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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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Light-induced qrf expression represses frq transcription and regulates light resetting of the clock. (a) Strand-specific RNA-Seq result of the frq locus showing the overlapping frq and qrf transcripts. (b) Sequence alignment of known LRE elements. The qLRE and the mutated regions in the qrf promoter are shown. (c) Diagrams showing the chromosomal modifications in the indicated loci in the frq10;frqWT and frq10;frqqLRE mut strains. C, Cla I; B, Bgl II; E, EcoR V. (d) Strand-specific RT-qPCR results showing the expression levels of frq and qrf in indicated strains in LL. Error bars are standard deviations (n=3). Asterisks indicate P value < 0.01. n.s. indicates that the difference is not statistically significant. frq-RT and qrf –RT represent the non-RT reaction control. (e) Diagram describes the strategy used to obtain the knock-in strains by homologous recombination. (f) Strand-specific RT-qPCR results showing the expression levels of frq and qrf in the indicated knock-in strains in LL. (g) Western blot results showing the FRQ expression levels in the indicated strains in LL. The densitometric analysis of western blot results from three independent experiments is shown at right. Error bars are standard deviations. Asterisks indicate P value < 0.01. (h) Phase response curves of circadian conidiation rhythms of the indicated knock-in strains after 2 min of light pulse at different circadian time (CT) points.
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Figure 1: Light-induced qrf expression represses frq transcription and regulates light resetting of the clock. (a) Strand-specific RNA-Seq result of the frq locus showing the overlapping frq and qrf transcripts. (b) Sequence alignment of known LRE elements. The qLRE and the mutated regions in the qrf promoter are shown. (c) Diagrams showing the chromosomal modifications in the indicated loci in the frq10;frqWT and frq10;frqqLRE mut strains. C, Cla I; B, Bgl II; E, EcoR V. (d) Strand-specific RT-qPCR results showing the expression levels of frq and qrf in indicated strains in LL. Error bars are standard deviations (n=3). Asterisks indicate P value < 0.01. n.s. indicates that the difference is not statistically significant. frq-RT and qrf –RT represent the non-RT reaction control. (e) Diagram describes the strategy used to obtain the knock-in strains by homologous recombination. (f) Strand-specific RT-qPCR results showing the expression levels of frq and qrf in the indicated knock-in strains in LL. (g) Western blot results showing the FRQ expression levels in the indicated strains in LL. The densitometric analysis of western blot results from three independent experiments is shown at right. Error bars are standard deviations. Asterisks indicate P value < 0.01. (h) Phase response curves of circadian conidiation rhythms of the indicated knock-in strains after 2 min of light pulse at different circadian time (CT) points.

Mentions: The transcription factors WHITE COLLAR (WC) -1 and -2 form a complex that activates frq transcription in the dark (DD) and mediates light-induced frq transcription for light-resetting of the clock by binding to light-responsive elements (LREs) on the frq promoter11-13. 3′ RACE and RNA sequencing showed that frq and qrf transcripts almost completely overlap (Figure 1a and Extended Figure 1a). In the wc mutants, frq expression was nearly abolished but qrf transcript was observed at ∼25% of wild-type levels (Figure 1b), indicating that both WC-dependent and -independent mechanisms mediate qrf transcription. WC complex binds to the qrf promoter region14. frq constructs with point mutations in each of the five putative binding sites in the qrf promoter were individually introduced into a frq10 (frq and qrf ) strain15. Mutation of only one site, qLRE, dramatically reduced qrf level (Figure 1c and Extended Data Figure 1b-d). In the frqqLREmut strain, the qrf level was comparable to that of the wc mutant. In a qLRE knock-in strain (frqKI(qLREmut), Extended Data Figure 1e), qrf levels were also much lower than in the control knock-in strain and WC binding at the qrf promoter was completely abolished (Figure 1c-d and Extended Data Figure 1f), indicating that qLRE is the only WC binding site in the qrf promoter.


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)

Light-induced qrf expression represses frq transcription and regulates light resetting of the clock. (a) Strand-specific RNA-Seq result of the frq locus showing the overlapping frq and qrf transcripts. (b) Sequence alignment of known LRE elements. The qLRE and the mutated regions in the qrf promoter are shown. (c) Diagrams showing the chromosomal modifications in the indicated loci in the frq10;frqWT and frq10;frqqLRE mut strains. C, Cla I; B, Bgl II; E, EcoR V. (d) Strand-specific RT-qPCR results showing the expression levels of frq and qrf in indicated strains in LL. Error bars are standard deviations (n=3). Asterisks indicate P value < 0.01. n.s. indicates that the difference is not statistically significant. frq-RT and qrf –RT represent the non-RT reaction control. (e) Diagram describes the strategy used to obtain the knock-in strains by homologous recombination. (f) Strand-specific RT-qPCR results showing the expression levels of frq and qrf in the indicated knock-in strains in LL. (g) Western blot results showing the FRQ expression levels in the indicated strains in LL. The densitometric analysis of western blot results from three independent experiments is shown at right. Error bars are standard deviations. Asterisks indicate P value < 0.01. (h) Phase response curves of circadian conidiation rhythms of the indicated knock-in strains after 2 min of light pulse at different circadian time (CT) points.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4214883&req=5

Figure 1: Light-induced qrf expression represses frq transcription and regulates light resetting of the clock. (a) Strand-specific RNA-Seq result of the frq locus showing the overlapping frq and qrf transcripts. (b) Sequence alignment of known LRE elements. The qLRE and the mutated regions in the qrf promoter are shown. (c) Diagrams showing the chromosomal modifications in the indicated loci in the frq10;frqWT and frq10;frqqLRE mut strains. C, Cla I; B, Bgl II; E, EcoR V. (d) Strand-specific RT-qPCR results showing the expression levels of frq and qrf in indicated strains in LL. Error bars are standard deviations (n=3). Asterisks indicate P value < 0.01. n.s. indicates that the difference is not statistically significant. frq-RT and qrf –RT represent the non-RT reaction control. (e) Diagram describes the strategy used to obtain the knock-in strains by homologous recombination. (f) Strand-specific RT-qPCR results showing the expression levels of frq and qrf in the indicated knock-in strains in LL. (g) Western blot results showing the FRQ expression levels in the indicated strains in LL. The densitometric analysis of western blot results from three independent experiments is shown at right. Error bars are standard deviations. Asterisks indicate P value < 0.01. (h) Phase response curves of circadian conidiation rhythms of the indicated knock-in strains after 2 min of light pulse at different circadian time (CT) points.
Mentions: The transcription factors WHITE COLLAR (WC) -1 and -2 form a complex that activates frq transcription in the dark (DD) and mediates light-induced frq transcription for light-resetting of the clock by binding to light-responsive elements (LREs) on the frq promoter11-13. 3′ RACE and RNA sequencing showed that frq and qrf transcripts almost completely overlap (Figure 1a and Extended Figure 1a). In the wc mutants, frq expression was nearly abolished but qrf transcript was observed at ∼25% of wild-type levels (Figure 1b), indicating that both WC-dependent and -independent mechanisms mediate qrf transcription. WC complex binds to the qrf promoter region14. frq constructs with point mutations in each of the five putative binding sites in the qrf promoter were individually introduced into a frq10 (frq and qrf ) strain15. Mutation of only one site, qLRE, dramatically reduced qrf level (Figure 1c and Extended Data Figure 1b-d). In the frqqLREmut strain, the qrf level was comparable to that of the wc mutant. In a qLRE knock-in strain (frqKI(qLREmut), Extended Data Figure 1e), qrf levels were also much lower than in the control knock-in strain and WC binding at the qrf promoter was completely abolished (Figure 1c-d and Extended Data Figure 1f), indicating that qLRE is the only WC binding site in the qrf promoter.

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