The formation of a fuzzy complex in the negative arm regulates the robustness of the circadian clock

Abstract

The circadian clock times cellular processes to the day/night cycle via a Transcription-Translation negative Feedback Loop (TTFL). However, a mechanistic understanding of the negative arm in both the timing of the TTFL and its control of output is lacking. We posited that the formation of negative-arm protein complexes was fundamental to clock regulation stemming from the negative arm. Using a modified peptide microarray approach termed Linear motif discovery using rational design (LOCATE), we characterized the interaction of the disordered negative-arm clock protein FREQUENCY to its partner protein FREQUENCY-Interacting RNA helicase. LOCATE identified a specific Short Linear Motif (SLiM) and interaction hotspot as well as positively charged islands that mediate electrostatic interactions, suggesting a model where negative arm proteins form a fuzzy complex essential for clock timing and robustness. Further analysis revealed that the positively charged islands were an evolutionarily conserved feature in higher eukaryotes and contributed to proper clock function.

Phosphorylation, disorder, and phase separation govern the behavior of Frequency in the fungal circadian clock

“…Note: Ref50 is a clustering method utilized by UniProt and is defined in (Steinegger, et al, 2018). While this work was in preparation, conceptually similar analyses were carried out by Jankowski et al, 2022 (Jankowski, et al, 2022).…”

“…The FRQ functional analogs share physicochemical characteristics with FRQ, particularly an enrichment of hydrophilic residues, increases in Gly and Pro content and a depletion of hydrophobic residues, even though their positional sequence conservation with FRQ is low (Fig. S14 and S15; see also (Jankowski, et al, 2022; Pelham, et al, 2021)). Similar amino acid composition patterns among FRQ, dPER and hPER1, include the most common residues in each protein and its UniRef50 cluster (sequences with at least 50% identity with the target protein) being serine, proline and glycine (∼30% of total sequence) accompanied by a high depletion in aromatic residues (only ∼7.5%) (Fig.…”

“…However, in addition, FRQ and its homologs are unusual in their high Arg and Asp content, which tend to separate in charge blocks throughout their sequences (as indicated by high kappa values, Fig. S15; see also (Jankowski, et al, 2022)). Such charge clustering correlates with LLPS (Somjee, et al, 2020).…”

Acetylation of WCC is dispensable for the core circadian clock but differentially regulates acute light responses inNeurospora

“…There exist two independent DNA elements in the promoter of the central circadian pacemaker gene frequency (frq): In constant darkness, WCC drives circadian expression of frq by binding to the Clock box (C-box) DNA element (7), while it associates with the Proximal Light-Response Element (pLRE) to activate frq transcription upon light exposure (8,9). FREQUENCY (FRQ), the gene product of frq, nucleates formation of a multi-component complex, FFC (FRQ-FRH complex), with FRH (FRQinteracting RNA helicase) (10)(11)(12) and CKI (Casein Kinase I) (13). In circadian cycles, FFC promotes phosphorylation of WCC at over 95 residues, most of which are involved only in downregulation of circadian amplitudes (expression levels of circadian genes) but not in determining period length (14).…”

“…FREQUENCY (FRQ), the gene product of frq , nucleates formation of a multi-component complex, FFC (FRQ-FRH complex), with FRH (FRQ-interacting RNA helicase) (10–12) and CKI (Casein Kinase I) (13). In circadian cycles, FFC promotes phosphorylation of WCC at over 95 residues, most of which are involved only in downregulation of circadian amplitudes (expression levels of circadian genes) but not in determining period length (14).…”