New genes, new tricks, part 2

In my previous post, I marvelled over the strange and unexpected way duplicated genes behave in fruit flies. The second study I wanted to discuss is also about new fruit fly genes gaining new functions, but unlike the other one, it’s about new genes that didn’t come from pre-existing genes.

Reinhardt et al. (2013) wasn’t the best written paper I’ve read, and I had some difficulty figuring out exactly what was going on in places, but there is some interesting stuff in there nonetheless.

The authors investigated six recently evolved new ?protein-coding genes in Drosophila. They wanted to know how they came about and managed to stick. For example, did they first originate as non-coding RNA genes? Did they gain a function through their RNA copies alone before they began to encode a protein? Or did they first awaken from the no man’s land between old genes with protein-coding potential already present?

This harkens back to one of the papers about new genes that I’d previously discussed. Xie et al. (2012) found that the genes for several human-specific proteins began life (and function?) as RNA genes expressed in particular tissues in ancestral primates. What about the six fly genes the new study investigated?

Reinhardt et al.‘s illustration of the two routes to protein-coding geneness is below. Starting with an inactive stretch of DNA (black line), you need two things: (1) an “on” switch or promoter (green box), which causes the transcription of RNA (blue) from the region, and (2) a sequence that can be translated into a decent length protein (an open reading frame or ORF, pink box). These two can theoretically appear in either order.

Before we get into the meat of the paper, let’s borrow the Drosophila family tree from the 12 genomes project page:

D. melanogaster, third from the top, is the species that has been used for every variety of biological investigation for over a hundred years, and also the focus of this study. However, the other species were also used for comparison, to see exactly where and how the genes originated.

Five of the six genes had a relatively long history, with similar sequences being found in D. yakuba and erecta or even further out in D. ananassae. Three of them were not only there in those species, but could also potentially make a nice protein. In two genes, the sequence or part of it was recognisable all the way to ananassae, but it only had long sensible ORFs in melanogaster itself.

In terms of activity… well, first of all I think they screwed up Figure 2. Supposedly, the names of the species in which transcription of these genes was detected are bolded, but actually, all the names are bolded in all the trees, which doesn’t agree with what they say (or with the green dots signifying the origin of transcription in the same figure). Anyway, assuming the bolding was a mistake and the green dots are in the right place, it sounds like four of the six genes were already active in the common ancestor of melanogaster and yakuba or earlier, while another two were only turned on in the melanogaster/sechellia/simulans lineage.

The order of events varies from gene to gene: four genes had good solid ORFs right from the start, while two were transcribed before they were suitable protein templates. The authors note that we can’t actually be sure whether or not the first four developed an ORF before they became active. To be certain of that, we would need more distantly related species with a matching ORF that isn’t transcribed, but in all four cases the species lacking expression of the gene also totally lack any trace of the sequence. So, while the remaining two genes provide positive evidence for the transcription-first scenario, the jury is still out on the ORF-first option.

In D. melanogaster, the presence of the protein product was confirmed for the four genes with the oldest ORFs. The two youngest may still be translated: the protein data came only from embryos, and in fact all six genes contain short signals that are normally associated with the transport of proteins to specific parts of the cell. You might reason that a gene that never makes a protein doesn’t need such signals, but nevertheless, the authors couldn’t positively confirm the existence of these proteins without data from other life stages.

Where these genes are active brings us back to a common theme we encountered in the previous post. In adult D. melanogaster, all six are most strongly expressed in the testicles, and the products of one of them are exclusive to those organs. Likewise, male larvae show more expression of all six genes than females do. The other species show basically the same pattern.

What do these genes do? Actually, do they do anything? Being expressed, even being translated to protein, doesn’t necessarily equate to having a function. Luckily, “function” is not terribly difficult to test for in fruit flies. There are lots of clever tricks that allow you to manipulate their genes and look at the consequences. In this case, Reinhardt et al. bred flies where these genes were turned off. If I understood them correctly, they managed to do this for five genes, four of which resulted in very dead flies. Weirdly, for all four, the affected flies died at the same life stage, just before hatching from the pupa.

With a different strategy that produced only partial knock-down of the genes, they got themselves some grown-up survivors, which allowed them to test the effect of the genes on male fertility (a sensible question given where these genes are most active). Out of three knock-downs with surviving adults of both sexes, only one showed a serious effect, and that was the one that produced generally crappy, short-lived weakling males anyway, so while these genes are active in the testicles and they might disproportionately affect males, they don’t seem to have much to do with fertility per se.

In general, the results sound like new genes that come from random bits of DNA can very quickly become essential to the organism, and it also sounds very much like an overabundance of transcripts in the testicles doesn’t mean that that’s where their function lies – it’s probably more that all kinds of things are expressed in testicles, and these genes are still expressed there because that’s how they started their lives.

Something big missing from the study is actually testing when these genes became functional – we’re told when they became expressed and when they started making a protein, but without manipulating them in relevant non-melanogaster species, it’s impossible to tell whether either of those means function. *disappointed pout*

And what’s up with those four genes that were necessary for the flies’ survival? The knock-downs all did their killing at the same stage. I don’t know what to think about that, and the authors don’t really offer an explanation beyond describing control experiments to make sure the deaths weren’t an unfortunate side-effect of the manipulation itself. Is there something about the development of adults that attracts new genes? Is the process of metamorphosis especially sensitive to even minor mess-ups? (More sensitive than early embryonic development?) Intuitively, I’d find the first possibility more likely, but gods know intuition is a poor guide to reality…

***

References:

Reinhardt JA et al. (2013) De novo ORFs in Drosophila are important to organismal fitness and evolved rapidly from previously non-coding sequences. PLoS Genetics 9:e1003860

Xie C et al. (2012) Hominoid-specific de novo protein-coding genes originating from long non-coding RNAs. PLoS Genetics 8:e1002942

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