A large number of plant genes are aligned with partially overlapping genes in antisense orientation. Transcription of both genes would therefore favour the formation of double-stranded RNA, providing a substrate for the RNAi machinery, and enhanced antisense transcription should therefore reduce sense transcript levels. We have identified a gene pair that resembles a model for antisense-based gene regulation as a T-DNA insertion into the antisense gene causes a reduction in antisense transcript levels and an increase in sense transcript levels. The same effect was, however, also observed when the two genes were inserted as transgenes into different chromosomal locations, independent of the sense and antisense gene being expressed individually or jointly. Our results therefore indicate that antagonistic changes in sense/antisense transcript levels do not necessarily reflect antisense-mediated regulation. More likely, the partial overlap of the two genes may have favoured the evolution of antagonistic expression patterns preventing RNAi effects. 1. Introduction The expression of genetic information involves a sequence of molecular processes, including transcript synthesis, processing, turnover, transport and translation. Eukaryotes have developed regulatory systems at each of these levels, which can all contribute to the expression efficiency and profile of a gene. Key components in this context are natural antisense transcripts (NATs), for which a regulatory role has been demonstrated at most levels [1]. Work on yeast and animals has identified model genes that illustrate the regulatory influence of antisense transcription on sense transcript synthesis, processing, or stability. This has led to the proposal that NATs have evolved to comprise a second tier of gene expression in eukaryotes [2]. Most of our current knowledge about the mechanistic aspects of antisense-mediated gene regulation derives from work in the fungal or animal field. In plant research, antisense-mediated gene regulation systems are poorly characterised, which is contrasted by an unexpectedly high number of NATs in plants [3–5]). Among 26,939 annotated Arabidopsis genes, 3,027 contain antisense transcripts that are jointly expressed with their sense transcript partner. For another 7,598 genes, sense and antisense transcripts are both detected in specific tissues [3]. The abundance of NATs very strongly suggests that they are part of regulatory systems. The few examples, for which natural plant antisense transcripts have been documented to modify sense transcripts, are based on RNA
References
[1]
C. Vanhée-Brossollet and C. Vaquero, “Do natural antisense transcripts make sense in eukaryotes?” Gene, vol. 211, no. 1, pp. 1–9, 1998.
[2]
J. S. Mattick, “Non-coding RNAs: the architects of eukaryotic complexity,” EMBO Reports, vol. 2, no. 11, pp. 986–991, 2001.
[3]
K. Yamada, J. Lim, J. H. Dale et al., “Empirical analysis of transcriptional activity in the Arabidopsis genome,” Science, vol. 302, no. 5646, pp. 842–846, 2003.
[4]
X. J. Wang, T. Gaasterland, and N. H. Chua, “Genome-wide prediction and identification of cis-natural antisense transcripts in Arabidopsis thaliana,” Genome biology, vol. 6, no. 4, p. R30, 2005.
[5]
C. H. Jen, I. Michalopoulos, D. R. Westhead, and P. Meyer, “Natural antisense transcripts with coding capacity in Arabidopsis may have a regulatory role that is not linked to double-stranded RNA degradation,” Genome biology, vol. 6, no. 6, p. R51, 2005.
[6]
O. Borsani, J. Zhu, P. E. Verslues, R. Sunkar, and J. K. Zhu, “Endogenous siRNAs derived from a pair of natural cis-antisense transcripts regulate salt tolerance in Arabidopsis,” Cell, vol. 123, no. 7, pp. 1279–1291, 2005.
[7]
S. Katiyar-Agarwal, R. Morgan, D. Dahlbeck et al., “A pathogen-inducible endogenous siRNA in plant immunity,” Proceedings of the National Academy of Sciences of the United States of America, vol. 103, no. 47, pp. 18002–18007, 2006.
[8]
J. M. Alonso, A. N. Stepanova, T. J. Leisse et al., “Genome-wide insertional mutagenesis of Arabidopsis thaliana,” Science, vol. 301, no. 5633, pp. 653–657, 2003.
[9]
A. F. Tissier, S. Marillonnet, V. Klimyuk et al., “Multiple independent defective Suppressor-mutator transposon insertions in Arabidopsis: a tool for functional genomics,” Plant Cell, vol. 11, no. 10, pp. 1841–1852, 1999.
[10]
R. L. Scholl, S. T. May, and D. H. Ware, “Seed and molecular resources for Arabidopsis,” Plant Physiology, vol. 124, no. 4, pp. 1477–1480, 2000.
[11]
K. D. Edwards, P. E. Anderson, A. Hall et al., “FLOWERING LOCUS C mediates natural variation in the high-temperature response of the Arabidopsis circadian clock,” Plant Cell, vol. 18, no. 3, pp. 639–650, 2006.
[12]
Z. Vejlupkova and J. E. Fowler, “Maize DNA preps for undergraduate students: a robust method for PCR genotyping,” Maize Genetics Cooperation Newsletter, vol. 77, pp. 24–25, 2003.
[13]
D. Swarbreck, C. Wilks, P. Lamesch et al., “The Arabidopsis Information Resource (TAIR): gene structure and function annotation,” Nucleic Acids Research, vol. 36, supplement 1, pp. D1009–D1014, 2008.
[14]
A. Peragine, M. Yoshikawa, G. Wu, H. L. Albrecht, and R. S. Poethig, “SGS3 and SGS2/SDE1/RDR6 are required for juvenile development and the production of trans-acting siRNAs in Arabidopsis,” Genes and Development, vol. 18, no. 19, pp. 2368–2379, 2004.
[15]
M. Stam, R. De Bruin, R. Van Blokland, R. A. L. Van Der Hoorn, J. N. M. Mol, and J. M. Kooter, “Distinct features of post-transcriptional gene silencing by antisense transgenes in single copy and inverted T-DNA repeat loci,” Plant Journal, vol. 21, no. 1, pp. 27–42, 2000.
[16]
R. P. Hellens, E. Anne Edwards, N. R. Leyland, S. Bean, and P. M. Mullineaux, “pGreen: a versatile and flexible binary Ti vector for Agrobacterium-mediated plant transformation,” Plant Molecular Biology, vol. 42, no. 6, pp. 819–832, 2000.
[17]
E. Zubko and P. Meyer, “A natural antisense transcript of the Petunia hybrida Sho gene suggests a role for an antisense mechanism in cytokinin regulation,” Plant Journal, vol. 52, no. 6, pp. 1131–1139, 2007.
[18]
K. E. Shearwin, B. P. Callen, and J. B. Egan, “Transcriptional interference—a crash course,” Trends in Genetics, vol. 21, no. 6, pp. 339–345, 2005.
[19]
E. M. Prescott and N. J. Proudfoot, “Transcriptional collision between convergent genes in budding yeast,” Proceedings of the National Academy of Sciences of the United States of America, vol. 99, no. 13, pp. 8796–8801, 2002.
[20]
S. K. Crosthwaite, “Circadian clocks and natural antisense RNA,” FEBS Letters, vol. 567, no. 1, pp. 49–54, 2004.
[21]
C. Kramer, J. J. Loros, J. C. Dunlap, and S. K. Crosthwaite, “Role for antisense RNA in regulating circadian clock function in Neurospora crassa,” Nature, vol. 421, no. 6926, pp. 948–952, 2003.
[22]
J. Dekker, K. Rippe, M. Dekker, and N. Kleckner, “Capturing chromosome conformation,” Science, vol. 295, no. 5558, pp. 1306–1311, 2002.
[23]
A. K. Linnemann, A. E. Platts, and S. A. Krawetz, “Differential nuclear scaffold/matrix attachment marks expressed genes,” Human Molecular Genetics, vol. 18, no. 4, pp. 645–654, 2009.
[24]
M. Shogren-Knaak, H. Ishii, J. M. Sun, M. J. Pazin, J. R. Davie, and C. L. Peterson, “Histone H4-K16 acetylation controls chromatin structure and protein interactions,” Science, vol. 311, no. 5762, pp. 844–847, 2006.
[25]
K. Havas, A. Flaus, M. Phelan et al., “Generation of superhelical torsion by ATP-dependent chromatin remodeling activities,” Cell, vol. 103, no. 7, pp. 1133–1142, 2000.
[26]
E. Zubko, A. Kunova, and P. Meyer, “Sense and antisense transcripts of convergent gene pairs in Arabidopsis thaliana can share a common polyadenylation region,” PLoS One, vol. 6, no. 2, Article ID e16769, 2011.
