Article 4: The RNA-World Hypothesis is paradoxical!
Is RNA-World Hypothesis Real?
Was it possible that LUCA was preceded by a world of RNA-based autonomously living organisms?!
Lets explore the plausibility of the RNA-world hypothesis!
Replication and Mutation today
In a previous session we explored what is mutations and how living cells, based on DNA, have sophisticated error detection, correction, and repair systems to overcome its adverse effects (Check it here: https://youtu.be/lsRGSsDguWA). We explored how DNA together with its enzymes are finely engineered to provide such functionality, making it the solid foundation of the storage of the information needed to build the systems of life, and reproduce new living cell by reliably replicating this information.
In a following session, we dived deep in the alleged history of life, back to LUCA (the Last Universal Common Ancestor), and considered the various scenarios for what it was, and what it was preceded with, and showed that those scenarios, will inevitably lead to paradoxes if we consider the facts that we observe today concerning RNA-based and DNA-based organisms!
RNA-world Hypothesis
To complete the picture, we need to dive even further, towards the alleged first living systems that preceded LUCA (whatever LUCA was), where one of the prevalent hypothesis is one called “The RNA-world hypothesis”.
According to this hypothesis, RNA stored both genetic information and catalyzed the chemical reactions in primitive cells. Only later in evolutionary time did DNA take over as the genetic material and proteins become the major catalyst and structural component of cells. (Read, Molecular Biology of the Cell. 4th edition: https://www.ncbi.nlm.nih.gov/books/NBK26876/ ).
Catalysis for life!
Life processes cannot exist without catalytic reactions (Read, Autocatalytic Networks at the Basis of Life’s Origin and Organization: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6315399/ ). A reaction under enzymatic catalysis will take place trillions of times faster and more efficient than without a catalyst. In other words, without a catalyst, the reaction will not occur in any beneficial form!
The reason why DNA is not proposed to be the starting point of life is that it is so complex as we have touched upon before, with machinery of transcription and translation and error correction that are not reasonable to have risen at the outset. Instead, hypotheses propose that life started based on RNA, the simpler version of genetic material, which has certain properties that made it a candidate of which is “auto-catalysis”.
Catalysis is “the process of increasing the rate of a chemical reaction by adding a substance known as a catalyst. Catalysts are not consumed in the reaction and remain unchanged after it”. (Def: https://www.merriam-webster.com/dictionary/catalysis ). Auto-catalysis is simply “Catalysis of a reaction by one of its products”.
If RNA possesses efficient auto-catalysis, for long enough strands, and with very-low error rates, then we can start to look further… but is this the case?
In an RNA-world, we expect to find organisms that have RNA, with proper genetic information encoded on it, and RNA-machinery to replicate it; i.e. we need to start from a point where only RNA can faithfully replicate RNA! If you ask “Where did the RNA come from?”, well, I agree with you, and we will address that in an upcoming session, so we will park this aside for the purpose of this session, because getting meaningful RNA remains a mystery of its own!
RNA autocatalysis: Could RNA have just replicated itself?
To make this inference of RNA sustaining its own replication, we look at processes already today in operation, since we do not have historical facts (fossils) for RNA or any other genetic material that is billions of years old, and we would be otherwise speculating.
The existing RNA-based systems are viruses. We will rule out DNA viruses, and viruses that depend on first creating a DNA template from their RNA to perform their replication also known as retro-viruses, because we already noted that RNA needs to come first to respect the complexity gradient. So, remains is RNA viruses!
First question would be: do we have an RNA virus, or any living creature, where the very RNA genetic material self replicates using auto-catalysis?
The answer is: No! From processes in operation we cannot find RNA replicated by merely by itself, but rather by a molecular machine external to it. (Read: Replication and Expression Strategies of Viruses: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7158166/ ).
But, should we look for setups that can be arranged in the lab, will we find something different? Well, first, we need to say that we shouldn’t consider it evidence unless we can establish it occurs in nature, otherwise it will be really out of context, since what we are interested in is actually what is applicable to nature, not what is a product of human manufacturing. But having said that, it is still the case the RNA self replicators that are built in the lab are not useful for life as they breakdown after just tens of nucleotides apart from errors resulting from infidelity!
I am uploading here a 2020 paper with my own comments on for your reading, you can also find it in the literature review 4 section: RNA-2007-Zaher-1017-26 (Artificial Ribozyme, with notes). You might also be interested to pursue further reading with the following two papers, (DOI: 10.1261/rna.548807, DOI: 10.1126/science.1060786).
Now, we are obliged to rule out the center piece of the RNA world hypothesis, yielding it invalid from the outset, however, I would like to proceed for the sake of completeness and to give the argument its full breadth,
RNA replication through enzymes?
So, lets now consider that maybe RNA-world lifeforms used an enzyme to replicate the RNA genetic code. So, we need to find out if it is possible to replicate RNA without the need for autocatalysis. We seek processes already in operation and referring to the above literature, we find that RNA can be indeed replicated by an enzymatic molecular machine called RNA-Dependent-RNA-Polymerase (RdRp), also know as RNA Replicase.
But the bad news is that RdRp is an essential protein encoded in the genomes of all RNA-containing viruses with no DNA stage, i.e. RNA viruses. Some eukaryotes also contain RdRp. (For more about RNA Replicase, Read wiki here: https://en.wikipedia.org/wiki/RNA-dependent_RNA_polymerase ).
Obviously, the whole point of an RNA-world is evading the matter of having DNA and Proteins as the starting point in the first place, and since RdRp is proteins not a Ribozyme (RNA enzyme), then the hypothesis is dead once again!
You might though spend a minute pondering the sophistication of the structure of RdRp!
Proteins
Proteins are the dominant machines and catalysts of modern cellular life. They are translated from mRNA by molecular machines called Ribosomes which catalyze the process, linking amino acids that correspond to the coded codons, producing the peptide chains that will become the proteins. But the produced proteins will not be useful until they are folded into a functional 3d structure, through the protein coding process. The protein coding is facilitated by enzymes in the cytoplasm, of which are the Ribosomes themselves which can also be attached to the Endoplasmic Reticulum. So, the invocation of proteins in the cellular environment will call for a myriad of enzymes and cellular machinery that evolutionary biologists agree are not appropriate at the primordial forms of life, which is the reason for having hypothesis like RNA-world, to eliminate the complexity driven from DNA and Proteins and seek an exit to smoother evolutionary gradualism which starts at the simpler end; RNA!
This is a short video showing how a protein folding (Six Microseconds of Protein Folding). The following video gives a good illustration of the protein folding process and how the complex shapes of proteins make their functions possible. (Watch: What is a Protein? Learn about the 3D shape and function of macromolecules). We will further address proteins and enzymes in a future session.
Ribosomes, Transcription, and Proteins!
Now, even if there was a way for RNA to exist, can it be expressed into the necessary proteins? Well, as mentioned above, it needs Ribosomes, which does this function in modern cells; translates RNA to proteins!
Ribosomes consist of two major components: the small and large ribosomal subunits. Each subunit consists of one or more ribosomal RNA (rRNA) molecules and many ribosomal proteins (RPs or r-proteins).
Proteins form 35%-63% of Ribosomes (RNA forms 37-65%), (Read: Ribosomes – Definition, Structure, Size, Location and Function (microscopemaster.com)). For example, prokaryotic ribosomes have 65% RNA and 35% Protein. But, we have established before that we don’t have proteins in RNA-world, so we cannot have Ribosomes! Meanwhile, Ribosomes are critical in the function of Protein folding, which causes produced proteins to get their 3D shapes, and hence acquire their proper function, so another paradox of which came first, the Ribosome or the protein! (Read about Ribosomes: https://en.wikipedia.org/wiki/Ribosome ).
At this point, the RNA hypothesis should be totally dead, yet we stretch ourselves till the end of the line, to explore the whole landscape of paradoxes!
There is another implicit paradox here, that we will further explore later, which is the matter of the proteins, which need ribosomes to be produced, and then folded, however, the ribosomes themselves contain many proteins in their structure, creating another chicken and egg paradox!
Now, even if we accept that all of the above was available to the RNA world organisms in one way or the other, in a way that we are not able o imagine now, lets give this hypothesis the final test and see if RNA will have the ability to capture all the needed information to make the paradigm work!!!
The minimum genome?
At this point, to have RNA-based life that is functioning, we have already violated the RNA world hypothesis by having to code for RdRp which has proteins, so once again we invoked the ribosomes to produce the proteins needed for the RdRp, with all what comes with that of having to code for the ribosomes with all their proteins and ribozymes on the RNA genetic material plus other enzymes needed to complete the process successfully!
But, to have life, do we only need replication? Of course not! we at least need the following minimum requirements of life: (Read: Systems Biology Perspectives on Minimal and Simpler Cells: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4187685/ ):
1. Energy, i.e. metabolism: ability to consume food and extract chemical energy!
2. Reproduction: Replication: copying genetic material with all the necessary machinery needed for that.
3. Shelter: a membrane that keeps the cell together to be able to maintain homeostasis!
To achieve all of the above, we look for an organism that just fulfills the minimum requirements and has the smallest genome. (Read, under minimum Genome: As mentioned above, the smallest natural genome capable of autonomous growth or laboratory cultivation in pure culture and also in a defined medium (24) is the one of M. genitalium, with 580 kb (13): Systems Biology Perspectives on Minimal and Simpler Cells ).This pushes the minimum genetic length to support life to 580K bases!
PS: Prokaryotic organisms termed Candidatus that has a smaller genome is not considered autonomously living by mainstream science. (Same reference under title: Minimal Genome)
How big can RNA be?
So, can RNA grow that large and support itself?
Well, the average RNA virus genome size is just 9Kb, and the maximum is 29kb (Read abstract: The evolution of genome compression and genomic novelty in RNA viruses )
So, it does not seem possible (from observing RNA genomes we see around us) that there has ever been an RNA life, pre or post-LUCA!!
Lets think of this:
If we stretch our imagination, that somehow, those essential components were to be found in an RNA based genome, to support the minimal requirements of life, of Metabolism, Membrane, and Replication, then we should have a minimum genome of 580Kb… However, what we see around us is that the average viral RNA genome is 9kb, and the maximum is ony 29kb… and this is not because it is a virus, as some DNA-based viruses, like the mimi-virus, has a genome that is as big as ~1.1Million bases (Read full article in nature mag: Discovery of the Giant Mimivirus ), or the Megavirus which goes up to 1.2Milion bases (Read: Genomics of Megavirus and the elusive fourth domain of Life ), so clearly, a short genome is not a virus feature, but rather an RNA feature!
Is this my opinion only, no! Read it here in the abstract of this paper: The evolution of genome compression and genomic novelty in RNA viruses and we quote:
The two most striking attributes of RNA viruses are their small size and their high mutation rate. The average genome length of a family is only 9 kb, with the longest being the Coronaviridae at 29 kb. Viral polymerases (RNA-dependent RNA replicases and reverse transcriptases) have a high misincorporation frequency and lack a proofreading 3′ to 5′ exonuclease domain (Steinhauer et al. 1992); there is also no mismatch repair, even in double-stranded RNA viruses. This leads to mutation rates in the order of 10−4 per base per round of replication (Drake and Holland 1999; Mansky 2000; Crotty et al. 2001), several orders of magnitude higher than those found in DNA-based life forms (Drake et al. 1998). The deleterious effects of most mutations in RNA viruses are well studied (Sanjuan et al. 2004; Elena et al. 2006).
These two attributes have been linked most recently by Holmes (2003), who suggests that the genome size of an RNA virus is limited by its mutation rate. This argument is derived from the inverse relationship, first identified by Eigen (1971), expected between the size of any replicating molecule (its information content) and its mutation (error) rate. For example, a hypothetical 1-Mb RNA virus (the size of the largest DNA virus) with a mutation rate similar to that of known RNA viruses would be unable to replicate without incurring lethal mutations
End Quote.
And this is not just a hypothesis, but it is a theory that provides useful predictions that we make use of to treat virus infections, as the paper goes on:
Indeed, the idea that RNA viruses exist near a so-called error threshold, determined by a function of their genome size and mutation rate (Nowak 1992), lies behind the development of drug therapies that artificially elevate the viral mutation rate—referred to as lethal mutagenesis (Crotty et al. 2001).
I also find this article to be worth reading in this context: Why are RNA virus mutation rates so damn high? (plos.org).
Conclusion: An RNA based living cell that has the minimum requirements of life could not have existed!
Kicking the can further?
The theory will get out of one paradox, only by pushing the can further back, appealing to ideas like that RNA-world organisms worked together in a community manner with genetic material within the “populations”, or even pushing-the-can further back by talking about a preRNA world! (Read: A Pre-RNA World Probably Predates the RNA World: https://www.ncbi.nlm.nih.gov/books/NBK26876/ ).
Some have recognized those fundamental problems, yet, they would appeal to another pushing-the-can argument of co-evolution, once more, based on speculation (Read, Did Gene Expression Co-evolve with Gene Replication? https://link.springer.com/chapter/10.1007%2F978-3-319-95954-2_16).
Does this regress have an end, and does it solve the problems by getting even more simpler and simpler to escape complex problems or do the questions and gaps get bigger as ToE tries to escape them?!
I leave you with this question!
RNA-world 4 Paradoxes
A main problem with the ToE is that upon solving a problem, it will break up the problem into pieces, and then attempt to solve each piece alone, finding solution and examples for the validity of those solutions, each alone. This is not a bad strategy, under the condition that the solutions add up, are not mutually exclusive or paradoxical! However, the individual solutions will not add up together in a coherent overall picture, and even worse, in many cases the individual solutions are in contradiction, so when you add them together, you get a paradox, instead of a solution!
The problem:
We need a base for life that has enough genetic information, a catalytic replication effect, a suitably low error rate to maintain this information, together with an energy source and an enclosure for the process to take place.
Paradox #1:
We realize that the existing genetic material today are DNA w/EC, plus RNA, and protein enzymes is too complex, so this piece is solved alone taking one step back to RNA-only-world for coding and enzymes.
But we are struck by the fact that RNA cannot auto-catalyze itself faithfully, so, we hv assume the existence of enzymes that have protein components (RdRp – RNA Relicase), leading to a paradox.
Paradox #2:
We still don’t have an error correction mechanism, so we appeal to short genetic material, but this material is not even sufficient to properly code for all the enzymes that we already assumed they exist, because mutation will destroy it, leading to another paradox!
Paradox #3:
We then consider that we need a cellular environment with metabolism for energy and a cell membrane for this to be even considered life, apart from starting to work! Meanwhile, metabolism as a chemical reaction in the cell will push mutation rate even more, making the absence of error correction and repair even more aggravated! Yet, now we have to assume that the genetic code codes for all the enzymes for the metabolism, and the cell membrane materials and mechanisms, but then again, we know that the RNA genome needed for that is an order of magnitude bigger that the maximum observed RNA genome, pushing the organism beyond a safe mutation threshold that is lethal to its survival!
Paradox #4:
Some would then try to address this problem by breaking life in small organisms that work in a community each having a fragment of what is needed and cooperating for the outcome, but this means that none of those are actually living, facing again the same problem of finding an origin of life, and there is no evidence for it or similar analogues other than viruses, and we know that viruses do not do that, but rather infect hosts to replicate and are otherwise inert! Pushing the can is just not a solution! At the end, regardless how you reach to the RNA organism that will then develop into LUCA, once you put it together as an RNA organism in an RNA world, you are back to the same above 3 paradoxes once more!
Closing
The RNA-World hypothesis, which is widely endorsed by evolutionary biologists lacks the merits of accepting it, whether theoretically or practically looking at processes today in operation – RNA World Paradox!
