A Double-Stranded-RNA Response Program Important for RNA Interference Efficiency

Choudhary, Swati; Lee, Heng-Chi; Maiti, Mekhala; He, Qun; Cheng, Ping; Liu, Qinghua; Liu, Yi

doi:10.1128/mcb.00186-07

Cited by 73 publications

(80 citation statements)

References 62 publications

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“…RNA Analyses-Total RNA was acquired and quantitative real time PCR experiments were performed as described previously (36,37). For these experiments, the ␤-tubulin gene was used as a control.…”

Section: Methodsmentioning

confidence: 99%

Regulation of the Activity and Cellular Localization of the Circadian Clock Protein FRQ

Cha

Yuan

Liu

2011

Journal of Biological Chemistry

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Eukaryotic circadian clocks employ autoregulatory negative feedback loops to control daily rhythms. In the filamentous fungus Neurospora, FRQ, FRH, WC-1, and WC-2 are the core components of the circadian negative feedback loop. To close the transcription-based negative feedback loop, the FRQ-FRH complex inhibits the activity of the WC complex in the nucleus by promoting the casein kinases-mediated WC phosphorylation. Despite its essential role in the nucleus, most FRQ is found in the cytoplasm. In this study, we mapped the FRQ regions that are important for its cellular localization. We show that the C-terminal part of FRQ, particularly the FRQ-FRH interaction domain, plays a major role in controlling FRQ localization. Both the mutation of the FRQ-FRH interaction domain and the down-regulation of FRH result in the nuclear enrichment of FRQ, suggesting that FRH regulates FRQ localization via a physical interaction. To study the role of FRQ phosphorylation, we examined the FRQ localization in wild-type as well as an array of FRQ kinase, FRQ phosphatase, and FRQ phosphorylation site mutants. Collectively, our results suggest that FRQ phosphorylation does not play a significant role in regulating its cellular localization. Instead, we find that phosphorylation of FRQ inhibits its transcriptional repressor activity in the circadian negative feedback loop. Such an effect is achieved by inhibiting the ability of FRQ to interact with WCC and casein kinase 1a. Our results indicate that the rhythmic FRQ phosphorylation profile observed is an important part of the negative feedback mechanism that drives robust circadian gene expression.The eukaryotic circadian oscillators are comprised of autoregulatory negative feedback loops (1-5). Despite the evolutionary distance between the filamentous fungus Neurospora crassa and higher eukaryotes, their circadian oscillator mechanisms share remarkable similarities (6 -8). In N. crassa, two PAS (PER-ARNT-SIM) domain-containing transcription factors, WC-1 and WC-2, form a complex (WCC) 2 that activates the transcription of the frq gene by binding to its promoter (9 -13). FRQ protein binds FRH to form the FRQ-FRH complex (FFC), which acts as the negative element in the circadian negative feedback loop (14 -17). To close the circadian negative feedback loop, FFC decreases frq mRNA-levels by promoting frq mRNA degradation and by inhibiting frq transcription. To repress frq transcription, FFC inhibits WCC activity by recruiting casein kinase 1a (CK-1a) and CKII to phosphorylate the WC proteins, resulting in a decrease of WCC DNA binding activity and an increase of WCC nuclear export (9, 10, 14 -24). Like the animal PER proteins, FRQ is progressively phosphorylated upon its synthesis and becomes extensively phosphorylated before its disappearance, resulting in robust oscillations of its level and phosphorylation profile (25). Under normal physiological conditions, FRQ is phosphorylated by CK-1a, CKII, and PKA (21,22,(25)(26)(27)(28). On the other hand, FRQ is also dephosphorylated and st...

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Section: Methodsmentioning

confidence: 99%

Regulation of the Activity and Cellular Localization of the Circadian Clock Protein FRQ

Cha

Yuan

Liu

2011

Journal of Biological Chemistry

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“…Previously, we demonstrated that DNA damage agents, such as histidine, HU, or ethyl methanesulfonate, are able to induce the production of qiRNA and QDE-2 protein expression in Neurospora crassa (12,27). In addition, DNA damage-induced QDE-2 expression is abolished in the mutants that are defective in quelling and qiRNA pathways (12).…”

Section: A Genetic Screen Identified Rtt109 To Be Essential For the Dmentioning

confidence: 97%

Histone H3K56 Acetylation Is Required for Quelling-induced Small RNA Production through Its Role in Homologous Recombination

Zhang

Yang

Sun

et al. 2014

Journal of Biological Chemistry

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Background: Quelling and DNA damage induce small RNA from repetitive DNA regions. Results: Genetic Screen identified RTT109 as a critical component of the siRNA production pathway in Neurospora. Conclusion: RTT109 acts as a histone H3K56 acetyltransferase to mediate siRNA biogenesis through its role in homologous recombination. Significance: This study identifies a new link between DNA damage response and small RNA production at the chromatin level.

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“…dsal-1, wild-type (301-6, his-3), qde-1 ko (his-3), qde-2 ko (his-3), and dicer dko (his-3) strains were generated previously (Choudhary et al 2007;Maiti et al 2007). The qde-1 ko Ku80 ko his-3 strain was generated in this study by crossing qde-1 ko and Ku80 ko his-3 strains.…”

Section: Strains Growth Conditions and Constructsmentioning

confidence: 99%

Mechanism of siRNA production from repetitive DNA

Yang

Liu

2015

Genes Dev.

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RNAi is a conserved genome defense mechanism in eukaryotes that protects against deleterious effects of transposons and viral invasion. Repetitive DNA loci are a major source for the production of eukaryotic small RNAs, but how these small RNAs are produced is not clear. Quelling in Neurospora is one of the first known RNAi-related phenomena and is triggered by the presence of multiple copies of transgenes. Here we showed that DNA tandem repeats and double-strand breaks are necessary and, when both are present, sufficient to trigger gene silencing and siRNA production. Introduction of a site-specific double-strand break or DNA fragile site resulted in homologous recombination of repetitive sequences, which is required for gene silencing. In addition to siRNA production, the quelling pathway also maintains tandem repeats by regulating homologous recombination. Our study identified the mechanistic trigger for siRNA production from repetitive DNA and established a role for siRNA in maintaining genome stability.

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A Double-Stranded-RNA Response Program Important for RNA Interference Efficiency

Cited by 73 publications

References 62 publications

Regulation of the Activity and Cellular Localization of the Circadian Clock Protein FRQ

Regulation of the Activity and Cellular Localization of the Circadian Clock Protein FRQ

Histone H3K56 Acetylation Is Required for Quelling-induced Small RNA Production through Its Role in Homologous Recombination

Mechanism of siRNA production from repetitive DNA

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