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Hypothetical CHARMs Meet Occam’s Razor

May 10, 2011

In this post, biopolyverse continues the previous theme of hypothetical biological forms going under the coined acronym of CHARMs (Cell-Harbored Autonomously Replicating Molecules in the most fundamental and broad circumstances; Cell-Harbored Autonomous RNA Molecules in the scenario regarded as most feasible). These molecular entities would in effect be (perhaps among other activities) self-replicating ribozyme polymerases. Here, I offer criticisms of the notion, which takes us on a somewhat philosophical journey at times. When a hypothesis is raised, evidence for its falsification should be sought, if that can be found…..

Putting CHARMs to Rest?

Problems with the CHARM hypothesis can be placed in three broad categories:   (1) How CHARMS could avoid being ‘seen’ by their host cells; (2) Problems with CHARMs co-ordinating their replication with the host cell cycle; and (3) The evolution of CHARMs towards greater replicative efficiency, which might select against CHARM self-replication. These issues would exist even if a CHARM was purely commensal (causing no significant harm) in its host cell.

(1) How CHARMs Could Induce Alarms

To control intracellular viral parasites or foreign transposable genetic elements, complex organisms must evolve ways of distinguishing such unwanted invaders from their own legitimate biosystems. This comes down the classic ‘self / non-self’ required of immune systems, only taken down to the molecular level. One ‘non-self’ feature which can alert host recognition mechanisms is double stranded RNA, generated during the life-cycles of diverse RNA viruses, or through host mechanisms acting on single-stranded RNAs transcribed from mobile DNA elements. Such double-stranded RNA activates RNA interference (RNAi) pathways, leading to cleavage of corresponding single-stranded (foreign target) RNA from which the activating RNA duplexes were derived. This effect is now routinely used for directed ‘knock-down’ of any gene of interest, through the artificial transfer or in situ generation of specific siRNA (short interfering RNA) duplexes. (This was alluded to in the previous post, and also earlier). If the double-stranded RNAs are long enough (>25 bp), another arm of innate immune responses is activated in vertebrate organisms, that of the antiviral and immune-activating interferon proteins.

As a consequence of their hypothesized self-replicative function, a single-stranded RNA CHARMs would necessarily possess a complex folded structure, with numerous (although not necessarily long) regions of duplex formation. But even if this was not enough to act as a signal to host systems, CHARM replication itself would involve transient formation of RNA duplexes (as shown in a diagram in the previous post), and a clear opportunity for flagging of recognition to the host cell.  Endogenous RNA molecules are constantly turned over by degradative enzymes (ribonucleases), so CHARMs would also need to either escape their attention somehow, or replicate at a rate which maintained their survival over a constant loss through ribonucleolytic degradation.

The prospects for a newly invading CHARM to escape host detection (and probable elimination) would not seem very good. But a CHARM with a very long evolutionary history within a host organism might have attained some form of ‘accommodation’ with its host, or a devious means for evading host recognition. Given the ingenuity of viral and viroid systems, this restriction is not necessarily a fatal flaw, but it does mandate another level of complexity into the hypothesis.

(2) CHARM Replication Rates

To become established as a stable commensal replicator, CHARMs would need to co-ordinate their own replication rate with their host cell’s division cycle. If CHARM replication significantly exceeded that of its host, a continuous elevation of CHARM copy number would occur with each host cell division generation, which would be likely to eventually result in detrimental effects. (That is to say, an observable host cell disease phenotype would tend to occur). On the other hand, slower CHARM replication than its host division cycle would result in eventual loss of the CHARM replicator. (Daughter host cells would partition without any CHARM copies).

This question of replication rates brings up an issue not specifically dealt with until now: the type of host cell involved. While prokaryotic cells (bacteria and archaea) vary greatly between species in their average generation times, when nutrients are not lacking cell division can occur in as little as 20 minutes. Complex eukaryotic cells, on the other hand, typically require much greater time periods to complete division – sometimes on the order of days. Clearly, a CHARM which was intrinsically slow in its replication would be ‘left behind’ in a fast-growing prokaryotic environment, but perhaps not in a eukaryotic system. This issue brings to mind arguments regarding the origin of genetic intervening sequences, or introns (referred to in passing in a previous post). Such intronic sequences are very common in eukaryotes, but relatively rare in prokaryotes. (In particular, prokaryotes lack the complex enzymatic machinery – the ‘spliceosome’ – necessary to excise the spliceosomal class of introns). The ‘introns early’ school of thought holds that introns were a universal feature during very early cellular evolution, but were largely lost from prokaryotes. Why should that be the case? One opinion holds that with large fast-growing bacterial populations, even a very slight fitness loss owing to introns would favor their loss. With relatively small populations, this pressure would be significantly less in complex (especially multicellular) eukaryotes. Also, in eukaryotes there are clear precedents where intronic sequences have acquired certain useful functions (such as harboring specific genetic control elements), which would favor their preservation. So, if CHARMs were hypothesized to be hold-overs from remote times, then complex eukaryotes might be the best place to consider finding them, by analogy with the ‘introns-early’ theory.

An orthogonal CHARM would be definition be decoupled from communication with its host cell replication cycle. But unless by some exceedingly fine chance its inherent replication rate happened to be ‘just right’ for matching its host, the above arguments suggest that complete decoupling (and hence complete orthogonality, as argued previously) would be essentially impossible. So some kind of feed-back regulation of CHARM replication would be required, necessitating at least a minimal degree of CHARM-host interaction. Again, this is not an insuperable theoretical problem, but dictates another functional constraint.

(3) Evolving CHARMs

Some simple Darwinian principles can be applied to hypothetical host cell-CHARM evolutionary relationships. The term ‘commensal’ might seem to imply that while the occupying replicator of such a description has no deleterious effect on its host, neither does it have any negative effect. (In other words, the commensal agent is truly neutral). In practice, with a complex cellular metabolic balance-sheet, true neutrality in such circumstances may be very difficult to approach. And even a slight decrease in a host cell’s fitness would provide a selective advantage to competitor cells of the same type lacking CHARMs, and lead to the eventual displacement of their CHARM-bearing counterparts. This in turn would lead to the extinction of CHARMs unless they possessed some means for re-infecting new cell hosts. Of course, CHARM maintenance would be actively selected for if the opposite circumstances applied, where a ‘CHARM+’ phenotype provided even a slight survival or propagation advantage.

But evolutionary pressures would also be equally applicable to CHARM replicators themselves, although there are multiple factors to consider. A CHARM variant which replicated more efficiently would superficially seem at a selective advantage, but ultimately not if such a replicative boost killed its host cell. But since protein catalysis is usually more efficient than that achievable with RNA, a strong pressure might exist for CHARM self-replication to be superseded by reliance on host polymerases, with loss of then-superfluous RNA sequences mediating self-replication. (Such a process is analogous to the subsuming of once-independent organelle functions to the host, as noted in the previous post. And in such circumstances, the replicating RNA is no longer fully autonomous, and thus loses its ‘CHARM’ status. The product of this hypothetical CHARM-streamlining process has points of similarity with viroids, as previously considered.

If CHARMs conferred even marginal host benefits, over evolutionary time it might also be expected that the relevant RNA sequences would tend to be acquired as genomic host copies through reverse transcription events, where expression and regulation might be more efficient. Still, as noted in a previous Table, the existence of a genomic copy of a CHARM sequence would not formally rule out the possibility that a self-replicating CHARM could still be expressed in the same cell.

Apart from the above theoretical considerations, what might be done experimentally – at least in principle?

Testing For CHARMs?

In biology, a novel agent with even a truly neutral phenotypic effect may be revealed through modern intensive screening approaches, provided it falls within the net for what is being screened in the first place. This brings to mind the so-called ‘First Law of Directed Evolution’, which states, “You Get What You Screen (or Select) For”. In other words, you can only expect to find what your evaluation system will allow, and things may escape one’s detection if the system is improperly designed. There are both obvious and more subtle aspects of this dictum. In the ‘obvious’ sphere, a cellular screen based on RNA sequencing could not identify an unknown entity based on an entirely distinct informational macromolecule.

Within the less obvious domain, let’s take a look at a technology referred to in the previous post, high-throughput sequencing. Here, given very powerful means for rapid sequence determination and computational analyses which were unheard of a decade age, the problem can be in principle approached along the following simple lines: (1) Obtain a complete read-out of all RNA sequences present within a cell of interest; (2) Compare these sequences with the entire DNA genome of the same cell; and (3) Find any RNA sequences which are not represented within the host genome. If any of the latter are found, they are then isolated (by standard nucleic acid amplification procedures) and further analyzed. Any such confirmed candidates would certainly not automatically be CHARMs (they could be adventitious viral sequences, for example), and would only pass the final test if demonstrably capable of ribozyme-mediated self-replication. Easy enough? Well, a first caveat is to ensure that the RNA sampling is indeed global. Many studies have been performed towards cataloguing all cellular polyA+ RNAs, since these include almost all mRNAs expressed from functional genes, and certain non-coding RNAs as well. Still, non-biased RNA sampling can be instituted. In such cases, the great bulk of material by mass is ribosomal RNA, but if necessary such sequences can be physically depleted from the remainder through specific hybridization procedures. A more significant problem arises if (as noted above) a CHARM sequence is ‘mirrored’ by a genomic copy, even if such a copy is a non-functional (but closely similar) ‘pseudogene’. In such a case, the CHARM would no longer stand out as a non-genomic entity, and would merely fall into the large and growing class of non-coding RNAs.

What about other approaches? If the assumption is that a cellular RNA could self-propagate, then whole cellular RNA preparations (from a large number of potential sources) could be incubated in vitro with suitable buffering, protection from ribonucleases, and appropriate supplements. (The latter being ribonucleotide triphosphates in particular, but also metal ions and perhaps some simple organic cofactors). If it was postulated that CHARM self-replication required certain host protein assistance (perhaps RNA chaperones to assist folding of newly formed CHARM molecules), then a whole RNA-cellular protein mix could be employed. In a prolonged incubation, the mix could be regularly supplemented with fresh low-molecular weight material, or precipitated and reconstituted with fresh solutions. Whatever the conditions, a positive result would predict an enrichment of specific CHARMs at the end of the designated incubation period. How to detect them, given that such an ‘amplification’ might still be a very small blip against a very large general RNA background?. The solution would likely have to rely on a nucleic acid subtraction process (see References & Details).

All very well as armchair experiments…..but as in so many cases, since experiments can fail in so many ways, only a positive result would be useful (While very interesting!). With so many other things to do, it would be nice to have some firmer grounds for conducting such a search in the first place. And the caveats noted above are not encouraging.

Making Biological Predictions

There are many precedents in physics where theoretical predictions have been validated through real-world findings, most notably the observational confirmations of predictions made by Einstein’s Theory of Relativity. Although the complexity of biology renders this a much more problematic exercise, there been a few notable successes. An important one that comes to mind was the prediction by Francis Crick of an RNA adaptor (transfer RNA) in the ‘reading’ of the genetic code during protein synthesis. Systems biology (as discussed in the last post) is yet very limited in its predictive power, meaning that hypothetical entities such as CHARMs cannot be easily dismissed purely by in silico models. But on the other hand, if the existence of CHARMs does not explain known phenomena, the CHARM hypothesis itself can be challenged by invoking a simple and very old principle…

Qualms about CHARMs from Occam’s Razor

William of Occam (sometimes spelt Ockham) was an English medieval cleric and scholar who formulated a simple rule which has been inspirational in many fields. Although described in a number of different ways, it can be concisely presented as “Complexity should not be assumed unnecessarily”  (In the original, ‘complexity’ is usually given as ‘plurality’).  This principle has come to be known as ‘Occam’s Razor’, and is sometimes also known as the ‘Law of Parsimony’. To more modern ears, it might sound similar to the wise words, ‘If it ain’t broke, don’t fix it’, with respect to physical contrivances. In medicine, another somewhat analogous adage is ‘If you hear hoofbeats outside, think horses before you think zebras’ (simple and common explanations for a patient’s symptoms should be considered and ruled out before more exotic possibilities are entertained).

Could the received history of William of Occam himself be an illustration of applying Occam’s Razor? William is believed to have died in 1349 in Munich, where he had lived for many years as a guest of the emperor Ludwig of Bavaria. All things considered, the cause of his death cannot be stated with any confidence. Yet at that time, the bubonic plague (black death) was killing vast numbers of Europeans, and many sources simply state that William of Occam fell among the millions of victims of Yersinia pestis, the responsible bacterium. Are they in effect formulating the simplest hypothesis which fits the known facts to account for William’s demise? (In effect applying the Razor to the demise of poor William himself?).

But to return to biology, a hypothesis can be put forward to explain observations as they are available. From ancient times, an explanation for life invoked a mysterious ‘vital force’, more or less equivalent to the élan vital proposed by the philosopher Henri Bergson in the first part of the 20th century. In a similar vein, Chinese traditional medicine considers mysterious qi energy (and its ‘meridian lines’) as the basis for acupuncture. Old or not, such once-acceptable explanations now fall by the wayside in the light of modern molecular and cellular biology. But it is impossible to comprehensively disprove something as nebulous as élan vital or qi. With modern science, these notions dissolve into ghosts, and how can one prove (especially to the satisfaction of a ‘true believer’) that ghosts do not exist? But here Occam’s Razor simply rules that, lacking any evidence, both élan vital and qi are ‘unnecessary complexities’ and can be dismissed from any rational arguments.

Are CHARMs to be similarly cast aside? They certainly could be presented as a complexity tacked on to existing biosystems without any (as yet known) experimental justification. (Some might say, Is not Life already complex enough?) Also, when additional levels of complexity have to be introduced to sustain a logical proposition (such as the ‘ingenious means for evading host recognition’ by CHARMs noted above), then the case for invoking Occam’s razor becomes stronger.

And yet CHARMs are rather more respectable than ghosts. Unlike élan vital and qi, there is a broad scientific consensus that something like a CHARM must have once existed, in the form of a ribozyme self-replication during the existence of the RNA World. And some relics of this world certainly do persist, in the form of diverse ribozyme activities, some of which (such as RNase P and the ribosomal ribozyme peptidyl transferase) are absolutely essential for life. While these ribozyme activities thrive within modern cells, they do not directly provide any evidence towards the co-survival of CHARMs, for which the situation is undoubtedly rather different – as the conclusions of this post tend to bear out.

But this is not definitive either way. If, for the sake of argument, we accept that practically all the information required for a systems interpretation off biology is ‘out there’, it does not exclude the possible existence of other bio-entities existing in parallel or outside the defined system. And this is in line with a proper interpretation of Occam’s Razor, which was primarily applied to the world of logic and ideas. Real entities could indeed show apparent unnecessary complexity, attributable (in William’s day) to divine omnipotence. Let’s briefly examine this…..

Does Complex Biology Sometimes Dull Razors?

Occam’s Razor stands as a formal warning against invoking complexity when there is no need based on experimental data. But knowledge limitations themselves indicate that at least some hypothetical entities cannot be dismissed simply because they are not ‘needed’ as such. In the previous post, the complexity and seeming ad hoc nature of biology as a product of evolutionary accretion of a ‘parts list’ was discussed, in the context of the young science of Systems Biology. In essence, it remains exceedingly difficult to separate evolutionary constraints and history from functional necessity in the context of complex biosystems. Thus, it is possible that gene regulation by small RNAs may not be ‘needed’ from first principles (efficient protein-only systems might be designable with an advanced systems / synthetic biology), but they are certainly with us.

So while Occam’s razor might insist (if enough information was available) that protein-based gene regulation would suffice to explain biosystems, the reality is not so simple. Biology may thus provide examples of mixed systems which owe to evolutionary history at least as much as to efficiency of design. Therefore, the ‘simplest’ explanation is not necessarily the best, although we must careful exactly what we mean here. Certainly the simplest explanation from first principles might be the best (that is, use all-protein regulation if designing from the ground up), but this is not how biology is derived. If one already has a functional RNA repertoire as well as an evolving protein-based capacity, then perhaps the existing protein / RNA regulatory situation is the most efficient compromise with the tools available.

At very least, this exercise serves to demonstrate the determining the ‘simplest’ arrangement for an intricately organized system is itself anything but simple. In principle, a simplest-possible solution might have practical constraints which prevent it from being attained through biological evolution (although not necessarily through artificial intervention). Therefore, over-enthusiastic application of Occam’s Razor in biology may well backfire. Francis Crick himself (noted above for the perspicacity of his biological predictions) cautioned against Occam’s Razor as a ‘dangerous implement’ in biology, going on to say “It is thus very rash to use simplicity and elegance as a guide in biological research”. Indeed.

Once again, a parallel with the ‘First Law of Directed Evolution’ (‘You Get What You Screen / Select For’) can be made. With the exception of current ‘global’ biological projects noted above, biology has quite rightly normally focussed on specific targets and goals, in order to cut through the thickets of bio-complexity. For example, the specificity of antibodies renders them very valuable tools for ‘pulling down’ proteins or other factors associated with the antibody target itself, eliminating a vast amount of irrelevant biological ‘noise’. But the flip side of this is: if you don’t specifically look for something, you may be blind…and fail to see CHARMs or other ‘dark’ bio-entities lurking at the fringes of the known.

An analogy can be made with the Search for Extra-Terrestrial Intelligence, or SETI. This noble undertaking is also based on a hypothesis for as-yet undescribed entities (intelligent aliens). Here the underlying assumption is that contact with such intelligent species could be made by radio-communications using the technologies available with radio-astronomy. Recently, the lack of success to date has prompted more thoughts as to whether advanced societies would continue to use radio waves for informational transmission, in lieu of something else. Whatever that might be, if it was true, the same principle as above would hold. Seek, and ye shall find – but only if your eyes are attuned to the right wavelength, whether literally or metaphorically.

To conclude, a versical salute to William. Despite the important qualifications made by Crick and others, the Razor can still cut through much higher-level biological dross….

 William of Occam, be my friend

Abide with me until the end

You cannot have a stauncher praiser

I bow before your mythic razor

Long may its blade the truth defend.

(Judiciously applied, of course!)

References & Details

(in order of citation)

‘ ….. or through host mechanisms acting on single-stranded RNAs transcribed from mobile DNA elements.’   For a review of small RNA-based defenses, see Malone & Hannon 2009.

‘…..another arm of innate immune responses is activated ‘   ‘Innate immunity’ refers to a diverse range of responses (to foreign replicators) which are genomically ‘pre-programmed’. This can be distinguished from the adaptive immune systems of vertebrates, where processes of diversification, selection and amplification lead to the generation of immune receptors tailored to invading pathogens or molecules, including those which could never have been anticipated in advance.

‘……the antiviral and immune-activating interferon proteins.’   The antiviral interferons (IFNs) are grouped into three classes, Types I, II and III. There are multiple types of Type I proteins, of which IFN-α (13 different forms in humans) and IFN-β are the best known. Only one Type II representative (IFN-γ) is known, which is also a very important mediator in the immune system. The most recently discovered is Type III, of which IFN-λ is the representative. See Pestka et al. 2004 and Donnelly & Kotenko 2010.

‘……ingenious means for evading host recognition.‘  By the same token, a long evolutionary relationship can allow time for host counter-measures to evolve. And in turn, counter-counter measures by the foreign agent… or an evolutionary ‘arms race’ as documented in a great many circumstances. Such ‘arms races’ have been discussed by Richard Dawkins in his books, including The Blind Watchmaker (1988) Penguin Books, London.

‘…introns…. relatively rare in prokaryotes.’    The paucity of introns in the prokaryotic world, in strong contrast with eukaryotes, is a problem intimately connected with the origin of introns in general. In the strong version of the ‘introns late’ theory, the issue does not arise, since by this logic introns only arose to a large extent after the divergence of eukaryotes. Compromise versions of the early/late opinions have also been put forward incorporating features of both (see Koonin 2006). Alternatively, certain endogenous processes (that is, not mediated by foreign selfish mobile elements) have been postulated as governing the preponderance of introns in eukaryotes (see Catania et al. 2009).

‘…..were largely lost from prokaryotes…’   For more detail on the importance of organismal population size and intron loss, see Lynch 2006a; Lynch 2006b.

‘…the ‘spliceosome’ – necessary to excise one the spliceosomal class of introns…’    The spliceosome is a large ribonucleoprotein complex, which may in part operate through RNA-based catalysis (See Valadkhan 2007).

‘…..introns early’ school of thought….’  For an overview of this area, see Penny et al. 2009.

‘…..First Law of Directed Evolution….’   This ‘Law’ was noted in a report of scientific meeting on directed evolution in 1999 by Schmidt-Dannert & Arnold.

‘…..these include almost all mRNAs expressed from functional genes.’  In multicellular animals, histone mRNAs are not polyadenylated (see Marzluff 2005)

‘……certain non-coding RNAs as well.’  For a review of non-coding RNAs which includes polyadenylated forms, see Goodrich & Kugel 2009.

‘….some simple organic cofactors…’   Vitamin-like organic ‘coenzymes’ as catalytic cofactors are widely found among protein enzymes. The fact that many of these coenzymes are nucleotides (or derivable from nucleotides) led to an early proposal that these resulted from an earlier world of nucleic acid-based catalysis (White 1976), since supported by the discovery of RNA catalysis and the proposed RNA World.

‘…nucleic acid subtraction process…’ This kind of subtraction process ideally requires two sources which differ only in a target gene product of interest. If RNAs from the a control source (target gene -) are subtracted through a hybridization-based process from a target source itself (by definition, target gene +), then theoretically the remainder is just the sought-after target gene. In practice, of course, this is an enrichment process rather than an absolute purification, but it has been very productive in molecular biology (an example that comes to mind is the cloning of the T cell receptor by Mark Davis and colleagues [Hedrick et al. 1984]). In this armchair CHARM scenario, the control source would be a sample of the starting RNA and the ‘target’ source would be incubated RNA which had had opportunity for putative self-replicators to do their thing. Many more practical details would need to be added to this speculative experiment to render it capable of delivering the desired outcome, in principle.

‘…..the prediction by Francis Crick of an RNA adaptor…..’  Crick wrote this proposal for consumption by his colleagues, and it became influential despite not having been published in a normal scientific journal. This is described in Crick’s book What Mad Pursuit: A Personal View of Scientific Discovery (1988; Basic Books, New York).

William of Occam……was a medieval cleric….’   For more details, see Wildner 1999, or alternatively the full-text version from the Lancet source.

‘….to have died in 1349 in Munich… a guest of the emperor Ludwig of Bavaria.’   See Manfred Wildner’s comments on the 650th anniversary of William’s death in 1999.

‘…..the philosopher Henri Bergson…’ The French philosopher Henri Bergson (1859-1941) was very influential in his time, beyond his promulgation of the élan vital principle.  Ironically, he was born the same year that Darwin’s Origin of the Species was published.

‘…Chinese traditional medicine considers mysterious qi energy… the basis for acupuncture….’

For an overview of traditional Chinese explanations for acupuncture (qi energy and meridian lines of qi, etc. See Snake Oil Science by R. Barker Bausell (Oxford University Press, 2007). As an aside, this book should be required reading for anyone contemplating designing a clinical trial for the first time.

‘…..unnecessary complexity, attributable (in William’s day) to divine omnipotence.’   Wildner 1999.

‘…Francis Crick himself …..cautioned against Occam’s Razor….’   The Crick quote comes from the same book as noted above (What Mad Pursuit: A Personal View of Scientific Discovery (1988) Basic Books, New York)

An analogy can be made with……..SETI.’   SETI has been brought to public attention in considerable part by books and articles by one of SETI’s directors and leading proponents, the astronomer Seth Shostak (For example, Sharing the Universe ; Berkeley Hills Books, 1998). Sadly, SETI has recently become a victim of budgetary cut-backs.

An important distinction between SETI and any search for biological unknowns (whether CHARMs or anything else) is that biological knowledge is in principle a closed set (as discussed in a previous post), while (in principle, if certainly not in practice), SETI could continue indefinitely, ranging further and further across the cosmos until intelligent life was finally found…and then continue on to the next contact. But the two aims actually overlap, because if SETI was successful even once, by definition, at least one other biology (a Life X.0) would thereby exist. This would immediately broaden the meaning and scope of biology as it is currently understood.

Next post: Three weeks from now.

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