Escherichia coli cells, we measure, for each cell, the waiting time for the production of the first RNA under the control of PBAD promoter after induction by arabinose, and subsequent intervals between transcription events. We find that the kinetics of the arabinose intake system affect mean and diversity in RNA numbers, long after induction. We observed the same effect on Plac/ara-1 promoter, which is inducible by arabinose or by IPTG. Importantly, the distribution of waiting times of Plac/ara-1 is indistinguishable from that of PBAD, if and only if induced by arabinose alone. Finally, RNA production under the control of PBAD is found to be a sub-Poissonian process. We conclude that inducer-dependent waiting times affect mean and cell-to-cell diversity in RNA numbers long after induction, suggesting that intake mechanisms have non-negligible effects on the phenotypic diversity of cell populations in natural, fluctuating environments.Using a single-RNA detection technique in live  E. coli is, at a genome-wide scale, a relatively rare stochastic event  (1 and O2) and promotes the formation of a DNA loop that prevents access of RNA polymerases to the promoters in that region (PC and PBAD). When bound by arabinose, AraC binds instead to the adjacent I1 and I2 half-sites. The resulting configuration promotes transcription initiation at PBAD  and for the catabolism of arabinose (araBAD) ,22. In t at PBAD .In vitro measurements suggest that this process has at least two to three rate limiting steps , which is a single-copy F-based vector carrying a sequence coding for a monomeric red fluorescent protein (mRFP1) followed by a 96 binding site array under the control of PBAD (cloning information provided in Supplementary Methods) . The strain with plasmids PLtetO-1-MS2d-GFP and pIG-BAC (Plac/ara-1-mRFP1-MS2-96bs)   was usedS2-96bs) ] is a ge600 ≈0.3–0.5. To attain full induction of the MS2d-GFP reporter, cells were pre-incubated for 40 min with 100 ng/ml anhydrotetracycline . The same protocol was used for each strain.Cells were grown overnight at 30°C with aeration and shaking in Luria-Bertani (LB) medium, supplemented with antibiotics according to the plasmids. The cells were diluted in fresh M63 medium and allowed to grow until an optical density of ODFor microscopy measurements, cells were pelleted and re-suspended in ∼50 µl of fresh M63 medium. Afterwards, few microlitres of cells were placed between a 3% agarose gel pad made with medium and a glass coverslip before assembling the imaging chamber . Before the starting of the experiment, the chamber was heated to 37°C.Cells were visualized in a Nikon Eclipse  inverted microscope with C1 confocal laser-scanning system using a ×100 Apo TIRF objective. A flow of fresh, pre-warmed M63 medium containing the inducer was provided with a peristaltic pump at a rate of 1 ml/min. Images were taken once per minute for 2 h, and the laser shutter was open only during the exposure time to minimize photobleaching. The peristaltic pump was initialized at the same time as the collection of the time series. For image acquisition, we used Nikon EZ-C1 software. GFP fluorescence was measured using a 488 nm argon ion laser (Melles-Griot), 515/30 nm emission filter and a pixel dwell time of 3.36 µs .t based on the measurement at t-1. It allows reducing the number of required images at different z-planes for drift correction, thus minimizing photobleaching.An interacting multiple model filter-based autofocus strategy  was usedData and images were analysed using custom software written in MATLAB 2011b (MathWorks). Cells were detected from fluorescence images by a semi-automatic method described previously . In timeP-value of 0.01. Each jump corresponds to the production of a single RNA molecule , the total number of MS2d-GFP–tagged RNA molecules was extracted from the total spot intensity distribution, obtained from all cells in an image obtained at a given moment after induction. For this, the first peak of the obtained distribution is set to correspond to the intensity of a single-RNA molecule. The number of tagged RNAs in each spot can be estimated by dividing its intensity by that of the first peak , as well as the subsequent intervals between consecutive RNA productions, Δt, until cell division occurs or until the end of the measurement period , we, weBAD, t period D.0 can only be obtained from cells of the first generation , and as cells that do not divide in the first 2 h will not, in general, divide afterwards, we limited the measurement period to 2 h for simplicity. This was possible, as this period also proved to be sufficient to acquire enough samples of Δt.Given that values of t0.From cells born during the measurement period, we only extract intervals between consecutive RNA productions, not waiting times, as these contain inducers by inheritance. We detected no difference in the distributions of intervals obtained from such cells and cells already present when induction is initiated. Finally, we observed ∼0.2 RNA molecules per cell, at the moment preceding induction, because of spurious transcription events. Cells where a target RNA was already present at the start of the measurement were also not used to obtain values of tBAD (Supplementary Methods). Using 16S rRNA as reference gene, we observe similar trend in activity over time in the native promoter and in the one on the F-plasmid .First, we compared by quantitative polymerase chain reaction the RNA production from the F-plasmid and from the native gene under the control of Pl-arabinose (Supplementary Methods). In Supplementary Figure S4A and B, we show the temporal variation after induction in mean numbers of MS2d-GFP–tagged RNA molecules in cell populations and in the fluorescence intensity of RFP measured by plate reader, respectively.We next compare expression levels of the target gene, when assessed by independent methods, for two induction levels, namely, 0.1 and 1% l-arabinose is increased from 0.1 to 1%. The MS2d-GFP in vivo detection method shows a fold change of 1.74 between these same conditions, showing that the results from the two methods are in accordance. From this and the previous experiment, we also conclude that the MS2d-GFP tagging method accurately detects RNA production of the target gene, and that the target gene behaves similarly to the natural system.The plate reader measurements of mRFP1 levels, 2 h after induction in liquid culture, show a fold change of 1.67 times when Supplementary Figure S5, maximum induction is achieved for 1% arabinose. Here onwards, unless stated otherwise, we use this concentration to assess the kinetics of RNA production under the control of PBAD.We also assessed for what range of inducer concentrations is the target gene under full induction. We measured with the plate reader its expression for varying inducer concentration, 2 h after induction. From 0 and subsequent values of Δt. t0 is expected to include the time for arabinose to enter the cell via the intake mechanism, the time to find the promoter and release the repressor and also the time for the recruitment of the RNA polymerase and subsequent production of the first target RNA. The latter process includes events such as the closed and the open complex formation at the promoter region, as well as the elongation time. Both the elongation time and the time for MS2d-GFP to bind to a target RNA are expected to be negligible in comparison with the duration of the intake and of transcription initiation  of the distribution is 0.37. Similar conclusions were obtained from measurements of the in vivo kinetics of RNA production under the control of Plac/ara-1 and PtetA  is shown in nd PtetA ,10.P-value of 2.8 × 10−18, much smaller than 0.05, which allows rejecting the null hypothesis of similarity. We conclude that in the case of PBAD and the arabinose intake mechanism, the time of intake of inducers affects significantly both mean and standard deviation of RNA numbers in individual cells, long after induction. Finally, note that the difference between the distributions of t0 and Δt provides evidence that the activity of PBAD changes significantly with induction. Otherwise, these two distributions should not differ significantly, as they would both result, e.g. from spurious transcription events alone.The distributions in in vivo induction kinetics of PBAD. This study uses measurements of GFP levels in cell populations, whose expression is controlled by PBAD (inserted into a medium-copy vector) and a model to extrapolate the mean activation time of the promoter, after induction. Assuming a threshold for GFP levels to consider the promoter as active, the mean appearance time of GFP after induction was ∼960 s. By considering several features of the measurement system, including the mean maturation time of GFP, a value was then extrapolated for the expected activation time of the promoter, namely, ∼250 s. This does not include the time for transcription to be completed, once the closed complex is formed. This study thus predicts a faster mean initiation time than what our direct measurements indicate (∼3000 s). Two main reasons exist for this difference. First, in the mutant used previously . This window is set so as to maximize the number of data points that can be extracted from the measurements.We next assessed whether the distributions of tes of Δt A and B a0 and Δt values in individual cells to be −0.15. We calculated a P-value of 0.18 assuming no correlation as the null hypothesis, which implies that we cannot prove that the correlation is significant. This result is in line with , or by both inducers simultaneously  of t0 and of Δt for the two promoters, each of which in two induction schemes.We measured the distributions of tse alone . We usedac/ara-1 . Results0 and Δt by the K–S test, for each promoter in each condition  of the two promoters , we assume that they are independent so as to be able to estimate the standard deviation of the duration of the intake process alone .The intake time of an inducer, here named ‘tdiff are similar for PBAD and for Plac/ara-1, when induced with 1% arabinose. Namely, in both cases, we obtained a mean of ∼1400 s and a standard deviation of ∼1100 s. This is expected, given the usage of the same intake mechanism and inducer concentration. Importantly, when Plac/ara-1 is induced by IPTG, the standard deviation of tdiff is much smaller (∼700 s), whereas the mean is similar to when induced by arabinose (∼1400 s). This suggests that the intake of arabinose is a noisier process (concerning the uncertainty of the intake time) than the intake of IPTG. Finally, we find that in the case of PBAD, the concentration of arabinose affects the mean of tdiff significantly, as it equals ∼2000 s for 0.1% arabinose.The estimated mean and a standard deviation of t0 depend mostly on the intake of arabinose and on the first transcription initiation event at the start site of PBAD. Meanwhile, the distribution of intervals between consecutive RNAs is assumed to depend solely on the kinetics of transcription initiation (Because of being stochastic and thus variable in duration from one event to the next (i.e. it differs from one cell to the next), the intake process impacts on the diversity in RNA numbers of a cell population. This impact should decrease with time, after induction. We estimated the time during which the effect is tangible for each measurement condition. For this, we assume that values of titiation ,10,37.0 are modelled as d-step processes, each step with an exponentially distributed duration (Supplementary Methods) (The events determining Δt as well as tMethods) . From thMethods) ,38, as eMethods) . This asMethods)  that indMethods) . The comP-values of these tests are shown in Supplementary Table S1 and show that in all but one case, it is possible to find a model that cannot be distinguished from the empirical distribution, in a statistical sense.Assuming these numbers of steps and stable conditions (e.g. induction level), we searched for models that fit the distributions accurately enough so that the K–S test does not find differences between model and measurements. The BAD at 0.1% arabinose induction. This may be due to lack of sufficient data or because the model is unsuitable. Future studies are required to assert this. One explanation may be that, in this case, the distribution of intake times results from two distinct kinetics, one being the productions under induction and the other being spurious productions by promoters in the ‘non-induced’ state.The case for which we could not find a model that fits the measurements is that of P0. We do not have measurements of the degradation rate of the target RNA, as the tagging with MS2d-GFP ‘immortalizes’ it for the duration of the measurements  and when induced by arabinose (dotted line), because of differing intake kinetics.In all cases, reaching equilibrium in mean RNA numbers takes >1 h, except when assuming infinitely fast intake, in which case the time to reach equilibrium is <0.5 h. Thus, for a time length as long as 1–2 h, the intake process has a non-negligible contribution on the mean and the on the cell-to-cell diversity in RNA numbers of the cell populations. From From BAD induced by 1% arabinose  with other RNA degradation rates , within realistic intervals (We also tested models of PBAD to be produced, followed the introduction of the inducer in the media. Also, we measured the subsequent intervals between consecutive RNA productions. From the intervals between transcription events, we determined that RNA production under the control of PBAD is a sub-Poissonian process. Two recent studies reached similar conclusions for Plac/ara-1 and PtetA, for all induction conditions tested (We measured, at the single-cell level, how long it takes for the first RNA under the control of Ps tested ,10. We hBAD and of Plac/ara-1, for different induction conditions, we assessed the effect of the kinetics of the intake process on the mean and cell-to-cell diversity in RNA numbers of cell populations. Relevantly, this effect was found to be tangible for a long period after induction. Also, we verified that different intake mechanisms differ significantly not only in mean but also in the degree of variability of the intake time, and that this has a non-negligible effect on RNA population statistics.From the distributions of the time, it takes for the appearance of the first RNA in each cell when under the control of Pin vivo kinetics of intake of arabinose and of IPTG to be applicable to any gene of interest. Such studies should provide valuable insight into the adaptability of prokaryotic organisms to environmental changes and stress. They should also provide a better understanding of the observed cell-to-cell phenotypic diversity in E. coli when in fluctuating environments.Given the aforementioned data, and considering that natural environments are fluctuating, we expect the kinetics of cellular intake mechanisms to have a significant effect on the degree of phenotypic diversity of cell populations. Finally, we expect the methodology used here to assess the Supplementary Data are available at NAR Online: Supplementary Table 1, Supplementary Figures 1–7, Supplementary Methods and Supplementary References [39–43].Academy of Finland [257603 to A.S.R.]; the Finnish Funding Agency for Technology and Innovation [40226/12 to O.Y.-H.]; the Foundation for Science and Technology, Portugal [PTDC/BBB-MET/1084/2012 to A.S.R.]. Funding for open access charge: Academy of Finland .The Conflict of interest statement. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.


Caenorhabditis elegans, Drosophila, and mice clearly show that the RNA editing process is an absolute requirement for nervous system homeostasis and normal physiology of the animal. Understanding the mechanisms of editing and findings of edited substrates has provided a better knowledge of the phenotype due to defective and hyperactive RNA editing. A to I RNA editing is catalyzed by a family of enzymes knows as ADARs. ADARs modify duplex RNAs and editing of duplex RNAs formed by ncRNAs can impact RNA functions, leading to an altered regulatory gene network. Such altered functions by A to I editing is observed in mRNAs, microRNAs (miRNA) but other editing of small and long ncRNAs (lncRNAs) has yet to be identified. Thus, ncRNA and RNA editing may provide key links between neural development, nervous system function, and neurological diseases. This review includes a summary of seminal findings regarding the impact of ncRNAs on biological and pathological processes, which may be further modified by RNA editing. NcRNAs are non-translated RNAs classified by size and function. Known ncRNAs like miRNAs, smallRNAs (smRNAs), PIWI-interacting RNAs (piRNAs), and lncRNAs play important roles in splicing, DNA methylation, imprinting, and RNA interference. Of note, miRNAs are involved in development and function of the nervous system that is heavily dependent on both RNA editing and the intricate spatiotemporal expression of ncRNAs. This review focuses on the impact of dysregulated A to I editing and ncRNAs in neurodegeneration.RNA editing is an alteration in the primary nucleotide sequences resulting from a chemical change in the base. RNA editing is observed in eukaryotic mRNA, transfer RNA, ribosomal RNA, and non-coding RNAs (ncRNA). The most common RNA editing in the mammalian central nervous system is a base modification, where the adenosine residue is base-modified to inosine (A to I). Studies from ADAR (adenosine deaminase that act on RNA) mutants in  Environmental signals provoke changes in gene expression in a mechanism that includes epigenetic-mediated gene regulation . EpigeneFigure 1).Numerous classes of regulatory non-coding RNA (ncRNA) molecules contribute to the intricate biological system organization and gene regulatory networks that collectively allow normal functioning of the CNS. Dysregulation of these complex gene regulatory networks plays a significant role in the pathogenesis of common neurodegenerative disease such as Alzheimer’s disease (AD), Parkinson disease (PD), Huntington disease (HD), and amyotrophic lateral sclerosis ALS; . In eukaNon-coding RNAs either receive or transmit information , which iAdenosine (A) to inosine (I) conversion in mRNA transcripts (A to I RNA editing) is catalyzed by a family of enzymes known as ADARs. Since inosine has the same base pairing properties as guanosine (G), the transcription and translational machinery recognizes I as a G. Hence, a silent mutation is created at the level of mRNA. In general, A to I editing most frequently targets repetitive RNA sequences located within the introns and 5′ and 3′ UTR to alter both sequence and structure of RNA. A to I editing is widespread and essential for normal life and development . The ADAA receptor , and the potassium voltage-gated ion channel KV1.1. It is of note that, they all play an important role in nerve cell function  buried within its enzyme core , and IP6 is required for ADAT activity  and regulates alternative splicing and editing of the 5HT2CR  to change codon recognition. Interestingly, a mutation in the “editing” domain of a specific aminoacyl-tRNA synthetase results in mischarged tRNAs, intracellular accumulation of misfolded proteins in neurons, and induction of the endoplasmic reticulum-mediated unfolded protein stress response with associated neurodegeneration . tRNAs aThe expansion of trinucleotide repeats caused by RNA-mediated mechanisms is associated with neurodegenerative diseases . DramatiSeveral forms of spinocerebellar ataxia (SCA) are also implicated in different RNA-mediated pathological mechanisms. SCA8 results from CTG expansion of the 3′ UTR of an untranslated antisense RNA with partial overlap with the Kelch-like 1 (KLHL1) gene . UtiliziRNA is a carrier of information and plays a central role in regulating development. A variety of regulatory non-protein-coding RNA molecules form complex multi-layered biological systems. A gene regulatory network that allows normal functioning of the CNS governs this complex system. A deregulated complex gene regulatory network plays a significant role in common neurodegenerative diseases. Furthermore, the list of known ncRNAs implicated in mammalian brain health and disease is growing. RNA can alter the information in the genetic code without altering the hard-wired DNA through splicing and RNA editing. ADAR substrates involved in RNA editing mechanisms provide functional complexity. RNA editing mediates the environmental cues by transmitting information to the epigenome. This mechanism connects the environment to the genome and plays important roles in a broad range of processes, from evolution to learning and memory. A to I RNA editing, besides altering protein function, also has the potential to alter splice site choice, miRNA target diversity, miRNA processing, and perhaps chromatin architecture. Furthermore, RNA editing alters RNA structure and thereby could potentially impact the biological functions of multiple types of ncRNAs. Therefore, RNA editing, RNA modification, small and long ncRNAs, and their complex regulatory network lead to a unifying theme of RNA-mediated regulatory circuitry for normal brain function.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


These isolated fractions were then used for analysis of global mRNA stability and de novo transcription by hybridization to a K. brevis microarray. Global K. brevis mRNA half-lives were calculated from the ratio of newly transcribed to pre-existing RNA for 7086 array features using the online software HALO . Overall, mRNA half-lives were substantially longer than reported in other organisms studied at the global level, ranging from 42 minutes to greater than 144 h, with a median of 33 hours. Consistent with well-documented trends observed in other organisms, housekeeping processes, including energy metabolism and transport, were significantly enriched in the most highly stable messages. Shorter-lived transcripts included a higher proportion of transcriptional regulation, stress response, and other response/regulatory processes. One such family of proteins involved in post-transcriptional regulation in chloroplasts and mitochondria, the pentatricopeptide repeat (PPR) proteins, had dramatically shorter half-lives when compared to the arrayed transcriptome. As transcript abundances for PPR proteins were previously observed to rapidly increase in response to nutrient addition, we queried the newly synthesized RNA pools at 1 and 4 h following nitrate addition to N-depleted cultures. Transcriptome-wide there was little evidence of increases in the rate of de novo transcription during the first 4 h, relative to that in N-depleted cells, and no evidence for increased PPR protein transcription. These results lend support to the growing consensus of post-transcriptional control of gene expression in dinoflagellates.Dinoflagellates possess many physiological processes that appear to be under post-transcriptional control. However, the extent to which their genes are regulated post-transcriptionally remains unresolved. To gain insight into the roles of differential mRNA stability and  A similar magnitude of transcriptome restructuring is found concurrent with the entry into stationary phase in K. brevis (29%) Alexandrium minutum (36%) Phytoplankton are essential primary producers, responsible for up to 70% of the world's oxygen production. Dinoflagellates are a major component of both marine and freshwater phytoplankton and, as approximately half are photosynthetic, they are key contributors to the base of aquatic food webs. However, many dinoflagellates also produce potent toxins and are responsible for harmful algal blooms (HABs). As HABs have significant ecological, human health, and economic impacts de novo transcription in K. brevis utilizing biosynthetic labeling of newly transcribed RNA.The steady-state abundance of mRNAs in a cell is determined by their relative rates of transcription and degradation de novo transcription and then monitoring the decay of specific messages over time. A disadvantage of this approach is that transcription inhibition has been shown to artificially stabilize many messages, leading to over estimation of RNA half-lives de novo synthesis rates must be much higher for short-lived transcripts than for stable transcripts. Therefore, RNA half-lives can be calculated from the ratios of either newly synthesized/total RNA, pre-existing RNA/total RNA, or newly synthesized/pre-existing RNA, and the duration of labeling RNA stability is often determined using transcription inhibitors to block K. brevis and to examine the role of de novo transcription in achieving short-term changes in transcript abundances by microarray analysis. Overall, K. brevis RNA half-lives were unusually stable, with a median half-life of 33 h, although as in most organisms studied, half-lives ranged over two orders of magnitude, from 42 minutes to greater than 144 h. Consistent with measurements from other organisms, there is a predominance among highly stable messages of genes that are involved in core cellular processes, including energy metabolism and transport. Shorter-lived transcripts include many genes involved in transcriptional regulation, stress response, and other response/regulatory processes. Microarray analysis of newly synthesized RNA following nitrate addition to N-depleted cultures revealed little change in the rate of de novo transcription of genes found previously to increase in abundance within 4 h of nitrate addition In this study we applied biosynthetic nucleotide labeling to characterize global mRNA stability in K. brevis (Wilson isolate) were maintained in 1 L bottles in f/2 medium using 20 µm filtered, autoclaved natural seawater  with the following modifications: ferric sequestrene was substituted for EDTA·Na2 and FeCl3·6H2O and 0.01 mM Na2SiO3 was added. Nitrogen-limited cultures were adapted to 10 µM nitrate, approximately 1.1% of the nitrate concentration in nutrient replete cultures, by a minimum of six serial log phase transfers prior to experimental treatments. N-depleted cultures were achieved by allowing these cultures to enter stationary phase. Illumination from cool white lights was supplied at approximately 175–215 µmol photons·m−2·s−1 on a 16∶8 h light∶dark cycle at 25±1°C. All experiments were initiated and cultures harvested during the light phase of the diel cycle.Batch cultures of K. brevis is amenable to biosynthetic labeling of newly synthesized RNA by 4-thiouracil  or 4-thiouridine , cultures were exposed to 0.02, 0.05, or 0.2 mM 4tU (in DMSO) or 0.2, 1, or 2 mM 4sU (in water) for 5 m to 20 h. To determine the half-lives of K. brevis mRNAs, one liter log-phase cultures (n = 6) were exposed to 0.2 mM 4tU for 2 h. To determine the transcriptional response to increased nitrate concentrations, sodium nitrate was used to add 155 µM NO3 to 6 N-depleted 1 L cultures, as in Morey et al. g for 10 m and total RNA was extracted using Tri-Reagent  according to the manufacturer's protocol with ethanol precipitation. RNA was resuspended in nuclease-free water, treated with Promega's  RQ1 RNase-free DNase, and further purified using an RNeasy mini column according to manufacturer's protocol . RNA was then quantified using a NanoDrop ND-1000  and qualified on an Agilent 2100 Bioanalyzer .To determine if −1 Biotin-HPDP in DMF were added to the labeling reaction per 1 µg of RNA. The labeling was carried out in 10 mM Tris (pH 7.4) and 1 mM EDTA in a final volume 5X that of the Biotin-HPDP in the reaction. RNA was then precipitated by the addition of 1/10 the reaction volume of 5 M NaCl and 1.1 volumes of isopropanol and centrifugation at 20,000× g for 20 m at room temperature. The pellet was washed in 75% EtOH, centrifuged at 20,000× g for 5 m at room temperature, resuspended in nuclease-free water, and quantified and qualified as described previously.Total RNA was biotinylated according to the methods of Dölken et al. K. brevis RNA, total RNA was biotinylated, heat denatured, centrifuged at 20,000× g for 1 m to remove free biotin, and bound to positively charged nylon membrane  with alkaline binding buffer  using a slot blot apparatus To determine how quickly 4tU is incorporated into Following biotinylation, total RNA was fractionated into two pools, newly synthesized thiolated RNA and pre-existing non-thiolated RNA using Dynabeads MyOne Streptavidin C1 magnetic beads . One microliter of beads was used per 160 ng of RNA and manufacturer's protocols were followed for immobilization of nucleic acids with the addition of 0.1% Tween 20 to the binding and washing buffer (BWT). Following immobilization, the supernatant containing unlabeled RNA was saved and, following isopropanol precipitation, constituted the pre-existing RNA fraction. The beads were then washed three times in 3X the original volume of beads with 1X BWT at 65°C, followed by 3 washes at room temperature and a final wash with 1∶10 1X BWT at room temp. Biotinylated RNA was released from the beads by adding 1X volume of 5% β-mercaptoethanol and incubating 5 m at room temperature with rotation. The supernatant was saved and another 1X volume of 5% β-mercaptoethanol was added to the beads and incubated at 60°C for 10 m with occasional mixing. This supernatant was added to the previous elution and, following isopropanol precipitation, constituted the newly synthesized RNA fraction.K. brevis oligonucleotide microarray containing 10,263 unique 60-mer gene probes A www.bio.ifi.lmu.de/software/HALO) Half-Life Calculation: Normalized data for each biological replicate of total, pre-existing, or newly synthesized RNA were uploaded to the freely available software HALO . Based on the Rosetta error model designed for the Agilent platform, a composite array was generated for newly-synthesized RNA at each time point following N-addition (1 and 4 h) from triplicate arrays , in which the data for each feature underwent a normalization, intensity averaging, and error estimation based on data from the replicate arrays making up the composite Blast2GO Annotation: Blast2GO K. brevis were grown to mid-log phase under standard culture conditions (described above). Triplicate tubes were then treated with either 2 µg·mL−1 actinomycin D or DMSO carrier for 12, 24 or 48 h. Total RNA was harvested at each time point as described above, quantified using a NanoDrop ND-1000  and duplicate reverse transcription reactions were carried out using 100 ng total RNA with an oligo(dT) primer using Ambion's RETROscript Kit . Primer pairs specific for the contig of interest were designed  was assigned at the beginning of the logarithmic phase of PCR amplification and the difference in the Ct values of the actinomycin D treated and carrier control samples were used to determine the relative expression of the gene in each time point.Tube cultures (25 mL) of designed  for qPCRToxoplasma gondii and yeast have demonstrated the ability to utilize 4tU as a substrate for RNA synthesis a priori knowledge of the pyrimidine salvage pathway in the dinoflagellate, we first assessed the ability of K. brevis to incorporate 4tU and/or 4sU into newly synthesized RNA. Cultures of K. brevis were exposed to increasing concentrations of 4tU  or 4sU  over a range of times (5 m to 20 h). These concentrations have previously been shown to yield measurable incorporation into RNA by T. gondii (4tU) K. brevis mRNA in as little as 5 minutes  with highest homology to UPRT in Aureococcus , as well as a gene encoding a dual domain UK-UPRT protein  with highest homology to Arabidopsis thaliana . UK-UPRT fusion genes encoding bifunctional proteins with both UK and UPRT activities are found across eukaryotic the tree of life, but are notably absent from the nearest neighbors to dinoflagellates, the apicomplexans, except for Cryptosporidium parvum, and have not previously been identified in dinoflagellates K. brevis transcriptome, it appears that K. brevis contains a pyrimidine salvage pathway that utilizes UPRT activity.Methods for labeling and purifying newly synthesized RNA amenable to high-throughput downstream analyses have been employed in a variety of eukaryotes using either 4-thiouracil (4tU) or 4-thiouridine (4sU)  minutes . The K. K. brevis total RNA profiles with prominent ribosomal peaks at approximately 41 and 44 s and numerous smaller RNAs visible. Both newly synthesized and pre-existing fractions of K. brevis RNA were of high quality and amenable to downstream applications such as microarray and qPCR.When the newly synthesized RNA was separated from pre-existing RNA by thiol-specific biotinylation, we observed expected bioanalyzer profiles , whereinK. brevis, we utilized the method of Dölken et al. K. brevis were labeled with 4tU for 2 h and total RNA was biotinylated and separated into 3 fractions: total RNA, pre-existing RNA, and newly synthesized RNA. Each fraction from each replicate was Cy3 labeled and hybridized to an Agilent custom microarray for K. brevis containing 10,263 unique 60-mer gene probes. This data has been deposited in the Gene Expression Omnibus (GEO) as accession # GSE46174. An array p-value of p≤0.0001 was required on all 18 arrays for inclusion of a probe in half-life calculations, resulting in 7102 probes used for the analysis in the freely available software HALO To establish the global distribution of RNA half-lives in Overall, extremely stable messages were observed, with a mean of 40.6 h and median of 33.3 h for the 7086 features . The minK. brevis transcripts calculated in this study exceeded that of other organisms reported to date. Global analyses of transcript stability report median mRNA half-lives of 2.4 m in the cyanobacteria ProchlorococcusE. coliArabidopsis 3.8 h K. brevis with an average cell cycle of approximately 3 days (4320 m) K. brevis, approximately 13% of features showed a half-life of at least 3 days, or at least one generation time. It has been noted in trypanosomes that messages present in high copy number tend to be more stable, while rarer messages have short half-lives K. brevis microarray employed in these studies was constructed using 10,263 unique contigs obtained from Sanger sequencing, it is possible that the transcriptome features arrayed are biased towards the more highly expressed transcripts. With a genome size of 1×1011 bp, K. brevis is predicted to possess approximately 75,000 expressed genes K. brevis using the Illumina sequencing platform resulted in over 75,000 unique contigs . Thus, the absence of extremely short-lived mRNAs in this study may not preclude their occurrence in K. brevis; rather those sequences may not be represented on the microarray employed. A similar analysis using RNA-seq may reveal a missing population of shorter-lived messages. It is also plausible that the unusually long half-lives reported here reflect another characteristic of dinoflagellate physiology: circadian regulation. Many processes in dinoflagellates are under circadian control, including bioluminescence Lingulodinium polyedrum, translation also appears to occur almost exclusively during the subjective night The median half-life of K. brevis, we utilized Fisher's Exact tests for enrichment in Blast2GO on the binned K. brevis mRNA half-life data. Significant enrichment was only observed among mRNAs with a half-life of at least 3 days  and stress responses are among the least stable messages, enabling the cell to rapidly alter their abundance through alternative synthesis and degradation. In contrast, genes involved in housekeeping functions  are overall among the most stable messages, alleviating the need to continually synthesize new messages for constitutive processes t 3 days . Indeed,t 3 days . In contK. brevis is a highly represented family of pentatricopeptide repeat (PPR) containing proteins. PPR proteins are sequence-specific RNA binding proteins involved in post- transcriptional processing of organellar RNAs, including editing, splicing, translation, and stability ArabidopsisK. brevis have much shorter half-lives than the transcriptome as a whole. These short half-lives for PPRs are likely indicative of their essential roles in regulatory functions in chloroplasts and mitochondria Among the genes involved in RNA processing in K. brevis by post-transcriptional mechanisms Lingulodinium polyedrum varied somewhat depending on the time of day at which the transcription inhibitor thiolutin was added, but all were on the order of several hours as assessed by northern blot analysis To independently assess half-lives of selected RNAs, we used the traditional approach of blocking transcription with actinomycin D and following transcript levels over a time course of 12, 24 and 48 h by qPCR. We selected two cytoskeletal genes, α-tubulin and β-tubulin, one cell cycle gene, PCNA, previously shown to be regulated in K. brevis transcriptome's response to nutrient addition found only 4.4% of array features significantly changing over a 48 h time course following nitrate addition to N-depleted K. brevis cultures de novo transcription and or to changes mRNA stability. Therefore, in the current study we examined de novo transcription by 4tU incorporation into newly synthesized RNA over two 1 h periods (0–1 h and 3–4 h) following N addition in comparison with that in the N-depleted cultures This data has been deposited as GEO Accession # GSE46175.The presence of the SL on all nuclear encoded gene transcripts examined in dinoflagellates, along with the observation of post-transcriptional regulation of a number of processes studied, has led to the hypothesis that gene regulation in dinoflagellates is dependent largely on post-transcriptional processes During the first hour post N-addition only 0.2% of array features were significantly different in the newly transcribed RNA pool when compared to the N-depleted cells similarly labeled for 1 h  . HoweverK. brevis, is capable of incorporating 4-thiouracil into newly transcribed RNA, making it amenable to studying RNA stability using biosynthetic labeling approaches. By microarray analysis, K. brevis appears to possess highly stable messages relative to other eukaryotic models. Further analyses using deeper transcriptome surveys via RNA-seq may refine this estimation of median half-life. In addition, the potential contribution of circadian accumulation and turnover of messages must be addressed. Nonetheless, in keeping with conserved properties in other eukaryotes, the dinoflagellate's most stable messages are largely involved in core processes such as energy generation and transport, whereas regulatory processes, including transcription regulation and stress response, are more highly represented among the shorter-lived transcripts. In particular, PPR repeat proteins involved in organellar RNA regulation are highly represented among the short-lived mRNAs, consistent with their rapid increase in abundance following nutrient stimulation. However, their apparent lack of transcriptional activation, along with the presence of the SL on PPR protein messages, lends support to the growing consensus of post-transcriptional control of dinoflagellate gene expression.Insight into mRNA stability is important to understanding gene regulation, particularly in poorly characterized and divergent eukaryotes. Here we demonstrate that the pyrimidine salvage pathway of the dinoflagellate, This publication does not constitute an endorsement of any commercial product or intend to be an opinion beyond scientific or other results obtained by the National Oceanic and Atmospheric Administration (NOAA). No reference shall be made to NOAA, or this publication furnished by NOAA, to any advertising or sales promotion which would indicate or imply that NOAA recommends or endorses any proprietary product mentioned herein, or which has as its purpose an interest to cause the advertised product to be used or purchased because of this publication.Figure S1Regression plots used for normalization in HALO. Following 2 h incubation with 0.2 mM 4-thiouracil, total RNA was biotinylated and bead purified. Total, pre-existing, and newly synthesized RNA fractions were hybridized to a custom oligonucleotide microarray. Linear regressions of the ratios of newly synthesized RNA to total RNA and pre-existing RNA to total RNA are conducted in HALO prior to half-life calculations. The regression of the raw uncorrected data (A) and regression-normalized data (B) used for half-life calculations are shown.(TIF)Click here for additional data file.Figure S2Karenia brevis mRNA half-lives calculated from additional ratios. Following 2 h incubation with 0.2 mM 4-thiouracil, total RNA was biotinylated and bead purified. Total, pre-existing, and newly synthesized RNA fractions were hybridized to a custom oligonucleotide microarray and mRNA half-lives were calculated for 7086 features using HALO. Following normalization by linear regression, newly synthesized RNA to total RNA (A) or pre-existing to total RNA (B) ratios were used to calculate half-lives.(TIF)Click here for additional data file.Table S1Primers used for quantitative real-time PCR.(XLSX)Click here for additional data file.Table S2Half-lives for 7086 features passing quality filters based on newly synthesized RNA to pre-existing RNA ratios.(XLSX)Click here for additional data file.


Two important hallmarks of RNA silencing in plants are (1) its ability to self-amplify by using a mechanism called transitivity and (2) its ability to spread locally and systemically through the entire plant. Crucial advances have been made in recent years in understanding the molecular mechanisms of these phenomena. We review here these recent findings, and we highlight the recently identified endogenous small RNAs that use these advantageous properties to act either as patterning signals in important developmental programs or as a part of regulatory cascades. RNA silencing is a recently identified mechanism important for the transcriptional and posttranscriptional control of genes and genomes in eukaryotes –4. It alRNA silencing involves processing of dsRNA by DICERs or DICER-LIKEs to produce small RNA (sRNA) duplexes, capture of the guide siRNA strand by ARGONAUTE (AGO) proteins to form RNA-induced silencing complexes (RISCs) and recognition of homologous target DNA or RNA sequences by RISCs –19. Arabidopsis thaliana by four specific DICER-LIKE enzymes (DCLs), are involved in sRNA, that is, small interfering (si)RNA and micro (mi)RNA, production , 9963, 9Arabidopsis leaf primordial TAS3a precursor RNA is another example. It is exclusively produced in the L1 and L2 adaxial (upper) leaf layers [TIR/AFB2 auxin receptor (TAAR) transcripts in the regulation of auxin signaling homeostasis and of leaf morphogenesis [f layers , 100–103f layers , 104–106f layers  highlighogenesis , 92. Howogenesis , 92. In some cases, the biogenesis of sec-siRNAs extends towards regions upstream and downstream of the initial target site, a phenomenon called “transitivity” –109. TraA reason for this differential susceptibility for transitivity might be that the fragments created by RISC-directed cleavage are not only substrates for RDRs but also for exonucleases lanaceae , 95 and lanaceae . Like TAS RNAs, the corresponding PHAS RNAs are either targeted by double hits, with only one of them leading to slicing while the other is anchored  or a sinRecent bioinformatics works have suggested that at least 4 novel TAS families exist in grapevine although they need to be experimentally validated and their biological role remains to be clarified .We discussed previously that the targeting of RNAs by miRNAs and siRNAs can lead to the production of ta-siRNAs, siTAARs, and pha-siRNAs. These secondary sRNAs could initiate further layers of sRNA production and form extensive cascades and networks of gene regulation. Bioinformatics and molecular evidence for this was reported in , 121. Arabidopsis [Recent works by Rajeswaran et al.  have shobidopsis . The worThe cascade is known to further continue at least from TAS2 and TAS2D6, giving rise to siR2140 , which tThe frequent cases of pha-siRNA production from NB-


Hepatitis E virus (HEV) causes acute or fulminant hepatitis in humans and is an important public health concern in many developing countries. China has a high incidence of HEV epidemics, with at least three genotypes  and nine subtypes  so far identified. Since genotype 3 and the newly identified subtype 4i have been exclusively limited geographically to Shanghai and its neighboring provinces, the epidemiology of HEV infections within the municipality, a major industrial and commercial center, deserves closer attention.EF570133], together with five full-length swine and human HEV genomic sequences, all emanating from Shanghai, were retrieved from GenBank. Consistent with the primary role of genotype 4 in China overall, analysis of the sequences revealed this to have been the dominant genotype (58/65) in Shanghai. Six HEV subtypes  were also represented. However, although subtype 4a is the dominant subtype throughout China, subtype 4i (29/65) was the most prevalent subtype among the Shanghai sequences, followed by subtypes 4d (10/65) and 4h (9/65). Subtypes 4h, 4i and 4d were found in both swine and humans, whereas 4b was found only in swine and subtype 4a only in humans.A total of 65 sequences, 60 located within the HEV SH-SW-zs1 genome [GenBank:Six different swine and human HEV subtypes have so far been documented in Shanghai. More molecular epidemiological investigations of HEV in swine, and particularly among the human population, should be undertaken. Hepatitis E virus (HEV), the causative agent of acute or fulminant hepatitis in humans, is an important public health concern in many developing countries. It is estimated that about two billion people, or one-third of the world population, live in areas where HEV is endemic and are therefore at risk of infection . The disHepeviridae. At least four mammalian HEV genotypes have been recognized   together with five full-length swine and human HEV genomic sequences, all derived from Shanghai, were retrieved from GenBank as of June, 2011. All the sequences were aligned with Clustal W (version 1.8), and the sequence percent identity was calculated using Lasergene . Phylogenetic trees were constructed by the neighbor-joining method , suggesting that 4a subtypes in Shanghai were also zoonotic. Human subtype 4b HEV has a more limited distribution in China than the swine counterpart , was reported recently in neighboring Jiangsu Province [Genotype 3 HEV strains were geographically limited to Shanghai and neighboring provinces, and seven sequences were classified into subtype 3b. No human genotype 3 HEV sequences were among those from Shanghai although one strain, EChN22 [GenBank:Province . The EChNo genotype 1 HEV representatives were found in this study, possibly due to the limited number (10/65) of human HEV sequences available.In summary, six different HEV subtypes, i.e. 3b, 4a, 4b, 4d, 4h and 4i, have so far been shown to be prevalent in Shanghai, with genotype 4 and subtype 4i the dominant forms. Only subtypes 4h, 4i and 4d were found in both swine and human hosts, whereas subtypes 4a and 4b were confined to either human or swine populations, respectively. A recent epidemiological investigation has shown that the average incidence of HEV RNA positives among the swine population of Shanghai was approximately 20.0%, and that serum IgG positives reached 72.18% . HepatitHEV: Hepatitis E virus; ORF: Open reading frameThe authors declare that they have no competing interests.YMZ, FYS, RSY, YSZ and ZL participated in the study design, YMZ, SJD and FSS performed the sequence analysis, and all authors participated in writing and revising the manuscript. All authors have read and approved the final version.


Hypoxia is an important microenvironmental factor that induces cancer metastasis. Hypoxia/hypoxia-inducible factor-1α (HIF-1α) regulates many important steps of the metastatic processes, especially epithelial-mesenchymal transition (EMT) that is one of the crucial mechanisms to cause early stage of tumor metastasis. To have a better understanding of the mechanism of hypoxia-regulated metastasis, various hypoxia/HIF-1α-regulated target genes are categorized into different classes including transcription factors, histone modifiers, enzymes, receptors, kinases, small GTPases, transporters, adhesion molecules, surface molecules, membrane proteins, and microRNAs. Different roles of these target genes are described with regards to their relationship to hypoxia-induced metastasis. We hope that this review will provide a framework for further exploration of hypoxia/HIF-1α-regulated target genes and a comprehensive view of the metastatic picture induced by hypoxia. Hypoxia is a critical microenvironmental factor that is demonstrated to induce tumor metastasis -5. HypoxOne well-studied aspect of hypoxia-induced metastasis is that hypoxia induces various transcriptional regulators  to cause EMT -16. The Hypoxia/HIF-1α is originally shown to regulate the expression of metalloproteases including matrix metalloprotease-1 (MMP1) and MMP3 to induce metastasis . In contVarious receptors, receptor-activated kinases, small GTPases, and transporters are regulated by hypoxia/HIF-1α to play a significant role in cancer metastasis. For example, chemokine receptor 4 (CXCR4) is one of the early identified hypoxia/HIF-1α target genes that mediates hypoxia-induced angiogenesis and metastasis . SimilarVarious adhesion or surface molecules, membrane proteins, and miscellaneous proteins are regulated by hypoxia/HIF-1α and involved in hypoxia-induced metastasis. For breast cancer, angiopoietin-like 4 (ANGPTL4) and L1 cell adhesion molecule (L1CAM) are the two important molecules that mediate vascular metastasis of hypoxic breast cancer cells to the lungs . ANGPTL4Various microRNAs are shown to be regulated by hypoxia/HIF-1α. A recent review describing the various microRNAs regulated by hypoxia provides a concise summary of the roles of these microRNAs in metabolism, DNA damage response, and angiogenesis . Howeverin vitro and in vivo metastatic assays to demonstrate their contribution to tumor metastatic activity. The linkage between metabolism and tumor metastasis remains to be further explored. The concept will also apply to other hypoxia/HIF-1α-regulated targets that appear not to be directly regulating metastasis.Hypoxia is one of the most important environmental factors that induce cancer metastasis. Hypoxia-regulated biological effects have been extensively investigated. It is obvious that hypoxia regulates key steps of the metastatic process. Through outlining various targets regulated by hypoxia/HIF-1α to mediate tumor metastasis, the review wishes to provide a comprehensive guide according to the various categories described. A summary of all these targets is listed in Table From this review, it appears that different categories of targets can contribute to the metastatic process. Judging from the various steps involved in tumor metastasis, it is not surprising. However, it will need further investigation to figure out the exact mechanisms of how these targets contribute to metastasis. In addition, the interacting roles played by these targets to mediate metastasis will also require an integrated approach to combine molecular biology and systems biology in order to put all these players into a more comprehensive picture. Furthermore, novel hypoxia/HIF-1α-regulated targets that can mediate metastasis will need to be identified in order to complete the full picture of tumor metastasis induced by hypoxia/HIF-1α. Finally, elucidation of the functions of these targets mediating metastasis will provide the possibility of using these targets as therapeutic venues to improve the treatment of hypoxia-induced tumors.HIF-1: Hypoxia-inducible factor-1; EMT: Epithelial-mesenchymal transition; ARNT: Aryl hydrocarbon receptor nuclear translocator; EWS-FLI1: Ewing Sarcoma-Friend leukemia virus integration 1; CDX2: Caudal type homeobox 2; SMAD7: SMAD family member 7; JMJD2B (KDM4B): Lysine-specific demethylase 4B; ACE: Angiotensin converting enzyme; CX3CR1: Chemokine (C-X3-C motif) receptor 1; IRS-2: Insulin receptor substrate 2; glut-1: Glucose transporter type 1; MDR1: Multidrug resistance protein 1; ID2: Inhibitor of differentiation/DNA binding 2; MMP: Matrix metalloprotease; LOX: Lysyl oxidase; ADAMTS1: A disintegrin and metalloproteinase with thrombospondin motifs 1; pdcd4: Programmed cell death 4; ANGPTL4: Angiopoietin-like 4; L1CAM: L1 cell adhesion molecule; RECK: Reversion-inducing-cysteine-rich protein with kazal motifs; STAT3: Signal transducer and activator of transcription 3; DAPK: Death-associated protein kinase; KLF4: Kruppel-like factor 4; Hsulf-1: sulfatase 1; XPA: Xeroderma pigmentosum, complementation group A; SF/HGF: Scatter factor/hepatocyte growth factor.The authors declare that they have no competing interests.KJW completed the final draft and revision of the manuscript. Both authors read and approved the final manuscript.


It is now believed that in the origin of life, proteins should have been "invented" in an RNA world. However, due to the complexity of a possible RNA-based proto-translation system, this evolving process seems quite complicated and the associated scenario remains very blurry. Considering that RNA can bind amino acids with specificity, it has been reasonably supposed that initial peptides might have been synthesized on "RNA templates" containing multiple amino acid binding sites. This "Direct RNA Template (DRT)" mechanism is attractive because it should be the simplest mechanism for RNA to synthesize peptides, thus very likely to have been adopted initially in the RNA world. Then, how this mechanism could develop into a proto-translation system mechanism is an interesting problem.Here an explanation to this problem is shown considering the principle of "replication parsimony" --- genetic information tends to be utilized in a parsimonious way under selection pressure, due to its replication cost . Because a DRT would be quite long even for a short peptide, its replication cost would be great. Thus the diversity and the length of functional peptides synthesized by the DRT mechanism would be seriously limited. Adaptors (proto-tRNAs) would arise to allow a DRT's complementary strand  to direct the synthesis of the same peptide synthesized by the DRT itself. Because the C-DRT is a necessary part in the DRT's replication, fewer turns of the DRT's replication would be needed to synthesize definite copies of the functional peptide, thus saving the replication cost. Acting through adaptors, C-DRTs could transform into much shorter templates  and substitute the role of DRTs, thus significantly saving the replication cost. A proto-rRNA corresponding to the small subunit rRNA would then emerge to aid the binding of proto-tRNAs and proto-mRNAs, allowing the reduction of base pairs between them (ultimately resulting in the triplet anticodon/codon pair), thus further saving the replication cost. In this context, the replication cost saved would allow the appearance of more and longer functional peptides and, finally, proteins. The hypothesis could be called "DRT-RP" ("RP" for "replication parsimony").The scenario described here is open for experimental work at some key scenes, including the compact DRT mechanism, the development of adaptors from aa-aptamers, the synthesis of peptides by proto-tRNAs and proto-mRNAs without the participation of proto-rRNAs, etc. Interestingly, a recent computer simulation study has demonstrated the plausibility of one of the evolving processes driven by replication parsimony in the scenario.An RNA-based proto-translation system could arise gradually from the DRT mechanism according to the principle of "replication parsimony" --- to save the replication cost of RNA templates for functional peptides. A surprising side deduction along the logic of the hypothesis is that complex, biosynthetic amino acids might have entered the genetic code earlier than simple, prebiotic amino acids, which is opposite to the common sense. Overall, the present discussion clarifies the blurry scenario concerning the origin of translation with a major clue, which shows vividly how life could "manage" to exploit potential chemical resources in nature, eventually in an efficient way over evolution.This article was reviewed by Eugene V. Koonin, Juergen Brosius, and Arcady Mushegian. As it was stated by Crick et al., "the origin of protein synthesis is a notoriously difficult problem" . Dating Indeed, proteinacious components involved in the modern translation system, including those in the ribosome, aminoacyl-tRNA synthetases (AARSs), and translation factors, have been either evidenced or implied to be absent originally -13. ThusAs it was pointed out, evolution should be continuous , "is myoin vitro selection experiments searching for RNA aa-aptamers, it was revealed that RNA could bind an amino acid with significant specificity [The "invention" of DNA in the RNA world seems to be not difficult to understand at the aspect that RNA could be "reverse transcribed" into DNA under the mechanism of template-directed synthesis via base-pairing. "Unfortunately", there is not such a simple mechanism for RNA to synthesize proteins. However, in cificity -23. ThenThis mechanism has been suggested in the "Direct RNA Template (DRT)" hypothesis ,22. TherHere, by "replication parsimony" I mean that informational sequences in genetic molecules have a tendency to be utilized in a parsimonious way under selection pressure, due to the cost involved in their replication. The principle should be very important at least in early stages of life. For instance, it is well known that there is little non-coding DNA in prokaryote genomes. In some viruses there are even overlapping genes coding for different proteins. In an RNA world, the saving of replication cost should have been particularly important because the synthesis of RNA precursors (i.e. nucleotides) and the replication of RNA would have to be catalyzed by ribozymes , which sNow, let us envision the actual structure of a "direct RNA template" (DRT) considering the principle of replication parsimony. First, an aa-binding site in a DRT should have a short sequence, just like a simple aa-aptamer, containing a hairpin loop, a following little stem, an internal (or bulge) loop and another following little stem -21. NextHow DRTs could appear is a side but important issue for an intact description of the scenario. I have discussed it in Appendix 1, in which it is suggested that DRTs in the compact form mentioned above were not only favored in principle of replication parsimony, but also very likely to have just been the DRTs emerging in the very beginning. In the following, when I mention "DRT(s)", I would mean "compact DRT(s)" of this kind (but note: this does not mean that DRTs must be in this compact form to render the following scenes possible).In the scene of the DRT mechanism, any specific peptide would cost a large source for the replication of the corresponding DRT (even if in the compact form) --- an aa-binding site would consist of quite a number of nucleotide residues , and there would be additional spacer sequences to allow the folding. Accompanying the increase of the diversity of peptides for various functions, as well as the increase of the length of peptides for more efficient functions, the replication burden would become greater and greater. Hence, it would be inevitable for the RNA organisms to "look for" a solution to achieve "replication parsimony", under natural selection.As exemplified by contemporary organisms, a good solution to achieve the replication parsimony is the introduction of adaptors (i.e. tRNA like molecules). In this way, the spacer sequences between the aa-binding sites on a DRT would no longer be necessary --- the stereochemical limitation mentioned above, would be compensated by the spatial stretch of the adaptors Figure , Left. IHowever, the initial appearance of the adaptors is a problem, considering the myopia of evolution. Apparently, a single adaptor molecule would not work. Two or more adaptor molecules for one kind of amino acids seem also to be of no use, unless homogenous peptides would have some advantages. That is, adaptor molecules should be introduced as a set, being multiple both in number and species. Their emergence could not be attributed to only one or several mutation events. Therefore, there should be an explanation on their original source before they were recruited into the proto-translation system. Interestingly, it was supposed that a tRNA-like structure at 3' end of some single stranded RNA viruses is a relic of the RNA world, wherein it might have served as a "genomic tag" ,29. SuchIn another hypothesis, the "Coding Coenzyme Handles (CCH)", it was suggested that the adaptors might appear primordially as handles for ribozymes to utilize specific amino acids as coenzymes ,30. A haNow let us envision the scenario along the line concerning the idea of DRTs. In the DRT hypothesis, it was speculated abstractly that adaptors might have arisen from some aa-binding sequences "escaping" from the RNA templates ,22,31. Iin trans), provided that the RNA contains an aa-binding site and by some folding one of its chain end could be "located" near the aa-binding site. Quite a few types of such RNAs, with a size only a little longer than typical tRNAs, were derived independently in in vitro selection experiments [Charging an amino acid onto an RNA's chain end seems to be an easy thing, if only the amino acid could be brought adjacent to the chain end, a reaction of which is well within the capacity of ribozymes -37. Whereriments . The rateriments . For a DOn a C-DRT, subsequences between "proto-tRNA binding sites" Figure  would be4 M-1 to 103 M-1. Indeed, as it was suggested [So far, a proto-tRNA would still recognize a proto-mRNA by quite a few nucleotide residues Figure . At thisuggested ), beforeIn an experimental research concerning RNA-catalyzed aminoacylation of RNA, it was reported that a specific aa-binding RNA domain contains a cognate codon-anticodon pair . StatistCertainly, considering the diversity of aa-binding sites for the same amino acids ,31 and rThe mechanism of the base-pair-reduction is a side issue, but it is important for the scene described here to be tenable. Here it should be noted that, without a proteinacious AARS, an intact aa-binding site would still be important for a proto-tRNA to charge its cognate amino acid, while some residues in the site would no longer be necessary in the subsequent template-recognition, due to the degeneration of the recognition loop. Then how could this seemingly contradictory situation be settled? I have discussed it in Appendix 3.The emergence of an RNA functioning like the large subunit rRNA(s), namely "proto-ls-rRNA(s)", would be easier to understand. Supposed that two aa-proto-tRNAs were compactly aligned on a proto-mRNA with the aid of the proto-ss-rRNA Figure , the pepIt should be noted that though the modern small subunit rRNA (e.g. 16 S rRNA) and large subunit rRNAs (e.g. 23 S rRNA) are complex molecules composed of nucleotide residues over one or more thousand, biochemical and genetic researches have suggested that their primitive form in the RNA world might have been much simpler, composed of nucleotide residues only around one hundred or even less . A recenThereafter, a mechanism of the proto-rRNAs' translocation on a proto-mRNA (composed of triplet codons then) should have emerged, with the advantage of avoiding repeated disassembly and assembly of the proto-rRNAs when the proto-translation reaction turned to subsequent codons.Accompanying the emergence of the whole proto-translation system, with much shorter template RNAs and greater efficiency than that in the case of the DRT mechanism, more and longer useful peptides would emerge under natural selection. Finally real proteins with great structural and chemical diversity would emerge, many of which would act as independent functional molecules, taking the role of most functional RNAs. Wherein, certainly, the proto-translation system would incorporate proteinacious components gradually, and evolve towards the modern translation system. The primary life was thus leaving from the RNA world and arrived in the RNP world.The scenario described here is open for experimental work at several key scenes. First, it is possible to demonstrate a (compact) DRT mechanism Figure . We mighin silico results like this, some experiments (e.g. based on in vitro molecular evolution) could be designed to test these evolving processes involving "replication parsimony", at various aspects.It may be not easy to demonstrate experimentally the evolving processes driven by replication parsimony in the scenario, which should have lasted a rather long period in early stages of life. However, it could be expected that computer simulation would help us to evaluate the plausibility of these processes. As a parallel example, our previous computer simulation studies concerning the origin of the RNA world demonstrated that the limitation of replication resources should have been an important selective pressure for the evolution in the RNA world -48. MoreAs mentioned in the "Background", there have been a number of hypotheses on the scenario about the origin of translation (protein synthesis) in the RNA world. Here I will compare the present one with fourteen of them, which are deemed representative Table .th hypothesis in Table Usually, the hypotheses were quite different on various aspects of the scenario. For example, even the views on the relationship between primordial peptides and the emergence of proto-translation system were not consistent. A recent hypothesis asserted that the driving force for the emergence of a proto-translation system is associated with the double strand-dissociation in the RNA replication process, having nothing to do with primordial peptides . In other words, without adaptors, no peptides could be synthesized. In Hp1 (DRT) and Hp5, it was asserted that initially, peptides could be synthesized without adaptors. In the former, the advantage of the introduction of adaptors was to "adapt amino acids to" their binding sites on DRTs, while in the latter, to improve "the stability and spatial precision" of the binding of amino acids on the ribozyme facilitating their ligation. According to Hp4 and Hp14, proto-tRNAs was first involved in the RNA replication, in which the advantage of their emergence was implemented. In agreement with Hp1 and Hp5, the present hypothesis (DRT-RP) asserts that primordial peptides were synthesized without adaptors. However, it states that the advantage of the introduction of the adaptors should be to exploit C-DRTs to synthesize the peptides originally synthesized only by their corresponding DRTs, thus saving the replication cost for the DRTs (because a C-DRT is a necessary part in the replication of its corresponding DRT).Emergence of adaptors: the origin of proto-tRNAs and proto-mRNAs" for the explanation), which has been adopted in several other hypotheses . The other two explanations came from the "Coding Coenzyme Handles (CCH)" hypothesis (Hp3) (see above in the section "Emergence of adaptors: the origin of proto-tRNAs and proto-mRNAs" for the explanation) and Hp5 . The DRT hypothesis (Hp1) had an abstract explanation, which stated that the adaptors should have "escaped" from DRTs. The present hypothesis (DRT-RP) formulates a detailed explanation for the "escaping" " hypothesis Hp2)  and Hp7. However, if this is not the case, the direct advantage for their introduction might not be associated with this aspect. For example, it was stated in Hp12 that the direct advantage for the initial appearance of proto-mRNAs is to enhance the stability of proto-tRNAs' binding on an "ancestral peptidyl transferase ribozyme" (a primitive form of proto-ls-rRNA). In the present hypothesis (DRT-RP), the advantage of the evolving process from C-DRTs Figure  to protoUnlike proto-tRNAs, proto-mRNAs could be introduced into the translation system "independently". Thus, their original source has not an urgency to be interpreted clearly, especially considering that they might not be long, corresponding to those short primordial peptides. Nevertheless, there have been some abstract explanations to this point . Wherein, it was usually implied (more or less) that proto-mRNAs might have arisen from RNAs with a random sequence. The DRT hypothesis (Hp1) is an exception, which suggested that proto-mRNAs might have come from the specific RNAs, DRTs, which could "direct" the synthesis of primordial functional peptides. The present hypothesis (DRT-RP) agrees to this viewpoint largely, but explains in a clear way that proto-mRNAs should have actually come from complementary chains of DRTs (i.e. C-DRTs).Due to solid evidence on the role of rRNAs in the modern translation systems, there seems to be little controversies on the advantage of the introduction of proto-rRNAs . Wherein, the proto-ss-rRNA would stabilize the association of proto-tRNAs with a proto-mRNA, and the proto-ls-rRNA(s) would facilitate the peptidyl transference between proto-tRNAs. The present hypothesis (DRT-RP) agrees with this viewpoint, but emphasizes that with the stabilization function, the proto-ss-rRNA would allow the reduction of base pairs between proto-tRNAs and proto-mRNAs. Thus, proto-mRNAs could become shorter . In other words, they could have been derived from some ordinary ribozymes acting in the RNA world, via some "ordinary mutation events" Therefore, an explanation for their original source seems to be not so urgently needed as it has often been deemed. On this consideration, such an explanation is not clearly offered in the present hypothesis. Certainly, if there should indeed be a speculation on the original source of the proto-rRNAs, I tend to agree the suggestion in Hp9, namely that proto-rRNAs  might have derived from group I intron .The original source of rRNAs has attracted much attention , often occupying an important role in the hypotheses. The hypothesis of "Triplet Replicase (TR)"  stated that proto-rRNAs should have come from an RNA replicase, which cut the anticodon triplets from proto-tRNAs to be used as substrates in the template-directed RNA replication. Similarly in some degree, Hp14 also asserted that proto-rRNAs should have had its origin in the RNA replication. It stated that the proto-ls-rRNA(s) should have derived from an RNA polymerase replicase (using monomers as substrates), while the proto-ss-rRNA should have derived from a partner of this replicase, which could prevent the annealing of the copy and template strands (thus facilitating a new turn of replication). Hp5 asserted that the proto-ls-rRNA(s) should have derived from a ribozyme using two or more amino acids as coenzymes, but did not offer a clear explanation of the original source of the proto-ss-rRNA. Hp9 speculated that proto-rRNAs might have derived from group I intron. Hp10 stated that proto-rRNAs might have derived from an ancient form of a bacterial RNA, named "tmRNA", which contains separate domains that can serve as a tRNA and an mRNA. The DRT hypothesis (Hp1) asserted that proto-rRNAs might have derived from intermediate subsequences between aa-binding sites on a DRT, because these intermediate subsequences had a structure, and thus a function, to facilitate the ligation of amino acids on the aa-binding sites. However, it seems hard to imagine how these intermediate subsequences could still function in such a way when they "escaped" from the DRT, as independent RNA molecules. Moreover, it seems that DRTs would be most likely to have emerged in a compact form (Appendix 1), in which these intermediate subsequences should be just short "spacer sequences", perhaps without substructures . The main problem comes from that there is not evidence shown the affinity to be strong enough to ensure the specificity, and that the coenzyme handles do not seems to be good adaptors with sufficient spatial stretch to allow their charged amino acids to reach each other . The problem is a critical one for the hypotheses assuming that the specificity of primordial peptides is important (Hp1-9), and at least a necessary one that should be solved sooner or later in the scenario described in others (Hp10-14). However, only a few of them addressed the problem directly . The CCH hypothesis (Hp3) attributed the specificity to the chemical affinity between amino acids and their cognate anticodon . TheAs mentioned above, the issue of "formation of aa-tRNA specificity" Table  is just However, some surprisingly, one could find that the present (DRT-RP) hypothesis tend to adopt a different view from the coevolution theory. That is, the initial amino acids that entered the genetic code should be complex, biosynthetic amino acids instead of simple, prebiotic amino acids. In this hypothesis, it is believed that the retention of amino acids in protocells for their repetitive usage was important and could be achieved by RNA-binding. Such RNA-binding of amino acids would result in the origin of DRTs (see Appendix 1) and perhaps also that of adaptors (see Appendix 2). Apparently, complex amino acids, which were "biosynthesized" within the protocell, would be more precious than simple amino acids, which were available in the prebiotic circumstance. Additionally, complex amino acids would be more important considering their possible function, e.g. as coenzymes of ribozymes. Along this logical line, the first aa-repository RNAs, including RNAs with multiple aa-binding sites (subsequently evolved into DRTs) and single aa-aptamers (subsequently developed into adaptors), should have just emerged for the retention of those complex amino acids. As a consequence, the first peptides should have been composed of complex amino acids, and the anticodons/codons for the complex amino acids should have emerged first in the genetic code. Later, accompanying the exhaustion of simple amino acids in the prebiotic circumstance, they also had to be "biosynthesized" within protocells and the retention of them would become important. Then aa-aptamers for these simple amino acids would emerge and, subsequently, develop into corresponding specific adaptors Thereafter, these simple amino acids would find their place in the peptides, perhaps acting as structural elements to allow the emergence of more complex functional peptides and, finally, proteins. Such a scene is consistent with the present evidence that only the aa-aptamers for complex amino acids, e.g Arg, His, Ile, Phe, Trp and Tyr, have been found to be enriched with cognate anticodons (/codons) ,23,31. TThe present hypothesis (DRT-RP) provides a major clue for the scenario on the origin of translation in the RNA world --- it should have started with the Direct RNA Template (DRT) mechanism ,22, and 1) The DRT mechanism was the simplest mechanism for RNA to synthesize peptides, and thus very likely to have been adopted initially, but the increase of the diversity and the length of the peptides would be seriously limited because the replication cost of DRTs was great, even if the DRTs could be organized in a most compact way;2) Adaptors (proto-tRNAs) would arise to allow the exploitation of the complementary strand of a DRT  to synthesize the same functional peptide synthesized by the DRT, thus saving the replication cost ;3) Acting through the adaptors , these C-DRTs could transform into much shorter templates , and substitute the role of original template RNAs (DRTs), thus significantly saving the replication cost;4) The proto-rRNA corresponding to small subunit rRNA (proto-ss-rRNA) would then emerge to aid the binding of the proto-tRNAs on the proto-mRNAs, allowing the reduction of base pairs between them (ultimately resulting in the triplet anticodon/codon pair), thus further saving the replication cost.In this context, the replication cost saved would allow the appearance of more and longer functional peptides and, finally, proteins. The logic is simple, clear and reasonable. Especially, it is well consistent with the fundamental idea concerning the scientific interpretation of the origin of life --- chemistry in nature does not exist with an aim to support life, instead, life would "manage" to exploit potential chemical resources , eventually in an efficient way over evolution, under the power of natural selection.The author declares that they have no competing interests.The answer that initially RNA would bind amino acids just in order to make beneficial peptides would fall back on the assumption that evolution worked like an engineer, thus not appropriate. A feasible answer could be formulated as the following. Amino acids might have acted as coenzymes to offer some missing functional groups for ribozymes ,30, or aIn the repository RNAs, there might be a portion containing multiple aa-binding sites, for which the benefit was that one turn of RNA replication would produce more aa-binding sites (like the situation of tandem-organized rRNA gene clusters or histone gene clusters in modern genomes). However, if the multiple aa-binding sites were organized one by one as shown in Figure For amino acids to be ligated to form a peptide, some sort of energy source was required. In an RNA-based protocell, active nucleotides, e.g. NTPs, would be abundant for the synthesis of RNA. The reaction of NTPs with amino acids would form aa-NMPs, which could be catalyzed by ribozymes . There mComparing with single amino acids, some peptides might be more efficient to aid the activities of ribozymes ,76, or tIt is assumed here that adaptors would come from the degradation or partial replication of DRTs, however, the adaptors would have to be aminoacylated before they could conduct their function. Therefore, in a view of "evolution has no foresight", an alternative explanation on the original source of the adaptors, already charged with cognate amino acids, might be more convincing.In the aa-repository RNAs (see Appendix 1), beside the ones containing multiple aa-binding sites, which could have developed into DRTs, there could also be single aa-aptamers. If, as it was assumed, retention of amino acids within protocells was very important , such siTowards RNA-based translation: the origin of proto-rRNAs" for details), and that the binding between the adaptors and a C-DRT  for each kind of amino acids in a protocell, or that different aptamers for the same amino acid would adopt similar sequences. The first assumption is reasonable considering that aptamer molecules for one specific amino acid might have descended from a common ancestor RNA by duplication. The second assumption may also be reasonable considering that sequences of different RNA binding sites for the same amino acid seem enriched with its anticodon(s) ,23 , the mechanism seems not easy to appear considering that evolution "has no foresight". Interestingly, it was revealed that the intron within some tRNA precursors in plastids, mitochondria and bacteria is a larger, self-splicing one (the group I or group II intron) [The base pairs between a proto-tRNA and a proto-mRNA could reduce when the proto-ss-rRNA emerged, aiding their binding to each other. The mechanism of the base-pair-reduction needs an interpretation. Here it should be noted that an intact aa-binding site would still be important for a proto-tRNA to charge its cognate amino acid, while some residues in the site would no longer be necessary in the subsequent template-recognition. Then how could this seemingly contradictory situation be settled? Actually, the structure of the proto-tRNAs' aa-binding site (the aa-aptamer domain) is very similar to that of the intron-contained anticodon loop of some modern tRNA precursors in eukaryotes and archaea , particu intron) -80, whic intron) ,81 and p intron) . Then, win cis "aminoacyl-tRNA synthetase" ribozyme for the proto-tRNAs. There has been experimental work suggesting that group I intron can catalyze the aminoacyl transfer reaction [Now let us turn back to an earlier stage, before the emergence of the proto-ss-rRNA. The self-aminoacylation state of proto-tRNAs might be not very stable Figure , Left anreaction . More inreaction -85. In tIf the model shown in Figure in cis "arg-tRNA synthetase" ribozyme. Further, such an association might bear some potential supports for the hypothesis stating that even proto-rRNAs  themselves might have evolved from ancient group I introns [Comparing the structure of the self-aminoacylation state Figure . Interes introns .I am grateful to the reviewers for their thoughtful analysis and critique of my manuscript. I think that this manuscript, which addresses a challenging problem full of controversies, is very suitable to appear in this journal, which has a policy to publish reviewers' comments and authors' responses together with a manuscript. In my response to the reviewers below, I have omitted some minor points brought to my attention , correcting them directly in the manuscript instead.Reviewer 1: Eugene V. Koonin, National Center for Biotechnology Information, NIH, USAThis manuscript addresses what I believe to be the ultimate problem in the study of the origin of cells: the emergence of the translation system. Any informed discussion of this fundamental enigma is of interest, and this one is no exception. I find it equally evident that the decisive breakthrough remains elusive, and even the direction in which one should proceed to find it is less than obvious. Again, this manuscript is no exception because, in my opinion, it makes very little progress towards a plausible solution.Author's response: Yes, the emergence of the translation system is almost the most challenging problem in the origin of life. According to the RNA world hypothesis, RNA could both self-replicate (via Watson-Crick base-pairing) and act as functional molecules in an early stage of life. The key issue is how a completely different set of molecules, namely proteins, could emerge to substitute the functional role of RNA. Apparently, an RNA-based "proto-translation system", perhaps including proto-tRNAs, proto-mRNAs and proto-rRNAs, would be too complicated to emerge at one step. Though a number of hypotheses have attempted to explain those intermediate steps, they lacked a major clue. Just as the reviewer said, "even the direction in which one should proceed to find it is less than obvious". The present hypothesis is just an effort to illustrate the direction, i.e. if starting from the DRT mechanism, which should be the simplest and most direct mechanism for RNA to synthesize peptides, the proto-tRNAs, proto-mRNAs and proto-rRNAs  could have all emerged due to the advantage to save the replication cost of RNA templates for corresponding peptides. At this point, the manuscript should not only have a sense making "very little progress towards a plausible solution".Moreover, to me, key aspects of Wentao Ma's hypothesis remain rather obscure. The cornerstone of the discussion seems to be the "replication parsimony principle" that is introduced as follows: "Here, by "replication parsimony" I mean that informational sequences in genetic molecules have a tendency to be utilized in a parsimony way under selection pressure, due to the cost involved in their replication." At this point, I am lost as to the meaning of this definition. The subsequent text seems to suggest that the "replication parsimony" principle is a combination of the minimization of replication cost in terms of energy and supply expenditure, and avoidance of the "error catastrophe" by minimizing the genome size. If so, these are well known consideration, and there is no need to introduce a new "principle".Author's response: Yes, the reviewer's understanding on the meaning of "replication parsimony" in this hypothesis is quite right. These may be well known considerations, but no previous hypotheses have applied these considerations to address the problem of the origin of translation. In this context, the replication cost or the room within the size-limited genome which could be saved would allow the appearance of more and longer functional peptides and, finally, proteins. Then, accompanying the appearance of proteinacious enzymes, the proto-organisms would be more efficient in metabolism and replication, and less limited in the genome size, hence evolving towards modern organisms. Certainly, here I define such a "principle" is mainly for the convenience to mention it in the text, not to introduce a new "principle".In the main part of the article which is the presentation of the hypothesis, Wentao Ma suggests that the evolution of adaptors (tRNAs in the modern translation systems) could have been driven by the advantage of compact nucleotide sites for interaction between polynucleotides and amino acids. Because of stereochemical reasons, at the stage of Direct RNA Templating (DRT), RNA-amino-acid interactions would require oligonucleotides approximately the size of the aptamers that have been shown by the Yarus lab and others to interact with some cognate amino acids, especially Arg. These experiments suggest that about 10-15 nucleotides would be required. By contrast, the adaptor mechanism allows the use of only 3 nucleotides whereas the adaptors themselves are a relatively small expenditure as they can be recycled.Author's response: Yes, this is one of the two main points that the replication parsimony would make sense in the scenario (see below for the second point)." In principle", this idea makes sense but there are several gaping holes here. First, the plausibility of DRT is very dubious at best. Appendix 1 attempts to provide some explanations but they effectively amount to previously published arguments for potential roles of amino acids and/or non-coded peptides in evolving primordial systems. These are not bad arguments However, the real problem is that DRT is extremely challenging mechanistically, and no new solutions are offered. I would note that even specific binding of individual amino acids by oligonucleotides that is taken here more or less for granted is a difficult problem as the aptamer experiments are not really compelling, perhaps, with the sole exception of arginine.Author's response: Yes, I agree that the plausibility of DRT is under debate. However, the DRT mechanism is the simplest and most direct mechanism for RNA to synthesize peptides, and is very likely to have been adopted initially in the RNA world. The key issue is, as mentioned by the reviewer, whether the evidence for specific binding of individual amino acids by oligonucleotides is compelling. Now, updated evidence on the aa-aptamers has confirmed the specific binding for some complex amino acids, e.g. Arg, His, Ile, Phe, Trp and Tyr [The compatibility with theories on the origin of the genetic code", newly added). and Tyr . Remarka and Tyr   stereochemical models.Author's response: Yes, but I think the stereochemical model shown in Figure Fourth, the current article does not even attempt to address the origin of the ribosome whereas to me any discussion of the origin of translation that does not consider ribosomes is woefully incomplete.Author's response: In this hypothesis, the origin of proto-rRNAs, including the proto-ss-rRNA and the proto-ls-rRNA(s), has been discussed. I think that the reviewer meant that the original source of the proto-rRNAs is not addressed. The reason is that I believe that the proto-rRNAs could have derived from ordinary ribozymes acting in the RNA world, considering that both the proto-ls-rRNA(s) and proto-ss-rRNA could have been small functional RNAs with a sequence length around one hundred nucleotides or even less [Towards RNA-based translation: the origin of proto-rRNAs"). Unlike proto-tRNAs, which should be introduced "as a set" (being multiple both in number and species), the proto-rRNAs "stand alone" in the proto-translation system, and the necessary to explain their original source is not so apparent .ven less ,45 . The author proposes self-splicing introns as aminoacylation enzymes, but fails to mention self-splicing introns as possible precursors of rRNAs in cis, reminiscent of the extant tmRNA [Other alternatives should be discussed or at least mentioned, such as rRNA evolving from an amino acid tagged RNA that also served as a template nt tmRNA . Alternant tmRNA .Author's response: Yes, in this hypothesis, I have not answered directly about the question about the precursors of ribosomal RNAs . The reason is that I believe that the proto-rRNAs could have derived from ordinary ribozymes acting in the RNA world, considering that they might have originally included only one hundred nucleotides or even less [Towards RNA-based translation: the origin of proto-rRNAs" and the newly added section "A comparison with other hypotheses on the origin of translation"; also see my response to reviewer 1). If there must be a tendency, I would like to agree the viewpoint mentioned by this reviewer -- self-splicing introns as possible precursors of rRNAs [A comparison with other hypotheses on the origin of translation"  iAuthor's response: No, apparently, tRNAs with covalently bound amino acids would emerge in the translation system sooner or later. I do not mean to exclude "amino acids covalently bound to nucleic acids", instead, to suppose that the synthesis of initial peptides should have relied on the DRT mechanism and developed into this way only later.In any event, the DRT model is problematic as discussed previously by Wolf and Koonin in reference  and woulAuthor's response: Yes, I admit that the DRT model is under debate . However, it is attractive on at least two points: first, it is based on available evidence from aa-aptamer experiments; second, it is the most simple and direct mechanism for RNA to synthesize peptides, which was very likely to be adopted initially in the RNA world, considering that evolution "has no foresight". On the possible route towards DRTs described in Appendix 1, the appearance of concatenation of multiple amino acid binding sites was not attributed to the reduction of the replication cost, but to the efficiency of the replication. Surely, the replication parsimony could not be the only driving force in evolution . In fact, the incorporation of spacer sequences would even increase the replication cost, but this disadvantage could have been outweighed by the improvement of replication efficiency for these aa-binding sites (i.e. one turn of replication for a number of such sites). Further, as the reviewer mentioned, the advantage of short peptides synthesized on the RNAs with multiple aa-binding sites would strengthen the advantage of this organizational form of multiple aa-binding sites.A significant problem is the transition from the non-covalent aa-binding pocket to the covalent tag or handle position Figure . ClearlyAuthor's response: Yes, this problem is significant. The present hypothesis addresses the problem by supposing the self-aminoacylation of aa-aptamers and then extending this idea by supposing a self-splicing intron as in cis aa-tRNA synthetase (Appendix 3). Indeed, as the reviewer pointed out, the attraction of this scene is that the intron can work modularly on all tRNAs without significantly inflating genome size, which is consistent with the principle of replication parsimony. Additionally, it avoids the assumption of a separate set of aminoacyl-tRNA synthetase ribozymes . The 3-k et.al. . Base paHow is the scheme in Figure Author's response: The operational code determines the aa-tRNA specificity nowadays for some amino acids. This fact has brought some speculation that it might also have had some role in the initial formation of aa-tRNA specificity during the origin of proto-translation system in the RNA world [NA world . Especiain cis aminoacyl-tRNA synthetase ribozyme, the discussion in [in trans). The latter, as I have mentioned above, should explain both the formation of the specificity between amino acids and the ribozymes, and that between the ribozymes and cognate tRNAs. This "separate assignment" of the aa-tRNA specificity is (much) more complicated, thus (much) less likely to have occurred in the RNA world, considering that "evolution can not work like an engineer". Again, I suggest that the "separate assignment" of the aa-tRNA specificity should have occurred after (or accompanying that) the role of the group I introns was substituted by p-AARSs.Additionally, by contrast with the scene in Figure ssion in  assumed Reviewer 3: Arcady Mushegian, Stowers Institute, Kansas City, USAThe related questions of the origin of translation and the origin of genetic code have been examined from various angles for decades, with the seminal book by C. Woese appearing as early as 1968 and the power of comparative genomics applied to the problems in the 21st century. There is so much said and achieved that even the review of the existing theories, their strengths and weaknesses would take more than a brief review. In the pages of Biology Direct, the study of Wolf and Koonin  that theAuthor's response: Yes, I agree with the reviewer on that "there is so much said and achieved that even the review of the existing theories, their strengths and weaknesses would take more than a brief review." For a quick start to introduce the main idea of the present hypothesis, I did not comment on various previous hypotheses in the part of "Background", except the DRT hypothesis, which is tightly related with the scenario described here. In the part "The presentation of the hypothesis" where the key issue, the origin of adaptors, was addressed, I discussed the strengths and weaknesses of another two popular hypotheses, the GT (Genomic Tag) and CCH (Coding Coenzyme Handles). According to the opinions of this reviewer (see below) and reviewer 1, in the part "Implication of the hypothesis", I have extended the discussion to some other representative hypotheses (the section "A comparison with other hypotheses on the origin of translation"). Among these hypotheses, the Hp5 [The compatibility with theories on the origin of the genetic code"), and a surprising deduction is reached --- complex, biosynthetic amino acids should have entered the genetic code earlier than simple, prebiotic amino acids, which is opposite to the common sense and the assertion in the coevolution theory [ the Hp5  and Hp14 the Hp5  . Therefore, as mentioned above, I have added the section "A comparison with other hypotheses on the origin of translation" and the section "The compatibility with theories on the origin of the genetic code"."Replication parsimony" is in my view an unfortunate expression: "parsimony" is a preference for an explanation that is "least complex", usually understood as "consisting of the smallest number of distinct events". The "replication parsimony" discussed by the author is more along the lines of minimizing the replicon size. Moreover, I am not sure why this would be a major evolutionary force at the early stages of emergence of translation, when counterbalanced by the functional needs.Author's response: Yes, the "replication parsimony" discussed here means "minimizing the size of RNA templates for the synthesis of corresponding peptides", to save replication cost of these templates. Perhaps "replication economy" is a better expression, but I am not quite sure because "economy" has a lot of other meanings. So I have kept the word "parsimony". Surely, the "replication parsimony" is not the only driving force in evolution. Even more, it should not be the major driving force in evolution because it would be counterbalanced by the functional needs (just as the reviewer said), which is just the reason why the life system and its genome could have been becoming more and more complicated. However, when the functional needs (for the first peptides) could be realized by a mechanism (the proto-translation system) that significant lessened the corresponding replication needs (comparing with the DRT mechanism), the principle of "replication parsimony" would work . More explicitly, the replication cost  whic