Skip to main content

Thank you for visiting You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Role for antisense RNA in regulating circadian clock function in Neurospora crassa


The prevalence of antisense RNA in eukaryotes is not known and only a few naturally occurring antisense transcripts have been assigned a function1,2,3,4. However, the recent identification of a large number of putative antisense transcripts5 strengthens the view that antisense RNAs might affect a wider variety of processes than previously thought. Here we show that in the model organism Neurospora crassa entrainment of the circadian clock, which is critical for the correct temporal expression of genes and their products, is controlled partly by an antisense RNA arising from a clock component locus. In a wild-type strain, levels of antisense frequency (frq) transcripts cycle in antiphase to sense frq transcripts in the dark, and are inducible by light. In mutant strains in which the induction of antisense frq RNA by light is abolished, the time of the internal clock is delayed relative to the wild-type strain, and resetting of the clock by light is altered. These data provide an unexpected link between antisense RNA and circadian timing and provide a new example of a eukaryotic cellular process regulated by naturally occurring antisense RNA.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type



Prices may be subject to local taxes which are calculated during checkout

Figure 1: Expression of sense (S) and antisense (AS) RNA transcripts from the N. crassa frq locus.
Figure 2: Altered expression of sense (S) and antisense (AS) frq transcripts in mutant strain frq10frqccg-2.
Figure 3: Circadian and molecular phenotype of frq10frqccg-2 after a light (LL) to dark (DD) transfer.
Figure 4: The response of frq10frqccg-2 and frq10KAJ128 to light pulses.


  1. Kumar, M. & Carmichael, G. G. Antisense RNA: function and fate of duplex RNA in cells of higher eukaryotes. Microbiol. Mol. Biol. Rev. 62, 1415–1434 (1998)

    CAS  PubMed  PubMed Central  Google Scholar 

  2. Eddy, S. R. Non-coding RNA genes and the modern RNA world. Nature Rev. Genet. 2, 919–929 (2001)

    Article  CAS  Google Scholar 

  3. Avner, P. & Heard, E. X-chromosome inactivation: counting, choice and initiation. Nature Rev. Genet. 2, 59–67 (2001)

    Article  CAS  Google Scholar 

  4. Sleutels, F., Zwart, R. & Barlow, D. P. The noncoding Air RNA is required for silencing autosomal imprinted genes. Nature 415, 810–813 (2002)

    Article  ADS  CAS  Google Scholar 

  5. Ambros, V. MicroRNAs: tiny regulators with great potential. Cell 107, 823–826 (2001)

    Article  CAS  Google Scholar 

  6. Dunlap, J. C. Molecular bases for circadian clocks. Cell 96, 271–290 (1999)

    Article  CAS  Google Scholar 

  7. Cermakian, N. & Sassone-Corsi, P. Multilevel regulation of the circadian clock. Nature Rev. Mol. Cell Biol. 1, 59–67 (2000)

    Article  CAS  Google Scholar 

  8. Hastings, M. H. Circadian clockwork: two loops are better than one. Nature Rev. Neurosci. 1, 143–145 (2000)

    Article  CAS  Google Scholar 

  9. Aronson, B. D., Johnson, K. A., Loros, J. J. & Dunlap, J. C. Negative feedback defining a circadian clock: autoregulation of the clock gene frequency. Science 263, 1578–1584 (1994)

    Article  ADS  CAS  Google Scholar 

  10. Merrow, M. W. & Dunlap, J. C. Intergeneric complementation of a circadian rhythmicity defect: phylogenetic conservation of structure and function of the clock gene frequency. EMBO J. 13, 2257–2266 (1994)

    Article  CAS  Google Scholar 

  11. Crosthwaite, S. K., Dunlap, J. C. & Loros, J. J. Neurospora wc-1 and wc-2: transcription, photoresponses, and the origins of circadian rhythmicity. Science 276, 763–769 (1997)

    Article  CAS  Google Scholar 

  12. Cheng, P., Yang, Y. & Liu, Y. Interlocked feedback loops contribute to the robustness of the Neurospora circadian clock. Proc. Natl Acad. Sci. USA 98, 7408–7413 (2001)

    Article  ADS  CAS  Google Scholar 

  13. Collett, M. A., Dunlap, J. C. & Loros, J. J. Circadian clock-specific roles for the light response protein WHITE COLLAR-2. Mol. Cell. Biol. 21, 2619–2628 (2001)

    Article  CAS  Google Scholar 

  14. Froehlich, A. C., Liu, Y., Loros, J. J. & Dunlap, J. C. White Collar-1, a circadian blue light photoreceptor, binding to the frequency promoter. Science 297, 815–819 (2002)

    Article  ADS  CAS  Google Scholar 

  15. Denault, D. L., Loros, J. J. & Dunlap, J. C. WC-2 mediates WC-1-FRQ interaction within the PAS protein-linked circadian feedback loop of Neurospora. EMBO J. 20, 109–117 (2001)

    Article  CAS  Google Scholar 

  16. Merrow, M. et al. Circadian regulation of the light input pathway in Neurospora crassa. EMBO J. 20, 307–315 (2001)

    Article  CAS  Google Scholar 

  17. Cheng, P., Yang, Y., Heintzen, C. & Liu, Y. Coiled-coil domain-mediated FRQ-FRQ interaction is essential for its circadian clock function in Neurospora. EMBO J. 20, 101–108 (2001)

    Article  CAS  Google Scholar 

  18. Aronson, B. D., Johnson, K. A. & Dunlap, J. C. Circadian clock locus frequency: protein encoded by a single open reading frame defines period length and temperature compensation. Proc. Natl Acad. Sci. USA 91, 7683–7687 (1994)

    Article  ADS  CAS  Google Scholar 

  19. Crosthwaite, S. K., Loros, J. J. & Dunlap, J. C. Light-induced resetting of a circadian clock is mediated by a rapid increase in frequency transcript. Cell 81, 1003–1012 (1995)

    Article  CAS  Google Scholar 

  20. Loros, J. J. & Dunlap, J. C. Genetic and molecular analysis of circadian rhythms in Neurospora. Annu. Rev. Physiol. 63, 757–794 (2001)

    Article  CAS  Google Scholar 

  21. Bell-Pedersen, D., Dunlap, J. C. & Loros, J. J. The Neurospora circadian clock-controlled gene, ccg-2, is allelic to eas and encodes a fungal hydrophobin required for formation of the conidial rodlet layer. Genes Dev. 6, 2382–2394 (1992)

    Article  CAS  Google Scholar 

  22. Johnson, C. H. Forty years of PRCs: what have we learned? Chronobiol. Int. 16, 711–743 (1999)

    Article  CAS  Google Scholar 

  23. Heintzen, C., Loros, J. J. & Dunlap, J. C. The PAS protein VIVID defines a clock-associated feedback loop that represses light input, modulates gating, and regulates clock resetting. Cell 104, 453–464 (2001)

    Article  CAS  Google Scholar 

  24. Catalanotto, C., Azzalin, G., Macino, G. & Cogoni, C. Involvement of small RNAs and role of the qde genes in the gene silencing pathway in Neurospora. Genes Dev. 16, 790–795 (2002)

    Article  CAS  Google Scholar 

  25. Bernstein, E., Caudy, A. A., Hammond, S. M. & Hannon, G. J. Role for a bidentate ribonuclease in the initiation step of RNA interference. Nature 409, 363–366 (2001)

    Article  ADS  CAS  Google Scholar 

  26. Sauman, I. & Reppert, S. M. Circadian clock neurons in the silkmoth Antheraea pernyi: novel mechanisms of period protein regulation. Neuron 17, 889–900 (1996)

    Article  CAS  Google Scholar 

  27. Gotter, A. L., Levine, J. D. & Reppert, S. M. Sex-linked period genes in the silkmoth, Antheraea pernyi: implications for circadian clock regulation and the evolution of sex chromosomes. Neuron 24, 953–965 (1999)

    Article  CAS  Google Scholar 

  28. Johnson, K. A. Molecular Characterization of the Circadian Clock Locus Frequency of Neurospora crassa. Thesis, Dartmouth College (1993)

    Google Scholar 

  29. Roenneberg, T. & Taylor, W. Automated recordings of bioluminescence with special reference to the analysis of circadian rhythms. Methods Enzymol. 305, 104–119 (2000)

    Article  CAS  Google Scholar 

  30. McClung, C. R., Fox, B. A. & Dunlap, J. C. The Neurospora clock gene frequency shares a sequence element with the Drosophila clock gene period. Nature 339, 558–562 (1989)

    Article  ADS  CAS  Google Scholar 

Download references


We thank K. Gull, A. Loudon and S. Oliver for critical reading of the manuscript; C. Heintzen for discussion and critical reading of the manuscript; and J. Miller for technical assistance. This work was supported by grants from the BBSRC, NIGMS, NIMH and NSF.

Author information

Authors and Affiliations


Corresponding author

Correspondence to Susan K. Crosthwaite.

Ethics declarations

Competing interests

The authors declare that they have no competing financial interests.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Kramer, C., Loros, J., Dunlap, J. et al. Role for antisense RNA in regulating circadian clock function in Neurospora crassa. Nature 421, 948–952 (2003).

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI:

This article is cited by


By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.


Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing