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Transcriptional interference by antisense RNA is required for circadian clock function

Nature volume 514, pages 650–653 (2014)Cite this article

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Abstract

Eukaryotic circadian oscillators consist of negative feedback loops that generate endogenous rhythmicities1. Natural antisense RNAs are found in a wide range of eukaryotic organisms2,3,4,5. Nevertheless, the physiological importance and mode of action of most antisense RNAs are not clear6,7,8,9. frequency (frq) encodes a component of the Neurospora core circadian negative feedback loop, which was thought to generate sustained rhythmicity10. Transcription of qrf, the long non-coding frq antisense RNA, is induced by light, and its level oscillates in antiphase to frq sense RNA3. 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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Figure 1: WC-mediated light-induction of qrf represses frq to regulate light resetting of the clock.
Figure 2: qrf expression is required for circadian rhythmicities.
Figure 3: Mutual inhibition of frq and qrf transcription forms a double negative feedback loop that is required for clock function.
Figure 4: qrf transcription results in pol II stalling, premature termination of transcription, and chromatin modifications.

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Sequence Read Archive

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RNA-Seq data are available at the Sequence Read Archive under accession number SRP030415.

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Acknowledgements

We thank J. Cha, Y. Dang and H. Yuan for technical assistance, and B. Li for critical comments. Supported by grants from the National Institutes of Health to Y.L. (GM068496, GM062591) and N.L.G. (GM081597), the Welch Foundation (I-1560) to Y.L., the Cancer Prevention Research Institute of Texas (RP101496) to Z. X., and the Biotechnology and Biological Sciences Research Council (BBS/S/C2005/13012) to S.R.A. and S.K.C.

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Authors and Affiliations

  1. Department of Physiology, The University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, Texas 75390, USA,

    Zhihong Xue, Qiaohong Ye & Yi Liu

  2. Faculty of Life Sciences, University of Manchester, Manchester M13 9PT, UK,

    Simon R. Anson & Susan K. Crosthwaite

  3. Department of Clinical Sciences, The University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, Texas 75390, USA,

    Jichen Yang & Guanghua Xiao

  4. Department of Plant and Microbial Biology, University of California, Berkeley, 94720, California, USA

    David Kowbel & N. Louise Glass

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Contributions

Z.X., S.R.A., S.K.C. and Y.L. designed experiments. Z.X., S.R.A., J.Y. and D.K. performed experiments. Q.Y. provided technical support. Y.L., Z.X., S.R.A., N.L.G. and S.K.C. analysed data. Y.L., Z.X. and S.K.C. wrote the paper.

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Extended data figures and tables

Extended Data 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;frqqLREmut strains. C, ClaI; B, BglII; E, EcoRV. d, Strand-specific RT–qPCR results showing the expression levels of frq and qrf in indicated strains in LL. Error bars show standard deviations (n = 3). **P < 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 describing 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 the right. Error bars show standard deviations. **P < 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.

Extended Data Figure 2 qrf expression is required for circadian rhythmicities.

a, Strand-specific RT–qPCR results showing the expression levels of qrf in the indicated knock-in strains at DD24. b, Race tube analyses of the frq10;frqWT and frq10;frq.aq strains in medium containing 0% glucose and 0.17% arginine with the indicated concentrations of QA in DD. The lack of glucose in medium is known to allow more efficient expression from the qa-2 promoter. The black lines on race tubes indicate the daily growth fronts. c, The unnormalized luciferase activity of the experiments in Fig. 2c. d, The normalized result of Fig. 2c on a ×10 scale, showing that fluctuation of the luciferase activity in the frq10;frq.aq strain is random and not rhythmic. e, The phases of the first conidiation band in DD of the race tube results shown in Fig. 2b. f, Western blot analysis showing the FRQ expression profiles in the frq10;frq.aq strain in the presence and absence of QA in DD at the indicated time points. g, Northern blot analysis showing frq expression profiles in the frq10;frq.aq strain in DD at the indicated time points. The densitometric analysis is shown below.

Extended Data Figure 3 Regulation of qrf expression.

a, WC complex does not bind to the qrf promoter in DD. WC-2 ChIP assays showing the relative levels of WC binding at the frq (dLRE) and qrf (qLRE) promoters at the indicated time points in DD. b, Strand-specific RT–qPCR results showing the levels of frq and qrf at DD24 in the indicated strains. c, The unnormalized luciferase activity of the experiments in Fig. 3b. d, The normalized luciferase activity of the Pqrf-luc construct in Fig. 3b shown on a ×10 scale. e, Densitometric analyses of three independent northern blot results, indicating that levels of qrf transcripts are decreased in the frq9 strains as a result of increased frq expression.

Extended Data Figure 4 Rhythmic transcription of frq drives rhythmic qrf expression and mathematical modelling of the Neurospora circadian oscillator.

a, The unnormalized luciferase activity of the experiments in Fig. 3e. b, The unnormalized luciferase activity of the wild-type strain carrying the Pmin-luc construct. Results of two independent transformants are shown. c, Mathematical modelling 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 equation (8), which includes the inhibition of qrf transcription by frq. The rate constants used in the simulations are listed below.

Extended Data Figure 5 Neither RNAi nor DNA methylation pathways have a significant role in the clock.

a, b, Strand-specific RT–qPCR results showing the induction of frq after a light pulse are similar in the indicated strains at DD24. b, Strand-specific RT–qPCR results showing the induction of frq after a light pulse is similar in the indicated strains at DD24. c, Strand-specific RT–qPCR results showing similar expression levels of frq and qrf in the indicated strains in LL. d, A table showing the period lengths of the conidiation rhythms in the wild type and in different RNAi mutants. e, Strand-specific RT–qPCR results showing that the induction of frq after a light pulse is similar in the indicated strains at DD24. f, Strand-specific RT–qPCR results showing that the induction of frq after a light pulse is similar in the indicated strains at DD24. Error bars show standard deviations (n = 3).

Extended Data Figure 6 Characterization of the qrf action on frq expression.

a, Diagram showing the chromosomal modifications in the frqqLREmut;qrf strains that allow the expression of qrf in trans. The frqqLREmut 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 show standard deviations. *P < 0.05 (n = 3). d, Northern blot results showing that expression of qrf in trans in the frq10;frqqLREmut;qrf strains does not repress frq expression. Densitometric analysis of the northern blot results is shown at the right. e, WC-2 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 a lack of statistical significance (n = 3). f, WC-2 ChIP assays showing the relative WC-2 binding levels at the frq promoter in LL in the indicated strains. The wc double mutant (wccDKO) was used as a negative control for the ChIP assays. n.s. indicates a lack of statistical 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. Nascent nuclear RNA was used. 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.

Extended Data Figure 7 Modified Pol II CTD distribution profiles of a locus lacking antisense transcripts.

a, Top panel: strand-specific RNA-Seq results of the NCU01953 locus. Bottom panel: the positions of primers used in ChIP assays and the position of the riboprobe used in northern blot analysis. b, ChIP assays showing the relative enrichment of Pol II Ser 5 phosphorylation, Pol II Ser2 phosphorylation, and H3K36me3 at the NCU01953 locus in the wild-type strain. The scale on the y axis is the enrichment percentage of immunoprecipitation (IP) over input. IgG was used as the mock control for IP.

Extended Data Figure 8 Modified Pol II CTD distribution profiles at the frq locus.

a, Diagram showing the positions of primers at the frq locus used for ChIP assays and the positions of riboprobes used for northern blot analyses. b, ChIP assays showing the relative enrichment of histone H3 and Pol II CTD in the frq locus. The scale on the y axis is the enrichment percentage of immunoprecipitation (IP) over input. IgG was used as the mock control for IP. c, ChIP assays showing the relative enrichment of Pol II Ser 5 phosphorylation and Pol II Ser 2 phosphorylation (ChIP data in Fig. 4b were normalized by Pol II CTD ChIP results in b), and H3K36me3 (ChIP data in Fig. 4b were normalized by histone H3 ChIP results in b). *P < 0.05, **P < 0.01, ***P < 0.001 (n = 3).

Extended Data Figure 9 Complete abolition of qrf expression changes the distributions of modified pol II CTD to a normal profile.

a, Diagram showing the positions of primers used for ChIP assays. b, ChIP assays showing the relative enrichment of Pol II Ser 5 phosphorylation, Pol II Ser 2 phosphorylation, and H3K36me3 in the frq locus in the frq10;frq.aq strain in medium with 2% glucose. Under these conditions, qrf expression is completely abolished (shown in Fig. 2a). c, ChIP assays showing the relative enrichment of Pol II Ser 5 phosphorylation and Pol II Ser 2 phosphorylation (ChIP data in b were normalized by Pol II CTD ChIP results), and H3K36me3 (ChIP data in b were normalized by histone H3 ChIP results).

Extended Data Figure 10 Mechanism of qrf action on frq expression.

a, Densitometric analyses of three independent experiments shown in Fig. 4c. b, Densitometric analyses of independent experiments shown in Fig. 4d. c, Northern blot analysis using a N-term probe (marked in Extended Data Fig. 8a) specific for the 5′ half of qrf transcripts in the WT and WT;dsrrp44 strains in LL. d, Northern blot analysis using a N-term probe (marked in Extended Data Fig. 7a) specific for the 5′ half of NCU01953 transcripts in the WT;dsrrp44 strain in LL.

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Xue, Z., Ye, Q., Anson, S. et al. Transcriptional interference by antisense RNA is required for circadian clock function. Nature 514, 650–653 (2014). https://doi.org/10.1038/nature13671

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