Mammalian circadian oscillators are driven by a transcription-translation feedback loop where CLOCK:BMAL1 activity is repressed by the PER:CRY complex. While transcriptional regulation by PER is well established, the role of circadian feedback in co- and post-transcriptional processes remains unclear. Here, we used Nanopore long-read direct RNA sequencing (dRNAseq) and quantitative mass spectrometry (qMS) to uncover a critical function of PERs in alternative splicing (AS) regulation in the liver. Our expanded long-read transcriptome revealed significant changes in rhythmic expression of annotated transcripts, novel isoforms of known genes, and previously unannotated genes, with widespread perturbations in Per1-/-;Per2-/- (PerKO) livers. Rhythmic AS events were restricted to a distinct subset of transcripts, and splicing entropy - a metric of AS complexity - displayed oscillations in only a limited number of pathways, primarily those associated with glucose homeostasis and cellular responses to insulin. In PerKO livers, however, we detected increased isoform complexity and altered splicing entropy across a broad range of pathways linked to cell growth, morphogenesis, ER-associated degradation (ERAD), insulin response and histone methylation. Biochemical analyses and qMS data indicate that these changes are not due to mis-expression of splicing factors, but rather stem from altered nuclear abundance and chromatin retention of a few Serine-Arginine-rich splicing factors (SRSFs). In particular, SRSF3 acts proximal to the core-clock by defining both the period and amplitude of cellular rhythms. Our findings highlight a critical role for PER proteins in shaping the circadian liver proteome by integrating rhythmic transcription with the regulation of a complex and dynamic splicing landscape.
