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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 primers positions at the frq locus used for ChIP assays and riboprobes positions used for Northern blot analyses. (b) The ChIP assays showing the relative enrichment of histone H3 and Pol II CTD in the frq locus. Scale on y axis is the enrichment percentage of immunoprecipitation (IP) over input. IgG was used as the mock control for IP. (c) The ChIP assays showing the relative enrichment of Pol II Ser5 phosphorylation and Pol II Ser2 phosphorylation (ChIP data in Figure 4b normalized by Pol II CTD ChIP results in panel b), and H3K36me3 (ChIP data in Figure 4b normalized by Histone H3 ChIP results in panel b). Asterisks indicate differences that are statistically significant (*P<0.05, **P<0.01, ***P<0.001).
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Figure 8: (a) A diagram showing the primers positions at the frq locus used for ChIP assays and riboprobes positions used for Northern blot analyses. (b) The ChIP assays showing the relative enrichment of histone H3 and Pol II CTD in the frq locus. Scale on y axis is the enrichment percentage of immunoprecipitation (IP) over input. IgG was used as the mock control for IP. (c) The ChIP assays showing the relative enrichment of Pol II Ser5 phosphorylation and Pol II Ser2 phosphorylation (ChIP data in Figure 4b normalized by Pol II CTD ChIP results in panel b), and H3K36me3 (ChIP data in Figure 4b normalized by Histone H3 ChIP results in panel b). Asterisks indicate differences that are statistically significant (*P<0.05, **P<0.01, ***P<0.001).

Mentions: After transcriptional initiation, RNA polymerase II CTD is phosphorylated at serines 2 and 5 with Ser 5 and Ser 2 phosphorylation enriched near the 5′ and 3′ ends of transcribed regions, respectively22. Similar Ser 5 and Ser 2 phosphorylation profiles were seen at a Neurospora locus without antisense transcripts (Extended Data Figure 7a-b). In contrast, both modifications of CTD peaked at the same position in the middle of the transcribed frq region (Figure 4b, Extended Data Figure 8). Mutation of the qLRE, which reduces qrf expression, resulted in decreases of both Ser 2 and Ser 5 phosphorylation. Phosphorylation of pol II can trigger histone H3K36 methylation23. H3K36me3 enrichment at the frq locus peaked at the same position as did CTD phosphorylation, and the qLRE mutation reduced H3K36me3 (Figure 4b, bottom panel). In the frq10;frq.aq strain minus QA, in which qrf expression is completely abolished, the distributions of CTD phosphorylation and H3K36me3 on frq resemble the control locus lacking antisense transcription (Extended Data Figure 9a-c). These results suggest that pol II stalls in the middle of frq locus due to convergent transcription9.


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 primers positions at the frq locus used for ChIP assays and riboprobes positions used for Northern blot analyses. (b) The ChIP assays showing the relative enrichment of histone H3 and Pol II CTD in the frq locus. Scale on y axis is the enrichment percentage of immunoprecipitation (IP) over input. IgG was used as the mock control for IP. (c) The ChIP assays showing the relative enrichment of Pol II Ser5 phosphorylation and Pol II Ser2 phosphorylation (ChIP data in Figure 4b normalized by Pol II CTD ChIP results in panel b), and H3K36me3 (ChIP data in Figure 4b normalized by Histone H3 ChIP results in panel b). Asterisks indicate differences that are statistically significant (*P<0.05, **P<0.01, ***P<0.001).
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Related In: Results  -  Collection

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Figure 8: (a) A diagram showing the primers positions at the frq locus used for ChIP assays and riboprobes positions used for Northern blot analyses. (b) The ChIP assays showing the relative enrichment of histone H3 and Pol II CTD in the frq locus. Scale on y axis is the enrichment percentage of immunoprecipitation (IP) over input. IgG was used as the mock control for IP. (c) The ChIP assays showing the relative enrichment of Pol II Ser5 phosphorylation and Pol II Ser2 phosphorylation (ChIP data in Figure 4b normalized by Pol II CTD ChIP results in panel b), and H3K36me3 (ChIP data in Figure 4b normalized by Histone H3 ChIP results in panel b). Asterisks indicate differences that are statistically significant (*P<0.05, **P<0.01, ***P<0.001).
Mentions: After transcriptional initiation, RNA polymerase II CTD is phosphorylated at serines 2 and 5 with Ser 5 and Ser 2 phosphorylation enriched near the 5′ and 3′ ends of transcribed regions, respectively22. Similar Ser 5 and Ser 2 phosphorylation profiles were seen at a Neurospora locus without antisense transcripts (Extended Data Figure 7a-b). In contrast, both modifications of CTD peaked at the same position in the middle of the transcribed frq region (Figure 4b, Extended Data Figure 8). Mutation of the qLRE, which reduces qrf expression, resulted in decreases of both Ser 2 and Ser 5 phosphorylation. Phosphorylation of pol II can trigger histone H3K36 methylation23. H3K36me3 enrichment at the frq locus peaked at the same position as did CTD phosphorylation, and the qLRE mutation reduced H3K36me3 (Figure 4b, bottom panel). In the frq10;frq.aq strain minus QA, in which qrf expression is completely abolished, the distributions of CTD phosphorylation and H3K36me3 on frq resemble the control locus lacking antisense transcription (Extended Data Figure 9a-c). These results suggest that pol II stalls in the middle of frq locus due to convergent transcription9.

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