Tuesday, May 13, 2008

Some possibilities for novel mutation-checking mechanisms

Nature has set up a number of well-known mechanisms for keeping mutations in check. The first that comes to mind is the one by which mismatched base pairs are recognized, with the older, methylated strand serving as template. Despite these mechanisms, mutations do slip by at the rate of about 1/10^8 to 1/10^9 base pairs per generation1 in the human population.

Stepping into the realm of science fiction, one can try to imagine improvements. One could imagine an organism utilizing triple-stranded DNA. If such an organism actually existed, mismatches would easily be recognized because only 1 of the 3 strands would contain an error. The two consensus strands would be assumed to be correct, and the error in the other would be repaired.

More realistically, one might imagine a system that somehow compares DNA strands in cases where a gene has two or more copies. If a mismatch is found, the cell might be directed to apoptose. Up until recently, the comparison of two strands seemed a bit fanciful. However, with "RNA interference" and "RNA activation", we know of mechanisms whereby two strands from disparate locations in the genome may be compared.

In the case of RNAi, an mRNA that pairs with a smaller RNA strand (miRNA or siRNA) may be degraded or otherwise "silenced" in the cytoplasm. An RNAi mechanism that seeks out slight mismatches (via a single base pair difference) and then instructs the cell to self-destruct or fail to thrive is unknown. Such a mechanism is not horribly fanciful...it is well-understood that perfectly matching targets, imperfectly matching targets, and non-matching targets can have different fates.

In the case of RNA activation (RNAa), however, we have a known mechanism by which strand comparison can occur...instead of silencing, two properly matched strands interact to promote translation2. Mismatched strands result in poor translation, which may then hamper cell viability.

Should such an error checking system become established, what consequences might result? In addition to negative and neutral mutations, positive mutations would be minimized. On one hand, organisms inhibited from positive mutations will tend to be outcompeted over the long haul. On the other hand, genes love to be replicated with full fidelity. We have a battle, worthy of mathematical modeling, between the drive for fidelity and the need for improvement. Is it possible that the extinction of some species is being driven by such overactive strand-comparison systems? Conversely, rapidly evolving species might see a dearth of such mechanisms.

It has been documented that the deletion or alteration of some ultra-conserved DNA results in no obvious negative effects to the organism3. This seems odd, until one considers the possibility that the extraordinary persistence of such DNA may be due to an efficient strand-comparison system, not because it has any particular function, or because the existent version is tremendously better than any others. Such mechanisms do not benefit the organism and may be thought of as "ultra-selfish".

Determining whether ultraconserved sequences might be targets for "strand comparison" might be a tad tricky. When knockouts of ultraconserved sequences result in no apparent harm to the organism, strand comparison is a possibility. Computationally, in the relatively few cases where an miRNA targets an exon, one might expect that every third base pair in a reading frame would have a lower mutation rate than would be ordinarily expected. Conversely, sequences that are ultraconserved via the importance of various protein domains for which they code would tend to have a higher mutation rate in the third base pair.

Known cases of RNAi/a are restricted to relatively small strands of mature RNA (19-23 nt). Thus we have no means of ensuring the copy-fidelity of an entire gene via a single miRNA. There are cases, however, where multiple miRNAs target different areas of a gene.

In animals, miRNA's are often found in ultra-conserved introns4. This conservation may be explained as simply as follows: if a mutation forces a mismatch between the miRNA and its target, the germ cell fails to thrive. Note that the mutation may actually be beneficial, but is eliminated. Again, this points to the possibility of "ultra-selfish" RNAi/a.


Another sort of hypothetical mutation-detection system might involve tying sperm function to proteins that are used in other tissues. Imagine, for example, that a protein necessary for brain function is also necessary for sperm motility. The brain disfunction associated with this mutation is never seen because sperm carrying this mutation never reach their destination.

Proteins that have important functions in a wide variety of tissues might not be under pressure to be tied into sperm function. Why? Because even if the sperm reaches its destination with these lethal mutations, the embryo will not be viable. Little parental investment has been lost. On the other hand, genes whose effects might only manifest later in development might be candidates for "sperm function pleiotropy".

Genes tied to brain function, then, would seem to be likely candidates for this sperm function linkage. A good deal of parental investment may have been incurred by the time it is understood that a child is mentally handicapped to the extent that his/her chances for reproductive success are strongly diminished. It's interesting to note that some computational studies have shown a high overlap between brain mRNA and testis mRNA5.

Other studies have not shown a strong brain mRNA/testis mRNA overlap. My own perusal of gene expression data leads me to believe that "sperm function linkage" occurs only to a limited extent. Some of the inferred overlaps between testis and brain expression may be more related to the fact that neither tissue is particularly secretory, and both tissues maintain degrees of immune privilege and blood barriers. In other words, many of the observed similarities may be more a matter of what isn't expressed, than what is. Good candidates for "sperm function linkage" might be transcription factors, which needn't be expressed in large quantities, or miRNA's, for which no public tissue-specific data seems to be available at the moment.

One might object that males are typically profligate with sperm, and would not be under any additional evolutionary pressure to screen out, say, DNA that carries a brain-development mutation. This is, however, only true to the extent that the male of the species does not invest in the child's upbringing.

The linkage between sperm function needn't only relate to, say, the construction of flagellum. It could also involve all sorts of proteins involved in spermatogenesis. In fact, we only need to overlap one important early function with a later developmental function to reduce the likelihood that a mutation would be preserved in further generations . Here, we have a pressure that would tend to favor reuse of proteins, rather than the creation of novel ones. Sperm would be under particular pressure to adopt these overlaps because we here have a mechanism whereby mutations are eliminated before any parental investment.

Some candidates for "sperm function linkage":

206581_at: basonuclin
209443_at: serine (or cysteine) proteinase inhibitor, clade A (alpha-1 antiproteinase, antitrypsin), member 5
220744_s_at: WD repeat domain 10
207144_s_at: Cbp/p300-interacting transactivator, with Glu/Asp-rich carboxy-terminal domain, 1
220110_s_at: nuclear RNA export factor 3
214183_s_at: transketolase-like 1
206310_at: serine protease inhibitor, Kazal type 2 (acrosin-trypsin inhibitor)

The extent to which sperm cells are subject to apoptosis is notable, with apoptosis sometimes occuring in "waves"6. The most common explanation of this phenomenon is that the apoptosis maintains a crucial ratio of Sertoli cells and pre-meiotic germ cells, but the issue is far from settled7. Of the 50 genes under strongest positive selection in the human genome, a good number code for sperm functions"8. Could some sort of "weeding out" process be involved here? With somatic cells, mutations can be masked via diploidy. Conveniently, this isn't the case with spermatids, which are haploid. The task of mutation-checking is thus simplified.

Note that, unlike the "strand comparison" system surmised above, beneficial mutations aren't necessarily entirely eliminated in sperm-function linkage. However, it is known that testis-specific mouse genes do evolve quite a bit more rapidly than testis genes that are also expressed elsewhere9. In other words, these genes may have more freedom to mutate because of the absence of pleiotropy.

The overlapping proteins might have structural importance for sperm function, but they might also simply activate other enzymes in a chain. Oddly, in sperm function linkage, we have a mechanism by which evolution may favor a degree of complexity that would otherwise seem unnecessary. Complexity is favored because the individual enzymes in a long chain are "tested" along the way. Here, some proteins or substrates necessary for sperm function are only produced through Rube Goldberg contraptions. However, the gun that's fired as part of a ludicrously long chain of events that results in the garage door being opened is perfectly useful when you want to shoot a thief. Including the gun in the Goldberg device means that it's likely to function correctly when you need it.

As a side note, it's interesting that "intelligent design" proponents are fond of citing cilia as an example of the sort of assembly that couldn't possibly be a result of natural selection. In addition to the usual, powerful arguments against this position, I'd argue for the possibility that evolution is doing its damnedest to make the process of creating functional sperm as ridiculously complex as possible.

One PLOS paper details a study of pleiotropy in C. Elegans, finding that 50% of genes involved in embryogenesis are pleiotropic. What's more, these genes tend to occupy central (rather than initial or final) positions in protein pathways10. Pleiotropy, when viewed in the light of evolution, is usually discussed as a constraining factor. That is, a single mutation may be beneficial or neutral in one tissue, but harmful in another, increasing the likelihood that pleiotropic gene mutations would be selected against. The effect of pleiotropy needn't always be one of constraint, however. For example, in the case of parsimonious pleiotropy11, "knowing" that a mutation that fails to harm sperm is also unlikely to harm the brain, the sperm may relax strict mutation checking controls a tad. Note that evolutionary pressures favoring pleiotropy tend to negate the popular notion of "modularity" in gene expression.

1. Rates of spontaneous mutation: http://www.genetics.org/cgi/content/full/148/4/1667

2. RNAa: Small dsRNAs induce transcriptional activation in human cells http://www.pnas.org/cgi/content/abstract/0607015103v1

3. More than 1 mb of non-coding DNA, much of it conserved between mice and humans, knocked-out: http://repositories.cdlib.org/cgi/viewcontent.cgi?article=3767&context=lbnl

4. Ultraconserved elements in insect genomes: A highly conserved intronic sequence implicated in the control of homothorax mRNA splicing. http://www.genome.org/cgi/content/full/15/6/800

5. In silico analysis indicates a similar gene expression pattern between human brain and testis: http://content.karger.com/ProdukteDB/produkte.asp?doi=10.1159/000076290

6. An early and massive wave of germinal cell apoptosis is required for the development of functional spermatogenesis. http://www.nature.com/emboj/journal/v16/n9/abs/7590214a.html

7. Involvement of apoptosis in the control of Sertoli and pre-meiotic germ cell numbers in the developing rabbit testis. http://www.blackwell-synergy.com/doi/abs/10.1046/j.1439-0272.2002.00464.x?cookieSet=1&journalCode=and

8. A Scan for Positively Selected Genes in the Genomes of Humans and Chimpanzees. http://biology.plosjournals.org/perlserv/?request=get-document&doi=10.1371/journal.pbio.0030170#JOURNAL-PBIO-0030170-B33

9. Rates of Protein Evolution Are Positively Correlated with Developmental Timing of Expression During Mouse Spermatogenesis http://mbe.oxfordjournals.org/cgi/content/full/22/4/1044

10. Systematic Analysis of Pleiotropy in C. elegans Early Embryogenesis. http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1000003

11. Seven types of pleiotropy. http://www.ijdb.ehu.es/web/paper.php?doi=9654038

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