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Transcriptional interference by antisense RNA is required for circadian clock function.

Xue Z, Ye Q, Anson SR, Yang J, Xiao G, Kowbel D, Glass NL, Crosthwaite SK, Liu Y - Nature (2014)

Bottom Line: Natural antisense RNAs are found in a wide range of eukaryotic organisms.Moreover, our results suggest that antisense transcription inhibits sense expression by mediating chromatin modifications and premature termination of transcription.Taken together, our results establish antisense transcription as an essential feature in a circadian system and shed light on the importance and mechanism of antisense action.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, The University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, Texas 75390, USA.

ABSTRACT
Eukaryotic circadian oscillators consist of negative feedback loops that generate endogenous rhythmicities. Natural antisense RNAs are found in a wide range of eukaryotic organisms. Nevertheless, the physiological importance and mode of action of most antisense RNAs are not clear. frequency (frq) encodes a component of the Neurospora core circadian negative feedback loop, which was thought to generate sustained rhythmicity. Transcription of qrf, the long non-coding frq antisense RNA, is induced by light, and its level oscillates in antiphase to frq sense RNA. Here we show that qrf transcription is regulated by both light-dependent and light-independent mechanisms. Light-dependent qrf transcription represses frq expression and regulates clock resetting. Light-independent qrf expression, on the other hand, is required for circadian rhythmicity. frq transcription also inhibits qrf expression and drives the antiphasic rhythm of qrf transcripts. The mutual inhibition of frq and qrf transcription thus forms a double negative feedback loop that is interlocked with the core feedback loop. Genetic and mathematical modelling analyses indicate that such an arrangement is required for robust and sustained circadian rhythmicity. Moreover, our results suggest that antisense transcription inhibits sense expression by mediating chromatin modifications and premature termination of transcription. Taken together, our results establish antisense transcription as an essential feature in a circadian system and shed light on the importance and mechanism of antisense action.

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qrf expression is required for circadian rhythmicities. (a) Strand-specific RT-qPCR results showing the expression levels of qrf in the indicated knock-in strains at DD24. (b) Race tube analyses of the frq10;frqWT and frq10;frq.aq strains in medium containing 0% glucose, 0.17% arginine with the indicated concentrations of QA in DD. The lack of glucose in medium is known to allow more efficient expression from the qa-2 promoter. The black lines on race tubes indicate the daily growth fronts. (c) The unnormalized luciferase activity of the experiments in Figure 2c. (d) The normalized result of Figure 2c in 10× scale showing fluctuation of the luciferase activity in the frq10;frq.aq strain is random and not rhythmic in the frq10;frq.aq strain. (e) A table showing the phases of the first conidiation band in DD of the race tube results shown in Figure 2b. (f) Western blot analysis showing the FRQ expression profiles in the frq10;frq.aq strain with/without QA in DD at the indicated time points. (g) Northern blot analysis showing frq expression profiles in the frq10;frq.aq strain in DD at the indicated time points. The densitometric analysis is shown below.
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Figure 2: qrf expression is required for circadian rhythmicities. (a) Strand-specific RT-qPCR results showing the expression levels of qrf in the indicated knock-in strains at DD24. (b) Race tube analyses of the frq10;frqWT and frq10;frq.aq strains in medium containing 0% glucose, 0.17% arginine with the indicated concentrations of QA in DD. The lack of glucose in medium is known to allow more efficient expression from the qa-2 promoter. The black lines on race tubes indicate the daily growth fronts. (c) The unnormalized luciferase activity of the experiments in Figure 2c. (d) The normalized result of Figure 2c in 10× scale showing fluctuation of the luciferase activity in the frq10;frq.aq strain is random and not rhythmic in the frq10;frq.aq strain. (e) A table showing the phases of the first conidiation band in DD of the race tube results shown in Figure 2b. (f) Western blot analysis showing the FRQ expression profiles in the frq10;frq.aq strain with/without QA in DD at the indicated time points. (g) Northern blot analysis showing frq expression profiles in the frq10;frq.aq strain in DD at the indicated time points. The densitometric analysis is shown below.

Mentions: Similar levels of qrf transcripts seen in the frqKI(WT) and frqKI(qLRE mut) strains at DD24 (Extended Data Figure 2a) indicate that qLRE does not regulate qrf expression in DD. In a strain (frq10;frq.aq) in which the promoter of qrf was replaced with the quinic acid (QA)-inducible qa-2 promoter, qrf expression was abolished in the absence of QA (Figure 2a). On addition of QA, qrf was induced but frq levels were significantly reduced, further indicating repression of frq by qrf.


Transcriptional interference by antisense RNA is required for circadian clock function.

Xue Z, Ye Q, Anson SR, Yang J, Xiao G, Kowbel D, Glass NL, Crosthwaite SK, Liu Y - Nature (2014)

qrf expression is required for circadian rhythmicities. (a) Strand-specific RT-qPCR results showing the expression levels of qrf in the indicated knock-in strains at DD24. (b) Race tube analyses of the frq10;frqWT and frq10;frq.aq strains in medium containing 0% glucose, 0.17% arginine with the indicated concentrations of QA in DD. The lack of glucose in medium is known to allow more efficient expression from the qa-2 promoter. The black lines on race tubes indicate the daily growth fronts. (c) The unnormalized luciferase activity of the experiments in Figure 2c. (d) The normalized result of Figure 2c in 10× scale showing fluctuation of the luciferase activity in the frq10;frq.aq strain is random and not rhythmic in the frq10;frq.aq strain. (e) A table showing the phases of the first conidiation band in DD of the race tube results shown in Figure 2b. (f) Western blot analysis showing the FRQ expression profiles in the frq10;frq.aq strain with/without QA in DD at the indicated time points. (g) Northern blot analysis showing frq expression profiles in the frq10;frq.aq strain in DD at the indicated time points. The densitometric analysis is shown below.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4214883&req=5

Figure 2: qrf expression is required for circadian rhythmicities. (a) Strand-specific RT-qPCR results showing the expression levels of qrf in the indicated knock-in strains at DD24. (b) Race tube analyses of the frq10;frqWT and frq10;frq.aq strains in medium containing 0% glucose, 0.17% arginine with the indicated concentrations of QA in DD. The lack of glucose in medium is known to allow more efficient expression from the qa-2 promoter. The black lines on race tubes indicate the daily growth fronts. (c) The unnormalized luciferase activity of the experiments in Figure 2c. (d) The normalized result of Figure 2c in 10× scale showing fluctuation of the luciferase activity in the frq10;frq.aq strain is random and not rhythmic in the frq10;frq.aq strain. (e) A table showing the phases of the first conidiation band in DD of the race tube results shown in Figure 2b. (f) Western blot analysis showing the FRQ expression profiles in the frq10;frq.aq strain with/without QA in DD at the indicated time points. (g) Northern blot analysis showing frq expression profiles in the frq10;frq.aq strain in DD at the indicated time points. The densitometric analysis is shown below.
Mentions: Similar levels of qrf transcripts seen in the frqKI(WT) and frqKI(qLRE mut) strains at DD24 (Extended Data Figure 2a) indicate that qLRE does not regulate qrf expression in DD. In a strain (frq10;frq.aq) in which the promoter of qrf was replaced with the quinic acid (QA)-inducible qa-2 promoter, qrf expression was abolished in the absence of QA (Figure 2a). On addition of QA, qrf was induced but frq levels were significantly reduced, further indicating repression of frq by qrf.

Bottom Line: Natural antisense RNAs are found in a wide range of eukaryotic organisms.Moreover, our results suggest that antisense transcription inhibits sense expression by mediating chromatin modifications and premature termination of transcription.Taken together, our results establish antisense transcription as an essential feature in a circadian system and shed light on the importance and mechanism of antisense action.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, The University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, Texas 75390, USA.

ABSTRACT
Eukaryotic circadian oscillators consist of negative feedback loops that generate endogenous rhythmicities. Natural antisense RNAs are found in a wide range of eukaryotic organisms. Nevertheless, the physiological importance and mode of action of most antisense RNAs are not clear. frequency (frq) encodes a component of the Neurospora core circadian negative feedback loop, which was thought to generate sustained rhythmicity. Transcription of qrf, the long non-coding frq antisense RNA, is induced by light, and its level oscillates in antiphase to frq sense RNA. Here we show that qrf transcription is regulated by both light-dependent and light-independent mechanisms. Light-dependent qrf transcription represses frq expression and regulates clock resetting. Light-independent qrf expression, on the other hand, is required for circadian rhythmicity. frq transcription also inhibits qrf expression and drives the antiphasic rhythm of qrf transcripts. The mutual inhibition of frq and qrf transcription thus forms a double negative feedback loop that is interlocked with the core feedback loop. Genetic and mathematical modelling analyses indicate that such an arrangement is required for robust and sustained circadian rhythmicity. Moreover, our results suggest that antisense transcription inhibits sense expression by mediating chromatin modifications and premature termination of transcription. Taken together, our results establish antisense transcription as an essential feature in a circadian system and shed light on the importance and mechanism of antisense action.

Show MeSH
Related in: MedlinePlus