Are Overapping Genes Real?

Bacteria belonging to the Pseudomonas family are a perennial favorite among bacteriology instructors (and students) because of the curious ability of some of its members to produce pigments that fluoresce under an ultraviolet light. If you're unlucky enough to get an infected cut on the arm while working in the garden, it's possible your cut will fluoresce under a black light. That's enough of a diagnosis to pronounce the infectious agent. 

Fluorescent colonies of Pseudomonas.

Silby and Levy, investigating the adaptation of the bacterium Pseudomonas fluorescens to soil, uncovered the existence of at least ten antisense genes in P. fluorescens. They went on to demonstrate experimentally that one of the genes, cosA, produces not just antisense RNA but an associated protein. Tellingly, Silby and Levy commented:

These findings suggest that current genome annotations provide an incomplete view of the genetic potential of a given organism.

The implication is that additional antitranscriptome genes remain to be found, not only in Pseudomonas but in other organisms.

There's a good reason they haven't been found yet. Overlapping genes are automatically rejected by many of the annotation programs that are commonly used to find, identify, and label genes in genome sequences. (The oft-used freeware Glimmer 2 program allows you to set the overlap-rejection threshold.) Many yet-to-be-discovered antisense genes have been deliberately and systematically obscured in published genomes.

Still, once in a while such genes do surface. For example, in Pseudomonas stutzeri A1501, we find a pair of overlapping genes at an offset of 3035137 on the chromosome (see illustration below).

Overapping genes in Pseudomonas stutzeri.

The top gene is annotated merely as a "hypothetical protein," while the underlying gene on the opposite strand is an aspartyl-tRNA synthetase. One's normal inclination is to dismiss a hypothetical protein as being unimportant, but this may not be wise. Twenty percent or more of bacterial genes are annotated as hypothetical proteins; common sense says they can't all be unimportant. In fact, in "Transcriptome Analysis of Pseudomonas syringae Identifies New Genes, Noncoding RNAs, and Antisense Activity" by Filiatrault et al. (2010), researchers found that 818 out of 1,646 protein genes in P. syringae annotated as "hypothetical proteins" were expressed under iron-limited conditions. Many (probably most) genes annotated as "hypothetical protein" are quite real and should probably be re-annotated as PUF: "protein of unknown function."

In this case, the "hypothetical protein" shown in yellow (above) turns up medium-strength protein-BLAST hits with other "hypothetical proteins" from other organisms, including a hit with an E-value of 3.0×10^-49 in Parasutterella excrementihominis YIT 11859 and a comparable hit on a predicted phosphatase/phosphohexomutase in Rothia mucilaginosa DY-18.

In this particular case, the hypothetical-protein gene lacks a strong upstream Shine Dalgarno sequence (a sequence preceding many genes that helps bind a ribosome to the mRNA). But so too does the gene on the opposite strand. (This is not unusual. The SD sequence is not required for translation and in fact, in about half of bacterial species, a Shine Dalgarno sequence is associated with fewer than 50% of genes.) Hence, the jury's out on whether the antigene is expressed. It could be that no protein is made from the top strand but the gene provides RNA-mediated control of the gene on the bottom strand. We won't know for sure until someone investigates.

In Pseudomonas aeruginosa strain PADK2_CF510, we find another instance of a bidirectional overlapping gene pair (see graphic below). In this case, the gene on the top strand (CF510_06030) encodes the large subunit of an isopropylmalate isomerase. The gene on the bottom strand (CF510_06025, shown in yellow) is annotated as "Flp pilus assembly protein TadG." It could very well be a misannotated non-gene. However, five genes away is FimV (CF510_06060), another pilus-assembly (motility) protein. Moreover, the gene marked TadG has a strong upstream SD sequence containing the canonical GGAGG motif. The gene above it has a weaker GGAAA motif.

P. aeruginosa has an overlap of an isopropylmalate isomerase gene and a gene for a motility protein. The latter is shown in yellow.

In previous posts, I've mentioned (and shown data for) the fact that in the overwhelming majority of protein-encoding genes (across every kind of genome), the first base of a codon tends to be purine-rich. One check of whether a bidi-overlap gene is "real" or not ought to be that the first codon base should be purine rich in both reading directions. This is, in fact, the case for the examples shown above. The aspartyl-tRNA synthetase gene for P. stutzeri has AG1 (1st base, purine) content averaging 59.8%, whereas its bidirectional partner gene ("hypothetical protein") has AG1 = 58.5%. The isopropylmalate isomerase of P. aeruginosa has AG1 = 65.9%, while its antisymmetric partner (TadG) has AG1 = 56.2%.

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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.