Molecular Mechanisms of Circadian Clocks and RNA Interference
Circadian clocks have been described in almost all organisms ranging in complexity from single cells to mammals and function to control daily rhythms in a variety of biochemical, cellular, physiological, and behavioral events. These rhythms have a period close to 24 hours (circadian) and persist in the absence of external time cues. One of the most important characteristics of circadian rhythms is that they can be synchronized or entrained by environmental signals, the strongest of which are light and temperature. In humans and mammals, circadian clocks control events such as sleep-wake and activity cycles, body temperature cycles, endocrine functions, and gene expression. Clinical consequences in humans including sleep disorders and depression can be observed when the clock malfunctions. The influence of a functional clock on temporal regulation is evident from the decreased performance of shift workers and the jet lag felt by long distance travelers.
Our lab is using filamentous fungus Neurospora crassa, one of the best studied model organisms for circadian clocks, to understand the molecular mechanisms of the circadian clock. In Neurospora, the circadian clock acts to control a variety of processes, and previous studies have shown that the Neurospora circadian clock is an auto-regulated negative feedback loop in which the frequency (frq) gene is an essential component. My laboratory is using molecular, biochemical, and genetic approaches to answer three general questions: 1) What are the components of the input pathways to the clock and how do environmental signals entrain the clock; 2) What are the genes that make up the oscillator and how are they regulated to generate rhythms and 3) How does the clock control rhythmic output events? In the long term, these studies will enable us to compare clock mechanisms of fungi with those of other eukaryotes and to help guide research in other organisms.
The production of double-stranded RNA (dsRNA) is known to elicit RNA interference (RNAi) in most eukaryotes and interferon response in mammals. RNAi and related pathways are evolutionarily conserved gene silencing mechanisms that regulate gene expression, development, genome stability, and host-defense responses. The filamentous fungus Neurospora crassa, an organism that broadly employs gene silencing in regulation of gene expression, offers a unique and powerful system for understanding the RNAi pathway and its function in eukaryotes. Using Neurospora as a model system, we have revealed the mechanism of the RISC activation process in the RNAi pathway. We also showed that dsRNA activates a novel signaling pathway to induce transcription of many genes in Neurospora, including most of the RNAi components, putative antiviral genes, and homologs of the interferon stimulated genes; this activation is analogous to the interferon response in mammals. Our current research is focusing on the understanding of the regulation of RNAi components and on the involvement of RNAi pathway in various cellular processes.
Cheng, P, Y. Yang, C. Heintzen, and Y. Liu (2001) Coiled-coil domain mediated FRQ-FRQ interaction is essential for its circadian clock function in Neurospora. EMBO J. 20: 101-108.
Cheng, P, Y. Yang, and Y. Liu. (2001) Interlocked feedback loops contribute to the robustness of the Neurospora circadian clock. Proc. Natl. Acad. Sci. USA 98:7408-7413
Yang, Y., P. Cheng, G. Zhi, and Y. Liu (2001) Identification of a calcium/calmodulin-dependent protein kinase that phosphorylates the Neurospora circadian clock protein FREQUENCY. J. Biol. Chem. 276: 41064-41072
Cheng, P, Y. Yang, K. H. Gardner, and Y. Liu. (2002) PAS domain mediated WC-1/WC-2 heterodimeric complex is essential for their circadian clock function and the stability of WC-1. Mol. Cell. Biol 22: 517-524
Yang, Y., P. Cheng, and Y. Liu. (2002) Regulation of the Neurospora circadian clock by casein kinase II. Genes & Development 15:994-1006.
He, Q.*, P. Cheng*, Y. Yang, L. Wang, K. H. Gardner, and Y. Liu. (2002) White Collar-1, a DNA-binding transcription factor and a light sensor. Science. 297: 840-843 (cover article, accompanied by a Perspective)
Froehlich, A. C., Y. Liu, J. J. Loros, and J. C. Dunlap. (2002) White Collar-1, a circadian blue light photoreceptor, binds to the frequency promoter. Science. 297: 815-819 843 (cover article, accompanied by Perspective)
Cheng, P, Y. Yang, L, Wang, Q. He, and Y. Liu. (2003) WHITE COLLAR-1, a multifunctional Neurospora protein involved in the circadian feedback loops, light sensing, and transcription repression of wc-2. J. Biol. Chem. 278:3801-3808.
Cheng, P., Q. He, Y. Yang, L, Wang, and Y. Liu. (2003) Functional conservation of light, oxygen, or voltage domains in light sensing. Proc. Natl. Acad. Sci. USA 100:5938-5943
Liu, Y., (2003) J. Biol. Rhythms Molecular mechanisms of entrainment of the Neurospora circadian clock. 18: 195-205.
Yang, Y., P. Cheng, Q. He, L. Wang, and Y. Liu (2003) Phosphorylation of FREQUENCY Protein by Casein Kinase II is Necessary for the Function of the Neurospora Circadian Clock. Mol. Cell. Biol 23:6221-6228
He, Q.*, Cheng, P.*, Y. Yang, Q. He, H. Yu, and Y. Liu. (2003) FWD1-mediated degradation of FREQUENCY in Neurospora establishes a conserved mechanism for circadian clock regulation. EMBO J. 22:4421-4430
Liu, Y., Q. He, and P. Cheng. (2003) Photoreception in Neurospora, A Tale of Two White Collar Proteins. Cell. Mol. Life Sci. 60:2131-2138.
Yang, Y., Q. He, P. Cheng, P. Wrage, Oded Yarden, and Y. Liu. (2004) Distinct roles for PP1 and PP2A in the Neurospora circadian clock. Genes & Development. 18:255-260
Min, H., Y. Liu, C. H. Johnson, and S. S. Golden. (2004) Phase Determination of Circadian Gene Expression in Synechococcus elongatus PCC 7942. J. Biol. Rhythms 19:103-112.
Liu, Y., Y. Yang. and Q. He (2005) Posttranscriptional regulation of the Neurospora circadian clock. Circadian Clocks in Eukaryotic Microbes. Edited by Fred Kippert. Published by Landes Bioscience.
Liu, Y.(2005) Analysis of posttranslational regulations in the Neurospora circadian clock. Methods in Enzymology. Circadian Rhythms, edited by Michael Young. 393:379-393
Cheng, P., Q. He, Q. He, L. Wang, and Y. Liu. (2005) Regulation of the Neurospora circadian clock by an RNA helicase. Genes & Development 19: 234-241.
He Q., H. Shu, P. Cheng, S. Chen, L. Wang, and Y. Liu. (2005) Light-independent phosphorylation of WHITE COLLAR-1 is important for its role in the circadian negative feedback loop. J. Biol. Chem. 280: 17526-17532.
He, Q., Cheng, P., Q. He, and Y. Liu. (2005) The COP9 signalosome regulates the Neurospora circadian clock by controlling the stability of the SCFFWD-1 complex. Genes & Development 19: 1518-1531.
He, Q, and Y. Liu (2005) Degradation of the Neurospora circadian clock protein FREQUENCY through the ubiquitin-proteasome pathway. Biochem Soc Trans. 33:953-956.
He, Q. and Y. Liu (2005) Molecular mechanism of light responses in Neurospora: from light-induced transcription to photoadaptation. Genes & Development 19: 2888-2899.
Huang, G., L. Wang, and Y. Liu (2006) Molecular mechanism of suppression of circadian rhythm by a critical stimulus. EMBO J. 25: 5349-5357.
Liu. Y. and D. Bell-Pedersen (2006) Circadian rhythms in Neurospora and other filamentous fungi. Eukaryotic Cell. 5: 1184-1193.
He, Q., J. Cha, Q. He, H. Lee, Y. Yang, and Y. Liu. (2006) CKI and CKII mediate the FREQUENCY-dependent phosphorylation of the WHITE COLLAR complex to close the Neurospora circadian negative feedback loop. Genes & Dev. 20:2552-2565.
Heintzen, C. and Y. Liu (2007) The Neurospora crassa circadian clock. Adv in Genetics, 58:25-66
Maiti, M., HC. Lee, and Y. Liu (2007) QIP, a putative exonuclease, interacts with the Neurospora Argonaute protein and facilitates conversion of duplex siRNA into single strands. Genes & Dev. 21: 590-600
Choudhary, S.*, HC Lee*, M. Maiti*, Q. He, P. Cheng, Q. Liu, and Y. Liu (2007) A double-stranded RNA response program important for RNAi efficiency. Mol. Cell. Biol. 27:3995-4005. (the cover article).
Guocun Huang, She Chen, Shaojie Li, Joonseok Cha, Chengzu Long, Lily Li, Qiyang He and Yi Liu. (2007) Protein Kinase A and Casein Kinases Mediate Sequential Phosphorylation Events in the Circadian Negative Feedback Loop. Genes & Dev. 21: 3283-3295.
Cha, J., G. Huang, J. Guo, and Y. Liu (2007) Posttranslational control of the Neurospora circadian clock. Cold Spring Harbor Symposia on Quantitative Biology 72: 185-91.
Cha J, Chang SS, Huang G, Cheng P, and Y. Liu (2008) Control of WHITE COLLAR localization by phosphorylation is a critical step in the circadian negative feedback process. EMBO J. 27: 3246-3255.
He Q., Tang C, and Y. Liu (2008) The roles of the ubiquitin-proteasome system in the control of circadian clocks. The Ubiquitin Proteasome System. Edited by Napoli and Wojcik. In press.
Jinhu Guo, Guocun Huang, Joonseok Cha, Yi Liu (2008) Protein analyses of the filamentous fungus Neurospora crassa. Molecular and Cell Biology Methods for Fungi. Edited by Amir Sharon. In press.
Lee HC, Chang SS, Choudhary S, Aalto AP, Maiti M, Bamford DH, Y. Liu. (2009) qiRNA is a new type of small interfering RNA induced by DNA damage. Nature 459(7244):274-277. Cover article.
Chi-Tai Tang, Shaojie Li, Chengzu Long, Joonseok Cha, Guocun Huang, Lily Li, She Chenand Yi Liu (2009) Setting the pace of the Neurospora circadian clock by multiple independent FRQ phosphorylation events. Proc. Natl. Acad. Sci. USA 106(26):10722-7.
Guo J, Cheng P, Yuan H, and Yi Liu (2009) The Exosome Regulates Circadian Gene Expression in a Posttranscriptional Negative Feedback Loop. Cell, 138:1236-1246.