insulin secretion. We propose that RNA missplicing in β cells contributes to the etiology of diabetes mellitus in conditions of circadian disruption
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
