Functional characterization of the molecular timer of the circadian clock of Neurospora crassa.
Circadian rhythms rely on cell-autonomous clocks that control the rhythmic expression of a large number of
genes. The core of eukaryotic circadian clocks consists of a negative feedback loop. The main clock
components are known and their interactions have been demonstrated in many cases. However, it is not clear
how these proteins measure time at the molecular level.
The frequency gene (frq) is a key element of the circadian clock of Neurospora. Expression of frq is
rhythmically controlled by the transcription factor WCC. FRQ, together with casein kinase 1a (CK1a) and FRQinteracting
RNA helicase (FRH), form the FFC complex. The FFC transiently interacts with WCC allowing CK1a
to phosphorylate and inactivate WCC and thereby inhibiting frq RNA synthesis. Over the course of a day, FRQ
becomes increasingly phosphorylated by CK1a, leading to its inactivation and degradation, and the onset of a
new circadian cycle1. Progressive hyperphosphorylation of FRQ is associated with circadian timekeeping.
To elucidate how these molecules measure time, we recently showed that CK1a and FRQ form a timing module
that supports slowly progressing hyperphosphorylation of FRQ on a circadian time scale in a temperaturecompensated
manner2,3. However, the conformational changes and associated functional changes induced by
multisite phosphorylation of FRQ are not yet known and will be investigated in the upcoming funding period.
FRQ is a dimer and consists largely of intrinsically disordered regions (IDRs). Predictions by AlphaFold2
suggest that the N-terminal dimerization domain of FRQ serves as a hub that organizes IDRs into a distinct
loop via long-range Beta−sheet interactions. The loop is divided into smaller loops by a long-range interaction of
elements that form a CK1a-binding domain. The organization into loops promotes a compact conformation of
FRQ. Each loop contains a cluster of phosphorylation sites. Interestingly, the conformational organization
predicted in an unbiased manner bring two verified nuclear localization signals of FRQ into spatial proximity,
which are separated in the primary sequence by about 450 amino acid residues. We will investigate how
phosphorylation of sites in these clusters affects the conformational organization of FRQ and the accessibility
of its NLSs. Molecular dynamics simulations identified sites critical for the organization of long-range Beta−sheet
interactions and the assembly of the CK1a-binding domain. Preliminary data show that mutations of these
sites lead to circadian arrhythmicity in vivo. We will use Förster resonance energy transfer (FRET)-based
analyses to study the organization of FRQ in loops and characterize the dynamics of conformational changes
in response to phosphorylation. We hypothesize that phosphorylation of these clusters triggers with a time
delay a molecular switch that affects the conformation and potentially oligomeric state of FRQ, thereby timing
its interaction with CK1a, WCC, nuclear shuttling, and degradation.
Prof. Dr. Michael Brunner (BZH Heidelberg)
Dr. Sigrid Milles (FMP Berlin), from 07/2024
Prof. Dr. Hans-Peter Herzel (Charité Berlin), until 06/2024