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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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(a) The unnormalized luciferase activity of the experiments in Figure 3e. (b) The unnormalized luciferase activity of the wild-type strain carrying the Pmin-luc construct. Results of two independent transformants were shown. (c) Mathematical modeling of the Neurospora circadian oscillator with the double negative feedback loop. The differential equations used in the model are shown. The model is identical to a previously developed model23 with the exception of equation 1, which in this case includes the inhibition of frq transcription by qrf, and the equation 8, which includes the inhibition of qrf transcription by frq. The rate constants used in the simulations were listed below.
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Figure 4: (a) The unnormalized luciferase activity of the experiments in Figure 3e. (b) The unnormalized luciferase activity of the wild-type strain carrying the Pmin-luc construct. Results of two independent transformants were shown. (c) Mathematical modeling of the Neurospora circadian oscillator with the double negative feedback loop. The differential equations used in the model are shown. The model is identical to a previously developed model23 with the exception of equation 1, which in this case includes the inhibition of frq transcription by qrf, and the equation 8, which includes the inhibition of qrf transcription by frq. The rate constants used in the simulations were listed below.

Mentions: To further investigate the regulation bewteen frq and qrf, we created a luciferase reporter construct (Pmin-luc-Pfrq), in which the luciferase sense mRNA is driven by a constitutive promoter17 and antisense luciferase mRNA is driven by the frq promoter (Figure 3e). Wild-type strains containing the Pfrq-luc or Pmin-luc (lacking antisense luciferase RNA) construct were used as controls. Luminescence in the Pmin-luc strain was arrhythmic but the Pmin-luc-Pfrq strain exhibited a robust circadian luminescence rhythm antiphase to that of the Pfrq-luc rhythm (Figure 3e and Extended Data Figure 4a-b). These results indicate that the antiphasic rhythm of qrf expression is driven by rhythmic frq transcription independent of RNA sequence. Therefore, frq and qrf transcription forms a double negative feedback loop that results in antiphasic rhythms of frq and qrf (Figure 3f).


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)

(a) The unnormalized luciferase activity of the experiments in Figure 3e. (b) The unnormalized luciferase activity of the wild-type strain carrying the Pmin-luc construct. Results of two independent transformants were shown. (c) Mathematical modeling of the Neurospora circadian oscillator with the double negative feedback loop. The differential equations used in the model are shown. The model is identical to a previously developed model23 with the exception of equation 1, which in this case includes the inhibition of frq transcription by qrf, and the equation 8, which includes the inhibition of qrf transcription by frq. The rate constants used in the simulations were listed below.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 4: (a) The unnormalized luciferase activity of the experiments in Figure 3e. (b) The unnormalized luciferase activity of the wild-type strain carrying the Pmin-luc construct. Results of two independent transformants were shown. (c) Mathematical modeling of the Neurospora circadian oscillator with the double negative feedback loop. The differential equations used in the model are shown. The model is identical to a previously developed model23 with the exception of equation 1, which in this case includes the inhibition of frq transcription by qrf, and the equation 8, which includes the inhibition of qrf transcription by frq. The rate constants used in the simulations were listed below.
Mentions: To further investigate the regulation bewteen frq and qrf, we created a luciferase reporter construct (Pmin-luc-Pfrq), in which the luciferase sense mRNA is driven by a constitutive promoter17 and antisense luciferase mRNA is driven by the frq promoter (Figure 3e). Wild-type strains containing the Pfrq-luc or Pmin-luc (lacking antisense luciferase RNA) construct were used as controls. Luminescence in the Pmin-luc strain was arrhythmic but the Pmin-luc-Pfrq strain exhibited a robust circadian luminescence rhythm antiphase to that of the Pfrq-luc rhythm (Figure 3e and Extended Data Figure 4a-b). These results indicate that the antiphasic rhythm of qrf expression is driven by rhythmic frq transcription independent of RNA sequence. Therefore, frq and qrf transcription forms a double negative feedback loop that results in antiphasic rhythms of frq and qrf (Figure 3f).

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