FRQ-Interacting RNA Helicase Mediates Negative and Positive Feedback in the Neurospora Circadian Clock

Mi Shi,Jennifer J Loros,Michael Collett,Jay C Dunlap

doi:10.1534/genetics.109.111393

Abstract

The Neurospora circadian oscillator comprises FREQUENCY (FRQ) and its transcription activator, the White Collar Complex (WCC). Repression of WCC's transcriptional activity by FRQ via negative feedback is indispensable for clock function. An unbiased genetic screen that targeted mutants with defects in negative feedback regulation yielded a fully viable arrhythmic strain bearing a novel allele of FRQ-interacting RNA helicase (frh), an essential gene that encodes a putative exosome component protein. In the allele, frh(R806H), clock function is completely disturbed, while roles of FRQ-interacting RNA helicase (FRH) essential for viability are left intact. FRH(R806H) still interacts with FRQ, but interaction between the FRQ-FRH(R806H) complex (FFC) and WCC is severely affected. Phosphorylation of WC-1 is reduced in the mutant leading to constantly elevated WCC activity, which breaks the negative feedback loop. WCC levels are considerably reduced in the mutant, especially those of WC-1, consistent both with loss of positive feedback (FRQ-dependent WC-1 stabilization) and with a reduced level of the FRQ-mediated WCC phosphorylation that leads to high WCC activity accompanied by rapid transcription-associated turnover. FRH overexpression promotes WC-1 accumulation, confirming that FRH together with FRQ plays a role in WC-1 stabilization. Identification of a viable allele of frh, displaying virtually complete loss of both negative and positive circadian feedback, positions FRH as a core component of the central oscillator that is permissive for rhythmicity but appears not to modulate periodicity. Moreover, the results suggest that there are clock-specific roles for FRH that are distinct from the predicted essential exosome-associated functions for the protein.

Highlights

The Neurospora circadian oscillator comprises FREQUENCY (FRQ) and its transcription activator, the White Collar Complex (WCC)
The qa-wc-1; qa-wc-2; frhR806H strain was cultured in 2% glucose (ÀQA condition) and moved to 0.1% glucose with 0.01 m quinic acid (QA) (1QA condition) and grown for 8 hr
Extracts were made and subjected to Western analysis and the result showed that WC-1 and WC-2 could be induced by QA. (B) Reduced WC-1 stability in frhR806H

Summary

Introduction

The Neurospora circadian oscillator comprises FREQUENCY (FRQ) and its transcription activator, the White Collar Complex (WCC). An unbiased genetic screen that targeted mutants with defects in negative feedback regulation yielded a fully viable arrhythmic strain bearing a novel allele of FRQ-interacting RNA helicase ( frh), an essential gene that encodes a putative exosome component protein. The inhibition combined with delayed turnover of the negative elements leads to oscillatory transcription factor activity Since those positive elements control expression of other genes (named clock-controlled genes, ccgs), the negative feedback loop gives rise to rhythmic expression of many ccgs that are involved in processes of metabolism, development, stress responses, etc. In the Neurospora clock, components of the FREQUENCY (FRQ)–White Collar Complex (WCC)-based oscillator have been identified by forward genetics, and negative feedback was proven by showing that FRQ overexpression would stop the clock (McClung et al 1989; Aronson et al 1994). It was shown that FRH interacts, albeit weakly, with exosome components and strains with reduced FRH have reduced frq mRNA stability (Guo et al 2009)

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Conclusion