Phosphorylation, disorder, and phase separation govern the behavior of Frequency in the fungal circadian clock
Year: 2024
Type: preprint
Abstract: Circadian clocks are composed of molecular oscillators that pace rhythms of gene expression to the diurnal cycle. Therein, transcriptional-translational negative feedback loops (TTFLs) generate oscillating levels of transcriptional repressor proteins that regulate their own gene expression. In the filamentous fungus Neurospora crassa, the proteins Frequency (FRQ), the FRQ-interacting RNA helicase (FRH) and Casein-Kinase I (CK1) form the FFC complex that represses expression of genes activated by the White-Collar complex (WCC). A key question concerns how FRQ orchestrates molecular interactions at the core of the clock despite containing little predicted tertiary structure. We present the reconstitution and biophysical characterization of FRQ and the FFC in unphosphorylated and highly phosphorylated states. Site-specific spin labeling and pulse- dipolar ESR spectroscopy provides domain-specific structural details on the full-length, 989- residue intrinsically disordered FRQ and the FFC. FRQ contains a compact core that associates and organizes FRH and CK1 to coordinate their roles in WCC repression. FRQ phosphorylation increases conformational flexibility and alters oligomeric state but the changes in structure and dynamics are non-uniform. Full-length FRQ undergoes liquid-liquid phase separation (LLPS) to sequester FRH and CK1 and influence CK1 enzymatic activity. Although FRQ phosphorylation favors LLPS, LLPS feeds back to reduce FRQ phosphorylation by CK1 at higher temperatures. Live imaging of Neurospora hyphae reveals FRQ foci characteristic of condensates near the nuclear periphery. Analogous clock repressor proteins in higher organisms share little position-specific sequence identity with FRQ; yet, they contain amino-acid compositions that promote LLPS. Hence, condensate formation may be a conserved feature of eukaryotic circadian clocks.
Source: eLife
Language: English
Subfield: Endocrine and Autonomic Systems
Field: Neuroscience
Domain: Life Sciences
Open Access status: gold
Estimated APC paid: $2,500
Awards: G1430952875, G1974059531, G2579886042, Role of DNA Methylation in Liquid-liquid Phase Separation-mediated Heterochromatin Formation, G440393388, G522049510, OPERATION OF CORNELL HIGH ENERGY SYNCHROTRON SOURCE (CHESS), G6429492609, G6843289442, National Biomedical Center for Advanced ESR Technology (ACERT), G8676815010, National Biomedical Resource for Electron-Spin Resonance Spectroscopy (ACERT), Molecular mechanisms of signaling systems responsive to light, redox and chemical environment, Genetic and Molecular Dissection of the Neurospora Clock
OpenAlex ID: W4391485810