Regulation of the Neurospora circadian clock by an RNA helicase

Abstract

The eukaryotic circadian oscillators consist of autoregulatory negative-feedback loops. FRQ, WC-1, and WC-2 are three known components of the negative-feedback loop of the Neurospora circadian oscillator. FRQ represses its own transcription by interacting with the WC-1/WC-2 complex and inhibiting WC's role in transcriptional activation. Here we show that all FRQ associates with FRH, an essential DEAD box-containing RNA helicase in Neurospora. The budding yeast homolog of FRH, Dob1p/Mtr4p, is a cofactor of exosome, an important regulator of RNA metabolism in eukaryotes. Down-regulation of FRH by inducible expression of a hairpin RNA leads to low levels of FRQ but high levels of frq RNA and the abolishment of circadian rhythmicities. FRH is associated with the WC complex and this interaction is maintained in a frq null strain. Disruption of the FRQ-FRH complex by deleting a domain in FRQ eliminates the FRQ-WC interaction, suggesting that FRH mediates the interaction between FRQ and the WC complex. These data demonstrate that FRH is an essential component in the circadian negative-feedback loop and reveal an unexpected role of an RNA helicase in regulating gene transcription. Endogenous circadian (daily) clocks control a wide variety of physiological and molecular activities in most eukaryotic and some prokaryotic organisms. At the molecular level, autoregulatory negative-feedback loops composed of positive and negative elements form the core circadian oscillators (Dunlap 1999;King and Takahashi 2000;Reppert and Weaver 2001;Young and Kay 2001). The rhythmic activation of transcription of the negative elements by the positive elements is thought to be the main basis for the generation of the endogenous rhythmicity.In the Neurospora frequency (frq)-white collar (wc)-based circadian negative-feedback loop, a heterodimeric complex formed by WC-1 and WC-2 (two PAS domaincontaining transcription factors) acts as the positive element and activates the transcription of frq by binding to its promoter (Crosthwaite et al. 1997;Cheng et al. 2001b;Loros and Dunlap 2001;Froehlich et al. 2003). FRQ proteins (large FRQ [lFRQ] and small FRQ [sFRQ] resulting from alternative translation initiation) form homodimeric complexes and function as the negative elements in the loop by repressing their own transcription (Aronson et al. 1994a;Garceau et al. 1997;Liu et al. 1997;Cheng et al. 2001a). To close the negative-feedback loop, FRQ forms a complex with the WC proteins and prevents WC from binding to the frq promoter and activating frq transcription (Cheng et al. 2001a;Denault et al. 2001;Merrow et al. 2001;Froehlich et al. 2003). In strains lacking a functional FRQ protein, the negativefeedback loop is impaired, resulting in high frq mRNA levels (Aronson et al. 1994a;Merrow et al. 1997;. How FRQ inhibits the activity of WC complex is unclear. In frq null strains, in addition to their loss of circadian rhythmicities, less conidia and aerial hyphae are produced than a wild-type strain (Aronson et al. 1994b), suggesting that FRQ has funct...

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

“…The native FFC had an apparent M r of B670 kDa. Although all FRQ is bound to FRH 7 , only B15% of FRH were in complex with FRQ ( Fig. 1a and Supplementary Fig.…”

“…This demonstrates that FRH stabilizes FRQ and suppresses low-level phosphorylation of FRQ by yeast kinases. Mtr4p, the yeast orthologue of FRH 7 , does not interact with FRQ ( Supplementary Fig. 3b) and does not substitute for the stabilizing function of FRH.…”

“…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 . The remaining portions of FRQ are predicted to be unstructured.…”

“…FRQ interacts with CK1a 6 and forms a tight complex with FRQ-interacting RNA helicase (FRH), an essential DEAD box-containing RNA helicase 7 . DEAD box-containing RNA helicases comprise a large superfamily of ATPases whose functions are largely unknown 8,9 .…”

Molecular mechanism of light responses in Neurospora: from light-induced transcription to photoadaptation

“…In the dark, a heterodimeric WCC (D-WCC) binds to the C-box (the distal LRE) of the frq promoter, activating its transcription (Crosthwaite et al 1997;Cheng et al 2001b;Froehlich et al 2003;Dunlap and Loros 2004). On the other hand, FRQ and FRH (FRQinteraction RNA helicase), the negative elements in the loop, repress the transcription of frq by inhibiting WCC activity (Aronson et al 1994;Cheng et al 2001aCheng et al , 2005Denault et al 2001). The WC phosphorylation in the dark appears to be an important event in this process.…”