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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 transcription results in pol II stalling, premature transcription termination and chromatin modifications. (a) Strand-specific RT-qPCR results showing the levels of intron-containing frq after dark to light transfer. (b) ChIP assays showing the relative enrichment of pol II Ser 5, Ser 2, and H3K36me3 in the frq locus in LL. IgG was used as the mock control for IP. (c-d) Northern blot analyses showing the levels of frq and qrf in LL. (e-f) Northern blot analysis in LL. frq-N term and frq-C term are specific for the 5′ or 3′ half of the frq transcripts, respectively (shown in Figure 4b). The addition of QA results in rrp44 silencing. The ratios between truncated and full-length frq transcripts from three independent experiments in (f) are shown. Error bars are SD. *P<0.05, **P<0.01.
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Figure 14: qrf transcription results in pol II stalling, premature transcription termination and chromatin modifications. (a) Strand-specific RT-qPCR results showing the levels of intron-containing frq after dark to light transfer. (b) ChIP assays showing the relative enrichment of pol II Ser 5, Ser 2, and H3K36me3 in the frq locus in LL. IgG was used as the mock control for IP. (c-d) Northern blot analyses showing the levels of frq and qrf in LL. (e-f) Northern blot analysis in LL. frq-N term and frq-C term are specific for the 5′ or 3′ half of the frq transcripts, respectively (shown in Figure 4b). The addition of QA results in rrp44 silencing. The ratios between truncated and full-length frq transcripts from three independent experiments in (f) are shown. Error bars are SD. *P<0.05, **P<0.01.

Mentions: WC binding to the frq promoter initiates WC-dependent frq transcription, but the qLRE mutation did not affect WC binding at the frq promoter (Extended Data Figure 6e-f). However, However, levels of frq pre-mRNA were significantly elevated in the qLRE mutants (Figure 4a and Extended Data Figure 6g). Moreover, qrf expression in the frq10;frq.aq strain did not affect frq RNA stability (Extended Data Figure 6h). These results suggest that qrf regulates frq after transcriptional initiation.


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 transcription results in pol II stalling, premature transcription termination and chromatin modifications. (a) Strand-specific RT-qPCR results showing the levels of intron-containing frq after dark to light transfer. (b) ChIP assays showing the relative enrichment of pol II Ser 5, Ser 2, and H3K36me3 in the frq locus in LL. IgG was used as the mock control for IP. (c-d) Northern blot analyses showing the levels of frq and qrf in LL. (e-f) Northern blot analysis in LL. frq-N term and frq-C term are specific for the 5′ or 3′ half of the frq transcripts, respectively (shown in Figure 4b). The addition of QA results in rrp44 silencing. The ratios between truncated and full-length frq transcripts from three independent experiments in (f) are shown. Error bars are SD. *P<0.05, **P<0.01.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4214883&req=5

Figure 14: qrf transcription results in pol II stalling, premature transcription termination and chromatin modifications. (a) Strand-specific RT-qPCR results showing the levels of intron-containing frq after dark to light transfer. (b) ChIP assays showing the relative enrichment of pol II Ser 5, Ser 2, and H3K36me3 in the frq locus in LL. IgG was used as the mock control for IP. (c-d) Northern blot analyses showing the levels of frq and qrf in LL. (e-f) Northern blot analysis in LL. frq-N term and frq-C term are specific for the 5′ or 3′ half of the frq transcripts, respectively (shown in Figure 4b). The addition of QA results in rrp44 silencing. The ratios between truncated and full-length frq transcripts from three independent experiments in (f) are shown. Error bars are SD. *P<0.05, **P<0.01.
Mentions: WC binding to the frq promoter initiates WC-dependent frq transcription, but the qLRE mutation did not affect WC binding at the frq promoter (Extended Data Figure 6e-f). However, However, levels of frq pre-mRNA were significantly elevated in the qLRE mutants (Figure 4a and Extended Data Figure 6g). Moreover, qrf expression in the frq10;frq.aq strain did not affect frq RNA stability (Extended Data Figure 6h). These results suggest that qrf regulates frq after transcriptional initiation.

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