It is now popularly accepted that an “RNA world” existed in early evolution. During division of RNA-based protocells, random distribution of individual genes (simultaneously as ribozymes) between offspring might have resulted in gene loss, especially when the number of gene types increased. Therefore, the emergence of a chromosome carrying linked genes was critical for the prosperity of the RNA world. However, there were quite a few immediate difficulties for this event to occur. For example, a chromosome would be much longer than individual genes, and thus more likely to degrade and less likely to replicate completely; the copying of the chromosome might start at middle sites and be only partial; and, without a complex transcription mechanism, the synthesis of distinct ribozymes would become problematic.Inspired by features of viroids, which have been suggested as “living fossils” of the RNA world, we supposed that these difficulties could have been overcome if the chromosome adopted a circular form and small, self-cleaving ribozymes (e.g. the hammer head ribozymes) resided at the sites between genes. Computer simulation using a Monte-Carlo method was conducted to investigate this hypothesis. The simulation shows 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; and both circularity and self-cleavage are important for the spread of the chromosome.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). The strategy suggested here is very simple and likely to have been used in this early stage of evolution. By demonstrating the possibility of the emergence of an RNA chromosome, this study opens on the prospect of a prosperous RNA world, populated by RNA-based protocells with a number of genes, showing complicated functions.This article was reviewed by Sergei Kazakov (nominated by Laura Landweber), Nobuto Takeuchi (nominated by Anthony Poole), and Eugene Koonin. In modern cells, genes coding for different proteins can exist in the same DNA molecule (chromosome) and are replicated together. This “arrangement” is important to avoid “gene loss” during cell division. In prokaryotes there is only one chromosome. Although there are more chromosomes in eukaryotes, their numbers are still quite limited compared with the number of genes. Indeed, even for appropriate distribution of this limited number of chromosomes between offspring cells, a very complicated mechanism has evolved. It is now popularly accepted that in the early evolution of life, there was a stage called “the RNA world” ,2, in whTheoretical studies have indicated that replicators with different functions may coexist/cooperate in a cell-like container -5. A repHowever, there would be quite a few difficulties for the emergence of a chromosome. First, the RNA chromosome would be much longer than the single RNA genes and would thus run much more risk of degradation (chain breaking). Second, because of the significantly longer chain, its full replication would be much slower. Certainly, as mentioned above, the chromosome can be expected to be a better template (thus favoring the replication) because of the interference of individual “ribozyme domains” with each other in folding. However, the effect of this factor may be still limited (perhaps sequence-dependent). Third, the copying of the chromosome may start at some sites in the middle of the chain, and thereby would not result in full replication. Fourth, at this early stage of evolution, without a complicated mechanism of “transcription” , the synthesis of distinct gene products from the chromosome would become problematic. Compared with these difficulties, the selective advantages mentioned above are apparently more indirect. Considering that evolution is continuous and cannot “see advantages in the future”, these difficulties would have to have been be overcome first.A simple strategy is implied in certain small plant pathogenic RNAs (viroids or viroid-like satellite RNAs), which were suggested to be plausible candidates as living fossils of the RNA world . A viroiThe first three difficulties mentioned above can be overcome  by circularity. First, by circularity, the chromosome could evade the degradation starting from its chain ends, and this would compensate to some degree the disadvantage concerning degradation due to its longer chain. The end-degradation may be caused by the easier breaking of terminal phosphodiester bonds than internal ones, or by the spontaneous decay of terminal nucleotide residues (see the Discussion section for detailed explanations). If such end-degradation was strong, such compensation may be expected to be significant. Second, the circular topology is expected to further impede  the folding of distinct “ribozyme domains” in the chromosome, making the chromosome more suitable to act as a template in the replication. Third, there would be no issue concerning the starting point of copying . The fouIn order to determine the plausibility of this deduction, we conducted a computer simulation study. The simulation shows that an RNA chromosome can spread (increase in quantity and be sustained) in the system when it adopts a circular form and its linear “transcripts” (corresponding to the “sense strand”) is readily broken at the sites between genes. Here we suggest that circularity plus self-cleavage could have been used as a strategy for the emergence of a chromosome in the RNA-based protocell Figure .We investigated a hypothetical RNA chromosome that included four genes coding for a replicase ribozyme , a nucleotide synthetase ribozyme (“Nsr”), a nucleotide precursor synthetase ribozyme, (“Npsr”) and an amphiphile (membrane component) synthetase ribozyme (“Asr”). The approach of the simulation is similar to those used in our previous work studying Rep , Nsr 1919, Asr , and thePCRTT, PLRTT, and FIB described below) are associated with the topic of this study. The chromosome (as a circular RNA) may become a template with PCRTT (the probability of a circular RNA turning to a template), which should be higher than PLRTT (the probability of a linear RNA turning to a template), with which individual ribozymes (as linear RNAs) may become templates. Linear “transcripts”  times. This assumption represents the self-cleaving effect. It should be noted that the self-cleaving effect would not occur in the circular chromosome because steric constraints would inhibit the folding and function of the embedded self-cleaving ribozymes, as well as that of the ribozymes corresponding to those “genes”. This is consistent with the findings in viroids, wherein the embedded hammerhead ribozyme is, in fact, inactive in the circular RNA chromosome, but would work during the rolling-circle replication, when it resides in the long linear RNA, between the genome copies , where n is the quantity of inner impermeable ions, including nucleotides and RNA (measured by the number of nucleotide residues), and b is the quantity of amphiphiles within the membrane. Wherein, b/2 (there are two layers in the membrane) is a “scale” representation of the surface area of the membrane. Consequently, (b/2)3/2 is a scale representation of the cellular space. Thus, n/(b/2)3/2 is a representation of the concentration of the ions. FOP × n/(b/2)3/2 represents the consideration for the “osmotic pressure effect”; a higher concentration of the inner impermeable ions would cause the protocell to be more swollen, and thus amphiphiles within the membrane are less likely to leave [The probability of an amphiphile leaving the membrane is assumed to be to leave .PNPP)y] / [1 + FDE × n/(b/2)3/2], where n is the quantity of inner impermeable ions and b is the quantity of amphiphiles within the membrane 3/2, which represents the consideration of the limiting effect of the cellular space on the influx of nucleotide precursors. When b equals to LAM (the lower limit of the number of amphiphiles to form a protocell membrane), y equals to 1. When the b increases, meaning that the cellular space increases correspondingly, the probability of a nucleotide precursor permeating into the protocell would become greater. FDE × n/(b/2)3/2 represents the consideration of the effect of Donnan’s equilibrium [PNPPP)y] / [1 + FDE × n/(b/2)3/2]. The probability of an amphiphile precursor permeating into a protocell is assumed to be 1-(1-PAPP)y, wherein the effect of Donnan’s equilibrium is not considered because amphiphile precursors are assumed to be uncharged molecules.The probability of a nucleotide precursor permeating into a protocell is assumed to be  would become problematic.” Furthermore it is proposed that “inspired by features of viroids, which have been suggested as living fossils of the RNA world… these difficulties could have been overcome if the chromosome adopted a circular form and small, self-cleaving ribozymes (e.g. hammerhead ribozymes) resided at the sites between genes.”Yes, this is just the central idea of our paper. The reviewer’s description is quite right, thanks.The idea about the emergence of circular RNAs in RNA World is reasonable but it is not novel. For example, we state that “Circular RNAs have several features that could have been useful at an early stage of evolution: (a) they remain in one piece after a single random cleavage event, allowing recovery of their structure through re-ligation of their ends; (b) they can serve as a template for rolling circle replication and amplification; and (c) they have fewer conformational degrees of freedom compared to their linear counterparts, and therefore can bind to other molecules (or fold) with less entropic cost” .Yes, the emergence of circular RNAs in the RNA world may have other advantages, like those stated in the work coming from the reviewer’s group . The novIn this manuscript, Ma et al. offered three novel features (or factors) supporting the emergence of circular RNAs:“First, by circularity, the chromosome could evade the degradation starting from its chain ends, and this would compensate to some degree the disadvantage concerning degradation due to its longer chain. The end-degradation may be caused by the easier breaking of terminal phosphodiester bonds than internal ones, or by the spontaneous decay of terminal nucleotide residues. If such end-degradation was strong, such compensation may be expected to be significant.”– – The proposed “end-degradation” factor is purely speculative and is not based on known properties of RNA. Rather, it is feasible that RNA degradation under prebiotic conditions, in the absence of nucleases, was catalysed by low or high pH as well as by metal ions in combination with high temperatures; none of these conditions lead to preferential cleavage of the terminal rather than internal internucleotide bonds in polynucleotides , (1) the more exposure of the terminal phosphodiester bonds to some possible prebiotic peptides that cleaved RNA , (2) theAs to “the occurrence of ligating enzymes that can re-ligate the cleavage to restore the circular form of RNA chromosomes”, mentioned by the reviewer, we think that the idea is interesting and could be considered in our future study, but is perhaps beyond the scope of our topic here.“Second, the circular topology is expected to further impede  the folding of distinct ribozyme domains in the chromosome, making the chromosome more suitable to act as a template in the replication … because of the interference of individual ‘ribozyme domains’ with each other. However, the effect of this factor may be limited (and perhaps sequence-dependent).”– – This factor is feasible, but it definitely would limit the sequence diversity of these RNA chromosomes.Yes, the logic is: (1) an RNA chromosome (even a linear one) with linked “ribozyme domains” was expected to be a better template because of the interference of the domains with each other (concerning base-pairing), preventing the formation of tight structures as in free ribozymes; (2) however, the interference may be limited, depending on the RNA sequence of the ribozymes; (3) a circular topology is expected to further impede  the folding of distinct ribozyme domains in the chromosome, making the chromosome more suitable to act as a template in the replication.“Third, there would be no issue concerning the starting point of copying” from a large circular RNA chromosome template.– – It is true that a circular template would allow copying its entire sequence independently of the starting point through rolling-circle-amplification (RCA). It is a valid and good point.Yes, thanks; but note that the replication mechanism assumed in the present model is not that of RCA.“Fourth …if the ‘sense strand’ of the chromosome tends to be broken at the sites between genes, corresponding ribozymes may be produced this way. A hammerhead ribozyme catalyzing self-cleavage may have existed at these sites.”– – Hammerhead is a very effective ribozyme and with high probability would compromise the circularity of such RNA chromosomes by cleaving them. It is more likely that only smaller, less effective ribozymes could realistically be considered as part of circular chromosomes. Moreover, the consensus sequence of the in-cis cleaving hammerhead ribozyme is about 30 nt long, and the author’s computer modeling limits the length of the entire chromosome to this size. The latter demonstrates a serious problem with the author’s approach, since the model does not include longer chromosomes, which may behave differently from the small ones.Firstly, perhaps there is a misunderstanding here. Yes, hammerhead is a very effective ribozyme, but they would only work when the circularity is break, as mentioned in the second paragraph of the Results section: “It should be noted that the self-cleaving effect would not occur in the circular chromosome … This is consistent with the findings in viroids, wherein the embedded hammerhead ribozyme is, in fact, inactive in the circular RNA chromosome, but would work during the rolling-circle replication, when it resides in the long linear RNA, between the genome copies ,12.” A sSecondly, indeed, the lengths of the self-cleaving ribozyme and the RNA chromosome assumed in the simulation are much shorter than those that were possible in reality. However, such a case is somewhat a commonplace for computer simulation. In a computer simulation, sometimes simplifications or unrealistic assumptions have to be adopted in order to show the underlying mechanisms in a clear mode or even, as in this case, merely to avoid cumbersome computation. The key issue is that to what extent that the simplifications or assumptions may affect the results, influencing the relevance of the simulation to the realistic situation. It should also be admitted that we cannot say with certain that the behavior difference mentioned by the reviewer would not exist. Nonetheless, we should take an open mind at cases like this, appreciating the valuable message transferred by the simulation. For example, in traditional, popularly used “replicator” models in the field of the origin of life . Then, when we find the appearance of a phenomenon is sensitive to the setting of some parameters/factors, we judge that these parameters/factors may have been important for the mechanism of the phenomenon  if its length is sufficiently long”. In both the two opinions, it seems that if a preliminary circular RNA chromosome appeared in the RNA world, it may have been quite short, and cannot evolve to significant longer ones. If so, DNA may have arisen very early in the RNA world, serving as the carrier of genetic information -- for the appearance of more and larger genes. However, there may still be other issues. For example, DNA may be more suitable to act as template, but longer DNA chromosomes cannot yet escape the problem of strand dissociation, perhaps more serious because single chain DNA, unlike RNA, can hardly self-fold to prevent its annealing with its complement. If it is difficult to imagine a ribozyme that acted as a helicase, perhaps -- proteins emerged earlier (before DNA), helping with the strand dissociation? But how could proteins have emerged before the emergence of a large genome, especially considering the complexity of the translation mechanism? Certainly, that is already beyond the scope of our topic here.In the response to one of my comments, the authors wrote “… note that the replication mechanism assumed in the present model is not that of RCA.” However, RCA is the only mechanism (in the absence of helicases) to generate single-stranded RNA amplicons that then could be cleaved by the encoded ribozymes. As I described in another comment: “For example, the dissociation between sense/template and antisense/transcript RNA strands would be hampered due to a high thermostability of RNA-RNA duplexes. Without strand dissociation, the ribozyme genes and self-cleaving units cannot fold into their active conformation”. So without including RCA or an alternative mechanism for template-transcript strand separation into the model, the ribozyme units would remain in dsRNA form and could not fold into active conformation.Indeed, as the reviewer said: “RCA is the only mechanism (in the absence of helicases) to generate single-stranded RNA amplicons that then could be cleaved by the encoded ribozymes”. However, it seems that this mechanism is heavily depended upon the existence of an efficient RNA polymerase . We doubt if an efficient RNA polymerase ribozyme could exist in this early stage of the RNA world – instead, the RNA replicase ribozyme in the present model is a template-dependent ligase with a relatively low efficiency (please refer to one of our previous papers ). PerhapAs to the dissociation of the RNA strands, there would be no serious problems if the circular RNA chromosome was short. As mentioned by the reviewer: “… the duplex formed by a small circular template is stiff and does not allow the formation of a duplex longer than half of the length of the circle ”. This pIn their paper, Ma et al. investigate the origin of chromosome-like molecules in RNA-based protocell systems using computer simulations. The authors consider two key factors that seem relevant to the origin of such molecules: circular RNA and self-cleaving ribozymes (such as seen in viroids). The authors assume that circularization unfolds an RNA molecule and thereby renders it more suitable as a template; in addition, it removes the chain ends and so increases the resistance of an RNA molecule against hydrolytic (exo-) degradation. Moreover, the authors suggest that self-cleaving ribozymes can be used to implement a primitive form of “transcription” in a wide sense  if such ribozymes intervene multiple other ribozymes that are concatenated with each other. Using computer simulations, the authors show that protocells containing such primordial chromosomes can survive if both circularization and sufficiently high self-cleavage activity are assumed.Both the ideas of circularization and primitive transcription based on self-cleavage are novel and worthy of consideration in exploring the potentials of RNA-based evolvable systems. Particularly intriguing is the possibility that these ideas might be directly tested by experiments (at least in terms of their basic premises).Yes, the reviewer’s summary about our work is correct and accurate, thanks.The authors’ analysis of their model, however, focuses only on the question of whether primitive chromosomes can survive when they are introduced into a model system. From a theoretical point of view, this leaves unanswered two crucial questions regarding the evolution of primitive chromosomes. First, exactly what selective advantage enables protocells containing chromosomes to out-compete those lacking chromosomes? Second, can primitive chromosomes emerge through evolution in a protocell system where chromosomes are originally absent?PBB < <PNDE) and function as templates nearly two order of magnitude more frequently than unit ribozymes (PCRTT> > PLRTT) on the basis of their circular structure. Taken together, circularization provides about a 1000-fold advantage for chromosomes, which apparently far outweighs the 16-fold disadvantage described above. Interestingly, the model shows that such an excess advantage is necessary for survival of primitive chromosomes  in a time step is 0.1, then after a time step, for an 8-nt unit ribozyme, the probability that it remains intact is (1–0.1)(8–1), i.e. about 0.48; for an 32-nt chromosome, the corresponding probability is (1–0.1) (32–1), i.e. about 0.,038. Thus, the disadvantage of the chromosome seems to be already over 12-fold. While we enjoy ourselves on this calculative way which seems to be more clever and rigorous, suddenly we found that, in this way, the quantity about the disadvantage may change dramatically accompanying the value alteration of PBB. Which would be a more appropriate way? It seems difficult to envision one at once. The calculation of the advantage concerning the replication would be more sophisticated, if still feasible.Firstly, we doubt the validity of the review’s consideration in quantifying and comparing the advantages and disadvantages. Can advantage and disadvantage from different respects be judged in quantity and compared with each other in such a simple way? Furthermore, even they could be calculated in quantity, it seems that the calculative way could not be that simple. For example, as to the judgment “chromosomes are four times longer than unit ribozymes, which leads to a four-fold disadvantage for chromosomes over unit ribozymes in terms of … decay” (in fact judgments like this “originated” very early ) – if thIf the reviewer’s consideration of advantage-disadvantage quantification and comparison is problematic, his consequent consideration for the interpretation to the “phenomenon” that “chromosomes must have an extremely large additional advantage provided by the circular structure in order to survive in a protocell system” would loss its point of origin. However, indeed, according to our inference in the Background section and the result of the computer simulation, the advantage for a chromosome in respect of assortment load (i.e. avoid gene-loss during protocell division)  is not ePBB would act on each phosphodiester bond, whereas PNDE would act only on terminal nucleotide residues. Therefore, we cannot say “chromosomes decay nearly one order of magnitude slower than unit ribozymes (PBB < <PNDE)”. Second, self-cleaving effect would not occur in the circular form of the chromosome, as mentioned in text , the authors consider that primitive chromosomes occasionally appear through random ligation, recombination, and circularization of ribozymes by chance (Discussion section). This assumption might not be reasonable because there seems to be a large gap in terms of complexity between the assumed chromosomes and unit ribozymes. This assumption can be (and should be) tested by the current model, which seems to incorporate all the elementary steps that are necessary for the emergence of first primitive chromosomes in a chromosome-less system . What would happen if simulations are started without assuming primitive chromosomes at the beginning?Indeed, the evolutionary origin of primitive chromosomes is a problem. However, it is another problem. Our topic here is “if a chromosome has come into being, could it sustain in the context of those mentioned difficulties? Our results showed that if the chromosome adopted a strategy of circularity and self-cleavage, it may overcome the difficulties, spreading in the system. Admittedly, to be a sufficient condition, the problem of the evolutionary origin of the chromosome should be demonstrated.In the current model, we introduced four unit ribozymes for the sake of “a more representative form of a chromosome with linked genes” (mentioned in the first paragraph of the Results section). The evolutionary origin of such a chromosome from a system with the four unlinked ribozymes seems indeed a large gap. However, in reality, things may not have to occur this way, in one step. For example, in a primitive system with only two kinds of functional ribozymes favoring the protocell containing them , and then test the smaller gaps with modified models.If we explain these issues in the main text, apparently, it would seem too verbose. Therefore, thanks for the reviewer’s insightful comment on this point and certainly, also the chance offered by the journal for us to show our response to this comment.I have a few more comments. First, the authors assume that circularization destabilizes the folding of RNA molecules and thereby renders them more suitable as templates. However, such destabilization might depend on the length of RNA molecules: a circular RNA might behave nearly as a linear molecule  if its length is sufficiently long. Given that viroids are only a few hundred nucleotides long, which is about the same length as the template-directed RNA polymerase ribozyme , one neeAs we mentioned above in our response to Reviewer 1, this opinion, as well as the one appeared in the last comment of Reviewer 1, implies that the circular RNA chromosome may have been short. As to the length of the polymerase ribozyme , we haveFurthermore, we would like to talk a little more about the role of circularity to avoid the folding of RNA molecules. In fact, this role may have been somewhat stronger than the reviewer envisioned. For an efficient ribozyme, the correct folding should be very important, just like a protein enzyme. As mentioned in the text, those linked unit ribozymes in a chromosome might interfere with each other in folding, rendering the chromosome a better template. However, the two unit ribozymes at the two ends of a linear chromosome would still have a free “tail” each, which might fold “inwards” to the chromosome chain and entail the in-situ formation of the “correct” (compact) structures of these two unit ribozymes. Considering template-directed copying is perhaps more likely to start from the chain ends, this impact may have been significant. That is, circularity may also have been important in respect of avoiding this impact, by “removing the free tails”. This effect, beyond the pure steric impediment caused by the circularity, is apparently not influenced by the length of the RNA chromosome.Second, the authors argue that terminal phosphodiester bonds are more sensitive to hydrolysis than internal  phosphodiester bonds because terminal base stacking is less stable than internal base stacking. This argument seems to imply that circular molecules are more vulnerable to hydrolysis than linear molecules because, as the authors assume, circularization destabilizes the folding of RNA molecules. But, this is the opposite of the assumed effect of circularization.This point belongs to those issues difficult to clarify only by deduction. For example, it may be argued that though circular molecules, in a single chain form, might be more vulnerable to hydrolysis than linear ones, they are actually more likely to appear in a double chain form since they may act as better templates than linear ones. Apparently the base-stackings in a double chain would be more (and stronger) than those in a single chain – no matter how much it tends to self-fold.Third, the model seems to assume that the existence of ribozymes changes the equilibrium constants of chemical reactions such as the conversion reaction between nucleotide precursors and nucleotides. Is this reasonable?PNF, whereas the rate that a nucleotide decays into a nucleotide precursor is represented by PND. When a nucleotide synthetase ribozyme (Nsr) exists and acts upon a nucleotide precursor, the rate that this precursor would transform into a nucleotide is PNFR (> > PNF). Indeed, it seems here that the equilibrium constant is changed. However, the reaction catalyzed by the Nsr is actually not the original non-enzymatic reaction. The reactive path has been changed by the incorporation of some high-energy factor, just like in modern organisms (e.g. ATP as a common high-energy factor). If enzymatic reactions are the same as the original non-enzymatic reactions, that is, degradations are speeded as well as syntheses, how could our “living” system be sustained? The reason why here equilibrium constants appear to have been changed by ribozymes is that the events concerning the potential high-energy factor were omitted in our model – “the energy problem was simplified in the simulation…”, as stated in the earliest paper of our group that reported the usage of this kind of Monte-Carlo model system to simulate early evolution in the RNA world [No, we have not assumed that the existence of ribozymes changes the equilibrium constants of chemical reactions. For example, the rate that a nucleotide precursor forms a nucleotide is represented by NA world .Here Wentao Ma and colleagues continue their series of modeling studies of various aspects of the putative primordial RNA world. The current installment is based on the old idea of Diener that viroids are relics of the RNA world. Certainly, this is an attractive idea given that these are indeed the simplest known replicators, and they are stable small RNAs which is important for RNA World models. The new idea introduced by Wentao Ma and colleagues is the self-cleavage of a viroid-like circular RNA molecule into smaller molecules with various potential activities, via the action of built-in hammerhead ribozymes. There is no precedent for this among modern RNAs but neither is there anything biologically or chemically implausible about this model. There is actually little doubt that such a molecule can be produced experimentally, and its properties will be of interest. Certainly, a merit of the model.Thanks to the reviewer for the remark. In particular, indeed, such a molecule might be produced experimentally in future, and its properties will be interesting.Wentao Ma then proceeded to develop a Monte Carlo-type model of a replicator system consisting of such viroid-like molecules and claim that the result revealed robustness and viability of this type of replicators systems. This reviewer's attitude to this kind of models is ambivalent. On the one hand, it is clear that  everything depends on parameterization, and with some combinations of parameters, nearly any outcome is possible. On the other hand, the model is not useless because it shows that such replicator systems can exist in a large domain of the parameter space.Yes, the number of parameters in the model is great, and this may bring about an impression that any outcome of the simulation may be possible, depending on the values of the parameters. We would like to defend this situation from three aspects. First, as the reviewer mentioned, “the model is not useless because it shows that such replicator systems can exist in a large domain of the parameter space”  contains a viroid-like region and a protein-coding region ,37. Its Thus, there is nothing overtly wrong with the scenario put forward by Wentao Ma and colleagues, and moreover, I think it is more ingenious than many other models that circulate in the origin of life field. Except perhaps for one fundamental issue: all of this conceivably could have happened as outlined in the model, only there is not a shred of evidence it actually happened this way. A general problem with models of primordial evolution, not to be held against the authors.Thanks to the reviewer for his praise. As to the fundamental issue mentioned, we agree with the reviewer. But we have an additional remark about this issue. The origin of life is a problem of history. The process occurred in such remote past that perhaps we will never know exactly what happened actually. But perhaps this does not matter. Our scientific studies on this history are largely (if not completely) aimed at associated rules. In fact, rules behind phenomena are the shared concern of all scientific fields. Compared with how an event in this process may have happened, we are, in some degree, more interested in how it could have happened. Certainly, if we could know quite a few plausible ways for the event, we would be more interested in whether it happened this way or that way in reality. Unfortunately, this is usually not the case in the field of the origin of life. Therefore, in general, on the one hand we should try to collect evidence relevant to the process, but on the other we should appreciate that any plausible mechanisms we find concerning possible events in this process would be quite valuable.The authors declare that they have no competing interests.WTM conceived of the study, did the simulation work and wrote the manuscript. CWY and WTZ participated in the simulation work. All authors read and approved the final manuscript.Five supporting figures for the paper.Click here for file


Schizosaccharomyces pombe and Saccharomyces cerevisiae), a plant , and protists . For all species studied to date, including those in this report, only a small fraction of the theoretically possible circular RNA isoforms from a given gene are actually observed. Unlike metazoans, Arabidopsis, D. discoideum, P. falciparum, S. cerevisiae, and S. pombe have very short introns (∼100 nucleotides or shorter), yet they still produce circular RNAs. A minority of genes in S. pombe and P. falciparum have documented examples of canonical alternative splicing, making it unlikely that all circular RNAs are by-products of alternative splicing or ‘piggyback’ on signals used in alternative RNA processing. In S. pombe, the relative abundance of circular to linear transcript isoforms changed in a gene-specific pattern during nitrogen starvation. Circular RNA may be an ancient, conserved feature of eukaryotic gene expression programs.An unexpectedly large fraction of genes in metazoans  express high levels of circularized RNAs containing canonical exons. Here we report that circular RNA isoforms are found in diverse species whose most recent common ancestor existed more than one billion years ago: fungi ( Drosophila genes and constitute the major RNA isoform from hundreds of these genes C. elegans and fruit flies; and confirmed by other groups in human and mouse cells Until recently, circular RNA isoforms have largely gone unnoticed, with some notable exceptions Circular splicing of RNAs appears to be regulated: for many genes, the ratio of circular to linear transcripts and the relative abundance of differentially spliced circular isoforms is cell-type specific. Introns are retained in some circular RNAs, adding an additional layer of complexity to the circular RNA transcriptome Splicing is a ubiquitous feature of eukaryotic gene expression, and it is now thought that the “Last Common Eukaryotic Ancestor” (LECA) was relatively intron-rich and had complex spliceosome machinery and splicing signals Schizosaccharomyces pombe, the protist Plasmodium falciparum and the plant Arabidopsis thaliana, respectively. In addition, we prepared an RNase R treated RNA-Seq library from Dictyostelium discoideum and performed the same bioinformatic analysis described above.We mined RNA-Seq libraries from three species  gave circle-specific PCR products. The success rate in P. falciparum was lower; only two candidates gave a circle-specific PCR product, six gave no product . While for S. pombe and Arabidopsis we were able to test RNA from conditions identical to those in the datasets used for discovery, we could not be sure of this for P. falciparum because the dataset had little sample annotation.PCR products from circle-specific primers were resolved by agarose gel electrophoresis. In Arabidopsis circular-RNA candidates, so it was not further pursued). In all the other cases, the sequence results confirmed the expected circle-junction sequence  primers amplified DNA with products of expected size, while outward facing primers failed to give a specific product  mrps16 in S. pombe) studied here, the circular isoform was present at significantly lower abundance than its corresponding linear isoform.We also report in Arabidopsis thaliana and depleted of ribosomal RNA, followed by cDNA synthesis using a mix of oligo(dT) and random hexamers as primers . By the criteria described in the preceding section, we validated three genes with evidence of circular RNA expression: a circle consisting of only exon 3 of NPY4, a gene implicated in gravitropic response; an exon 6-7-8 circle in EMB2423, a telomere-length regulation protein homolog; and an exon 4–5 circle in CYP87A2, a cytochrome P450 enzyme. For CYP87A2, we also identified circles composed of exon 5 only and exons 4-5-6; in addition, some circular isoforms used alternative 5′ and 3′ splice sites; the latter phenomenon was seen in EMB2423 as well . For CYP87A2, encoding a cytochrome P-450, we did indeed detect spliced transcripts that skipped exons 4 and 5 (i.e. with sequence reads representing an exon 3 - exon 6 junction), precisely the exons present in the predominant circular isoform identified for this gene. For the additional circular isoforms of this gene's transcripts  we found no evidence of the complementary exon-skipping linear transcripts. Nor did the other two Arabidopsis genes we chose for validation show circular RNA exon-skipping reciprocity: we found no evidence for exon-skipping RNA isoforms of NPY4, and although one transcript isoform of EMB2423 skipped exon 2, this skipped exon had no obvious relevance to the observed circular isoforms, which comprised exons 6, 7 and 8.It has been proposed that some circular RNA isoforms could be produced secondarily by splicing from a lariat excised during exon-skipping, as it was observed that the exons present in some circular isoforms of a rat cytochrome P-450 gene were precisely those lacking in alternative mRNA transcripts arising from exon-skipping P. falciparum, we found evidence for several circular RNA species. All were represented at low levels in the RNA-Seq data. The abundance of the candidate circular RNA species relative to their linear counterparts varied among libraries (which we presume represented different life-cycle stages). All required many PCR cycles for detectable amplification.In a simple bioinformatic search for circular RNAs using 60 nt RNA-Seq libraries from PF11_0156, a putative serine/threonine protein kinase. This predicted circular isoform was confirmed using outward facing primers in exon 4; in addition, we also detected a circle composed only of exon 4 . We identified 3 putative circular RNAs which were all subsequently verified by qRT-PCR as being RNase R-resistant  exponential growth in complete minimal medium, and 2) after 24 hours of nitrogen starvation, and performed careful quantitative analysis of this data. Upon nitrogen starvation, S. pombe cells divide twice (decreasing in size), and either mate then undergo meiosis, or else arrest in a stress-resistant quiescent state S. pombe and 3% of the 1374 genes with 2 or more introns. The majority of these candidate circles were represented by single reads , pub1 (a ubiquitin ligase), ypt5 (a Rab GTPase), and SPAC57A7.13 (a putative splicing-related RNA-binding protein). All four gave circle-specific RT-PCR products of the expected size and sequence, and all were resistant to RNase R treatment , we obtain a rough estimate of ∼1.4 molecules of circular RNA per cell in exponential growth, and ∼4.2 circles per cell in nitrogen starvation. The true circle abundance may be higher; circles may be systematically under-represented in these data-sets as the majority of circles are smaller than the approximately 200 nt fragment size targeted for library construction . By multiplying by the number of mRNAs per cell as estimated by Marguerat tion see . In any S. pombe. As documented by Marguerat et al., cell size and total RNA content per cell decrease dramatically during nitrogen starvation; the majority of linear mRNAs decrease their copy number per cell mrps16, pub1, ypt5, and SPAC57A7.13 ; data from human cells is consistent with this idea We used quantitative RT-PCR to examine the relative abundance changes of individual RNA species during a time-course of nitrogen starvation of C57A7.13 . The cirS. cerevisiae, very few of its genes contain introns; of these only ten have more than one intron . We found evidence for circular transcript isoforms from the majority of these two-intron genes: products of the size expected from the predicted circular RNA isoforms, amplified by PCR using the outward-facing primers, resistant to RNAseR treatment of the template RNA, with exon junction sequences consistent with circular splicing , two protists  and a plant . In addition to the data presented here, we have also preliminary bioinformatic evidence of circular RNA in several other organisms: the protist Plasmodium yoelii; the alga Chlamydmonas reinhardtii; and the filamentous fungus Neurospora crassa . The occurrence of circular RNA with similar structural features in eukaryotes that diverged more than 1 billion years ago suggests that this aspect of gene expression is either deeply conserved, or the result of repeated convergent evolution.We have found evidence for circular RNA throughout the eukaryotic tree of life. Previous studies have reported circular RNA isoforms in mammals, fish, worms, and insects. In this study, we found clear evidence for RNA circles in two fungi  undergo exon skipping. Similarly, only a small minority – 254 (4.5%) – of genes in P. falciparum have been reported to undergo alternative splicing The expression of numerous circular RNA isoforms in Sry gene may likewise function to bind miR-138 P. falciparum lacks known siRNA or microRNA pathways S. cerevisiae has specifically lost these pathways Recent reports on circular RNA in animals have proposed that they can function as microRNA sponges. CDR1 antisense transcript (CDR1as) is a circular RNA in mouse and human brain that contains more than 70 binding sites for the microRNA miR-7 and may suppress its activity. The mouse testis-specific circle of the S. pombe, the amount of linear mRNA per cell decreases dramatically During nitrogen starvation in S. cerevisae, S. pombe and Arabidopsis will complement the ongoing work on circular RNAs in humans and other metazoans.Overall, the identification of circular RNAs in these diverse organisms challenges prevailing ideas about how circular RNAs are generated and what roles they may play. Their wide phylogenetic distribution multiplies the opportunities for investigation of these novel molecules. In particular, we predict that many circular RNAs may be regulated during development and environmental responses; exploring these in experimentally tractable organisms like S. pombeS. pombe mutant for the debranching enzyme Dbr1. In addition to abundant reads in lariats, they observed 11 exon-scramble reads but did not do any further validation. Two of these reads correspond to genes identified in this study, mrps16 and ypt5. The mrps16 junction is identical to the one we describe, while their ypt5 isoform consisted of exon 4, intron 4, and exon 5 (distinct from the exon 8 – exon 7 isoform we describe). We did not identify their other exon-scramble genes in the analysis presented in this paper, nor did they identify exon-scramble reads for pub1 and SPAC57A7.13. However, we subsequently analyzed the data from these authors pub1 in addition to a total of 36 genes, in more than 50 reads  were generated by taking L – 15 bases at the 3′ end of exon i and L – 15 bases at the 5′ end of exon j. In cases where the length of exon i or exon j was shorter than L –15, we padded out using tandem repeats of exon i – exon j (mimicking the expected product of “rolling-circle” reverse transcription of a circular RNA). In P. falciparum and S. pombe, all reads were mapped to this database without previous filtering, under bowtie conditions as below. Due to the extent of annotated alternative splicing in Arabidopsis and Dictyostelium discoideum, only reads that failed alignment to the annotated genome and transcriptome were mapped to the database.For each organism with RNA-Seq reads of length :The following RNA-Seq data, genome annotations, readlength, and bowtie alignment flags were used: -v 3 was used for reads exceeding 50nt and –m 1 was used for organisms which lack extensive annotated alternative splicing http://www.broadinstitute.org/annotation/genome/plasmodium_falciparum_spp/MultiDownloads.html; read length  = 60, bowtie flags: -v 3 -m 1.Data was downloaded from the study: ERP001849. Annotations were downloaded from: ftp://ftp.ebi.ac.uk:21/pub/databases/pombase/pombe/GFF/; read length  = 50; bowtie flags -v 1.Data was downloaded from the study: ERP001483. Annotations were downloaded from: L = 40 and the flags –v 1 and –m 1.We also analyzed data from SRR927118, SRR927119 using the same annotations; reads were 43 nt and all alignments were performed with flags –trim5 3, reads failing alignment to the genome with flag –v 3 were aligned to the custom database as described above using the effective read length of ftp://ftp.arabidopsis.org/home/tair/. Only the first 10 annotated exons per transcript were used to generate the junctional database to conserve computational resources for the small minority of genes with >10 exons. Reads failing alignment to the genome and the transcriptome (TAIR10_cdna_20101214_updated) under flags –v 3 were aligned to the junctional database; read length  = 100; bowtie flags -v 3.Data will be reported in detail in another study . Annotations were downloaded from: ftp://ftp.ensemblgenomes.org/pub/protists/release-18/fasta/dictyostelium_discoideum/ and http://dictybase.org/db/cgi-bin/dictyBase/download/download.pl?area=blast_databases&ID=dicty_primary_cds.gz . To estimate the number of circular RNAs per cell, this fraction was multiplied by the total number of mRNAs per cell .RNA sequencing data and estimates of total mRNA copies per cell from Marguerat S. pombe (972 h- strain) were grown in Edinburgh minimal medium (EMM) at 32°C with shaking to exponential phase (OD600 0.25-0.3). The cells were centrifuged, washed with EMM-N (EMM lacking ammonium chloride), and resuspended in warm EMM-N to the original culture volume. The nitrogen-starved culture was continued at 32°C with shaking. Culture aliquots taken at various timepoints, cells counted, centrifuged, and cell pellets frozen at −80°C. RNA was purified from cell pellets by the hot-phenol method Wild-type P. falciparum total RNA was a kind gift of Joe DeRisi and Danny Ebert. Total RNA was harvested from a schizont-stage (∼42–44 hours post-invasion) synchronized by 5% sorbitol treatment. Total RNA was extracted using Trizol  followed by acid phenol chloroform extraction.A.thaliana root was extracted using RNeasy Plant Mini Kit (Qiagen) according to the manufacture's instructions.Total RNA from cultured 2), 1 μl 0.1M DTT, 40 U murine Ribonuclease Inhibitor, and 1 μl reverse transcriptase  was added, and the reaction incubated at 25°C for 10 min, 42°C for 50 min, 45°C for 5 min, 50°C for 5 min, 85°C for 5 min, 4°C hold.1 microgram of total RNA was incubated with 5 U RNase R (Epicentre), 10 U murine Ribonuclease Inhibitor (New England Biolabs), in 1x RNase R buffer at 37°C for 30 min, then placed on ice. Mock reactions were the same but without addition of RNase R. cDNA synthesis followed without purification: 1 μl 1 mM EDTA, 1 μl dNTPs (10 mM each), and 1 μl random hexamer (100 μM) was added and the sample denatured at 65°C for 5 min, then placed on ice. 4 μl of 5× buffer ; quantitative PCR was done with Power SYBR Green Mix (ABI/Life Technologies) on an ABI 7900HT. qPCR Ct values were calculated automatically by the manufacturer's software. A list of PCR primers is given in PCR products were either Sanger-sequenced directly using the amplification primers, or cloned into a TOPO vector (Invitrogen/Life Technologies) and sequenced with vector primers.Total RNA was extracted from root tissue of seedlings (ecotype Col-0) using RNeasy Plant Mini Kit (Qiagen) according to the manufacture's instructions and quantified using the Qubit RNA Assay Kit (Invitrogen). Ribo-Zero Magnetic Kit (Plant Seed/Root) (Illumina) was used to remove rRNA from total RNA. The RNA was then fragmented using RNA Fragmentation Buffer (Kapa Biosystems). cDNA was prepared using SuperScript Double-Stranded cDNA Synthesis Kit (Invitrogen) using both random hexamer and oligo dT oligonucleotides. Library construction was performed using the KAPA HTP Library Preparation Kit (NEB) following the manufacture's instructions and amplified using custom-made barcoded adapters. Libraries were pooled together and sequenced on a HiSeq 2000 (Illumina) .Dictyostelium discoideum cells (orfj) grown axenically in 1.5xHL+FM medium. RNA was treated with DNase I, digested twice with RNase R, combined with similarly treated Neurospora crassa RNA, and then processed to a barcoded paired-end Illumina sequencing library using 1/4th of a normal ScriptSeq reaction (Epicentre). This library was included as 1/15th of a pool of libraries for a single Illumina MiSeq 150 nt paired-end run.Total RNA was extracted using TRIZOL from wildtype Figure S1RNase R resistance of circular and linear isoforms. a) Quantitation of RNase R resistance. Plotted here is the RNase R resistance of each isoform (the log2 fold-change in RNA abundance with RNase R treatment), measured by quantitative RT-PCR and taken as ΔCt  =  Ct(mock-treatment) – Ct(RNase R-treatment). All linear isoforms were sensitive to RNase R, showing a greater than 32-fold drop in abundance after RNase R treatment (ΔCt <−5). Circular isoforms show no significant decrease in abundance with RNase R treatment, and in many cases the signal increases (see main text). The absolute Ct for mock-treated RNA is also given, as an indicator of the comparative abundance of circular and linear isoforms. For S. pombe, data shown here is for exponential growth. b) Quantitation of RNase R resistance for two additional species, Dictyostelium discoideum and S. cerevisiae. Format is the same as a).(TIF)Click here for additional data file.Text S1PCR primers, Sanger sequencing. 1) List of primers used in PCR and qPCR. 2) Sanger sequences of detected circular RNA isoforms.(DOC)Click here for additional data file.Table S1S. pombe candidate circular isoforms, data of Marguerat et al. All candidate circular isoforms detected in S. pombe from the data of Marguerat et al.(TXT)Click here for additional data file.Table S2S. pombe candidate circular isoforms, data of Awan et al. All candidate circular isoforms detected in S. pombe from the data of Awan et al.(TXT)Click here for additional data file.


The LNCaP and C4-2B cell lines form an excellent preclinical model to study the development of metastatic castration-resistant prostate cancer, since C4-2B cells were derived from a bone metastasis that grew in nude mice after inoculation with the LNCaP-derived, castration-resistant C4-2 cells. Exome sequencing detected 2188 and 3840 mutations in LNCaP and C4-2B cells, respectively, of which 1784 were found in both cell lines. Surprisingly, the parental LNCaP cells have over 400 mutations that were not found in the C4-2B genome. More than half of the mutations found in the exomes were confirmed by analyzing the RNA-seq data, and we observed that the expressed genes are more prone to mutations than non-expressed genes. The transcriptomes also revealed that 457 genes show increased expression and 246 genes show decreased expression in C4-2B compared to LNCaP cells. By combining the list of C4-2B-specific mutations with the list of differentially expressed genes, we detected important changes in the focal adhesion and ECM-receptor interaction pathways. Integration of these pathways converges on the myosin light chain kinase gene (MLCK) which might contribute to the metastatic potential of C4-2B cells. In conclusion, we provide extensive databases for mutated genes and differentially expressed genes in the LNCaP and C4-2B prostate cancer cell lines. These can be useful for other researchers using these cell models. Prostate cancer (PCa) is the most frequently diagnosed cancer and third leading cause of death amongst men in Europe As an alternative, cell lines can be used as models to study the transition to metastatic castration-resistant PCa For these two cell lines, changes in karyotype and genomic copy numbers, some point mutations, insertions and deletions have been described, but the comparison of the exome sequences have not been reported yet et al.A first step to catalogue point mutations, insertions and deletions in the LNCaP cells was reported in Spans The LNCaP cell line was obtained from the American Type Culture Collection, while the C4-2B cells were a kind gift from Dr. M. Stallcup  Whole-exome capture of the LNCaP cells was performed using the SureSelect Human All Exon System (Agilent) according to the manufacturer's instructions. Paired-end, 100 bp long sequencing reads were generated using the GAIIx sequencer (Illumina). The exome capture of the C4-2B cells was performed using the SeqCap EZ Exome version 2 kit (Roche Nimblegen) and paired-end 100 bp long reads were generated using the HiSeq2000 (Illumina).http://www.bioinformatics.bbsrc.ac.uk/projects/fastqc/) and Picard (version 1.22) (http://picard.sourceforge.net/). Sequencing reads were aligned to the human reference genome  using BWA, where reads were trimmed when the quality was below 15 (version 0.5.9) http://bioinfogp.cnb.csic.es/tools/venny/index.html).Quality control was performed using FastQC software (version 0.10.1)  and treated overnight (18 h) with 1 nM R1881 (Perkin Elmer). The cells were collected and washed with PBS. The cell pellet was used to extract total RNA using the RNeasy Mini Kit from Qiagen. The quality and purity of the RNA was inspected on a Nanodrop ND-1000 Spectrophotometer. The integrity of the RNA was verified on the BioAnalyzer at the Genomics Core of UZ Leuven.After selection of polyA+ RNA, the RNA was converted into cDNA libraries using the TruSeq RNA Sample Preparation kit (Illumina). After sequencing paired-end short reads of 100 bp with the HiSeq2000 (Illumina), normalized gene counts  were calculated via the Tuxedo pipeline (Tophat – Cufflinks – CummeRbund) 5′-ACCCAAGGCCAACCG-3′ and reverse 5′-TGTACGTTGCTATCCAGGCTGT-3′, TMPRSS2 forward 5′-CCTGCATCAACCCCTCTAACTG-3′ and reverse 5′-AGGCGAACACACCGATTCTC-3′, IGF1 forward 5′-TGGATGCTCTTCAGTTCGTG-3′ and reverse 5′-TCATCCACGATGCCTGTCT-3′, IGF1R forward 5′-GTACAACTACCGCTGCTGGA-3′ and reverse 5′-TGGCAGCACTCATTGTTCTC-3′.cDNA was generated from RNA (1 µg) with Random Hexamer primers and RevertAid Reverse Transcriptase (Thermo Scientific). Quantitative Real Time PCR was performed using Platinum SYBR Green QPCR Supermix-UDG (Invitrogen). Results were normalized to the housekeeping gene β-actin and each sample was analyzed in triplicate. The sequence of the primers used are: β-actin forward http://www.ebi.ac.uk/ena/data/view/PRJEB4877. The sample accession numbers are ERS363578 and ERS363580 for whole exome sequencing data of LNCaP and C4-2B respectively. For the RNA-sequencing, the sample accession numbers are ERS363579 and ERS363581 for LNCaP and C4-2B cells respectively.Binary sequence alignment/map (BAM) files from whole exome sequencing data as well as RNA-seq data were deposited in the database of the European Nucleotide Archive with accession number PRJEB4877 and are accessible via http://ncbi.nlm.nih.gov/tools/primer-blast/) and obtained from Integrated DNA Technologies. Polymerase chain reactions were performed following standard protocols using Taq DNA polymerase (Thermo Scientific). Amplification of specific PCR fragments was confirmed by agarose gel electrophoresis. Sanger sequencing was performed at LGC Genomics. Sequence trace files were analyzed using Chromas Lite.Variants of interest were confirmed by Sanger sequencing of amplified PCR products. Primers specific to the region containing the variant to be tested were designed using the NCBI Primer-Blast , resulting in a high coverage of mRNA bases . As a meThe workflow for the detection and subsequent filtering of point mutations was similar to that used for the exome sequencing described higher. We found 1505 and 1882 mutations in LNCaP and C4-2B cells respectively, of which 1054 were detected in both cell lines ; 451 werA comparison of the allele-specific read counts from genome and transcriptome sequencing data of all detected point mutations can be used as a measure of the sequencing quality. The majority of mutations have a similar allele frequency in both DNA and RNA sequencing . Even thThe combination of both the exome and transcriptome sequencing resulted in a total of 2244 mutations common to both cell lines . MoreoveThe different types of transitions and transversions in the exomes and transcriptomes of LNCaP and C4-2B cell lines might give insight in the mutational processes that took place during the development of these cells. We observed that the predominant mutations (40–42%) in both cell lines were G-to-A and C-to-T transitions .The most prevalent type of RNA editing in higher eukaryotes is the conversion of adenosine to inosine. As inosine is read as a guanine after sequencing, this editing type manifests itself in RNA-sequencing as an A-to-G substitution In total, 80 mutations in the exome data from LNCaP (47) and C4-2B (33) were validated by manual Sanger re-sequencing . The genWe also detected and confirmed C4-2B specific mutations in CASP9, FLNB, POLR2A and STAT5A in genomic DNA and cDNA of C4-2B cells, but not in LNCaP cells. Finally, mutations in genes that are not expressed in LNCaP or C4-2B (KIT and GRAP) could only be confirmed on genomic DNA.et al. by comparing GATK with SAMtools, Atlas 2 and glftools In conclusion, the GATK UnifiedGenotyper for variant calling which we combined with our extensive filtering generated few false positives. Similar results were shown recently by Liu We next wanted to search for differentially expressed genes between the two cell lines, since these might provide clues to the mechanisms behind the evolution of LNCaP cells into C4-2B cells. Differential expression was called by the Tuxedo algorithm based on RNA-seq data of triplicates for each cell line, with additional filtering of log2-fold change>2 and q-value<0.001. All replicates were very similar, as can be seen in Our analysis resulted in the identification of 703 differentially expressed genes , of whicet al. already described some differentially expressed genes between LNCaP and C4-2B, but none of the genes they detected are differentially expressed in our data Fu LNCaP and C4-2B cells continue to be used in basic and preclinical research. We propose our databases of mutations and differentially expressed genes as important sources of inspiration for further research projects. In addition, these databases can now be checked for specific mutations before one starts using these cells to study any specific PCa-related pathway.in silico analysis of our data. Pathway-Express analysis of the C4-2B specific mutations combined with the 703 genes differentially expressed between LNCaP and C4-2B cells indicated that the most significant changes were found in the ECM-receptor interaction pathway and in focal adhesion. Both pathways converge in the upregulated expression of the myosin light chain kinase (MLCK) gene (This paragraph gives an example of a hypothesis based on CK) gene .C4-2B cells are derived from a bone metastasis in nude mice inoculated with cells originating from the LNCaP-derived, castration-resistant xenografts called C4-2. They are considered a useful preclinical model for metastatic, castration-resistant and androgen receptor positive PCa. Here, we provide for the first time comparative maps of the point mutations detected in the LNCaP and C4-2B cells. In addition, although transcriptome analyses of LNCaP and C4-2 have been reported, to our knowledge, this is the first transcriptome analysis of C4-2B cells.et al. already described a correlating high instability of satellite DNA in LNCaP cells C4-2B cells as well as LNCaP cells have a surprisingly high number of point mutations: 4373 and 2790 mutations respectively. Like in primary PCa and castration-resistant PCa samples, the mutational spectrum is dominated by G-to-A and C-to-T transitions et al., where a single PCa tumor harbored a frameshift mutation of the MSH6 gene among 996 other mutations The number of point mutations in our cell lines is much higher than the average 16–33 mutations detected in whole exomes of PCa samples For both the LNCaP and C4-2B cell line, we see that highly expressed genes more frequently contain point mutations than non-transcribed genes . This contradicts the general link between heterochromatin organization and higher regional mutation rates in human cancer cells We detected 1784 shared mutations in the exomes of LNCaP and C4-2B, and 2056 C4-2B-specific changes, which makes sense since the C4-2B cells are derived from the LNCaP cells. However, we also detected 404 LNCaP-specific changes, many of which were confirmed by our transcriptome sequences. Obviously, the LNCaP cells we analyzed have deviated from the LNCaP cells that were used originally to develop the C4-2B cells Our data can clearly lead to the hypothesis on the metastatic process that took place during the conversion of LNCaP to C4-2B cells. This is exemplified by the convergence of a number of affected pathways to an upregulation of MLCK. Indeed, there are several published links between MLCK and the metastatic process. Discriminant analysis of microarrays identified the MLCK gene as the most informative gene for the PCa genesis process In conclusion, our data clearly show that there are major differences in the number and distribution of mutations and gene expression between LNCaP and C4-2B cells. Since these cell lines are universally used to study the progression from non-metastatic to metastatic PCa, these data are crucial for researchers to correctly interpret their results when using these cell lines. Moreover, our databases will be very helpful in developing new investigational ideas.Figure S1FastQC quality control results of the per base qualities. Output results of the FastQC quality control software (version 0.10.1) are shown here for exome and transcriptome sequencing of LNCaP and C4-2B cells.(TIF)Click here for additional data file.Figure S2Normalized coverage by position. The average relative coverage is shown at each relative position along the transcript's length. LNCaP is depicted in green, while C4-2B is depicted in red. The x-axis represents the gene length normalized to 100%, where 0 is the 5′ end of each transcript and 100 is the 3′ end.(TIF)Click here for additional data file.Figure S3Heatmap of 703 differentially expressed genes. The heatmap shows the three replicates of each cell line, which are very similar. All differentially expressed genes were detected using the Tuxedo algorithm, with q<0.001 and log2-fold change >2 as cut-offs. It is clear that the majority of genes is upregulated in C4-2B compared to LNCaP, while a smaller group of genes is downregulated in C4-2B.(TIF)Click here for additional data file.Table S1Sequencing characteristics.(XLSX)Click here for additional data file.Table S2Filters used to identify point mutations in the exomes of LNCaP and C4-2B cells.(XLSX)Click here for additional data file.Table S3List of point mutations identified in the exome of LNCaP cells.(XLSX)Click here for additional data file.Table S4List of point mutations identified in the exome of C4-2B cells.(XLSX)Click here for additional data file.Table S5Filters used to identify point mutations in the transcriptomes of LNCaP and C4-2B cells.(XLSX)Click here for additional data file.Table S6List of 703 genes that are differentially expressed between LNCaP and C4-2B cells.(XLSX)Click here for additional data file.


RNA editing is a post-transcriptional process occurring in a wide range of organisms. In human brain, the A-to-I RNA editing, in which individual adenosine (A) bases in pre-mRNA are modified to yield inosine (I), is the most frequent event. Modulating gene expression, RNA editing is essential for cellular homeostasis. Indeed, its deregulation has been linked to several neurological and neurodegenerative diseases. To date, many RNA editing sites have been identified by next generation sequencing technologies employing massive transcriptome sequencing together with whole genome or exome sequencing. While genome and transcriptome reads are not always available for single individuals, RNA-Seq data are widespread through public databases and represent a relevant source of yet unexplored RNA editing sites. In this context, we propose a simple computational strategy to identify genomic positions enriched in novel hypothetical RNA editing events by means of a new two-steps mapping procedure requiring only RNA-Seq data and no a priori knowledge of RNA editing characteristics and genomic reads. We assessed the suitability of our procedure by confirming A-to-I candidates using conventional Sanger sequencing and performing RNA-Seq as well as whole exome sequencing of human spinal cord tissue from a single individual. RNA editing is a widespread post-transcriptional phenomenon through which primary RNA sequences are altered by nucleotide insertion/deletion or base conversion Inosine is commonly interpreted as guanosine by translation and splicing machineries other than sequencing enzymes. As a consequence, A-to-I modifications can alter codon identity and increase transcriptome as well as proteome diversity A peculiarity of RNA editing is that both the edited and unedited versions of affected transcripts are co-expressed in the same cell and the ratio between the two variants can be regulated by a variety of factors depending on tissue type or developmental stage.A-to-I modifications are prominent in human brain, mostly in untranslated mRNA regions, and indispensable for cellular homeostasis locus of origin in order to look for A-to-G mismatches. Indeed, a large number of A-to-I editing events have been detected by validation (through Sanger sequencing) of A/G mismatches revealed by multiple alignments of mRNA/EST sequences onto the reference genome The most common experimental procedure to discover novel RNA editing events requires the direct comparison between cDNA sequences and the corresponding genomic The advent of high-throughput sequencing technologies have provided unprecedented opportunities for genome-wide investigation of RNA editing aimed at finally obtaining a comprehensive inosinome map Diverse large-scale RNA editing investigations have been performed up to now showing the complexity of human inosinome, sometimes with questionable results likely depending on different computational strategies of data analysis a priori knowledge of RNA editing sites or the genomic sequences of the particular donor individual. Starting from a human RNA-Seq experiment our strategy provides a set of genome positions ranked according to their decreasing probability to be edited. We benchmarked this methodology by genuine short sequence reads, focusing on editing conversions in coding protein sequences. Independent Sanger sequencing as well as whole exome and transcriptome reads from human spinal cord of a single individual have been used to experimentally support newly detected A-to-I changes. Our results show that editing events can be reliably predicted de novo from RNA-Seq data and highlight that the accuracy of results is strongly biased by the mapping strategy and data quality. We discuss strengths and weaknesses of this approach.Although sequencing costs are rapidly decreasing, the concomitant whole genome and transcriptome sequencing from single individuals and tissues coupled to robust downstream statistical analyses, is still unpractical for large samplings. In contrast, public short read archives contain a huge amount of RNA-Seq data from a variety of human tissues and experimental conditions Illumina paired end RNA-Seq reads (2×50 bp) from SRP002274 study were downloaded from SRA archive using accessions: SRR039628, SRR039629, SRR039630, SRR039631, SRR039632 and SRR039633.http://hgdownload.cse.ucsc.edu/goldenPath/hg18/database/). ASPiCdb transcripts (version of 9/Jun/2009) in GTF format for hg18 human genome were obtained from the following web page: http://www.caspur.it/ASPicDB/.Ensembl (version of 9/Aug/2009), UCSC (version of 10/May/2009) and RefSeq (version of 15/Jul/2011) transcripts for human assembly hg18 were downloaded from UCSC genome browser  onto the assembled transcriptome including more than 370,000 transcripts from Ensembl, UCSC, RefSeq and ASPiCdb. Custom python scripts were then used to parse GSNAP results, filtering out inconsistent alignments due to reads with more than two Ns characters and very low quality scores. The same scripts converted transcript coordinates to genome coordinates producing a standard SAM file as output.Aligned reads were subsequently mapped onto the complete human genome (assembly hg18) by means of GSNAP (using as parameters: -B 5 -d hg18 -t 10 -s splicesites -a paired -O -A sam –no-sam-header) providing a list of exon-exon junctions extracted from the assembled transcriptome and avoiding the prediction of new splice sites. Next, genomic and transcriptomic alignments were compared using a custom python script in order to exclude discordant mappings. Final alignments were printed out in the standard SAM format and converted in the corresponding binary BAM by SAMtools.Exome reads from spinal cord tissue were aligned onto the complete human genome (version hg18) using GSNAP (with parameters: -d hg18 -B 5 -t 10 -a paired –pairmax-dna = 500 -n 1 -Q -O –nofails –query-unk-mismatch = 1 -A sam –no-sam-header) and requiring only unique mappings. Duplicated reads due to potential PCR artefacts were removed by SAMtools (rmdup program).Read alignments in SAM or BAM formats were converted in pileup format by SAMtools. Such file was parsed by a custom python script and traversed position by position in order to count the number of observed substitutions taking into account a minimum quality score per base of 25. In case of spinal cord RNA-Seq reads we increased the quality score cut-off to 30. The relative frequency of each observed substitution over their global number (versus the reference genome) was used as the empirical distribution of base substitutions for a given dataset  and human astrocytoma cell line U118 MG (HTB-15™), or U118 stably transfected with EGFP-ADAR2 cells were used in the study. The study was approved by the local ethic committee. The cells were grown in DMEM supplemented with 10% FCS and antibiotics, at 37°C in 5% CODNA and total RNA were extracted with specific kits (Roche and Invitrogen respectively), according to the manufacturer’s instructions. Each RNA sample was DNase treated  and quantified with the Agilent 2100 bioanalyzer (Agilent). cDNAs were generated by SuperScript II reverse transcriptase (Invitrogen) using random hexamers or specific primers (available upon request). Direct sequencing was performed on DNA and cDNA pools using standard Sanger procedure (Applied Biosystem kit). Direct sequencing was performed on gDNA and cDNA pools, and editing was calculated as described previously http://medschool.umaryland.edu/btbank/). DNA and RNA were isolated, using specific kits (Roche and Invitrogen) after tissue grinding with mortar and pestle. Quantification and integrity were checked by the Agilent 2100 bioanalyzer (Agilent).Human spinal cord samples were purchased from the NICHD Brain & Tissue Bank (University of Maryland - http://www.appliedgenomics.org/) using Illumina technology. Libraries were prepared from 2 µg of total RNA adopting a modified protocol based on miRNA sequencing . Paired-end reads of 101 bases long were obtained using the Illumina HiSeq2000 machine. Exome sequencing was also performed at IGA Institute from 6 µg of DNA using the Illumina TruSeq Exome Enrichment Kit (62M). Pair-end sequencing was performed on Illumina HiSeq2000.Directional RNA-Seq was performed at IGA Institute  we calculated the statistical support of observing RNA editing using the log-likelihood by perl scripts kindly provided by Iouri Chepelev.Potential RNA editing events can be identified looking for A-to-G mismatches in multiple alignments of expressed sequences  onto the reference human genome. A-to-G patterns could show different extents ranging from 0 (absence of editing) to 100% (fully edited). This is due to the concomitant presence of edited and unedited transcripts even though some apparent A-to-G substitutions could be caused by SNPs, somatic mutations or sequencing errors. In large-scale experiments involving data from next generation sequencing technologies, we may observe a large number of such A-to-G patterns, many of them being genuine RNA editing conversions. Looking at data from various RNA-Seq experiments, we reasoned that the identification of editing events could be facilitated by considering only those whose occurrence is statistically significant with regard to the empirically observed distribution of base substitutions. For example, given a multiple alignment of short reads onto the reference genome we can calculate empirically the frequency of A-to-G changes and employ such value to verify whether or not the observed A-to-G pattern is significantly different from the expected one by means of the Fisher exact test. Excluding known genomic SNPs and filtering A-to-G patterns by quality scores, we should be able to obtain a set of genomic positions enriched in RNA editing events .de novo RNA editing events. To assess the reliability of our working hypothesis and, thus, computational strategy, we downloaded from the SRA database the RNA-Seq experiment SRP002274 containing 23 millions of Illumina paired-end reads 50 bp long, obtained from human brain MAQC mRNA of seven individuals .A huge amount of deep sequencing data from entire transcriptomes are now freely available through public databases and could be employed to detect The first challenging task to address for predicting RNA editing events is to correctly align short reads onto the reference human genome reducing biases due to mappings in multiple genome locations or paralogous genes. To date there is no standard procedure to solve this issue and recent works focusing on DNA-RNA differences by deep sequencing take very different approaches and sometimes with questionable findings −27  An explanatory overview of our computational framework is depicted in The better performance of the last strategy with respect to the first and the second one, in the reliable identification of the editing sites, is supported by the observation that the fraction of known editing sites increases from 12/55 21.8%, , to 12/3In order to confirm the effectiveness of our strategy and, thus, demonstrate the specific enrichment of novel RNA editing events, we experimentally assessed the remaining 8/19 predicted editing positions. Two out of these eight editing events, in MRPL28 and ACP1 genes, were experimentally supported by parallel exome and transcriptome sequencing in human spinal cord (see below). The validation of the six remaining sites  was carried out by direct sequencing. In order to exclude potential SNPs at the predicted editing position, we sequenced both the gDNAs and the cDNAs from the same tissues and cell line, i.e. human brain tissue or astrocytoma cell lines. Briefly, total RNA was extracted and RT-PCR was performed using specific primers for the different transcripts, similarly the corresponding DNA portions were amplified and sequenced. The comparison of the gDNA and the cDNA traces revealed that four out of six candidates  were genuine RNA editing events .in silico for the ATP1A1 transcript was not supported by Sanger sequencing after comparison of both gDNA and cDNA despite different human sample tissues  and cell lines were used  falling in ZNF414 gene was shown to be a genuine SNP .In summary, 17 sites out of the 19 predicted candidates in SRP002274  resulted real RNA editing events, demonstrating the usefulness of our computational strategy in detecting bona-fide RNA editing sites from RNA-Seq data. This is potentially an underestimate, as invalidated events may occur in other tissues, cell types, physiological or pathological conditions.A further assessment of our strategy was carried out performing an RNA-Seq experiment on total RNA extracted from human spinal cord. In contrast with the above SRP002274 study, our deep sequencing was accomplished in spinal cord of a single human individual producing paired-end and strand oriented reads using the Illumina platform. Initially, by using GSNAP program, we mapped 40,277,555 RNA-Seq reads onto the human transcriptome and, then, mapped reads (80%) were also aligned to the complete reference genome. Both transcriptome and genome alignments were compared and concordant mappings (73%) were collected for downstream analyses. Multiple alignments of short reads were scanned site by site using SAMtools After mapping 33,180,939 exome reads onto the reference human genome and excluding potential PCR duplicates, we interrogated the resulting multiple alignments by SAMtools using the list of the 12 RNA editing candidates. Ten out of 12 sites were supported by independent exome reads taking into account only bases with quality scores of at least 30. However, only 6 out of 10 positions were covered by at least 5 reads  containing only adenines and thus strongly supporting the presence of editing . For sucExome and transcriptome reads from human spinal cord also confirmed editing candidates in MRPL28 and ACP1 genes from SRP002274 study (see previous paragraph). Indeed, both editing events in MRPL28 and ACP1 genes were supported by significant LLR of 176 and 30 respectively, suggesting a high probability of being real RNA editing events.Alu regions and 52 were already annotated in DARNED database as edited sites. In addition, 33 positions were confirmed as genuine RNA editing events by exome reads. Of the 21 sites in AEs, 8 were located in Alu elements and 2 confirmed as edited by exome data. Notably, the limited exome data support (35/177 positions) was due to lack of coverage in all cases. Such findings are consistent with the expected distribution of RNA editing events in different mRNA regions, being more frequent in 3′UTRs and Alu repeats, than in coding protein sequences or 5′UTRs. However only a small fraction of the detected sites have been validated as genuine RNA editing sites by exome data. This is quite expected, as exome sequencing platforms specifically enrich sequence reads mapping protein-coding exons.Although we focused on A-to-I editing in coding protein regions, we used the same computational strategy to predict potential events falling in untranslated regions (UTRs) or alternative exons (AEs) not represented in RefSeq transcripts. In particular, we detected 156 candidates in UTRs (154 in 3′UTR and 2 in 5′UTR) and 21 in AEs . InteresRNA editing is emerging as an important post-transcriptional process increasing the already complex gene expression dynamics in eukaryotic genomes ad hoc filters. Although these strategies overcome limitations of assembled transcriptome datasets, they might not be error free. Indeed, as demonstrated in our work, the mapping of short reads on the complete genome using both tools that do not map across exon-exon boundaries and tools that allow splice alignments, affects the prediction of RNA editing since ambiguous alignments are difficult to be detected and removed. In contrast with current mapping strategies we developed a new framework based on two mapping rounds, the first onto an extended transcriptome collection  and the second onto the complete genome. In this way read pairs mapping uniquely and consistently on transcripts and genome are maintained for downstream analyses, providing an accurate set of alignments. Minimizing biases due to read mapping, multiple alignments can be interrogated to select patterns of A-to-G changes. Excluding all known SNP positions, we can verify how many observed A-to-G conversions show a pattern significantly different from expected. In this way we greatly reduce the number of A-to-G candidates to investigate for RNA editing. The possibility of recovering reliable candidates is strictly affected by the quality of the original data set, read mapping and the use of filtering procedures to exclude SNPs. Without a priori information about known editing events and properties, it is not easy to distinguish genuine RNA editing events from polymorphisms or sequencing errors. However, we currently restrict the selection of significant A-to-G patterns to those in which all supporting bases have a quality score of at least 25 and an editing extent higher than 10%, mitigating potential sequencing errors. According to filtering procedures, therefore, we can provide a ranked list of positions enriched in RNA editing events, addressing experimental validations to a restricted number of candidates. To corroborate our computational strategy we predicted RNA editing candidates in two RNA-Seq experiments with different characteristics. In the SRP002274 study we verified the behaviour of our strategy in a data set enriched in editing events as well as SNPs. Indeed, this RNA-Seq experiment has been performed pooling diverse brain locations from seven different individuals. Results clearly confirm that our method, despite its simplicity, provides a set of reliable RNA editing candidates. Using stringent criteria we identify and confirm six new A-to-I changes in human brain in which four are recoding with potential functional consequences, as for example the RNA editing position at chr14:25987370 in NOVA1 that has been recently associated with protein stability In humans, RNA editing can expand the transcriptome and proteome diversity by the A-to-I and/or C-to-U conversions in primary RNAs. RNA editing changes by adenosine deamination have been investigated in detail during the last two decades using computational methodologies employing ESTs and full-length mRNAs other than intrinsic properties of RNA editing sites as their association to double RNA strands or evolutionary conservation Considering that our procedure requires only RNA-Seq data and, at the time of writing, the SRA database includes more than 4,200 entries of human transcriptomes, it could be used to investigate and discover new RNA editing events, improving our understanding of this basic molecular phenomenon. In addition, RNA-Seq data can now be produced in a timely and cost effective way greatly extending tissue types and experimental conditions to be tested.de novo RNA-Seq based approach not assuming any prior knowledge about RNA editing process, it requires genomic short reads to exclude genetic polymorphisms and assess the statistical significance of predictions by LLR test. In contrast, our approach is completely de novo and does not require a priori RNA editing information or genome reads from the same individual. Potential SNPs are filtered out using known annotations in polymorphism databases as dbSNP, making our method useful for any RNA-Seq experiment. In addition, our RNA editing workflow can be easily extended to take into account further filtering criteria – such as the presence of secondary RNA structures or evolutionary conservation. In the presence of genome and/or exome read, the method is still useful to single out genuine editing events after the exclusion of heterozygous genomic sites. Indeed, the p-value calculated by the Fisher exact test performs similarly to LLR test in which very low Fisher p-values correspond to highly significant LLR scores  using a minimum quality score of 25 and from spinal cord RNA-Seq experiment (b) using a minimum quality score of 30.(TIF)Click here for additional data file.Figure S2LLR score Vs Fisher Pvalue. Relationship between LLR scores and Fisher Pvalues on 180 genomic positions supported by at least 5 independent exome reads. LLR and Pvalues were calculated on transcriptome and exome data from spinal cord. Decreasing the Fisher Pvalue, and thus the probability observing a genuine RNA editing event is associated with increases in the corresponding LLR scores.(TIF)Click here for additional data file.Table S1List of significant A-to-G substitutions in protein coding regions detected in SRP002274 study aligning short reads onto the complete human genome (hg18 assembly) using Bowtie.(DOCX)Click here for additional data file.Table S2List of significant A-to-G substitutions in protein coding regions detected in SRP002274 study aligning short reads onto the complete human genome (hg18 assembly) using Tophat to take into account the spliced nature of RNA reads.(DOCX)Click here for additional data file.Table S3List of significant A-to-G substitutions in known introns and UTRs detected in transcriptome reads from spinal cord.(DOCX)Click here for additional data file.