The Ccr4-Not Protein Complex Regulates the Phase of the Neurospora Circadian Clock by Controlling WHITE COLLAR Protein Stability and Activity

Guocun Huang,Qiyang He,Jinhu Guo,Joonseok Cha,Yi Liu

doi:10.1074/jbc.m113.494120

Abstract

In the Neurospora circadian negative feedback loop, white collar 1 (WC-1) and WC-2 form the WC complex that activates frequency (frq) transcription. Here we show that Not1 is a WC-interacting protein and is important for maintaining WC levels. The not1 transcript displays a circadian oscillation with a similar phase as frq. Down-regulation of not1 leads to low levels of WC-1 and WC-2 and a delayed circadian phase as a result of increased protein degradation and increased WC activity. Protein purification of Not1 shows that it is part of the Neurospora Ccr4-Not complex. ccr4 is a clock-controlled gene and is regulated directly by the WC complex. Down-regulation of ccr4 results in a phase delay and period lengthening of the clock. Together, our findings suggest that the Ccr4-Not complex participates in the Neurospora clock function by interacting with and regulating the WC complex.

Highlights

The stability and activity of WC complex (WCC) is important for Neurospora circadian clock function
The presence of QA in the race tube, resulted in dramatic inhibition of cell growth and the loss of circadian conidiation rhythm for the dsnot1 strain. These results indicate that Not1 is critical for the cell growth of Neurospora and suggest that it may play a role in the Neurospora circadian clock
Previous studies have suggested that transcriptionally active WCCs are unstable and that active WCC leads to low WC levels [8, 10, 19, 20, 23, 42]

Summary

Background

The stability and activity of WCC is important for Neurospora circadian clock function. Conclusion: The Ccr4-Not complex regulates the Neurospora clock by controlling WCC stability and activity. Down-regulation of not leads to low levels of WC-1 and WC-2 and a delayed circadian phase as a result of increased protein degradation and increased WC activity. Frq and frh mutations or down-regulation of frh result in low levels of both WC-1 and WC-2 [11, 12, 21, 22] These results are consistent with the hypothesis that transcriptionally active WCs are unstable and that the positive feedback loop is, at least in part, due to the consequence of the FRQ-mediated negative feedback process [23]. FFC and VVD independently regulate the activity and stability of WCC by interacting with WC proteins. Our results uncovered an unexpected link between the circadian clock and the Ccr4-Not complex

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION