Conserved RNA Helicase FRH Acts Nonenzymatically to Support the Intrinsically Disordered Neurospora Clock Protein FRQ

Abstract

Summary Protein conformation dictates a great deal of protein function. A class of naturally unstructured proteins, termed Intrinsically Disordered Proteins (IDPs), demonstrates that flexibility in structure can be as important mechanistically as rigid structure. At the core of the circadian transcription/translation feedback loop in Neurospora crassa is the protein Frequency (FRQ), shown here shown to share many characteristics of IDPs. FRQ in turn binds to Frequency Interacting RNA Helicase (FRH), whose clock function has been assumed to relate to its predicted helicase function. However, mutational analyses reveal that the helicase function of FRH is not essential for the clock, and a region of FRH distinct from the helicase region is essential for stabilizing FRQ against rapid degradation via pathway distinct from its typical ubiquitin-mediated turnover. These data lead to the hypothesis that FRQ is an IDP and that FRH acts nonenzymatically, stabilizing FRQ to enable proper clock circuitry/function.

The RNA helicase FRH is an ATP-dependent regulator of CK1a in the circadian clock of Neurospora crassa

“…In the presence of FRH but absence of CK1a, FRQ was rather stable (t 1/2 B4 h) (Fig. 3b), confirming that FRH stabilizes FRQ 18 . Finally, in the presence of FRH and CK1a, FRQ displayed intermediate stability (t 1/2 B1.5 h) (Fig.…”

“…How can one CK1a molecule that is bound to a specific site in FRQ facilitate phosphorylation of a large number of sites distributed throughout the polypeptide chain 4,5 ? FRQ is an intrinsically unfolded protein 18,23 . The few predicted folded segments correspond to the N-terminal coiled-coil domain that facilitates oligomerization of FRQ, the two FCDs in the central portion, which interact with each other via a coiled-coil and the FRH interaction domain in the C-terminal portion of FRQ 7,19,23 .…”

“…Recently, it has been suggested that the ATPase of FRH is not required for the function of the circadian clock 18 . As FRH is essential for the viability of Neurospora, this conclusion was based on co-expression of two FRH alleles, one with a defect in the clock supplying the essential function of FRH, and the other with a defective ATPase.…”

“…As FRH is essential for the viability of Neurospora, this conclusion was based on co-expression of two FRH alleles, one with a defect in the clock supplying the essential function of FRH, and the other with a defective ATPase. To be conclusive, it is mandatory that FRH does not form a homodimer in its interaction with FRQ dimers, as dimerization of FRH could lead to the formation of a FFC bearing one helicase null FRH and one clock null FRH, thereby allowing for cross-complementation 18 . We show that a monomeric mutant version of FRQ interacts with one molecule of FRH.…”

“…However, molecular mechanisms underlying these functions are not known. Recently, it has been suggested that the ATPase of FRH is not required for its function in the clock 18 . This conclusion was based on the assumption that dimeric FRQ interacts with only one molecule of FRH, as in the genetic background used the interaction of a FRQ dimer with two molecules of FRH could lead to cross-complementation by two functionally distinct FRH alleles present in the same complex 18 .…”