The discovery of mechanisms that alter genetic information via RNA editing or introducing covalent RNA modifications points towards a complexity in gene expression that challenges long-standing concepts. Understanding the biology of RNA modifications represents one of the next frontiers in molecular biology. To this date, over 130 different RNA modifications have been identified, and improved mass spectrometry approaches are still adding to this list. However, only recently has it been possible to map selected RNA modifications at single-nucleotide resolution, which has created a number of exciting hypotheses about the biological function of RNA modifications, culminating in the proposition of the ‘epitranscriptome’. Here, we review some of the technological advances in this rapidly developing field, identify the conceptual challenges and discuss approaches that are needed to rigorously test the biological function of specific RNA modifications. Many recent reviews described the technical details of current RNA modification mapping approaches and also discussed the seminal studies in the field. Instead of reiterating the content of other reviews, we will only briefly introduce the technological advances for mapping RNA modifications, but will instead focus on conceptual and technical challenges that arise when studying complex biological systems involving only a few catalytic entities affecting a multitude of substrates. Because of the recent introduction of terms such as ‘RNA epigenetics’ and ‘epitranscriptomics’ into the field, we also aim to critically discuss their definition, especially in the light of an invoked similarity to the complexities observed for epigenetic gene regulation systems.Gene expression is a multi-layered process that starts with controlling access to particular sequence information encoded in DNA, followed by copying this information to RNA molecules, which then branch off into transferring their sequence information into polypeptides , or which function as non-coding RNAs (ncRNAs). Importantly, the functionality of RNA does not only rely on sequence information. While splicing, and in particular alternative splicing, can diversify RNAs, each and every RNA nucleoside can be chemically modified or even interchanged (RNA-edited). Presently, over 130 post-synthetic RNA nucleoside modifications can be distinguished in different kingdoms ,3. Moreo50 years –8, have 2.in vitro-transcribed 23S rRNA [in vitro-transcribed tRNAs lacking all modifications are functional in reconstituted protein translation assays [Understanding the development of organisms has been the focus of most experimental biology during the last 100 years. Ever since covalent RNA modifications were linked to amphibian oocyte development , concept23S rRNA , which e23S rRNA , but alsn assays . In addin assays ,20, indin assays –24. Four2.1.6A was mostly mapped to the last exon in the majority of cRNAs indicating regulation of 3′ UTR function [1A was enriched in the 5′ UTR and around start codons of human and mouse mRNAs [6A. Furthermore, while Ψ appeared to cluster in mRNA coding sequences [5C was mapped at 5′ and 3′ UTRs in mRNAs of a highly unstable cancer cell line [Arabidopsis [While physico-chemical detection methods can only report on the presence or absence of a modified nucleoside, adapted NGS technology provided evidence for the notion that virtually every RNA species, including lowly expressed RNAs, is decorated with specific RNA modifications. NGS data mining also pointed to commonalities in the local clustering of particular RNA modifications at functional sequence features, especially in cRNAs such as in untranslated regions (UTRs), transcriptional start sites, exons and introns. For instance, mfunction . By contse mRNAs ,27, indiequences , m5C wasell line , at tranell line , but alsell line  and in Abidopsis . NGS datbidopsis , and notbidopsis .2.2.6A, m6Am and m1A) [5C to various oxidation products by the activity of ten-eleven translocation family enzymes [3C) to 3-methyluridine (m3U) [The discovery of enzymatic activities that remove RNA modifications, although presently only documented as integral parts of adenosine methylation systems (reversing mand m1A) ,35–37, pand m1A)  or removand m1A) , m5C to  enzymes  or 3-metne (m3U) .2.3.6A (or m6Am) in 5′ UTRs of mammalian cRNAs, thereby promoting cap-independent translation [5C at specific positions in yeast tRNA [Exposure of cells or organisms to non-laboratory conditions revealed dynamic responses of RNA modification systems to various stresses. For instance, mass spectrometric analyses detected tRNA modification changes upon exposure to mechanistically different toxins , which anslation ,48. Alsoast tRNA , and theast tRNA . Similarast tRNA ,52. Thesast tRNA –56 bear 2.4.5C, m6A, m5U, s2U or Ψ suppressed signalling of innate RNA sensors such as human toll-like receptors TLR3, TLR7 and TLR8 [m18 in tRNA was sufficient to suppress immune stimulation through human TLR7, indicating that, beyond its primary structural role, 2'-O-Me acts as TLR7 signalling antagonist [5C, respectively, decreased binding of synthetic mRNAs to pattern recognition receptors  in human blood cells [RNA modifications contribute to immune system function by acting as discriminators between RNAs originating from different phyla. For instance, modified nucleosides such as mand TLR8 . Furtherand TLR8 . Supporttagonist ,60. Of ntagonist . Furthertagonist . These ftagonist ,64. Impotagonist . For insod cells . Developod cells –70. Furtod cells . In summ3.Understanding the biological impact of modified RNA nucleosides that are often non-abundant and respond dynamically to environmental conditions poses various conceptual challenges .Figure 3.1.6A-modified [The majority of documented RNA modifications map to abundant ncRNAs  . For insmodified , and higmodified . Of note3.2.Secondary products originating from mature RNAs that were considered to be metabolically stable might also be functionally relevant. For instance, stress-induced processing of various small RNAs  into smaller RNAs has been observed –78. Whet3.3.The high stoichiometry of some tractable RNA modifications, especially in ncRNAs, gave rise to the notion that RNA modifications are either present or absent from an RNA species. However, this binary view was challenged by findings showing that tRNA isoacceptors can be incompletely modified at specific positions –82. Rece3.4.Presently, the detection and functional analysis of RNA modifications uses and addresses entire cell populations. Given the low stoichiometry of cRNA modifications, key to assigning biological function will be quantifying actual copy numbers of individual RNAs that are modified, preferably in single cells. Single molecule detection using advanced hybridization and imaging techniques revealed that, on average, mammalian cells only contain 50–70 copies of an individual and well-expressed mRNA, while some mRNAs (i.e. coding for transcription factors) are only present in single-digit copy numbers ,84. Furt3.5.Gria2 gene mimicking an abundant editing event at the corresponding site in the mRNA [Gria2 allele raises the question as to the functional significance of the many other ADAR2 sites in coding regions, the elucidation of which may require further experimentation beyond the observation of loss-of-function phenotypes under standard laboratory conditions. Similar considerations apply to any multi-substrate RNA modification system, which presents an important conceptual challenge when interpreting RNA modification system mutant phenotypes.Deducing biological function often relies on interpreting the effects of gene knockouts or targeted mutations reducing catalytic activity of enzymes. In the case of RNA-modifying enzymes with multiple substrates, it is unclear how to assign mutant phenotypes to loss of modification in specific RNA species. For instance, the NOP/Sun2RNA methyltransferase family member 2 (NSUN2) targets the majority of nuclear-encoded tRNAs, but also other cRNAs and ncRNAs ,88. A gethe mRNA . By contthe mRNA –100. Int3.6.6A methyltransferase IME4 displayed more severe phenotypes than those caused by a catalytic mutant, suggesting that IME4 may have RNA methylation-independent functions [6A catalysis [6A methylation through oxidation, link to an autosomal-recessive lethal syndrome in humans [6A RNA methylation plays a role in the aetiology of metabolic diseases. However, a recent study showed that single-nucleotide polymorphisms (SNPs) in this FTO intron affect the promoter of IRX3, a transcription factor linked to the regulation of body mass in mice, and not FTO expression [ADAR1 null human ESCs rescued neuronal differentiation defects by affecting miRNA processing [Some RNA modification enzyme null mutant phenotypes are only partially recapitulated in mutants harbouring catalytically dead versions, indicating limits to simplified genotype-to-phenotype inference ,102. Forunctions ,104. Simatalysis . Null mun humans , and indn humans ,108, indpression . Even ADpression ,110. Supocessing . This suocessing ) and metocessing ), which ocessing . Therefo3.7.6A [5C to C, and various editing events by, for instance, reamination) unless one considers further modification of chemical groups as a kind of reversal which could account for neutralizing the impact of the original modification-intrinsic feature. In addition, the heritability of modified RNAs (through cell divisions or generations) has only recently been experimentally addressed. While RNA modifications have been detected in sperm-derived RNA [6A reader protein was shown to mediate accelerated degradation of maternal RNAs in zebra fish, thereby contributing to maternal–zygotic transition (MZT) [6A mapping studies showed widespread occurrence in every RNA sequence context [6A, m1A and m6Am in regulating mRNA splicing, export, stability and translation. In fact, the term ‘epitranscriptomics’ is now almost exclusively used for RNA modification systems that target cRNAs [1A demethylation in the control of protein translation is a reminder that this would be a mistake [The term ‘RNA epigenetics’ has been coined by invoking similarities between the reversibility of epigenetic DNA or protein modifications and the dynamic nature of one RNA modification, m6A . The pot6A ). Accordived RNA , a materon (MZT) . However context –121. Meaet cRNAs –125. Thi mistake . Further4.Determining the biological function of RNA modifications requires methodology that allows distinguishing unmodified from modified nucleosides, mapping their distribution and quantifying modified positions. For many years, RNA modifications could mostly be detected by means of their physico-chemical properties using chromatographic methods and mass spectrometry . While t4.1.Currently, only RNA editing events, which all involve deamination reactions, can be mapped directly because the identity of a deaminated nucleotide can be inferred from its cognate pairing during the reverse transcription reaction, which, when compared with the genomic reference sequence, allows for modification calling. The very first attempt to map RNA modifications on a transcriptome-wide scale made use of sequence information of expressed sequence tags and predicted thousands of A-to-I editing events in humans . Only th4.2.N-cyclohexyl-N′-(2-morpholinoethyl)-carbodiimidemetho-p-toluenesulfonate (CMCT), which introduces Ψ-CMC adducts that terminate cDNA synthesis, thereby revealing potential Ψ positions. This selective labelling chemistry has been combined with NGS to develop Ψ-Seq, Pseudo-seq and PSI-seq, which allow transcriptome-wide Ψ mapping [1A can be inferred indirectly both from aborted cDNA synthesis and from increased nucleotide misincorporation [5C mapping in RNA using NGS. Methylated cytosines are more refractory to deamination than non-methylated cytosines, which allows determining m5C in its sequence context by a method called RNA bisulfite sequencing [All indirect RNA modification-mapping methods without prior RNA enrichment rely on the differential reactivity of a canonical nucleoside when compared with its modified nucleoside. Many such methods exploit the fidelity and/or processivity of reverse transcriptases (RT) allowing ‘RNA modification calling’ at sites that interfered with RT . For ins mapping ,52,133.  mapping . In addi mapping . Indeed,poration . In addiquencing . Furtherquencing .4.3.6A sequencing  use cross-linking-induced mutation and RT truncation profiles to reveal the precise position of m6A [et al. [Various approaches aim at enrichment of modified RNAs before NGS. The most widely used employ antibodies against nucleic acids and their modifications. Because the majority of covalent RNA modifications involve methyl groups ,137, var-m6A-seq ) combinen of m6A ,139. Furn of m6A . In addin of m6A . Alternan of m6A . Also, r [et al. . The int [et al. ,144.5.Although major advances have been made in developing NGS-based modification mapping technologies, extensive bioinformatics analysis is needed for extracting experimental noise from potential RNA modification signatures. These algorithms employ user-defined stringencies/thresholds and use diverse statistical methods for ‘modification calling’. Bioinformatic output is often taken for granted and not further addressed using alternative methodologies.5.1.et al. [For instance, RT-mediated ‘modification calling’ relies on significant numbers of reads with specific mis-incorporations or terminations at particular positions. Still, the error rate of Illumina-based sequencing  might caet al. .5.2.7G antibodies cross-reacted with guanosine [7G antibody bound to the m7G-cap, while an average of three anti-m7G antibodies bound randomly to the remaining RNA molecule [6A antibody preparations were unable to distinguish m6Am (ribose-methylated m6A) from m6A in RNA [Importantly, RNA enrichment approaches are prone to the creation and inclusion of experimental artefacts. For example, antibody-based enrichment of modified RNA fragments critically depends on sufficient antibody specificity. Antibody specificity, although often taken for granted, is indeed a great concern in medical sciences . Seriousuanosine , and elemolecule . As manyA in RNA .5.3.A much-awaited alternative for single-molecule and single-nucleotide resolution mapping is direct RNA sequencing, which does not require RT. This so-called ‘nanopore’ technology was able to directly detect RNA modifications –159. Dir6.6A mapping results using MeRIP-seq and m6A-seq [5C in cRNA of mESCs when using RNA bisulfite sequencing [6A. One low-throughput method, named site-specific cleavage and radioactive-labelling followed by ligation-assisted extraction and thin-layer chromatography (SCARLET), quantifies m6A at candidate loci [6A-level and isoform-characterization sequencing (m6A-LAIC-seq), allows measuring m6A/A stoichiometry in a transcriptome-wide fashion [6A/A at 0.2–0.6% in cRNA. Estimates for m1A/A range from 0.015 to 0.054% in cell lines to 0.16% in tissues [5C/C from 0.025 to 0.1% [6Am/A [6A, raise questions as to whether only a few sequence-identical RNAs are modified at the same position or if they are modified only in a few analysed cells  pluripotency. It was reported that m6A methyltransferase mutants promote ESC self-renewal by maintaining their ground state [The choice of experimental design and model system will influence not only the detection of RNA modifications, but also how to address their dynamics and biological functions. This is highly relevant in the light of the fact that all known RNA modifications contribute to the fine-tuning rather than to the absolute molecular function/fate/destiny of an RNA. For instance, the use of fast-growing, immortalized cell culture systems, while useful for biochemical approaches, might be ill-suited for the robust detection and manipulation of context-dependent RNA modifications. For instance, A-to-I editing studies in most tissue culture cells require ectopic expression of ADARs . By contntiation . For insnd state , but alsnd state ,185, resnd state . In addind state ) indicatnd state , includi7.3.in vivo manipulation of single RNA modification sites (within a transcriptomic ‘ocean’) will make understanding mechanistic principles easier. Alternatively, further development of simplified in vitro reconstitution assays using specifically modified but homogeneous RNA species for functional tests will be useful for identifying mechanistic details such as site selection, protein-binding capabilities as well as structural consequences of particular RNA modifications. Importantly, parameters such as cap-dependency of translation, decapping efficiency, codon usage or splicing in the context of specific mRNA modifications have already been addressed using in vitro assays [However, it remains to be seen if o assays ,189,190,8.The explosion in RNA modification-related research during the last decade has mostly been fuelled by the success in identifying RNA modifications in their sequence context. First common conceptual denominators as to why RNA modification systems are so abundant and diverse have been delineated. These include that RNA modification systems facilitate discriminating RNAs from one another and that their activities become necessary when adjusting the functionality of specific RNAs to cellular processes (i.e. translation) that need fine-tuning, especially after environmental perturbation. However, neither transcriptome-wide maps of RNA modifications nor simply disrupting RNA modification systems by mutating writers/readers/erasers, while providing positional information and a diversity of phenotypes, will necessarily lead to a better understanding of the probably complex interplay of RNA modifications, their molecular consequences or their context-dependent regulation. Developing functional assays that incorporate positional information from existing high-throughput data will therefore be the challenge for future generations of RNA biologists.


Flaviviridae viruses  is a nucleotide triphosphatase (NTPase) -dependent superfamily 2 (SF2) helicase that unwinds double-stranded RNA while translocating along the nucleic polymer. Due to these functions, NS3 is an important target for antiviral development yet the biophysics of this enzyme are poorly understood. Microsecond-long molecular dynamic simulations of the dengue NS3 helicase domain are reported from which allosteric effects of RNA and NTPase substrates are observed. The presence of a bound single-stranded RNA catalytically enhances the phosphate hydrolysis reaction by affecting the dynamics and positioning of waters within the hydrolysis active site. Coupled with results from the simulations, electronic structure calculations of the reaction are used to quantify this enhancement to be a 150-fold increase, in qualitative agreement with the experimental enhancement factor of 10–100. Additionally, protein-RNA interactions exhibit NTPase substrate-induced allostery, where the presence of a nucleotide (e.g. ATP or ADP) structurally perturbs residues in direct contact with the phosphodiester backbone of the RNA. Residue-residue network analyses highlight pathways of short ranged interactions that connect the two active sites. These analyses identify motif V as a highly connected region of protein structure through which energy released from either active site is hypothesized to move, thereby inducing the observed allosteric effects. These results lay the foundation for the design of novel allosteric inhibitors of NS3.The C-terminus domain of non-structural 3 (NS3) protein of the  Flaviviridae  helicase that unwinds double stranded RNA while translocating along the nucleic polymer during viral genome replication. As a member of superfamily 2 (SF2) helicases, NS3 utilizes the free energy of nucleotide triphosphate (NTP) binding, hydrolysis, and product unbinding to perform its functions. While much is known about SF2 helicases, the pathways and mechanisms through which free energy is transduced between the NTP hydrolysis active site and RNA binding cleft remains elusive. Here we present a multiscale computational study to characterize the allosteric effects induced by the RNA and NTPase substrates  as well as the pathways of short-range, residue-residue interactions that connect the two active sites. Results from this body of molecular dynamics simulations and electronic structure calculations are highlighted in context to the NTPase enzymatic cycle, allowing for development of testable hypotheses for validation of these simulations. Our insights, therefore, provide novel details about the biophysics of NS3 and guide the next generation of experimental studies.Non-structural protein 3 (NS3) is a  Flaviviridae) are small (∼11 kilobases) positive-sense, single-stranded RNA (ssRNA) viruses that include members such as dengue (serotypes 1-4), Zika, West Nile, yellow fever, and Japanese Encephalitis viruses. The dengue virus (DENV) is a public health threat that causes serious morbidity and mortality globally [Flaviviruses , which plays a critical role in the viral replication cycle [Flaviviridae, possessing an N-terminal serine protease domain responsible for proteolytically cleaving the viral polyprotein during translation [One of the primary antiviral targets in on cycle –15. NS3 nslation  and a C-nslation –22. In anslation  for a renslation –26, demonslation  or RNA/Dnslation , suraminnslation  or aurinFlaviviridae NS3h have been classified as a superfamily 2 (SF2) helicase  where the NTPase cycle  are released. To date, it is unclear which stage(s) of the cycle are responsible for the translocation and unwinding functions of NS3h. Furthermore, the biophysical couplings between NTPase and helicase active sites are still poorly understood [The NS3/NPH-II subfamily of SF2 helicases exhibit both RNA-stimulated NTPase activity and NTPase-dependent helicase activity –22. Thesderstood .Flaviviridae NS3h is that of the Hepatitis C virus  [Flaviviridae NS3h, suggest that the NS3 enzyme tracks along the phosphodiester backbone of the nucleic oligomer, unwinding one base-pair per hydrolysis event [One of the better studied civirus) –40. Utilcivirus) –35 and scivirus) , 41, 42 is event . To explis event –35 or bais event , 39–41 pis event , 44.et al. reported a set of crystal structures of the DENV NS3h in important protein-substrate complexes of the NTPase cycle  simulations of the HCV NS3h-ssRNA systems in the presence and absence of ATP and ADP. The reported simulations were used to interrogate the thermodynamics of these substrate states with various conformations of the NTPase active site [Previous theoretical studies of helicases have focused on a broad range of enzymes such as PcrA (SF1) –49, tranive site . While tive site –56. TherWe report here a multiscale theoretical study of the DENV NS3h enzyme at each substrate state along the NTPase cycle. RNA-induced allostery on the NTPase active site is reported wherein the presence of an RNA substrate alters the positioning and dynamics of waters within the hydrolysis active site. Inspired by this observation, minimum energy electronic structure calculations are performed to investigate the energy landscape of the hydrolysis reaction. Additionally, investigations into NTPase substrate-induced allostery on the RNA-binding cleft suggest that NS3h interacts with RNA in a NTPase substrate-dependent manner. Umbrella sampling (US) simulations are performed to enhance the sampling of a proposed elementary step of the translocation mechanism observed during the unbiased simulations. Finally, analyses of the correlated motions between residues are used to identify allosteric pathways that connect the two active sites. It is through these pathways that we hypothesize that free energy released during the NTPase cycle is transduced to the RNA-binding cleft and utilized to perform work on the RNA. This study of the substrate states of DENV NS3h lays the foundation for further study of the NTPase cycle and marks the most complete picture of the molecular mechanism of the NS3 NTPase/helicase to date.et al. [i (2JLY), and ssRNA+ADP (2JLZ) model the pre-hydrolysis, post-hydrolysis, and product release states of the NTPase cycle, respectively. The Apo (2JLQ) and ATP (2JLR) substrate states are also simulated and used as experimental controls for our investigation into allostery.A subset of the crystal structures reported by Luo et al.  of the D2+ divalent cation is converted into a Mg2+. For the ATP crystal structure (2JLR), residues of the protease linker region were poorly resolved and so are transferred from the Apo (2JLQ) structure after aligning the neighboring amino acid backbones in both systems.The RNA-bound structures of DENV NS3h were crystalized as dimers of the protein . For thei, and ssRNA+ADP). The simulations are performed using the GPU-enabled AMBER14 software [χ OL3 [i (provided in Supplementary Information (SI); 2+ [μs, with the first 200 ns of simulation sacrificed to equilibration of the starting structures. Simulation of the ssRNA system is performed to 2 μs. For both the ATP and ssRNA+ATP systems, two 1.5 μs simulations are performed. The total amount of unbiased simulation reported here on the described structures is 12.5 μs.All-atom, explicit solvent MD simulations are performed for the six substrate states of DENV NS3 and presented in software , ff14SB software  parametee [χ OL3 , 60 parae [χ OL3 , ADP [61(SI); 2+  are also(SI); 2+ . The par(SI); 2+  is used i, and ssRNA+ADP systems, using the same protocol as the unbiased simulations with the addition of a bias. For each substrate state, a minimum of 22 sampling windows are simulated for 50 ns each with harmonic wells positioned every 0.5 Å and ranging from 3.50 to 14.00 Å. Harmonic force constants are 20 kcal mol-1 Å-2. Further simulation and additional windows are run in regions of collective variable space with poor sampling. The weighted histogram analysis method (WHAM) [μs.US simulations are performed to enhance sampling of a hypothesized elementary translocation event wherein the biased collective variable is the distance between the central carbon of the guanidinium group of Arg387 to the phosphorous atom of phosphate 4 in the RNA. These simulations are run for the ssRNA, ssRNA+ATP, ssRNA+ADP+Pd (WHAM)  is used ωB97X-D/6-31+G* level of theory [ωB97X-D functional is chosen due to its broad applicability [2+ ion, and seven water molecules. The amino acids are truncated at various positions , products (https://github.com/mccullaghlab/DENV-NS3h).Unless stated otherwise, analyses of MD trajectories are performed using Python 2.7 and the MDAnalysis module (version 0.15.0) . MatplotFor clarity, we present and discuss our results in three sections. The first and second sections independently report observed RNA-induced and NTPase substrate-induced structural allosteries, respectively. The focus of the RNA-induced allostery section is on the structural changes seen in the NTPase active site due to bound RNA. Similarly, the NTPase substrate-induced allostery section highlights changes seen in the structure and dynamics of the RNA-binding cleft due to the presence of different nucleotide substrates. In the final section, correlated motions between residues are used to highlight pathways through which these structural allosteric effects are induced.To date, no biophysical explanation has been proposed for the 10 to 100-fold increase in NTPase turnover rate observed for DENV NS3h in the presence of RNA . Crystalβ3β4; Thr244 to Glu255). The crystal structures of DENV NS3h from Luo et al. [α-helix 2 (α2). Transitions from “closed” to “open” conformations are not sampled during our MD simulations of the Apo and ATP systems demonstrating that the crystal structure conformations are minima in the solution phase free energy surfaces.The most marked structural difference between DENV NS3h with and without ssRNA is the change in conformation of the RNA-binding loop  and Val226, Ala228, Ala229 (α2). When in the “closed” conformation, this hydrophobic pocket is not formed and leaves the top of α2 exposed to solvent.The RNA-induced structural change of Lα2 is quantified by computing the root mean square deviation (RMSD) of the backbone atoms of α2 (residues 224 to 235) relative to the ssRNA+ATP crystal structure (2JLV). The distributions of this metric are presented in β3β4 and α2 stabilize the structural conformation of α2 where the top of the helix is pushed in towards the NTPase active site. Interestingly, Val227 and Met231 are the residues in α2 that have prominent positions in the NTPase active site. While these hydrophobic side chains likely have minimal influence on the hydrolysis reaction mechanism, their structural shift into the hydrolysis active site reduces the volume of the pocket.The structural deviation of Flaviviridae, motif II is the DEAH sequence (residues 284 to 287) where Asp284 and Glu285 are positioned in the rear of the NTPase active site. Luo et al. noted that the presence of RNA shifts the carboxylate group of Glu285 from a magnesium-bound position to a position more conducive to coordinating the lytic water [Motif II  is a set of highly conserved amino acid residues within NTPase enzymes and is known to play an important role in the catalysis of the hydrolysis reaction . In DENVic water . In thisγ-phosphorus atom. Structurally, with RNA bound, the carboxylate side chain of Glu285 is pulled away from the coordination sphere of the Mg2+ cation and is moved into plane with the terminal phosphoanhydride bond. In either position, Glu285 is observed to hydrogen bond with the lytic water yet, in the RNA-induced position, the lytic water is positioned in a more ideal environment for nucleophilic attack (quantified in the next section).Our MD simulations maintain these starting conformations and support the deduced importance of Glu285. Snapshots of the Glu285 positions in the ATP and ssRNA+ATP simulations are shown in Both Asp284 and Glu285 are major structural landmarks within the NTPase active site and have no direct interactions with the RNA substrate. Rather, the origin of the RNA-induced structural rearrangement of motif II residues is attributed to RNA-induced displacement of residues down the linear amino acid sequence, specifically Phe288 and Asp290. 2+ cation are minimally affected by the presence of RNA. Alternatively, waters within the NTPase active site are observed to be greatly influenced by the presence of bound RNA. For example, the average number of water molecules found within the NTPase active site decreases from 30.0±0.7 molecules for the Apo substrate state to 21.72±0.08 molecules in the ssRNA state. A similar but reduced trend is observed when comparing the ATP (15.0±0.2 water molecules) and ssRNA+ATP (12.8±0.4) simulations.RNA allosterically affects the positions of amino acids within the NTPase active site, yet it is unclear how these structural rearrangements influence the hydrolysis cycle. When comparing the ATP and ssRNA+ATP simulations, the positions and dynamics of the ATP molecule and MgThe translational and rotational dynamics of water molecules within the NTPase active site are also influenced by bound RNA, as shown graphically in γ-phosphorous atom (Pγ) of ATP is described by an attack angle of 180° with respect to the terminal phosphoanhydride  bond. The distance between the lytic water oxygen (Owat) and Pγ will decrease to a bonded distance of ∼ 1.7 Å over the course of this reaction. Therefore, the Pγ-Owat distance and Oβ,γ-Pγ-Owat angle are used as geometric collective variables that describe the nucleophilic attack of a lytic water. Projecting the positions of waters within the NTPase active site onto these two coordinates allows for comparisons of the positioning of catalytically relevant water in the ATP and ssRNA+ATP substrate simulations. The two-dimensional heat maps of this projection are shown in the SI  for both of the substrate states.Considering the hypothesized SN2 mechanism of the hydrolysis reaction , 77, ideγ-Owat distance less than 5.0 Å and an Oβ,γ-Pγ-Owat angle greater than 155° are defined as lytic. The probability of observing a frame with water in a lytic position is 72.93% ± 0.04% for the ATP system and 79.08% ± 0.03% for the ssRNA+ATP system.The difference between the probability densities for the ssRNA+ATP and ATP simulations is shown in β3β4, α2, and motif II. Although it is difficult to fully deconvolute the specific influences of these structural allosteries on the water molecules in the active site, we propose that the observed influence of RNA on number and dynamics of water molecules originates from the structural rearrangement of α2, where Val227 and Met231 become more prominent in the hydrolysis active site when RNA is bound. These hydrophobic residues not only exclude water molecules from the active site but also slow the translational and rotational motions of water molecules. This RNA-induced effect can be thought of as a entropic destabilization of the NTPase active site, where the RNA decreases the phase space that the water molecules can populate. Furthermore, the RNA-induced structural rearrangement of the Glu285 carboxylate group leads to the observed increased in probability of lytic water molecules. Through the backbone displacement of motif II residues, the Glu285 side chain is pulled away from the Mg2+ cation and into plane of the γ-phosphate group, thereby creating a local protein environment that stabilizes water molecules into more ideal positions for nucleophilic attack. This effect is interpreted as a direct destabilization of the lytic water in the hydrolysis reaction.In total, these results demonstrate that RNA affects the dynamics and positioning of waters within the NTPase active site. These effects are propagated from the RNA binding cleft to the NTPase active site through structural rearrangements of L2+, and Glu285 atoms.The impact of the RNA-induced repositioning of the lytic water on the hydrolysis reaction is investigated using density functional theory (DFT) calculations of an abbreviated NTPase active site where conformations are pulled from the unbiased MD simulations. Active site geometry optimizations are performed on the ATP and ssRNA+ATP substrate states where the hydrolysis reaction is modeled as a concerted SN2 mechanism using a reactant state (ATP*), a transition state (TS), and a product state TS − d(Owat − Hwat)ATP* = 0.12 Å). The product state was found following the TS in which the proton has completely transferred to Glu285, forming the HPO42- molecule.The free energy landscape of the ATP substrate state is presented in β,γ-Pγ-Owat angle of 161°. In the TS structure, the Pγ-Owat and Oβ,γ-Pγ distances become 2.03 Å and 2.39 Å, respectively, while the Oβ,γ-Pγ-Owat angle increases to 175°. Unlike in the ATP substrate state, the O-H bond distance of the lytic water is minimally perturbed when comparing the reactant and TS structures (ΔOwat-Hwat = 0.04 Å). Rather, the proton transfer step is completed during the transition from the TS to the product state. Overall, the calculated activation barrier height for the ssRNA+ATP substrate state is 27.6 kcal mol−1, corresponding to a 2.8 kcal mol−1 decrease in barrier height relative to the ATP substrate state landscape.Panel (B) of 2+ coordination sphere. Optimization of such a product state is infeasible due to the limited description of the protein environment in these DFT calculations. Additionally, it is assumed that the energy barriers of these subsequent events are much smaller than the hydrolysis reaction barrier and so, are disregarded in the current study.The overall reaction, e of RNA . While t2+ coordination sphere, Glu285 performs this function more effectively, stabilizing the lytic water at a shorter Pγ-Owat distance and a larger Oβ,γ-Pγ-Owat angle. Additionally, comparisons of the associative (Pγ-Owat distance) and dissociative  reaction coordinates suggest that the RNA-induced structural rearrangement of the active site leads to slight changes in the hydrolysis mechanism. For both substrate states, the change in Pγ-Owat distance from reactant to TS states  is larger in magnitude than the respective change in Oβ,γ-Pγ distance . These values suggest that the hydrolysis reaction proceeds through an asynchronous SN2 hydrolysis mechanism where the nucleophilic attack of the lytic water is enhanced by the local protein environment [wat-Hwat distance  and the OGlu285-Hwat distance  collectively suggest that the ssRNA+ATP TS structure is more reactant-like than the TS structure of the ATP substrate state.The calculated activation barrier heights of both substrate states are in good agreement with previous DFT studies of NTP hydrolysis in a protein environment –83 and iironment , 88. Furβ3β4, α2, and motif II affect the probability of water molecules to be located in lytic positions, and (2) the same RNA-induced structural changes alter the activation barrier of the hydrolysis reaction. To account for both effects, a reaction scheme is proposed to describe the NTPase function within NS3h, where a fast equilibrium exists between active site conformations with and without the presence of a lytic water. This equilibrium is followed by the slow, irreversible hydrolysis reaction. Using this scheme, the theoretical observed rate constant for the NTP hydrolysis reaction is kobs = Keqkhydrol. For both ATP and ssRNA+ATP substrate states, the Keq is defined as the respective ratio of probabilities of observing a MD frame with and without a lytic water. The hydrolysis rate constant is quantified using an Arrhenius rate equation where the Boltzmann factor accounts for the activation energy barrier observed in the electronic structure calculations. The ratio of observed rate constants is defined as the theoretical enhancement factor of the RNA-stimulated NTPase activity,Ea is the difference in activation energies between the ssRNA+ATP and ATP substrate states  by an order of magnitude on both sides. Even with this large range, the calculated enhancement factor maintains the qualitative narrative that RNA-induced structural allostery of Lβ3β4, α2, and motif II leads to the repositioning of the lytic water within the NTPase active site as well as affects the energetics of the hydrolysis reaction.Taking the ratio of the ssRNA+ATP and ATP rates results in a theoretical enhancement factor of 150, which is consistent with the experimentally observed enhancement factor (10 to 100) . Error ai) is observed in the crystal structures of DENV NS3h [i, and ssRNA+ADP) is used to interrogate protein-RNA interactions as well as identify protein structural changes that have NTPase substrate-dependent behaviors.Experimental studies have shown that the NS3h helicase functions (translocation and unwinding) are NTPase dependent, yet it is unclear which equilibrium states and/or dynamic events of the NTPase cycle are the source of the necessary free energy for these functions , 21. AllENV NS3h . Therefoβ3β4 are observed in crystal structures to have contact with the phosphodiester backbone of the nucleic oligomer [α-helix dipole moments  [For the RNA-bound substrate states modeled here, the RNA oligomer is seven residues long with the first five residues (5′ end) strongly bound within the RNA-binding cleft of the protein. The remaining two nucleic residues are poorly resolved in the crystal structures  and are oligomer , 45, yetoligomer . As obse 2, α2′)  are obselie analysis function in AMBER14 cpptraj [Nonbonding interaction energies are used to provide a quantitative description of the relative strength of the protein-RNA interactions. Comparisons of these data between substrate states of the NTPase cycle provide insight into the hypothesized NTPase dependence of protein-RNA interactions. The pair-wise sum of Lennard-Jones and short-range, unscreened electrostatic energies are calculated using the  cpptraj . Table 1-1) and ssRNA+ADP+Pi  structures have more stable protein-RNA interactions than the ssRNA  and ssRNA+ADP  structures. These results suggest that the presence of the γ-phosphate group (or Pi) in the NTPase active site has a stabilizing effect on the strongly bound phosphate groups of the RNA. This result is in direct disagreement with experimental results of HCV NS3 where it is observed that the protein has a decrease in affinity for nucleic oligomers in the presence of ATP [The totals of interaction energies between the protein and P1-4 demonstrate that the ssRNA+ATP  and P4  than the other three systems  and weaker interactions with P1 . This demonstrates that a bound nucleotide (ATP or ADP) causes a shift in protein-RNA interactions from 3′ (P3 and P4) to 5′ (P1) RNA residues.The interaction energies between protein atoms and individual phosphate groups presented in i, and ssRNA+ADP simulations, Arg387 is stable in the “up” conformation. In the ssRNA simulation, the “up” to “down” transition occurs once, after which no reverse transitions occur. Furthermore, after Arg387 transitions to the “down” conformation, two concerted events are observed to occur: (1) Lys388 (motif IVa) coordinates P1 and P2, taking up Arg387’s previous position, and (2) P4 partially unbinds from the RNA-binding cleft.Visual analysis of the protein-P3 and -P4 interactions in the ssRNA simulation highlighted a rare event where the guanidinium side chain of Arg387 (motif IVa) transitions from coordinating P1 and P2 to P3 and P4. The two conformations of this event are depicted in α2′). Lys388 is more solvent exposed than Arg387 and fluctuates around the phosphate groups of the RNA. The α2′ secondary structure is generally solvent exposed with its dipole-axis pointing towards P2. Historically, an arginine residue observed to coordinate adjacent phosphate groups of RNA has been termed an arginine fork [Arg387 and Lys388 are highly conserved residues in the NS3/NPH-II subfamily of SF2 helicases and have been classified as motif IVa residues  are -7.52±0.05 kcal mol-1, 13.885±0.05 kcal mol-1, 7.45±0.06 kcal mol-1, and 10.91±0.05 kcal mol-1 for the ssRNA, ssRNA+ATP, ssRNA+ADP+Pi, and ssRNA+ADP systems, respectively. Therefore, the Arg387 side chain states are observed to have a NTPase substrate-dependence where the ssRNA system energetically favors the “down” conformation while the ssRNA+ATP, ssRNA+ADP+Pi, and ssRNA+ADP substrate states favor the “up” conformation. Unbinding of P4 is not observed for US windows corresponding to the “down” conformation in the ssRNA+ATP, ssRNA+ADP+Pi, and ssRNA+ADP US simulations.Due to the concerted nature of the Arg387 transition and the unbinding of P4, we propose that the observed “up” to “down” transition is potentially an elementary step in the translocation mechanism of NS3h, where the guanidinium side chain conformations are NTPase substrate-dependent. Therefore, we investigate the thermodynamics of the side chain conformational states using one-dimensional US simulations for the ssRNA, ssRNA+ATP, ssRNA+ADP+PThese results support the hypothesis that the Arg387 side chain conformational states are NTPase substrate-dependent and exemplify a large shift in protein-RNA interactions. Considering the full NTPase cycle , Arg387 The current view of allosteric regulation focuses on signal transduction through complex, 3-dimensional networks, brought about by intrinsic structural and/or dynamic changes along pathways connecting two distal, non-overlapping active sites –98. Thesα2, motifs II and IVa) due to the observed structural rearrangements. Additionally, the correlation heat maps are used to identify segments of the protein that experience strong correlations with numerous other regions of the protein, such as motif V. While motif V does not experience substrate-induced structural rearrangements, the strong correlations between motif V and motifs in both the NTPase active site and RNA binding cleft are hypothesized to have functional importance in the signal transduction mechanism of allosteric regulation. Unlike the previous two sections, comparisons between substrate states (RNA-bound and NTPase substrate-bound) are not considered here. Instead, focus is given to the discussion of the residue-residue distance correlation analysis of the ssRNA+ATP substrate state.Dynamic network analyses, such as residue-residue correlations, have been used to identify allosteric pathways within proteins from simulation –100. A gβ-sheets in subdomains 1 (residues 188 to 326) and 2 (residues 327 to 481) produce the honeycomb patterns observed in the heat map, due to the extremely stable tertiary structure observed in NS3h. Lines drawn on α2, motifs II and IVa.As expected, there is strong correlation along the linear sequence of residues, as seen in α2/motif II off-diagonal region of the heat map and the structural features leading to the observed correlations between these two segments. Specifically, Asp284 and Glu285 of motif II experience correlated motions with most of α2 (residues 220 to 231) and especially strong correlations with Ala222, Pro223, and Thr224. Thr224 directly coordinates phosphate 3 of the RNA substrate. Therefore, panels (B) and (D) highlight a pathway through Glu285, Ala222, Pro223, and Thr224 that connects residues of paramount importance in the hydrolysis reaction (Glu285) with residues that directly coordinate the phosphodiester backbone of the RNA (Thr224).Panels (B) and (D) of α2, as shown in panels (C) and (E) of α2. Alternatively, from the perspective of α2, the guanidinium group of Arg225 has strong and maintained electrostatic interaction with the backbone carbonyl group of Arg387. Through this short-range interaction, Thr224, Arg225, and Ala226 experience correlated motions with residues 385 to 395 of motif IVa. As observed in π stacking-like structure, bookended by RNA phosphate 3 as well as residues in the NTPase active site (residues not shown for clarity but can be viewed in trajectories provided at https://hdl.handle.net/10217/186709).In a similar fashion, the motif IVa structure is observed to have strong correlations with residues in α2  with residues of motifs II  and IVa  act as channels through which free energy released from the NTPase cycle or helicase functions can be transferred from the NTPase active site to the phosphodiester backbone of the RNA (and vice versa). Structural or dynamic perturbation of these pathways, via mutation or small molecule binding, are hypothesized to affect the efficiency of energy transduction.Direct coupling between motifs II and IVa is minimal due to the large distances between the residues in both motifs, as shown in the respective off-diagonal region of α2. Structurally, the highly correlated nature of motif V is explained by the position of these residues in relation to the NTPase active site, RNA-binding cleft, and protein residues important in either active site, as shown in α-helix into loop). The backbone amide of Gly414 actively coordinates the lytic water of the hydrolysis reaction. The alcohol group of Thr408 coordinates phosphate 2 of the RNA. Therefore, the linear sequence of motif V is a direct pathway connecting the NTPase active site with the RNA-binding cleft. Limited structural changes are observed for this motif in the presence or absence of RNA or NTPase substrates. Rather, motif V is observed to have strongly correlated motions with the previously mentioned structural regions for all substrate states. Similar to the previously discussed pathways, motif V is hypothesized to be another pathway for free energy transduction from one active site to the other.The residue-residue correlation analyses are used to identify structural regions of NS3h that exhibited strong correlations with other regions of the protein. Particularly, this analysis highlighted residues 407 to 420 of motif V. β3β4, α2, and motif II. These RNA-induced structural changes lead to an entropic destabilization of the NTPase active site as well as a direct destabilization of the lytic water. Inspired from these results, electronic structure calculations were used to investigate the energetics of NTP hydrolysis reaction. The energetic landscapes obtained from the DFT calculations demonstrate that RNA decreases the activation barrier as well as affects the mechanism of the hydrolysis reaction. Combining these results into a kinetic model allowed for the calculation of a theoretical RNA-stimulated NTPase activity enhancement factor of 150, which qualitatively matches the experimentally observed enhancement factor. Therefore, results from MD and DFT calculations provide novel, multiscale insight into the RNA-induced allosteric effects that stimulate the catalysis of the NTP hydrolysis reaction in DENV NS3h.Through analyses of the reported simulations, molecular observables of RNA- and NTPase substrate-induced allostery were identified. Specifically, an RNA bound within the RNA-binding cleft affects the dynamics and positioning of water molecules within the NTPase active site. This allosteric influence is conferred from the RNA-binding cleft to the hydrolysis active site through structural rearrangements of Lγ-phosphate (or Pi) of the NTPase substrate was observed to strengthen the protein-RNA contacts. Furthermore, the localized nonbonding interaction energies demonstrate a large shift in protein-RNA contacts, originating in part from the side chain conformational states of Arg387. Results from US simulations demonstrate that the Arg387 side chain conformational states exemplify NTPase substrate-dependent protein-RNA interactions. With the purview of the NTPase cycle, transitions between conformational states leads to 3′ to 5′ translocation. Therefore, we hypothesize that the transition between Arg387 side chain conformations is an elementary step in the unidirectional translocation mechanism of NS3h along the phosphodiester backbone of RNA.Unlike RNA, the NTPase substrates are smaller perturbations to the NS3h structure and dynamics. Protein-RNA interaction energies were used to investigate the NTPase substrate-dependence of protein-RNA contacts in the unbiased MD simulations. From these analyses, the protein-RNA phosphodiester backbone interactions were observed to be NTPase substrate-dependent. The presence of the α2 with motifs II and IVa. The short-range, residue-residue interactions were presented that connect the RNA-binding cleft to the NTPase active site. Furthermore, the correlation heat maps allow for identification of regions of the protein that experience strong correlated motions with numerous other regions. Motif V is one such example, where the segment of 13 residues has strong coupled motions with seven other motifs in subdomains 1 and 2. This highly correlated nature suggests that motif V functions as a centralized communication hub that connects distal portions of the protein structure.Finally, consideration of these allosteric effects independent of one another provides an incomplete picture of the biophysics of the NS3h protein. Residue-residue correlation analyses were used to identify structural regions of the protein that experienced correlated motions with other regions. These analyses were used to describe the allosteric pathways that connect Complete modeling of a revolution of the NTPase cycle in the DENV NS3h presents a significant challenge for current computational methodologies. Rather, we have divided the cycle into equilibrium substrate states and dynamic events where the protein transitions from one substrate state to another. The simulations reported have modeled the important NTPase cycle substrate states, leading to novel insights into the function and underlying biophysics of the DENV NS3h enzyme with focus given to the allosteric connections between the RNA-binding cleft and NTPase active site. We hypothesize that the observed allosteric effects and pathways have important roles in the transduction of energy from one active site to the other during the dynamic events of the NTPase cycle. Therefore, the results reported have laid an initial foundation for theoretical investigations into the dynamic events of the NTPase cycle.Beyond further theoretical modeling of NS3h, transverse relaxation-optimized spectroscopy (TROSY) NMR, mutational biochemical studies, and targeted small molecule binding experiments can be envisioned to test the hypothesis and results presented. Nuclear magnetic resonance has been previously used to study dynamics within isolated HCV NS3 helicase subdomains –105, butS1 Filei, and ssRNA+ADP substrate states are presented. Cartesian coordinates of the optimized structures of the NTPase active site are tabulated.The file includes further details about simulation protocol and analyses performed on the MD simulations as well as structural figures of the atoms included in the electronic structure calculations. Additionally, residue-residue correlation heat maps of the Apo, ATP, ssRNA, ssRNA+ADP+P(PDF)Click here for additional data file.S2 Filei, as well as the frcmod.phosaa10 files are included. It is necessary to load these files into leap to obtain the Pi parameters and structure used in the reported simulations.Unzipping this file will create a directory containing the parameters and starting structures used for the reported unbiased simulations. Configuration files to run simulation in the AMBER software packages are also provided. Additionally, the structure prep file for inorganic phosphate, P(ZIP)Click here for additional data file.


Ginkgo biloba is one of the oldest seed plants and has an important evolutionary position. Determining the patterns and distribution of RNA editing in the ancient plant provides insights into the evolutionary trend of RNA editing, and helping us to further understand their biological significance.RNA editing is a posttranscriptional modification process that alters the RNA sequence so that it deviates from the genomic DNA sequence. RNA editing mainly occurs in chloroplasts and mitochondrial genomes, and the number of editing sites varies in terrestrial plants. Why and how RNA editing systems evolved remains a mystery. G. biloba and identified 255 editing sites, which is the highest number of RNA editing events reported in a gymnosperm. All of the editing sites were C-to-U conversions, which mainly occurred in the second codon position, biased towards to the U_A context, and caused an increase in hydrophobic amino acids. RNA editing could change the secondary structures of 82 proteins, and create or eliminate a transmembrane region in five proteins as determined in silico. Finally, the evolutionary tendencies of RNA editing in different gene groups were estimated using the nonsynonymous-synonymous substitution rate selection mode.In this paper, we investigated 82 protein-coding genes in the chloroplast genome of G. biloba chloroplast genome possesses the highest number of RNA editing events reported so far in a seed plant. Most of the RNA editing sites can restore amino acid conservation, increase hydrophobicity, and even influence protein structures. Similar purifying selections constitute the dominant evolutionary force at the editing sites of essential genes, such as the psa, some psb and pet groups, and a positive selection occurred in the editing sites of nonessential genes, such as most ndh and a few psb genes.The The online version of this article (doi:10.1186/s12870-016-0944-8) contains supplementary material, which is available to authorized users. Comparisons of the coxII transcript with homologous genes of other species showed that the open reading frame of this gene in Trypanosoma brucei shifts due to the addition of a nucleotide in the transcript, resulting in a new readable frame , and the primer sequences are listed in . The direct sequencing of cDNAs derived from these transcripts and of the corresponding genomic DNA (gDNA) was carried out by Sangon Biological Engineering Technology & Services .The primers of 82 TM Plant RNA Kit according to the manufacturer’s protocol. The tissue was disrupted and homogenized as above, and the gDNA was preliminarily eliminated with a gDNA filter. The flow-through at the very last step was mixed with the membrane-binding solution and then loaded into the HiBind RNA Mini column. Finally, RNA was washed with RWC buffer and RNA wash buffer to remove protein, polysaccharide and salt contamination. The total RNA was treated with DNaseI to remove gDNA contamination. The cDNA was synthesized according to the PrimeScript RT Reagent Kit protocol .The total RNA was extracted using E.Z.N.Ahttp://www.ebi.ac.uk/Tools/msa/clustalo/). The sequences were analyzed using SeqMan of the Lasergene software package (https://www.dnastar.com/t-seqmanpro.aspx). According to Mower and Palmer ; S. lycopersicum [AM087200]; C. sativus [AJ970307]; A. formosae [NC_004543.1]; G. hirsutum [DQ345959.1]; A. thaliana [NC_000932.1]; C. taitungensis [NC_009618]; A. capillus-veneris [AY178864.1]; T. aestivum [AB042240.3]; N. tabacum [Z00044.2]; Z. mays [NC_001666.2]; G. biloba [AB684440.1]. Most of the editing site information was acquired from GenBank and RNA (http://dna.kdna.ucla.edu/rna/index.aspx) databases. Some editing sites were found in the literature.The 12 species used for the sequence alignments are listed as follows: G. biloba chloroplast genome, and all of the editing sites were C-to-U conversions. Among the 255 editing sites, ycf3 , psbB (1391 and 1392 bp), rps14 (193 and 194 bp) and ndhD (1995 and 1996 bp) had two editing sites within one codon. RNA editing also created two new start codons in petL and rps8, and seven stop codons in ccsA, rps4, rps18, petD, petL, ndhC and ndhK , second (45) and third (5) codon positions. ndhD has the highest editing frequency, followed by ndhA, ndhB, ndhK, rpoC1, matK and rpoA. Additionally, ndhF has 18 partial editing sites, which is the highest number of partial editing sites in one gene. There are 16 silent editing sites, which cannot alter the corresponding amino acids, in 14 transcripts, ycf1, ycf3, ycf4, psbA, psbC, psbD, ndhD, ndhF, ndhK, petA, rpl2, rpoA, rpoB and chlN  and threonine to isoleucine (8.3%). The amino acids maintained their hydrophobic properties at 80 editing sites, and the highest rate occurred in proline to leucine (60.0%), followed by histidine to tyrosine (20.0%) and leucine to phenylalanine (1.3%). Only 13 and 7 editing sites caused amino acids to change from hydrophobic to hydrophilic and to maintain their hydrophilicity, respectively (Fig. Most RNA editing sites exist in the protein-coding regions and often cause corresponding amino acid alterations. In addition to 16 silent editing sites, there were 239 sites that resulted in corresponding codon changes in alanine 2.2% and tndhD-57 (Additional file ndhD, ndhE, ndhF, psbB and psbN, respectively, after the corresponding codons were edited (Fig. petB when the amino acid at the 212 codon position changed from proline to serine due to editing (Fig. In our attempt to understand whether RNA editing affects protein structure, we predicted the secondary structures of 82 proteins before and after editing using bioinformatics software. The results showed that many editing events might change the secondary structures of the corresponding protein. Most editing sites form a new α-helix structure in up- or down-stream regions around the editing codon (Additional file ted Fig. . In addiing Fig. .Fig. 5ThG. biloba transcripts have the highest C-to-U conversion rate among the three gymnosperms and the number of editing sites is nearly 10 times higher than in other seed plants. During the evolution of plants, the number of editing sites decreased from the highest number, 942, in A. formosae to 21–26 in the monocotyledons, O. sativa, S. officinarum, T. aestivum and Z. mays. The U-to-C conversions gradually vanished, and the percentage of editing in the second position increased from 58% in A. formosae, and 68% in A. capillus-veneris, to almost 100% in angiosperms. The silent editing sites decreased. There were 28 and 21 silent editing sites in A. formosae and A. capillus-veneris, respectively. However, they almost completely disappeared in seed plants (Table A comparison of chloroplast RNA editing events showed that the frequency and type of RNA editing were significantly variable among the major land plant groups, which included 11 angiosperms, 3 gymnosperms, 1 fern, 1 hornwort and 1 moss. C-to-U editing has been widely identified in these land plants, and U-to-C editing has been found only in hornwort and fern. Additionally, ndh and some psb genes were greater than zero (Fig. psa, a few psb and the pet genes studied, except for petB, were equal to zero (Fig. psa, psb and pet genes was faster than in the ndh gene group (Additional file psa, psb and pet gene families caused the editing sites to disappear. Moreover, the C-to-U editing at the mRNA level and the reverse mutations at the genome level can both increase codon conservation. For example, petA-329, psaA-725 and psbF-77 were edited in G. biloba, but they underwent a reverse mutation to T at the DNA level in A. thaliana, T. aestivum and Z. mays, causing an increase in the corresponding codon conservation in most of the species (Additional file To investigate the evolutionary tendency of RNA editing, the dN-dS values of the RNA editing sites in four photosynthesis-related gene families were calculated using the Z-test of selection in MEGA5.1 Beta software. The dN-dS values of most ero Fig. , indicatero Fig. , suggestVolvox globator to more than 900 in A. formosae. Over 300 chloroplast editing sites were known in early branching land plants, such as Anthoceros and Adiantum, but fewer sites were edited in angiosperm chloroplast RNAs (Fig. G. biloba contain 255 editing sites, which is by far the highest number of editing sites in a seed plant. A model for the evolution of editing in plant organelles proposed that RNA editing was of monophyletic origin, had a common ancestor with many editing sites during seed plant evolution, and that many of the original editing sites, particularly in seed plants, had been subsequently lost [G. biloba is one of the oldest seed plants and appeared in the Early Jurassic period, in which the CO2 concentration in the atmosphere may have reached high levels, accelerating climate warming [G. biloba to acquire many mutations at the DNA level and RNA editing recovered the equivalent genetic information. In addition, comparisons of editing events among three gymnospermaes, G. biloba, Pinus and Cycas, showed that a large number of unique editing sites of G. biloba had been lost in Cycas and Pinus (Fig. G. biloba may maintain a more ancestral version of the chloroplast genome than Cycas and Pinus. Moreover, G. biloba shares 11 and 3 editing sites with Cycas and Pinus, respectively, and three common editing sites, atpF-370, petB-634 and psbE-214, are shared among the three species (Fig. Except for the marchantiid subclass of liverworts, RNA editing has been observed in the chloroplasts of all of the investigated terrestrial plants. The number of C-to-U RNA editing sites in chloroplasts was variable among plants, ranging from 0 in NAs Fig. . In thistly lost . G. bilo warming . All of nus Fig. . G. biloies Fig. . This inG. biloba before and after editing using bioinformatics methods. One editing site changed the signal peptide, eight editing sites could create five new transmembrane regions, and one RNA editing event occurred in petB, which caused an existing transmembrane region to disappear. All of the newly created signal peptides and transmembrane regions might play important roles in the localization or formation of the proper spatial structures of the proteins, especially for membrane proteins. Until now, a great deal of experimental evidence supported the view that most of the unedited proteins had lower functional levels than the edited proteins. In peas, unedited acetyl-coA carboxylase carboxyl transferase β is not functional and cannot catalyze the synthesis of fatty acids [rpl2, the AUG initiation codon generated by a C-U editing of ACG is essential to seed development [Arabidopsis, an editing defect at atp1-C1178 has a strong impact on the assembly of the ATP synthase [G. biloba, two types mainly cause the conversion of amino acids from serine to leucine or phenylalanine and proline to leucine. The former might increase the hydrophobicity of the corresponding peptide and the latter did not change the peptide hydrophobicity, but it could recover the normal curl of the secondary structure or remove misfolding because proline is a helix-breaker.RNA editing, especially at the second codon position, can alter the encoding amino acid and change the protein primary, secondary or tertiary structures, which might be necessary for the protein function. We analyzed the secondary structures of 82 transcripts in ty acids . In maizelopment . In Arabsynthase . Of the G. biloba has many partial editing sites. Among the 255 editing sites, 73 partial editing sites were detected. Tseng et al. found that partial editing may regulate plastid gene expression by using a different editing frequency in the non-photosynthetic tissues of Arabidopsis [ndhB has different editing profiles in photosynthetic and non-photosynthetic organs in Z. mays [G. biloba might be associated with different tissues and developmental periods. Further experiments are needed.In addition, the chloroplast genome of bidopsis . Karcher Z. mays . Thus, mMarchantia polymorpha of the marchantiid subclass of liverworts [ndh, psa, psb and pet genes in 12 plant species revealed that the dN-dS values of psa, most of psb and the pet gene groups were nearly equal to zero (Fig. psa, psb and pet genes was faster than in the ndh gene group (Additional file psaI affects the association of psaL with the photosystem I core. Namely, the absence of psaI indirectly leads to a defect in photosystem I function [A. thaliana photosystem I gene, psaE, and observed several defective phenotypes, including a significantly increased light sensitivity and a decreased growth rate of ~50% under normal conditions [PsbJ in tobacco causes the photosynthetic performance to be drastically reduced, as well as an extreme hypersensitivity to light [psbN-F and psbN-R of N. tabacum were extremely light sensitive and failed to recover from photo inhibition [psa, psb and pet, most of them have abundant editing sites in ancient species, but many editing sites disappeared during plant evolution due to reverse mutations at the DNA level that restored codons to conserved amino acid residues. Those editing sites were probably essential for the structure and/or function of the encoded protein.The RNA editing phenomenon may be a relic of the ancient RNA world that is involved in primordial error correction, such as repairing UV damage or other uncertain factors at the transcriptional level. As a result, RNA editing appears in almost all land plants, except verworts . With evero Fig. . Additiofunction . Varottonditions . Additioto light . Salar Thibition . Fiebig ndh genes encode a thylakoid Ndh complex that purportedly acts as an electron feeding valve to adjust the redox level of the cyclic photosynthetic electron transporters [ndh group of genes [ndh genes also possessed the most editing sites and had the highest editing frequency. The ndh gene groups might be unessential for plants growing under normal conditions. Burrows et al. hypothesized that the ndh complex was dispensable for N. tabacum growth under optimal growth conditions [Ndh genes are absent in epiphytic plants [Phalaenopsis, Aphrodite and Erodium [P. thunbergii, most of the ndh genes are pseudogenes. Thus, we speculated that the RNA editing sites of the ndh genes might be randomly lost and that the loss rate was slow. Therefore, ndh genes could keep more editing sites than other gene groups in modern plants. For the ndh gene group, we found that RNA editing in ndhD, ndhF and ndhG might create obvious structural changes, which created a new transmembrane region or caused an existing transmembrane region to disappear after editing (Fig. Arabidopsis, the editing deficiency in ndhF was associated with a delayed greening phenotype [ndhB and ndhD affected the flow of cyclic electrons and enhanced disease resistance [ndh genes were unnecessary under standard growth conditions, editing was probably most important for the proper function of the NDH protein complex under stress conditions [ndh genes might improve photosynthesis and stress tolerance under harmful conditions, and they may display positive selection during evolution. Some psb and pet genes, which have a dN–dS bias greater than zero, such as psbE, psbF, psbH, psbJ, psbL, psbT, petB and petL may have similar evolutionary mechanisms. Thus, RNA editing may be a post-transcriptional regulatory process of ancient genes, as well as part of an evolutionary model with diverse evolutionary directions [The plastid sporters . By far of genes . In our nditions . Ndh genc plants  and are  Erodium . In P. ting Fig. . To a cehenotype . The decsistance . Althougnditions , 59. Duerections . We specG. biloba chloroplast genome, which is the highest number of RNA editing sites found in a seed plant. Most of the RNA editing sites can restore amino acid conservation, increase hydrophobicity, and even influence protein structures. Using the dN–dS method to predict the evolutionary mode of RNA editing, we found that similar purifying selection constituted the dominant evolutionary force at the RNA editing sites of psa, some psb and the pet groups, and a positive selection occurred in the RNA editing sites of most ndh, and a few psb and pet gene groups.In summary, 255 RNA editing sites were identified in the


Adenosine to inosine (A-to-I) RNA editing has been shown to be an essential event that plays a significant role in neuronal function, as well as innate immunity, in mammals. It requires a structure that is largely double-stranded for catalysis but little is known about what determines editing efficiency and specificity in vivo. We have previously shown that some editing sites require adjacent long stem loop structures acting as editing inducer elements (EIEs) for efficient editing.The glutamate receptor subunit A2 is edited at the Q/R site in almost 100% of all transcripts. We show that efficient editing at the Q/R site requires an EIE in the downstream intron, separated by an internal loop. Also, other efficiently edited sites are flanked by conserved, highly structured EIEs and we propose that this is a general requisite for efficient editing, while sites with low levels of editing lack EIEs. This phenomenon is not limited to mRNA, as non-coding primary miRNAs also use EIEs to recruit ADAR to specific sites.We propose a model where two regions of dsRNA are required for efficient editing: first, an RNA stem that recruits ADAR and increases the local concentration of the enzyme, then a shorter, less stable duplex that is ideal for efficient and specific catalysis. This discovery changes the way we define and determine a substrate for A-to-I editing. This will be important in the discovery of novel editing sites, as well as explaining cases of altered editing in relation to disease.The online version of this article (doi:10.1186/s13059-017-1324-x) contains supplementary material, which is available to authorized users. Complex organisms require a great diversity of gene products for proper development and function, particularly in the brain. This is achieved by the use of numerous co- or post-transcriptional processes, such as alternative splicing, alternative polyadenylation, and RNA editing. Adenosine-to-inosine (A-to-I) RNA editing is a highly conserved RNA modification process that occurs in all metazoan lineages . InosineA-to-I RNA editing is performed by the adenosine deaminases that act on RNA (ADAR) enzymes that recognize adenosines located in double-stranded RNA (dsRNA) to be deaminated into inosines . ADAR pr2+ [In general, there are two categories of A-to-I RNA editing determined by the structure of the RNA. Long double-stranded structures, commonly found in introns and 3′ UTRs, are subjected to hyper-editing of many adenosines in what appears to be a random manner –21. Most2+ . This ed2+ . HoweverA receptor [Another highly edited site changes a codon for isoleucine to methionine (I/M) in the Gabra-3 transcript, coding for the α3 subunit of the GABAreceptor . We havereceptor . This inIn the present study, we show that the efficiency of editing at the GluA2 Q/R site is dependent on a conserved 45 base pair (bp) long intronic stem structure acting as an EIE. The EIE of GluA2 can also induce editing at other selectively edited sites and even increase the efficiency of editing at sites of low efficiency. We further show that EIEs are commonly present adjacent to efficiently edited sites, while they are absent in proximity of sites of low editing efficiency. Moreover, EIEs are not limited to sites within coding sequence; editing within non-coding RNA such as pri-miRNAs can also be induced by EIEs. We therefore suggest that the use of EIEs is a general mechanism used by the enzyme to increase both editing specificity and efficiency.A-to-I editing is exceptionally efficient at the Q/R site of the transcript coding for glutamate receptor subunit GluA2 . The Q/RThe Q/R site of GluA2 has been shown to be highly edited in the brain during early embryogenesis, while most other sites show low editing levels at this stage. We speculated that the high level of editing at the Q/R site of GluA2 in the embryo might be explained by this being a high affinity site for ADAR2, requiring a lower amount of the editing enzyme for full catalysis compared to other sites. We wanted to investigate if the EIE contributes to the highly efficient editing at the Q/R site by attracting ADAR2 to the transcript. If so, a lower concentration of the ADAR2 enzyme should be required for efficient editing in the presence of the EIE compared to its absence. A titration of the ADAR2 expression vector 0–1.25 μg) was transfected into HEK293 cells together with a constant concentration (0.75 μg) of the GAQ/R or GAQ/R-ΔEIE reporter. Indeed, only 0.1 μg of transfected ADAR2 expression vector was enough to reach 83% editing in a co-transfected GA2Q/R reporter, while only 49% editing, on average, was seen in the GAQ/R-ΔEIE reporter using the same amount of transfected ADAR vector  for editing specificity and selectivity. Mutations from KKxxK to EAxxA in the two dsRBDs of ADAR2 were made to impair their RNA interaction as previously described . To compThe finding that a dsRNA stem structure, separated from the specific editing site by a larger internal loop, can work as an editing inducer made us look for EIEs in the vicinity of other highly edited ADAR substrates. In the kainate receptor subunit GluK2, over 90% of the transcripts are edited at the Q/R site in several different brain regions . As in GWe hypothesized that the sites with low editing efficiency lack adjacent dsRNA structures that could function as EIEs, while the sites showing high editing efficiency are flanked by conserved double-stranded structures, functioning as ADAR recruitment elements. To investigate this hypothesis, we analyzed substrates with conserved site selective editing and looked for the presence of stable, conserved stem structures in the vicinity. In total, 23 substrates were analyzed with sites of editing ranging from 7–100%  [1.1 is edited in no more than 25% of the transcripts in the adult mouse brain [1.1 (Kv1.1 WT) , editing increased to 20%. A similar increase in editing efficiency could be observed when the EIE from the Gabra-3 transcript was placed downstream of the I/V stem loop, and an increase to about 30% editing when the Gabra-3 EIE was inserted both upstream and downstream of the Kv1.1 substrate (G3 EIE.Kv1.1-G3 EIE) . These results indicate two things: i) editing at the I/V site in Kv1.1 is low due to the limited capability of the specific substrate to attract the ADAR2 enzyme; and ii) editing of the Kv1.1 RNA can be induced by the addition of stem structures in cis, ideal for ADAR recruitment.The mammalian potassium channel transcript Kvin (I/V) . This trse brain . As mentWT) Fig. . Only 5%WT) Fig. . StrikinIE) Fig. . TransieIE) Fig. . Here thIn the substrates analyzed so far in this study, the specific editing sites are located within coding sequence of mRNAs, giving rise to amino acid changes in the translated proteins. To determine if editing can be induced by EIEs also in non-coding sequences we analyzed editing within the human miR-376 cluster. This cluster has been shown to be highly edited in the mature sequence of several pri-miRNAs . A miRNATo investigate if one or several stem loop structures in the vicinity of pri-miR-376a2 act as EIEs, we did consecutive deletions of the stems in the cluster Fig. . FurtherWe have previously shown that EIEs consisting of long stem loop structures can attract ADAR1 and ADAR2 to facilitate catalysis within adjacent shorter stem structures , 30. In By in vitro analysis it has previously been shown that dsRNA flanked by internal loops larger than 6 nt are identified as helix ends by the ADAR1 enzyme, while smaller loops of up to 4 nt act as part of the helix . LimitinLike other dsRNA binding enzymes such as PKR, Staufen, and Drosha, the ADAR enzymes are thought to interact promiscuously with dsRNA via their dsRNA binding domains, as they recognize the minor groove in the sugar-phosphate backbone of the RNA without any direct contact with the nucleotide bases , 42. HenCo-crystallization of the ADAR2 deaminase domain together with an RNA substrate revealed that the deamination domain also specifically interacts with the RNA helix structure, covering 20 bp . During In mouse and human, editing has been shown to be regulated within both non-coding RNA and gene transcripts during development , 45–47. Interestingly, we also show that EIEs can induce editing in non-coding RNA. We and others have shown that several miRNAs in one specific cluster (miR379-410) in mouse are subjected to A-to-I editing in their target recognition sequence , 49. MosAn increasing amount of transcriptomics data provide evidence of A-to-I RNA editing playing an important role in specific tissues in response to external stimuli or stress as well as in developmental regulation and immunity. In order to verify these editing events, we need to know the structural requirements for substrate recognition. Revealing the mechanism and components required for efficient editing will contribute to an increased understanding of the variations in levels of A-to-I RNA modification. By understanding how a substrate is selected for editing we will also be able to discover new sites of editing as well as understand the cause of aberrant editing related to cancer progression as well as immunological and neurological disorders. Double-stranded RNA plays a key role in many biological functions in cells, including RNA interference, anti-viral immunity, and mRNA transport. Responsible for recognizing dsRNA are a class of dsRNA binding proteins (dsRBPs), including ADAR. Our novel way of explaining substrate selectivity and efficiency may therefore also relate to other dsRBPs, such as Staufen 1 and Drosha, for which little is known about the molecular mechanism underlying substrate recognition.The ADAR2 expression vector has been previously described , 50. TheAll plasmids and mutants were verified by Sanger sequencing (Eurofins MWG operon).GluA2 reporter constructs GA2Q/R, GA2Q/R ΔEIE, GA2Q/R-US EIE, GA2Q/R-US G3 EIE, and GA2Q/R-Δloop and Kv1.1 reporter constructs Kv1.1, Kv1.1-Q/R EIE, Kv1.-G3 EIE, and G3 EIE-Kv1.1-G3 EIE (0.75 μg) were co-transfected with the ADAR2 (100 ng) expression vector into HEK293 cells and grown in 12-well plates. For endogenous editing, the GluA2, GluK2, and Kv1.1 reporter constructs (100 ng) were transfected into HeLa cells grown in 12-well plates. The Gabra3 reporter constructs G3I/M, G3I/M-ΔEIE, G3I/M-DS, and GA2 EIE (0.75 μg) were co-transfected with ADAR1 or ADAR2 (1.25 μg) expression vectors into HEK293 cells and grown in 12-well plates. For endogenous editing, the Gabra3 (2 μg) reporter constructs were transfected into HeLa cells grown in 12-well plates.In the ADAR2 titration experiments a fixed amount of 0.75 μg reporter constructs GA2Q/R or GA2Q/R ΔEIE was co-transfected with varying amounts of ADAR2 expression vector  into HEK293 cells and grown for 48 h in 12-well plates. For the ADAR mutant experiments a fixed amount of 0.75 μg reporter constructs GAQ/R and GA2Q/R-Δloop was co-transfected with 0.8 μg ADAR mutant expression vector into HEK293 cells and grown in 12-well plates for 48 h. The ADAR protein level was controlled by western blot analysis. For the miR-376a2 editing reporter constructs, 2 μg of the constructs were transfected into HeLa cells grown in 12-well plates. LIPOFECTAMINE™ 2000 (Invitrogen) was used in all transfections. The transfection efficiency was comparable between separate experiments. As controls, co-transfections with an empty expression vector instead of ADAR2 were done for each experiment. RNA was isolated 48 h (HEK293 and miRNA constructs in HeLa) and 72 h (HeLa) after transfection using GenElute™ mammalian total RNA isolation (Sigma), and treated with DNase-1 Amplification Grade (Sigma). cDNA was generated using random hexamer deoxyoligonucleotides and SuperscriptII RT (Invitrogen). Negative control reactions without reverse transcriptase were performed in all RT-PCR experiments to exclude genomic DNA contamination. The following PCR was made using Taq (Invitrogen). Primers used for the PCR reactions were as follows: for GA2Q/R and GA2Q/R ΔEIE/Δloop reporters, FW 5′-cctggtcagcagatttagcc-3′, RE 5′-tgctagagctcgctgatcag-3′; for GA2Q/R-US EIE, FW 5′-ttgatcatgtgtttccctggt-3′, RE 5′-aaacacggtacccctccaag-3′; for GA2Q/R-US G3 EIE, FW 5′-aggaactcagcagggctatg-3′, RE 5′-gagaatatgcagcaaaaacacg-3′; for G3I/M, G3I/M-ΔEIE, and G3I/M-DS GA2 EIE, FW 5′-ggtgtcaccactgttctcacc-3′, RE 5′-gctgtggatgtaataagactcc-3; for GK2Q/R and GK2Q/R ΔEIE, FW 5′-gatagaatcttctcactgctat-3′, RE 5′-caaattgagacaggaaacagg-3′; for Kv1.1, Kv1.1-Q/R EIE, Kv1.-G3 EIE, and G3 EIE-Kv1.1-G3 EIE, FW 5′-aactttgtgcattttaggtc-3′, RE 5′-aaccttctgcattttatagcc-3′; for miR-367a2, FW 5′-taatacgactcactataggg-3′, RE 5′-ctgatggtggcttcagtcc-3′.To evaluate the level of edited transcripts, RNA from at least three independent experiments was sequenced. Editing was determined by measuring the ratio between the A and the G peak height in individual chromatograms using FinchTV. The percentage of editing was calculated as the peak height of G/(A + G) × 100.RNA secondary structure predictions were made through Mfold  and VienAdditional file 1: Figure S1.Figure S2. Titration of ADAR2 co-transfected with GA2Q/R or GA2Q/R-ΔEIE. (a) Sequencing chromatograms of RT-PCR products from ADAR2 co-transfections with GA2Q/R or GA2Q/R-ΔEIE. In each experiment, transfection of the reporter constructs was constant (0.75μg), while the concentration of ADAR2 was titrated (0-1.25μg). (b) Quantification of the Q/R editing efficiency in GA2Q/R (dots) and GA2Q/R-ΔEIE (squares) reporters when co-transfected with titrated ADAR2. Three individual experiments were done for each concentration. The mean value of the ratio between the A and G peak heights was calculated as percentage of editing. Error bars are standard deviation. Figure S3. (a) Sites of editing and average % editing in the GluA2 reporter GA2Q/R cotransfected with the mutant ADAR2-E488Q expression vector in HEK293 cells. Below, sites of editing in the GluA2 reporter with the internal loop deleted (GA2Q/R-Δloop) co-transfected with ADAR2-E488Q in HEK293. The average value of the ratio between the A and G peak heights from two separate experiments was calculated as percentage editing. (b) Western blot showing expression levels of different transiently transfected ADAR2 expression vectors shown in A and Figure 3. EV equals transfection of empty vector as control. Figure S4. Predicted RNA secondary structure of the EIE in mouse GluA2 and GluK2. Differences in the human sequences are indicated by arrows and base changes in blue. Edited adenosines in the mouse sequence are shown in read. (PDF 3470 kb)Quantification of editing efficiency at the Q/R sit from the different GA2Q/R reporters cotransfected with ADAR2 in HEK 293 cells. The mean value of the ratio between the A and G peak heights from three individual experiments are calculated as percentage of editing. Error bars are standard deviation. The value of GA2Q/R-ΔEIE was significantly different to the values of all the other reporters, the values of the GA2Q/R-US EIE and GAQ/R-US G3 EIE where not significantly different to the WT GA2Q/R reporter (P=0.05 two tailed student’s ttest). Additional file 2: Figure S5.Secondary structure predictions of the pre-mRNA sequence in the vicinity of the selectively edited sites listed in Table


The mystery of the origin of life can be divided into two parts. The first part is the origin of biomolecules: under what physicochemical conditions did biomolecules such as amino acids, nucleotides, and their polymers arise? The second part of the mystery is the origin of life-specific functions such as the replication of genetic information, the reproduction of cellular structures, metabolism, and evolution. These functions require the coordination of many different kinds of biological molecules. A direct strategy to approach the second part of the mystery is the constructive approach, in which life-specific functions are recreated in a test tube from specific biological molecules. Using this approach, we are able to employ design principles to reproduce life-specific functions, and the knowledge gained through the reproduction process provides clues as to their origins. In this mini-review, we introduce recent insights gained using this approach, and propose important future directions for advancing our understanding of the origins of life. There must have been many hurdles to overcome before complex living things arose on the early earth. The first hurdle was the birth of organic compounds. If the first life appeared in the hypothetical RNA world , the comHow can a set of molecules achieve self-replication, and how can such molecules then acquire the ability to evolve? A direct strategy for finding the answers to these questions is to construct an evolvable, self-replicating system in a test tube. This type of strategy is called the “constructive approach” ,11, in wThe constructive approach can be defined as an approach that attempts the construction of a target biological function, using defined molecules, in order to understand the conditions required to achieve that function. The key point in this approach is that it uses a defined set of molecules, rather than a complex mixture such as a crude cell extract. This is because complex mixtures make it impossible to identify important components and interactions required for the target function. The molecules used are not limited to those that could have existed on the ancient earth, and any nucleotide (DNA and RNA) or amino acid polymers can be used. However, the fact that a set of molecules is experimentally sufficient to achieve a target biological function does not simply imply that the same set of molecules was actually involved in achieving that biological function during the origin of life on earth. Therefore, this approach only proposes plausible physicochemical routes toward the origin of life.Various types of self-replicating systems have been constructed from defined molecules. The oldest example is a self-replicating RNA system developed by Spiegelman’s group in 1967 . They coSeveral types of protein-free self-assembling systems have been constructed. Joyce’s group has constructed a self-replicating RNA system by using a self-recombinase ribozyme and its fragments . Lehman’ReplicationInheritable variationSelectionDo the self-replicating systems mentioned above have the ability to evolve? For a molecular system to evolve, it must satisfy three requirements :ReplicaAmong the self-replicating systems mentioned, only Spiegelman’s RNA system, the SDA system, the 3SR system, and the TcRR system satisfied all three requirements. In these systems, inheritable variation was produced through the misincorporation of nucleotides into the RNA or DNA during polymerization. When replication in these systems was continued for many generations, mutant RNAs or DNAs that replicated faster came to dominate the population. This was the result of selection within these systems favoring faster replicating molecules. In contrast, there was no inheritable variation within the other self-replicating systems. This was because in protein-free self-ligating ribozyme systems, peptide systems, and vesicle systems, the molecules were not synthesized through polymerization. In this section, we describe in detail the evolution of the DNA and RNA within the four nucleotide-based systems. It is notable that in the field of directed evolution, there have been many studies using artificial selection methods, such as the systematic evolution of ligands by exponential enrichment (SELEX), to drive evolution in vitro ,38,39,40Spiegelman’s system contained the template RNA and the purified RNA replicase from bacteriophage Qβ. During this replication process, mutations and deletions were introduced to the RNA through replication errors, and non-homologous recombination, respectively . SpiegelEthidium bromide resistance was another example of evolution demonstrated using Spiegelman’s RNA system. The transfer experiment was repeated in the presence of a small amount of ethidium bromide, which partly inhibits RNA replication. They found that after 25 rounds of transfer, the template RNA had acquired three-point mutations, and was more resistant to ethidium bromide than the original RNA . These t4-fold higher self-ligation activity [In the 3SR system, the template RNA replicated via a DNA stage by using a reverse transcriptase and an RNA polymerase. During this process, mutations were incorporated through polymerization errors, and they were then inherited by all subsequent templates. Therefore, if a mutant RNA with higher replication ability appeared, it should have dominated the population after many rounds of replication. Wright and Joyce performed 300 rounds of serial transfer experiments using the ribozyme-based 3SR system B. The reactivity . In anotactivity . RNA-Z dThe template DNA in the SDA system C can alsIn the evolution of Spiegelman’s self-replicating RNA, the template RNA became smaller and lost all its originally encoded genes see . This waCompartmentalization of the reaction solution solved this problem. If the reaction was compartmentalized so as to have one RNA molecule per compartment, each template RNA could only use the replicase translated from itself B. Under We encapsulated the TcRR system into a water-in-oil emulsion with droplet sizes of approximately two micrometers in diameter, and performed the transfer experiment for 128 rounds . In contOther than providing a phenotype to genotype linkage, compartmentalization has another role in the TcRR system; it provides protection against selfish or parasitic replicators. We found that a small RNA often appeared spontaneously in the TcRR system during the uncompartmentalized reaction. The small RNA was generated from the template RNA, probably through recombination ,47. The One advantage of in vitro self-replicating systems is their simplicity . This enables detailed analysis of the evolutionary process at a level impossible when using complex organisms. One example is the drawing of a fitness landscape; the simplicity of the in vitro self-replication systems enables the depiction of their fitness landscapes at higher resolution. A fitness landscape is a visual representation of the relationship between genotype and fitness, and it is an important tool for understanding evolution in a system . In thesz-axis in We sequenced the RNA of more than 200 clones at 11 points in the first 32 rounds of a TcRR transfer experiment using a next-generation sequencing technology . We obseIn the previous sections, we have introduced several types of in vitro self-replication systems and described evolution within them. What have we learned from these experiments? First, we have learned that the ability to evolve is not a characteristic unique to living things. Transfer experiments using several systems have demonstrated that a set of molecules will autonomously evolve as long as it satisfies three requirements . The relatively easy reproduction of evolution in vitro is consistent with the possibility of an evolvable system having spontaneously formed on the ancient earth.The second lesson is that the evolutionary pattern differs among the self-replication systems. In the cases of Spiegelman’s RNA system and the SDA system, the sizes of the templates gradually decreased during evolution. In the cases of the ribozyme-based 3SR system and the TcRR system, the original template sizes were maintained, and the point mutations became fixed within the population. This difference is explained by the roles of the template RNA or DNA. The polynucleotides in Spiegelman’s system and the SDA system are only replication templates, and thus reducing their size is beneficial because smaller templates can be replicated faster. In contrast, to replicate themselves the templates of the ribozyme-based 3SR system and the TcRR system must encode an active self-ligating ribozyme or an active replicase, respectively. Therefore, large deletions that destroy the encoded ribozyme or gene do not occur, but point mutations are accumulated. 3 or 106 molecules of the protein are required to reach the same concentration in 10- or 100-µm-diameter compartments, respectively. If the affinity of an ancient protein for its ligand were lower, the required number of protein molecules would increase. Because ancient translation machinery was likely inefficient, a smaller size  would have been better for a primitive cell. Several cell-like compartments are reported to have possibly existed on the ancient earth, such as fatty acid vesicles [The third lesson is the importance of compartments for the evolution of multi-component self-replicating systems such as the TcRR system. This requirement suggests that a primitive life form composed of multiple molecules must have had a cellular structure to acquire the ability to evolve, and it implies that cellular structures may date back to the early origins of life. The size of a cellular structure is also important. The sizes of the first cellular structures would have depended on the concentrations and affinities of the internal molecules. For example, in the case of an extant protein with a high affinity for polynucleotides (Kd ~1 nM), one molecule is enough for the concentration to become more than the Kd value in a 1-µm-diameter compartment, while 10vesicles  and ice vesicles  (reviewevesicles ). FutureIn this mini-review, we introduced several examples of the constructive approach, focusing on self-replication and the ability to evolve. What is the next evolutionary hurdle for these self-replicating systems to overcome? One possible hurdle is the evolution of complexity. In the case of the TcRR system, only one replicase gene is encoded on the template RNA. Future template RNAs must encode more genes and acquire more complex replication systems to simulate the next stages of the early origin of life. In the case of the TcRR system, the largest problem is the severe structural restriction on the template RNA. Biochemical analysis reveals that a template RNA that is continuously replicable with the bacteriophage Qβ RNA replicase must have a strong secondary structure throughout . OtherwiEscherichia coli [Another direction is the introduction of the translation system into DNA-based self-replicating systems, such as SDA, in which the template double-stranded DNA is replicable without structural requirements. Some translation-coupled self-replicating DNA systems have been reported. These include a system based on genome replication in hia coli  and a syhia coli , althougThe development of a protein-free self-replication ribozyme is another important challenge. Although complete self-replication has not been achieved, recent advances in the past few decades have allowed for superior RNA polymerization activity of ribozymes ,24. In tSeveral other aspects of cellular function have been reproduced in vitro, although the efficiencies achieved have not yet been comparable to those in a living cell. These aspects include the growth and division of fatty acid vesicles or more complex liposomes ,63,64,65