How Antisense Genes Are Discovered

In the past ten years or so, a great deal of research has focused on antisense transcription of genes. Normally, RNA gets transcribed from one strand of DNA only. But it turns out, in many cases RNA also gets transcribed off the opposite strand of DNA (an antisense copy), either at the original gene (so-called cis transcription) or at a copy of the gene some distance away (trans transcription). The latter can be a pseudogene, or a normal copy of the gene.

Antisense transcripts occur very widely not only in human DNA but in bacteria, yeast, and (in fact) every place where scientists have looked, and places where they haven't looked. Some of the most interesting discoveries have happened when researchers weren't specifically looking for antisense transcripts but found them by accident. How does that happen? It happens in experiments involving IVET (in vivo expression technology), an important experimental technique for uncovering new genes.

IVET is a powerful gene manipulation strategy for discovering which genes in an organism (a pathogen, usually) are up-regulated or turned on during host infection. Let's say you're studying a new pathogen and you want to get an idea of which genes, in the pathogen, are turned on during the infection process. First, you need a strain of the organism that's disabled by virtue of lacking a working copy of a particular metabolic enzyme, say an enzyme needed for purine metabolism, e.g. purA. Secondly, you need a vector for inserting a promoterless copy of the working gene into the bacterium. What this usually means is, you need a plasmid (a small extra chromosome; many bacteria have them, and they can often be manipulated in the lab) on which to place a functional purA gene. The gene won't be expressed, however, if it lacks a suitable promoter region on the DNA upstream of the gene. That's good; that's what you want. You want to put a promoterless copy of the good gene on the plasmid, along with (this is crucial) a random chunk of DNA from the pathogen, inserted ahead of purA on the plasmid. In practice, it's easy to create a bunch of plasmids with this arrangement: a working copy of purA, and ahead of it, a random chunk of pathogen DNA. The idea is that you now attempt to infect a lab animal with the bacterium containing the plasmid. If the bacterium establishes infection in the animal, presumably it's because a random chunk of DNA happened to contain a promoter region (and associated downstream genes) that gets turned on during infection. If you now isolate the bacterium from the sick animal, you can look to see what kind(s) of genes got transduced into the bacterium.

IVET is a promoter trap technology for selecting bacterial genes that are specifically induced when bacteria infect a host organism. A plasmid vetor contains a random fragment of the chromosome of the pathogen (red) and a promoterless gene (selective marker, burgundy) that encodes an enzyme required for survival. Pooled plasmid-containing clones are inoculated into the mouse (B). Only those bacteria that contain the selective marker fused to a random gene that is transcriptionally active in the host are able to survive. After a suitable infection period, bacteria that express the marker are isolated from the spleen or other organs. The inclusion of a lacZY mutant gene (blue) allows post-selection screening for promoters that are active only in vivo. What you want are bacteria that are lac-positive only in the host environment, not "constitutive" (always-on).

Exactly this sort of technique was used by Silby, Rainey, and Levy to determine which genes were activated in Pseudomonas during colonization of soil. (The IVET technique can be adapted to any scenario in which an organism differentially expresses genes in its adaptation to a "host" environment, even if the environment is, in fact, a plant, or soil in this case, rather than a mouse.) They were looking to see which genes in Pseudomonas play an essential role in that organism's ability to thrive in soil, and they successfully identified more than 50 promoters (and associated fusions) that come alive during soil colonization. When they looked at 22 "soil genes" that got turned on, they found ten previously undescribed genes that were transcribed in the antisense direction from regions overlapping known genes. They called these ten genes "cryptic fusions" because of their un-annotated existence on the supposedly silent, antisense side of known genes.

Cryptic fusions discovered by Silby et al. are shown in grey, in their antisense orientation to known genes (darker grey).

It's not unusual to find that antisense transcripts are playing a regulatory role. When a gene gets transcribed in both directions, the resulting sense and antisense RNAs can combine (by Watson-Crick pairing) to form a double-stranded RNA product, preventing translation of the RNA into protein. But incredibly, sometimes an antisense RNA transcript encodes a legitimate protein (a protein that gets made off the antisense copy). Silby and Levy documented this for the previously unknown cosA gene in Pseudomonas. It seems likely additional antisense proteins await discovery. (Most studies stop at the level of identifying RNA products.)

The finding of antisense transcripts in IVET experiments is common. One of the authors of the Pseudomonas study (Rainey) had previously published a study of rhizosphere-induced genes in Pseudomonas but had not published the fact that 20% of genes found this way were in an antisense orientation to normal genes. Likewise, a 1996 study of Pseudomonas aeruginosa infection in the mouse (Pseudomonas is an opportunistic pathogen) found antisense activity. In fact, the first-ever paper on IVET (by Mahan et al., 1993) described finding antiscript products.

IVET has uncovered a previously unknown "antitranscriptome" world hidden inside living cells. Until we explore this world fully, we won't know how much undiscovered biology we've left on the table.

Bidirectionally Overlapping Genes

The occurrence of bidirectionally overlapping genes in bacteria is rare, and most such examples are dismissed as chimeric or representative of simple genome mis-annotation. After all, how can a gene make sense in one direction, but also make sense on the reverse-reading complementary strand of DNA? Such a situation is more than a mere palindrome. It's akin to the phrase:

Warsaw won, eh?
He now was raw.

The phrase has a sensical message in each direction, yet is not a mere bidi-symmetry of the "A man, a plan, a canal, Panama" kind. It defies credulity to believe a stretch of DNA spanning several hundred bases (several hundred "letters") could evolve to give a useful message in both directions. And yet, what is life itself, if not credulity-defying? Somehow, life began from primordial chemistry and evolved toward DNA genes coding for proteins. Is it so hard to believe that early replicant molecules (probably RNA) were transcribed and translated in both directions, and that some of the happy accidents survived? Is it so hard to believe that some proteins began life as reverse transcripts ("nonsense" proteins) that then evolved toward specialized functionality?

A bonafide example of a bidirectionally transcribed and translated gene was verified experimentally in 2008 by Silby and Levy, who were investigating the soil bacterium Pseudomonas fluorescens PF0-1. They found that the hitherto unknown cosA gene, which overlaps (on the opposite DNA strand) a gene for a fusaric acid resistance protein, is not only expressed as a protein but is required for soil colonization.

A section of P. fluorescens PF0-1 genome showing the existence of overlapping genes (note the yellow-colored segment, representing the cosA gene; the larger green gene above it, on the opposite strand, encodes a fusaric acid resistance protein). The overlapping genes have been shown experimentally to be expressed as protein.

Ironically, a month after Silby and Levy published their results, BMC Genetics published a study by Pallejà et al. looking at large gene overlaps in bacterial genomes. The Pallejà study concluded:

Among the 968 overlaps larger than 60 bps which we analysed, we did not find a single real one among the co-directional and divergent orientations and concluded that there had been an excessive number of misannotations. Only convergent orientation seems to permit some long overlaps, although convergent overlaps are also hampered by misannotations. We propose a simple rule to flag these erroneous gene length predictions to facilitate automatic annotation.

Silby and Levy argue that, to the contrary, current genome annotations are obscuring potentially important discoveries:

[Our] findings suggest that current genome annotations provide an incomplete view of the genetic potential of a given organism . . . In eukaryotes, the concept that genomes include numerous sense/antisense gene pairs is becoming increasingly obvious with genome-wide transcriptional studies in yeast [8] and Arabidopsis [10]. Antisense transcripts have been implicated in eye development [20] and control of entry into meiosis in yeast [21]. However, discussion of antisense transcription is limited to possible regulatory roles for antisense RNA [e.g. 8], without consideration of the possibility that they may specify proteins. Genome annotations do not routinely predict the existence of two protein-coding genes on opposite DNA strands, and in fact normally deliberately eliminate predicted overlaps. Moreover, small protein-coding genes can be missed by predictive algorithms. For example, the blr gene in E. coli specifies a 41 residue protein, and was discovered in a sequence believed to be intergenic [22]. The fact that antisense genes have been implicated in important biological functions indicates that more attention should be given to this emerging class of genes.

I happen to agree with Silby and Levy. It would be a shame if bidirectional overlaps in genomes are not investigated. The notion (furthered by Pallejà) that annotation software should suppress such findings automatically is repulsive. It's the kind of intolerant, rigid, dogmatic thinking science, quite frankly, doesn't need more of.