Circadian Proteomic Analysis Uncovers Mechanisms of Post-Transcriptional Regulation in Metabolic Pathways

Abstract

SUMMARY Transcriptional and translational feedback loops in fungi and animals drive circadian rhythms in transcript levels that provide output from the clock, but post-transcriptional mechanisms also contribute. To determine the extent and underlying source of this regulation, we applied newly developed analytical tools to a long-duration, deeply sampled, circadian proteomics time course comprising half of the proteome. We found a quarter of expressed proteins are clock regulated, but >40% of these do not arise from clock-regulated transcripts, and our analysis predicts that these protein rhythms arise from oscillations in translational rates. Our data highlighted the impact of the clock on metabolic regulation, with central carbon metabolism reflecting both transcriptional and post-transcriptional control and opposing metabolic pathways showing peak activities at different times of day. The transcription factor CSP-1 plays a role in this metabolic regulation, contributing to the rhythmicity and phase of clock-regulated proteins.

Post-Transcriptional Circadian Regulation in Macrophages Organizes Temporally Distinct Immunometabolic States

“…When we compared the oscillating transcriptome to the oscillating proteome, we found that the extent of post-transcriptional regulation in macrophages is greater than in any previously studied system, with only 15% of oscillating proteins pairing with an oscillating mRNA. Gene ontological analysis of these oscillating proteins suggested that this post-transcriptional regulation, unlike previously studied cell types, stems both from the regulation of translation as well as degradation (Hurley et al, 2018). In addition, we found that metabolism, particularly the process of ATP generation, was highly post-transcriptionally regulated.…”

“…68% of circadian transcripts did not result in a circadian protein and 86% of circadian proteins did not derive from a circadian transcript. As this discrepancy was considerably higher than has been previously reported, we wanted to rule out that this lack of overlap was due to the more lenient period cutoffs used for proteins to be considered circadian (Hurley et al, 2018). We therefore evaluated the overlap using the more or less lenient period cutoffs for both datasets (Supplemental Figures 3E & F) and found that the ratios remained skewed towards minority overlap, indicating strong evidence for post-transcriptional regulation in these BMDMs.…”

“…For ECHO-modeled protein periods, we broadened the period range of what we considered circadian to 18-30 hrs. to accommodate higher levels of technical noise attributable to proteomicsbased techniques (Hurley et al, 2018). Analysis of the proteome identified 1,778 proteins as circadian, meaning 29% of the detected proteome is under circadian control ( Figure 1C).…”

“…The extensive evidence of post-transcriptional circadian control described above led us to examine clock-timed biological processes that could contribute to this regulation. Past studies have shown that translation may be the key process underlying circadian post-transcriptional regulation and gene ontological enrichment analysis to examine the 250 genes rhythmic at both the mRNA and protein levels found that they were enriched for processes involved in translation (Bonferroni P-value = 2.72E-4, Fold enrichment = 5.18) ( Supplemental Table 1) (Ashburner et al, 2000;Hurley et al, 2014Hurley et al, , 2018Caster et al, 2016;Fabregat et al, 2018;Mi et al, 2019). Therefore, we examined the circadian proteome to determine if a significant proportion of the clock output has functionality in translation.…”

“…However, recent evidence has established that post-transcriptional and post-translational mechanisms play major roles in regulatory output from the circadian clock, resulting in circadianly oscillating proteins that are not derived from oscillating transcripts and vice versa (Reddy et al, 2006;Kojima, Shingle and Green, 2011;Chiang et al, 2014;Hurley et al, 2014Hurley et al, , 2018Mauvoisin et al, 2014;Robles, Cox and Mann, 2014;J. Wang et al, 2017;Green, 2018;Wang et al, 2018;Mauvoisin and Gachon, 2019).…”