Here, I report how the biological community reacted to my hypothesis and by what means scientists suggested to test its plausibility.
While my hypothesis was frequently cited in the biological literature with little or no comment, it was first critically examined in 1994 by Cheles-Flores [12], who pointed out that “a difficulty may be raised against the Diener hypothesis that viroids may be interpreted as molecular fossils of the RNA world,” in that viroids are known to exist only in angiosperms, whose first appearance was in the Cretaceous period. The author presented a scheme, based on cyanobacteria, which, after “extensive additional work by plant pathologists”, if successful, would “remove the importance, in the preservation of the relics of the RNA world, of the time of the first appearance of angiosperms” and thus show that “viroids could have been present during the major part of the duration of life on Earth.”
In a second 1994 paper [13], Chela-Flores expanded on these thoughts, but again asked the question whether it is possible to envisage a possible evolutionary pathway of the early replicators spanning the vast time span separating the first appearance of the angiosperms, late in the Mesozoic era (the Lower Cretaceous) from the most likely sub-eras in which the RNA world may have occurred, namely the Hadean/Early Archean. The author suggested that “through horizontal gene transfer, as well as through a series of symbioses in the precursor cells of the land plants, the genes of the replicases associated with RNA plasmids and other putative DNA-independent RNA replicators may have been transferred vertically, eventually becoming specific to the angiosperms.” However, no further report from Cheles-Flores has appeared; apparently, the proposed experimental work has not been performed or it was not successful.
In 1998, Jeffares. et al. [14] reported results of a theoretical study, in which the authors estimated—based on what they considered to be plausible parameters—which of many individual, extant RNAs may be relics of the RNA World and which are probably of more recent provenance. While the authors cited my 1989 hypothesis (as described in a 1993 book chapter [15]), they do not discuss it, or its relevance to their work.
Given the strong trend RNA --- > RNP --- > protein, Jeffares et al. examined the phylogenetic distribution of RNA in modern organisms for relics and thus developed a model for complexity in the RNA world. Candidates were RNAs which fit at least one of their criteria: “catalytic, ubiquitous (or at least conserved within the eukaryotic lineage…), or central to some aspect of metabolism.”
Most importantly, in the context of the present assessment, is Jeffares et al.’s conclusion that viroids, plant satellite RNAs, and “hammer heads” are, indeed, ancient relics of the RNA world. It is not clear, however, on which of the parameters this conclusion is based. Viroids are listed confusingly (together with “hammer heads”) at the bottom of table 2—titled “RNA functions in modern cells”— under the rubric “Function,“ as “Various,” under the rubric “Distribution” as “Plant satellite RNA,“ and under the rubric “In the RNA world?” as “In RNA world (see text)” but this referral in a footnote is not illuminating.
By the authors’ parameters, many, if not most, extant RNAs (or their precursors) were also already present in the RNA world, including precursors of the three major cellular RNAs: rRNA, mRNA, and tRNA.
A distinction must be made, however, between Jeffares et al.’s chosen criteria for relics and those chosen in my 1989 publication. Whereas Jeffares et al. developed their “model for the final complexity of the RNA World,”— just prior to the evolution of translation and proteins—it was actual properties of viroids, listed in 1989, which I considered to suit them for survival in a prebiotic “soup” far less hospitable than that envisioned by Jeffares et al., i.e., for an earlier stage in the RNA World.
Therefore, if correct, Jeffares et al.‘s results would not only be in accord with the relic hypothesis, but more accurately define the stage of the RNA World, in which viroids (or their precursors) could presumably have prospered. However, Jeffares et al.’s choice of parameters is, by necessity, subjective and any substitutions would likely alter the conclusions. Even given the existence of an ancient RNA World, there are problems with understanding how, without DNA or proteins, cellular life could have evolved. One of the major problems is the question as to how one and the same kind of RNA molecule could serve simultaneously as both information carrier and biocatalyst, which would require a combination of features: good “templating” ability (for replication) and stable folding (for ribozymes). This poses a paradox, because well folded sequences are poor templates for copying, but poorly folded sequences are unlikely to be good ribozymes [16].
In 2013, Ivica et al. [16] described a novel strategy to overcome this dilemma; it is based on G:U wobble pairing in RNA: Unlike Watson-Crick base pairs, wobble pairs contribute highly to the energetic stability of the folded structure of their sequence, but only slightly, if at all, to the stability of the folded reverse complement. Sequences in the RNA World might therefore combine stable folding of the ribozyme with an unstructured, reverse-complementary genome, resulting in a ‘division of labor’ between the strands.
The investigators demonstrated this strategy by use of computational simulations of RNA sequences (including 40 viroid sequences) and their folding as experimental models of early replication, “involving non-enzymatic, template-directed, RNA primer extension.” The investigators recognized the fact that “interestingly, viroid RNA sequences…show significant asymmetry in folding energy between the infectious (+) and template (-) strands due to G: U pairing, suggesting that this strategy may even be used by replicators in the present day world,” as well, as postulated, in the RNA world. If so, this viroid-suggested process should be amenable—beyond computer simulation—by experimentation with actual RNA molecules.
Also in 2013, Ma et al. [17] cited my 1989 paper and—“inspired by features of viroids,”—studied their properties in mathematical simulations. Ma et al. were particularly interested in determining whether the known structure of viroids, “their circularity and small, self-splicing ribozymes (e.g., the hammerhead ribozymes), could have been instrumental in helping them overcome problems in replication and stability.” Their study indicated “that an RNA chromosome can spread (increase in quantity and be sustained) in the system, if it is a circular one and its linear ‘transcripts’ are readily broken at the sites between genes; the chromosome works as genetic material and ribozymes ‘coded’ by it serve as functional molecules.” Ma et al. concluded that circularity and self-cleavage are important for the spread of the chromosome.” The authors concluded that “in the RNA world, circularity and self-cleavage may have been adopted as a strategy to overcome the immediate difficulties for the emergence of a chromosome (with linked genes).” While Ma et al. thus seemed to provide important evidence for the possible ancient nature of viroids, their conclusions are placed in doubt by the unknown significance of mathematical simulations to real-world evolutionary situations.
Forterre’s revolutionary proposal [18] to divide the biosphere according to organisms’ fundamental properties into two parts: capsid-encoding organisms (i.e., viruses) and ribosome-encoding cellular organisms. The author’s proposal is compatible with my 1989 hypothesis, except that viroids and other subviral agents belong to neither part, but must be accorded a new, third part, consisting of non-capsid, non-cellular life forms.
Theoretical studies [19] indicate that “selfish replicons (genetic parasites) inevitably emerge in any sufficiently complex evolving ensemble of replicators.” Indeed, genetic parasites seem to be truly ubiquitous: some such elements apparently are associated with all cellular life forms and mathematical models of the evolution of replicator systems—aimed at the reconstruction of the first stages in the history of life—invariably reveal partitioning into hosts and parasites [19]. It is therefore not surprising, that viroids, if viewed as survivors of the RNA World, would not be self-replicating, but would, like viruses, depend on host enzymes for their (autonomous) replication.
Koonin and Dolja [20] studied “the evolutionary relationships between typical viruses with different replication-expression strategies and capsidless genetic elements,” on the basis of which they proposed a paradigm of virus-world evolution that is in accord with Forterre’s model. The authors stated that “host-parasite arms races are a major formative factor in all evolution of life” and that “the simplest genomic parasites might be small RNA molecules that encoded no proteins and consisted primarily of cis signals for replication.” Koonin and Dolja [20] also described features of hepatitis delta virus (HDV), which “appears to be a derivative of a viroid that encodes a protein required for replication and virion formation, and is encapsidated into particles that consist of the capsid protein of the helper Hepatitis B virus” and that “most likely HDV evolved from a viroid-like ancestor by acquiring a protein-encoding gene from a still unknown source and adapting to use the capsid protein of the helper virus.”
Koonin and Dolja [21] concluded from a landmark, comprehensive review of all virus groups, that “among the parasites of modern organisms, viroids that cause many diseases of plants and satellites of plant RNA viruses show a striking resemblance to the putative primordial parasites.” However, “given that viroids so far have been identified only in plants,” the authors considered it “unlikely that viroids are direct descendants of the primordial parasites.” But then, the authors stated again: “Nevertheless, viroids seem to recapitulate the principle features of the selfish elements from the ancient RNA world”—thus leading to an internal contradiction, in that by one criterion (molecular properties), viroids “strikingly” appear to be descendants of primordial RNAs, whereas by another criterion (apparent evolutionary age), they clearly are not. Which is correct?