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Abstract Organismal physiology is widely regulated by the molecular circadian clock, a feedback loop composed of protein complexes whose members are enriched in intrinsically disordered regions. These regions can mediate protein-protein interactions via SLiMs, but the contribution of these disordered regions to clock protein interactions had not been elucidated. To determine the functionality of these disordered regions, we applied a synthetic peptide microarray approach to the disordered clock protein FRQ inNeurospora crassa. We identified residues required for FRQ’s interaction with its partner protein FRH, the mutation of which demonstrated FRH is necessary for persistent clock oscillations but not repression of transcriptional activity. Additionally, the microarray demonstrated an enrichment of FRH binding to FRQ peptides with a net positive charge. We found that positively charged residues occurred in significant “blocks” within the amino acid sequence of FRQ and that ablation of one of these blocks affected both core clock timing and physiological clock output. Finally, we found positive charge clusters were a commonly shared molecular feature in repressive circadian clock proteins. Overall, our study suggests a mechanistic purpose for positive charge blocks and yielded insights into repressive arm protein roles in clock function. 

Abstract Circadian rhythms broadly regulate physiological functions by tuning oscillations in the levels of mRNAs and proteins to the 24-h day/night cycle. Globally assessing which mRNAs and proteins are timed by the clock necessitates accurate recognition of oscillations in RNA and protein data, particularly in large omics data sets. Tools that employ fixed-amplitude models have previously been used to positive effect. However, the recognition of amplitude change in circadian oscillations required a new generation of analytical software to enhance the identification of these oscillations. To address this gap, we created the Pipeline for Amplitude Integration of Circadian Exploration suite. Here, we demonstrate the Pipeline for Amplitude Integration of Circadian Exploration suite’s increased utility to detect circadian trends through the joint modeling of the Mus musculus macrophage transcriptome and proteome. Our enhanced detection confirmed extensive circadian posttranscriptional regulation in macrophages but highlighted that some of the reported discrepancy between mRNA and protein oscillations was due to noise in data. We further applied the Pipeline for Amplitude Integration of Circadian Exploration suite to investigate the circadian timing of noncoding RNAs, documenting extensive circadian timing of long noncoding RNAs and small nuclear RNAs, which control the recognition of mRNA in the spliceosome complex. By tracking oscillating spliceosome complex proteins using the PAICE suite, we noted that the clock broadly regulates the spliceosome, particularly the major spliceosome complex. As most of the above-noted rhythms had damped amplitude changes in their oscillations, this work highlights the importance of the PAICE suite in the thorough enumeration of oscillations in omics-scale datasets. 

Alzheimer’s Disease (AD) is a neuroinflammatory disease characterized partly by the inability to clear, and subsequent build-up, of amyloid-beta (Aβ). AD has a bi-directional relationship with circadian disruption (CD) with sleep disturbances starting years before disease onset. However, the molecular mechanism underlying the relationship of CD and AD has not been elucidated. Myeloid-based phagocytosis, a key component in the metabolism of Aβ, is circadianly-regulated, presenting a potential link between CD and AD. In this work, we revealed that the phagocytosis of Aβ42 undergoes a daily circadian oscillation. We found the circadian timing of global heparan sulfate proteoglycan (HSPG) biosynthesis was the molecular timer for the clock-controlled phagocytosis of Aβ and that both HSPG binding and aggregation may play a role in this oscillation. These data highlight that circadian regulation in immune cells may play a role in the intricate relationship between the circadian clock and AD. 

The circadian clock is the broadly conserved, protein-based, timekeeping mechanism that synchronizes biology to the Earth’s 24-h light-dark cycle. Studies of the mechanisms of circadian timekeeping have placed great focus on the role that individual protein-protein interactions play in the creation of the timekeeping loop. However, research has shown that clock proteins most commonly act as part of large macromolecular protein complexes to facilitate circadian control over physiology. The formation of these complexes has led to the large-scale study of the proteins that comprise these complexes, termed here “circadian interactomics.” Circadian interactomic studies of the macromolecular protein complexes that comprise the circadian clock have uncovered many basic principles of circadian timekeeping as well as mechanisms of circadian control over cellular physiology. In this review, we examine the wealth of knowledge accumulated using circadian interactomics approaches to investigate the macromolecular complexes of the core circadian clock, including insights into the core mechanisms that impart circadian timing and the clock’s regulation of many physiological processes. We examine data acquired from the investigation of the macromolecular complexes centered on both the activating and repressing arm of the circadian clock and from many circadian model organisms.