"Antisense" is not the same thing as "nonsense" if there’s a reason for it. DNA is usually read in the "sense" direction — the direction that translates into a protein. The translation machinery can work in the opposite direction sometimes, though, producing an "antisense" RNA. Given our acquaintance with language, this would seem puzzling; what possible meaning could come from reading a paragraph backwards? Two recent papers show that cells can make sense out of antisense, by creating long noncoding RNA transcripts (lncRNAs) that act as switches.

Flowering

A paper in the Proceedings of the National Academy of Sciences shows a previously unknown role for antisense lncRNAs in flowering plants. Farmers and botanists have long known that cold weather is a prerequisite for "vernalization," or spring flowering, but how it works has been a mystery. Earlier research identified two epigenetic states that create a positive feedback loop, but that alone could not explain the response to cold. A gene named FRC that represses flowering needs to be repressed itself in order for a cold snap to switch on the flowering sequence.

In the paper, four researchers from the UK’s John Innes Centre studied a set of antisense lncRNAs collectively dubbed COOLAIR that are transcribed in cold weather. These RNAs mediate the coordinated switching of the two epigenetic states. Another pathway, the Polycomb complex 2, is also involved in cold-dependent silencing of FRC. Together, the players provide a "fail-safe" system that can switch on flowering in a cold snap of sufficient length, without over-reacting to induce "precocious" (early) flowering.

Long noncoding RNAs (lncRNAs) play important roles in chromatin regulation in higher eukaryotes. Studies analysing how lncRNAs influence Polycomb silencing suggested they specifically bind and recruit Polycomb Repressive Complex 2 (PRC2) to defined targets. However, the promiscuous binding of PRC2 to RNA has raised questions as to the mechanism by which lncRNA contributes to Polycomb silencing. We investigate the function of a set of cold-induced antisense transcripts in the Polycomb-dependent epigenetic silencing of a floral repressor gene in Arabidopsis [a common lab plant]. Through analysis of a transgene, in which antisense transcripts are no longer cold-induced, we show that these antisense transcripts are required for coordinated switching of opposing chromatin states. This mechanism links transcriptional shutdown by cold to long-term epigenetic silencing. (Emphasis added.)

The scientists apparently did not find "evolution" or "natural selection" worth mentioning. Mutations, however, were found to break this coordinated system.

Biological Clocks

Another role for antisense RNA was found in biological clocks. For a primer on biological clocks, see this short article from the National Institutes of Health, "Four Timely Facts About Our Biological Clocks," Number 1, "They’re incredibly intricate." Savor a bit of the intricacy in this Abstract from PNAS from a few months ago:

Circadian rhythms drive the temporal organization of a wide variety of physiological and behavioral functions in ?24-h cycles. This control is achieved through a complex program of gene expression. In mammals, the molecular clock machinery consists of interconnected transcriptional-translational feedback loops that ultimately ensure the proper oscillation of thousands of genes in a tissue-specific manner. To achieve circadian transcriptional control, chromatin remodelers serve the clock machinery by providing appropriate oscillations to the epigenome. Recent findings have revealed the presence of circadian interactomes, nuclear "hubs" of genome topology where coordinately expressed circadian genes physically interact in a spatial and temporal-specific manner. Thus, a circadian nuclear landscape seems to exist, whose interplay with metabolic pathways and clock regulators translates into specific transcriptional programs. Deciphering the molecular mechanisms that connect the circadian clock machinery with the nuclear landscape will reveal yet unexplored pathways that link cellular metabolism to epigenetic control.

One of those unexplored pathways involves antisense lncRNAs. A team of scientists from Texas, California and the UK, reporting in Nature recently, found that antisense RNA was required for circadian clock function in the fungus Neurospora. You can tell the antisense RNA’s name, because it’s the reverse of the normal RNA transcript name in this summary:

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.

This is really quite astonishing: sense and antisense transcripts balance each other in a double-feedback loop tied to the core feedback loop, but the antisense RNA is the one that sets the clock! The sense and antisense transcripts oscillate against each other, and yet environmental factors like light can push the "reset" button in order to provide "robust and sustained" biological clock.

And it’s not just in a fungus. The authors suspect that antisense RNA regulation is common in the animal kingdom as well. Their concluding paragraph states:

The mutual transcription interference of frq and qrf results in antiphasic oscillations of frq and qrf that resonate to achieve robust and sustained circadian gene expression. In the silkmoth and mouse liver, antisense per RNAs exist and were also found to oscillate in antiphase to sense RNAs, suggesting that a similar mechanism may function in animal circadian systems.

Like the other team, this group was able to break the system with targeted mutations, but otherwise had no use for "evolution" or "natural selection" in their work.

Two years ago, Casey Luskin quoted papers that suggested roles for antisense RNAs. Now we are seeing more examples of functional roles for what was previously thought to be genetic junk. These papers, describing examples of two unrelated genes in different species, show that antisense lncRNAs are not just partially helpful, but essential for proper regulation of key biological systems.

Imagine the challenge of designing codes that can generate functional information when read in either direction. The suggestion that regulatory roles for antisense RNAs are widespread in plants and animals raises the implications for intelligent design to a whole new level. Design-based research is poised to find more spectacular examples of "sense in the antisense" in coming years.

Image source: rebel rebel/Flickr.