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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) A diagram showing the chromosomal modifications in the frqqLRE mut;qrf strains that allow the expression of qrf in trans. The frqqLRE mut construct is at the his-3 locus, and qrf is expressed only from the csr-1 locus. The red dashed line indicates that the frq promoter region is deleted in the qrf construct to abolish frq expression. (b) Strand-specific RT-qPCR results showing that only qrf is expressed from the qrf construct in the frq10; qrf strain. (c) qrf expression does not repress frq transcription in trans. Strand-specific RT-qPCR results showing the levels of frq and qrf transcripts in the indicated strains in LL. Error bars are standard deviations. Asterisk indicates P value < 0.05 (n=3). (d) Northern blot results showing that expression of qrf in trans in the frq10;frqqLRE mut;qrf strains does not repress frq expression. Densitometric analysis of the northern blot results is shown at right. (e-f) WC ChIP assays showing the relative WC binding levels at the frq promoter in LL in the indicated strains. The wc double mutant (wccDKO) was used as a negative control for ChIP. n.s. indicates the lack of statistically significance (n=3). (g) Strand-specific RT-qPCR results showing that the mutation of the qLRE element in the qrf promoter results in significant increases in light-induced frq pre-mRNA expression. (h) Northern blot results showing that the stability of frq mRNA is not affected by the transcription of qrf. The frq10;frq.aq strain that can induce qrf expression in the presence of QA was used. Thiolutin, a transcription inhibitor30, was added in the culture to block frq transcription so that frq mRNA stability could be determined. Cultures were harvested at the indicated time points after the addition of thiolutin.
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Figure 6: (a) A diagram showing the chromosomal modifications in the frqqLRE mut;qrf strains that allow the expression of qrf in trans. The frqqLRE mut construct is at the his-3 locus, and qrf is expressed only from the csr-1 locus. The red dashed line indicates that the frq promoter region is deleted in the qrf construct to abolish frq expression. (b) Strand-specific RT-qPCR results showing that only qrf is expressed from the qrf construct in the frq10; qrf strain. (c) qrf expression does not repress frq transcription in trans. Strand-specific RT-qPCR results showing the levels of frq and qrf transcripts in the indicated strains in LL. Error bars are standard deviations. Asterisk indicates P value < 0.05 (n=3). (d) Northern blot results showing that expression of qrf in trans in the frq10;frqqLRE mut;qrf strains does not repress frq expression. Densitometric analysis of the northern blot results is shown at right. (e-f) WC ChIP assays showing the relative WC binding levels at the frq promoter in LL in the indicated strains. The wc double mutant (wccDKO) was used as a negative control for ChIP. n.s. indicates the lack of statistically significance (n=3). (g) Strand-specific RT-qPCR results showing that the mutation of the qLRE element in the qrf promoter results in significant increases in light-induced frq pre-mRNA expression. (h) Northern blot results showing that the stability of frq mRNA is not affected by the transcription of qrf. The frq10;frq.aq strain that can induce qrf expression in the presence of QA was used. Thiolutin, a transcription inhibitor30, was added in the culture to block frq transcription so that frq mRNA stability could be determined. Cultures were harvested at the indicated time points after the addition of thiolutin.

Mentions: We introduced a frq construct (qrf) with the frq promoter deleted into the frq10 and frq10;frqqLRE mut strains at the csr-1 locus (Extended Data Figure 6a). This transgene can express normal levels of qrf without detectable frq expression (Extended Data Figure 6b). In the frq10;frqqLRE mut;qrf strains, even though qrf expression was restored to normal levels, frq levels were not rescued (Extended Data Figure 6c-d), indicating that qrf regulates frq in cis.


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) A diagram showing the chromosomal modifications in the frqqLRE mut;qrf strains that allow the expression of qrf in trans. The frqqLRE mut construct is at the his-3 locus, and qrf is expressed only from the csr-1 locus. The red dashed line indicates that the frq promoter region is deleted in the qrf construct to abolish frq expression. (b) Strand-specific RT-qPCR results showing that only qrf is expressed from the qrf construct in the frq10; qrf strain. (c) qrf expression does not repress frq transcription in trans. Strand-specific RT-qPCR results showing the levels of frq and qrf transcripts in the indicated strains in LL. Error bars are standard deviations. Asterisk indicates P value < 0.05 (n=3). (d) Northern blot results showing that expression of qrf in trans in the frq10;frqqLRE mut;qrf strains does not repress frq expression. Densitometric analysis of the northern blot results is shown at right. (e-f) WC ChIP assays showing the relative WC binding levels at the frq promoter in LL in the indicated strains. The wc double mutant (wccDKO) was used as a negative control for ChIP. n.s. indicates the lack of statistically significance (n=3). (g) Strand-specific RT-qPCR results showing that the mutation of the qLRE element in the qrf promoter results in significant increases in light-induced frq pre-mRNA expression. (h) Northern blot results showing that the stability of frq mRNA is not affected by the transcription of qrf. The frq10;frq.aq strain that can induce qrf expression in the presence of QA was used. Thiolutin, a transcription inhibitor30, was added in the culture to block frq transcription so that frq mRNA stability could be determined. Cultures were harvested at the indicated time points after the addition of thiolutin.
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Figure 6: (a) A diagram showing the chromosomal modifications in the frqqLRE mut;qrf strains that allow the expression of qrf in trans. The frqqLRE mut construct is at the his-3 locus, and qrf is expressed only from the csr-1 locus. The red dashed line indicates that the frq promoter region is deleted in the qrf construct to abolish frq expression. (b) Strand-specific RT-qPCR results showing that only qrf is expressed from the qrf construct in the frq10; qrf strain. (c) qrf expression does not repress frq transcription in trans. Strand-specific RT-qPCR results showing the levels of frq and qrf transcripts in the indicated strains in LL. Error bars are standard deviations. Asterisk indicates P value < 0.05 (n=3). (d) Northern blot results showing that expression of qrf in trans in the frq10;frqqLRE mut;qrf strains does not repress frq expression. Densitometric analysis of the northern blot results is shown at right. (e-f) WC ChIP assays showing the relative WC binding levels at the frq promoter in LL in the indicated strains. The wc double mutant (wccDKO) was used as a negative control for ChIP. n.s. indicates the lack of statistically significance (n=3). (g) Strand-specific RT-qPCR results showing that the mutation of the qLRE element in the qrf promoter results in significant increases in light-induced frq pre-mRNA expression. (h) Northern blot results showing that the stability of frq mRNA is not affected by the transcription of qrf. The frq10;frq.aq strain that can induce qrf expression in the presence of QA was used. Thiolutin, a transcription inhibitor30, was added in the culture to block frq transcription so that frq mRNA stability could be determined. Cultures were harvested at the indicated time points after the addition of thiolutin.
Mentions: We introduced a frq construct (qrf) with the frq promoter deleted into the frq10 and frq10;frqqLRE mut strains at the csr-1 locus (Extended Data Figure 6a). This transgene can express normal levels of qrf without detectable frq expression (Extended Data Figure 6b). In the frq10;frqqLRE mut;qrf strains, even though qrf expression was restored to normal levels, frq levels were not rescued (Extended Data Figure 6c-d), indicating that qrf regulates frq in cis.

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