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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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Mutual inhibition of frq and qrf transcription forms a double negative feedback loop that is required for clock function. (a) Strand-specific RT-qPCR results showing the oscillations of frq and qrf in DD. Error bars are SD (n=3). (b) Luciferase reporter assay showing the normalized frq or qrf promoter activity after one day in DD in wild-type strains with the Pfrq:luc or Pqrf:luc construct. (c) Strand-specific RT-qPCR results showing the levels of frq and qrf in the frq10;frqqLRE mut strain at DD24 or 60 min after the dark to light transfer. (d) Levels of frq and qrf in the wild-type and wccDKO strains measured by strand-specific RT-qPCR in DD. (e) Luciferase reporter assays showing the normalized luminescence levels in a wild-type strain that carries the Pfrq.luc (black) or Pmini.luc.Pfrq (red) construct. (f) A model of the Neurospora oscillator. (g) Mathematical simulation of relative frq levels in DD without qrf (k19 =0) (left), with qrf (k16=0.5, k17=0.35, k19=0.1) (middle), and with qrf over-expression (k16 =0.91) (right).
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Figure 13: Mutual inhibition of frq and qrf transcription forms a double negative feedback loop that is required for clock function. (a) Strand-specific RT-qPCR results showing the oscillations of frq and qrf in DD. Error bars are SD (n=3). (b) Luciferase reporter assay showing the normalized frq or qrf promoter activity after one day in DD in wild-type strains with the Pfrq:luc or Pqrf:luc construct. (c) Strand-specific RT-qPCR results showing the levels of frq and qrf in the frq10;frqqLRE mut strain at DD24 or 60 min after the dark to light transfer. (d) Levels of frq and qrf in the wild-type and wccDKO strains measured by strand-specific RT-qPCR in DD. (e) Luciferase reporter assays showing the normalized luminescence levels in a wild-type strain that carries the Pfrq.luc (black) or Pmini.luc.Pfrq (red) construct. (f) A model of the Neurospora oscillator. (g) Mathematical simulation of relative frq levels in DD without qrf (k19 =0) (left), with qrf (k16=0.5, k17=0.35, k19=0.1) (middle), and with qrf over-expression (k16 =0.91) (right).

Mentions: qrf RNA oscillates in DD in the wild-type in antiphase to frq (Figure 3a)3 but WC-2 does not bind to the qrf promoter in DD (Extended Data Figure 3a). Moreover, the qLRE mutation did not affect either frq or qrf levels in DD (Extended Data Figure 3b), indicating that the WC complex does not regulate qrf transcription in DD. Crucially, a luciferase reporter (Pqrf-luc) driven by the qrf promoter showed that the qrf promoter activity is not rhythmic in a wild-type strain (Figure 3b and Extended Data Figure 3c-d).


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)

Mutual inhibition of frq and qrf transcription forms a double negative feedback loop that is required for clock function. (a) Strand-specific RT-qPCR results showing the oscillations of frq and qrf in DD. Error bars are SD (n=3). (b) Luciferase reporter assay showing the normalized frq or qrf promoter activity after one day in DD in wild-type strains with the Pfrq:luc or Pqrf:luc construct. (c) Strand-specific RT-qPCR results showing the levels of frq and qrf in the frq10;frqqLRE mut strain at DD24 or 60 min after the dark to light transfer. (d) Levels of frq and qrf in the wild-type and wccDKO strains measured by strand-specific RT-qPCR in DD. (e) Luciferase reporter assays showing the normalized luminescence levels in a wild-type strain that carries the Pfrq.luc (black) or Pmini.luc.Pfrq (red) construct. (f) A model of the Neurospora oscillator. (g) Mathematical simulation of relative frq levels in DD without qrf (k19 =0) (left), with qrf (k16=0.5, k17=0.35, k19=0.1) (middle), and with qrf over-expression (k16 =0.91) (right).
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Related In: Results  -  Collection

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

Figure 13: Mutual inhibition of frq and qrf transcription forms a double negative feedback loop that is required for clock function. (a) Strand-specific RT-qPCR results showing the oscillations of frq and qrf in DD. Error bars are SD (n=3). (b) Luciferase reporter assay showing the normalized frq or qrf promoter activity after one day in DD in wild-type strains with the Pfrq:luc or Pqrf:luc construct. (c) Strand-specific RT-qPCR results showing the levels of frq and qrf in the frq10;frqqLRE mut strain at DD24 or 60 min after the dark to light transfer. (d) Levels of frq and qrf in the wild-type and wccDKO strains measured by strand-specific RT-qPCR in DD. (e) Luciferase reporter assays showing the normalized luminescence levels in a wild-type strain that carries the Pfrq.luc (black) or Pmini.luc.Pfrq (red) construct. (f) A model of the Neurospora oscillator. (g) Mathematical simulation of relative frq levels in DD without qrf (k19 =0) (left), with qrf (k16=0.5, k17=0.35, k19=0.1) (middle), and with qrf over-expression (k16 =0.91) (right).
Mentions: qrf RNA oscillates in DD in the wild-type in antiphase to frq (Figure 3a)3 but WC-2 does not bind to the qrf promoter in DD (Extended Data Figure 3a). Moreover, the qLRE mutation did not affect either frq or qrf levels in DD (Extended Data Figure 3b), indicating that the WC complex does not regulate qrf transcription in DD. Crucially, a luciferase reporter (Pqrf-luc) driven by the qrf promoter showed that the qrf promoter activity is not rhythmic in a wild-type strain (Figure 3b and Extended Data Figure 3c-d).

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