Direct acting antivirals (DAAs) provide efficient hepatitis C virus (HCV) therapy and clearance for a majority of patients, but are not available or effective for all patients. They risk developing HCV-induced hepatocellular carcinoma (HCC), for which the mechanism remains obscure and therapy is missing. Annexin A2 (AnxA2) has been reported to co-precipitate with the non-structural (NS) HCV proteins NS5B and NS3/NS4A, indicating a role in HCC tumorigenesis and effect on DAA therapy.Surface plasmon resonance biosensor technology was used to characterize direct interactions between AnxA2 and HCV NS5B, NS3/NS4 and RNA, and the subsequent effects on catalysis and inhibition.KD = 30 nM), complex with NS5B, decreasing its catalytic activity and affinity for the allosteric inhibitor filibuvir. The RNA binding of the two proteins was independent and AnxA2 and NS5B interacted with different RNAs in ternary complexes of AnxA2:NS5B:RNA, indicating specific preferences.No direct interaction between AnxA2 and NS3/NS4A was detected, while AnxA2 formed a slowly dissociating, high affinity  contains supplementary material, which is available to authorized users. Although direct acting antivirals (DAAs) have improved the therapeutic options for hepatitis C virus (HCV) infections radically, there is still a need for continued research on disease mechanisms and new therapeutic strategies, as many patients do not have access to DAAs or are infected with genotypes insensitive to current drugs .The formation of the HCV replication complex is known to require various viral non-structural (NS) proteins as well as host cellular proteins. In addition, the HCV replication machinery is associated with a specific membrane alteration, “the membranous web”, derived from endoplasmic reticulum (ER) membranes. The NS proteins involved in replication  are anchored to the membrane via several integral or peripheral membrane binding domains . As our AnxA2 has been isolated from the HCV replication complex and shown to be recruited by NS5A to the HCV replication site . More spAlthough AnxA2 has been demonstrated to co-precipitate with the membrane-anchored NS5B and NS3/NS4A proteins .This study reveals that AnxA2 binds directly to NS5B and reduces its polymerase activity, thus providing a better understanding of how AnxA2 may be involved in the HCV life cycle and with implications for the design of novel allosteric inhibitors of NS5B.The ectodomain of NS5B NS5BΔ21) from genotype 1b was produced as earlier described, with the details of collection of blood samples, viral RNA extraction, cDNA formation, and cloning of NS5B 1b DNA into an expression vector published in  and meth from genBovine AnxA2 and an engineered variant containing mutations in helix C and in the CD loop  (mAnxA2) were expressed and purified as previously described .The 31-mer RNA (5’CGAUACUCCCUUUAUAUAACCAUCAAUCGCC 3′) used in SPR polymerase assay , was syn5′ ATTCGTTAATACGACTCACTATAGGG 3′ and. 5′ GGCGATTGATGGTTATATAAAGGGAGTATCGCCCTATAGTGAGTCGTATTA 3′.were used as a template for the 31 bp RNA synthesis. The primers were annealed by heating to 100 °C for 1 min in 50 mM Tris-HCl pH 7.4 and 100 mM KCl buffer. The RNA was synthesized using Riboprobe T7 kit  according to the manufacturer’s instructions. The synthesized RNA was purified with standard phenol/chlorophorm extraction procedure. Unincorporated nucleotides were removed using PD SpinTrap G-25 spin columns .The 8-mer RNA (5’GGG GAU UG-3′) used in interaction studies with AnxA2 and NS5B was purchased from Eurofins Genomics.v/v Surfactant P20) in the presence of 70 μM Ca2+ and 1 mM Mg2+, using a flow rate of 30 μl/min. AnxA2 or NS5B (genotype 1b BK) were immobilized on a CM5 chip by standard amine coupling using 5 mM maleate buffer pH 6 and NaAc buffer pH 6, respectively, as immobilization buffer. Multi cycle experiments with regeneration after each injection (with 2 M NaCl and 2 M MgCl2), or single cycle kinetic experiments, without regeneration between the injections, were performed with four or five concentrations of the analyte in HBS-EP running buffer. In experiments investigating the interplay between AnxA2, NS5B and RNA, AnxA2 was immobilized as above, and either 8-mer RNA or NS5B was injected to form a stable binary complex, followed by injection of the third interaction partner.SPR measurements were performed at 25 °C using a Biacore S51 or T200 instrument and the data was analyzed using Biacore T200 evaluation software version 1.0 . Experiments were performed using HBS-EP running buffer (0.01 M 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) pH 7.4, 0.15 M NaCl, 3 mM ethylene diamine tetra acetic acid (EDTA), 0.005% The sensorgrams were corrected for buffer bulk effects and unspecific binding of the samples to the chip matrix by blank and reference surface subtraction (subtraction of inactivated and deactivated flow cell channel or where NS5B was immobilized and surface inactivated by 3 × 30 s injections of 6 M guanidine-HCl).ka), dissociation rates (kd), dissociation constants (KD) and maximum binding responses (Rmax) were estimated by global non-linear regression analysis and a reversible 1-step interaction reaching steady-state at the end of the analyte injection or by fitting the sensorgram to a reversible 1-step interaction model. The analysis was based on report points taken at the end of analyte injections even if steady state had not been reached. In cases where the interaction mechanism was not established, the analysis is purely qualitative and any estimated KD-values are termed “apparent” (KDapp), only useful for discussions on the possible order of magnitude of affinities.The association rates (v/v Surfactant P20) in the presence of 70 μM Ca2+ and 1 mM Mg2+. The data was analyzed using Biacore T200 evaluation software version 1.0 . The NS3 based enzyme activity assay was performed according to the protocol described previously [A continuous de novo HCV NS5B polymerase assay, using surface plasmon resonance biosensor technology , was developed, as illustrated in Fig. eviously .2) was found to be suitable.To investigate a possible direct interaction between AnxA2 and NS5B polymerase, AnxA2 was immobilized on an SPR sensor chip and NS5B was run as analyte Fig. . The expTo confirm the interaction, the reverse experiment was performed, i.e. with NS5B immobilized on the chip and AnxA2 used as analyte. Moreover, to avoid artefacts potentially induced by a regeneration solution, single cycle kinetic (SCK) experiments were performed. This experimental approach confirmed the binding of AnxA2 to NS5B Fig. . To elimwild type (wt) AnxA2 and dissociated more rapidly   Table  are thusDapp for the interaction between AnxA2 and NS5B, based on two independent multicycle experiment where AnxA2 was immobilized and NS5B was run as analyte, was 30 nM. In the reverse experiment, the concentration of AnxA2 was too low relative to the KDapp for the affinity to be estimated.The mean KTo investigate if AnxA2 also interacts directly with the HCV NS3 protease, NS3 or the NS3/NS4A complex were run as analytes over sensor surfaces with immobilized AnxA2. However, no interactions were detected with either the NS3 protein at concentrations up to 270 nM or with the NS3/NS4A complex up to 500 nM  was first injected over AnxA2, providing a reference point for RNA-binding to AnxA2 Fig. . After rTo confirm this interpretation, the opposite experiment was performed, i.e. NS5B (50 nM) was injected over AnxA2 alone to get a reference value. Eight-mer RNA (100 nM) was then injected over the regenerated AnxA2 surface, creating a stable AnxA2-RNA complex Fig. . NS5B waFinally, injection of the 8-mer RNA and NS5B concomitantly (in ratio 1:1 or 2:1) over AnxA2 was compared to the response observed when injecting NS5B and the 8-mer RNA separately (data not shown). The response seen for the NS5B-RNA complex was identical to that resulting from the injection of NS5B alone, suggesting that the binding of the NS5B complex to the 8-mer RNA is disrupted upon NS5B binding to AnxA2.In the present study we used a sensitive, time-resolved, biophysical real-time method to investigate the proposed interactions between AnxA2 and HCV polymerase NS5B and serine protease NS3 in more detail. We could not confirm any direct interactions between AnxA2 and NS3 or the NS3/NS4A complex, or an influence of the presence of AnxA2 upon NS3 protease activity (data not shown). This may indicate that the previously observed link between AnxA2 and NS3/NS4A [D value in the nanomolar range, and with very slow dissociation kinetics, possibly indicating conformational changes. A practical consequence of the high stability of the complexes was that the sensor surfaces had to be regenerated using a relatively harsh procedure. Thus, visual inspection of the sensorgrams for the interaction between AnxA2 and NS5B, and data showing the effect of allosteric inhibitors on the interactions, suggest that it involves a conformational change in one or both of the two proteins, induced upon binding. Additional complexities prevented the identification of a suitable mechanistic model for global regression analysis of the sensorgrams, precluding an in depth mechanistic and quantitative kinetic analysis. Notably, the interaction between AnxA2 and NS5B interfered with the ability of NS5B to interact with the allosteric inhibitor filibuvir significantly. It also impaired the polymerase activity of NS5B seen by a significantly drop of the nucleotide incorporation rate.Specifically, we demonstrated that AnxA2 and NS5B interact directly with strong affinity Fig. , E1, havIn the literature, AnxA2 is described as an RNA binding protein , 26, but. However, the advantage of the current approach is that we can isolate certain interactions and study them in detail, without confounding factors. It is possible that the interactions and the characteristics we observe in vitro are modulated in an in vivo system. For example, viral replication may require that AnxA2, or the complex, also interacts with specific lipids to ensure that this process occurs at the correct site. To obtain a better understanding of the biological relevance and specificity of RNA binding to NS5B and AnxA2 in vivo, it would be of interest to base future studies on designed RNA constructs derived from the HCV RNA genome. Furthermore, human AnxA2 should ideally be used instead of bovine AnxA2, although the protein is highly conserved and the mammalian species only differ by a few amino acids [The presented analysis of the interactions and interplay between AnxA2, NS5B and RNA should bear in mind that the experiments were performed in vitro using a biosensor and isolated proteins in a biological buffer system. Although we have attempted to simulate physiological conditions, it is clearly a simpler system than the corresponding system in vivono acids .wt AnxA2. However, the non-ideal shape of the sensorgrams indicated that the interaction was not stable, potentially due to a low stability of the mAnxA2 surface, which can be explained by structural perturbations introduced by mutating AnxA2, indicated by a lower apparent transition temperature for mAnxA2 (~48 °C) than the native form (~55 °C) [wt AnxA2.The studies investigating the interplay between AnxA2, NS5B and RNA showed that binding of AnxA2 to NS5B did not prevent AnxA2 from binding to the 8-mer RNA simultaneously Fig. , E3-E4. (~55 °C) . However(~55 °C) . Due to NS5B, like AnxA2, interacts with RNA . Howevermyc mRNA to the perinuclear region [Previous studies of the role(s) of AnxA2 in the life cycle of HCV have suggested that AnxA2 may play an important role in several processes, ranging from replication complex formation to virus particle assembly , 5. It wr region . Howeverr region . In thisTaken together, it appears that the binding of AnxA2 to NS5B reduces the inherent and important structural flexibility of NS5B and locks the protein in a conformation, with impaired ability to interact with both substrates and inhibitors. This is interesting from a HCV drug discovery perspective as novel drugs may be designed with a similar mode of action, potentially targeting the AnxA2-NS5B interaction interface and thus preventing NS5B polymerase activity. To be able to understand how to potentially modulate, stabilize or mimic the interaction between AnxA2 and NS5B to inhibit NS5B polymerase activity by a small molecular drug, it is of relevance to further elucidate the structural details of this interaction both with respect to structural conformation of the proteins involved and information of the exact protein regions important for binding.The stable complex formation between AnxA2 and HCV NS5B, in this study shown to influence the catalytic activity of NS5B and its sensitivity to allosteric inhibitors, may indeed also disturb normal cellular functions of AnxA2, for example its role in mRNA transport and translation in the host cell . InteracThe biophysical approach taken in this study was suitable for investigating direct interactions between two non-structural proteins of HCV (NS5B and NS3), AnxA2, RNA and filibuvir. It was possible to elucidate the complex interplay between these molecular species and confirmed interactions between AnxA2 and NS5B, but not with NS3. The data suggest that AnxA2 may have an important role in HCV infections and the development of HCC. This is relevant also for design of new allosteric inhibitors targeting NS5B.


Acanthaster planci from 2006 to 2010, and Cyclone Oli in 2010, yet by 2015 some coral populations were approaching pre-disturbance sizes. Using long-term study plots, we quantified population dynamics of spawning Pocillopora spp. along the north shore of Moorea between 2010 and 2014, and considered evidence that population recovery could be supported by self-seeding. Results scaled up from study plots and settlement tiles suggest that the number of Pocillopora spp. colonies on the outer reef increased 1,890-fold between 2010 and 2014/2015, and in the back reef, 8-fold between 2010 and 2014/2015. Assuming that spawning Pocillopora spp. in Moorea release similar numbers of eggs as con-generics in Hawaii, and fertilization success is similar to other spawning corals, the capacity of Pocillopora spp. to produce larvae was estimated. These estimates suggest that Pocillopora spp. in Moorea produced a large excess of larvae in 2010 and 2014 relative to the number required to produce the recruits found in the back reef and outer reef in 2010 and 2014, even assuming that ∼99.9% of the larvae do not recruit in Moorea. Less than a third of the recruits in one year would have to survive to produce the juvenile Pocillopora spp. found in the back and outer reefs in 2010 and 2014/2015. Our first order approximations reveal the potential for Pocillopora spp. on the north shore of Moorea to produce enough larvae to support local recruitment and population recovery following a catastrophic disturbance.Coral reefs in Moorea, French Polynesia, suffered catastrophic coral mortality through predation by  Dramatic reductions in population size of tropical scleractinians , and pooAmong the data required to project changes in coral population size are estimates of reproductive features including fecundity, fertilization success, and the likelihood that larvae will form benthic recruits. Among scleractinians, a diverse array of sexual and asexual modes of reproduction support recruitment , but folGenetic techniques are effective in quantifying the spatio-temporal scales over which coral populations are connected , but theAcanthaster planci , together with surveys conducted in January 2015, to inform first order approximations of the potential of larvae from spawning Pocillopora spp. in Moorea to support local population recovery. Wherever possible, we use empirical data in our calculations, but rely on reasonable assumptions where suitable data are absent. Below we separate our presentation based on what we have measured versus what we have assumed.Moorea, French Polynesia, lends itself to the use of first order approximations focused on coral population growth, because reefs in this location have been extensively studied e.g., , and recr planci  and Cyclthin 5 y , and theack reef , and seaack reef . Exampleack reef , and froack reef . In the 2 to a depth of 17 m , and a back reef with a hard-bottom area of ∼4.58 km2, as determined from Google Earth (accessed in March 2015). To evaluate the potential for Pocillopora spp. populations along this shore to increase through self-seeding, we compared the growth of populations between 2010 and 2014 with their inferred capacity to produce larvae and support local recruitment. Most empirical data came from two sites on the outer and back reefs (http://mcr.lternet.edu), and analyses of coral cover from 2005–2014 were used to contextualize a contrast of Pocillopora spp. populations between surveys conducted in April/May 2010 and mostly in April/May 2014. These sites are representative of the north shore . P. verrucosa, P. meandrina, P. effusus, P. woodjonesi, P. eydouxi, P. damicornis, and two unknown haplotypes are found in the region  placed randomly along a 40-m transect at 10-m and 17-m depth at two sites. In the back reef, community structure was quantified annually  along four, 5 m axes centered on five bommies site−1 (n ∼ 100 photoquadrats site−1 y−1). In both habitats, sampling occurred at LTER 1 and 2, with additional sampling at three sites in the back reef that were close to LTER 1 and 2. The LTER sites were established in 2005 to sample the north shore with annual frequency, and three additional sites are sampled every 3–4 y along the outer reef of the north shore to explore the generality of the results obtained from LTER 1 and 2. Outer reef photoquadrats were analyzed for benthic cover using CPCe software .On the outer reef, community structure was quantified annually in April/May from 2005–2014 using photoquadrats  placed horizontally at both sites on the outer reef (10 m and 17 m depth), and at three sites in the back reef . Tiles were replaced every 5–7 months in January/February and August/September, and upon recovery, were bleached, dried, and scored for scleractinian recruits. Recruits were identified to family level based on corallite structures, and here the density of pocilloporids is reported. The number of pocilloporid recruits on each tile was standardized to the area of the lower surface (225 cm2) where >90% of recruits were found, and the mean recruitment at each sampling within each year summed to estimate annual recruitment.Pocillopora spp., the abundances of juvenile Pocillopora spp. (colonies ≤ 4 cm diameter) and the abundance of all Pocillopora spp. colonies were measured. On the outer reef, juvenile colonies were quantified at 10-m depth during April/May using quadrats (0.5 × 0.5 m) placed at random positions along the same transects used for the photoquadrat sampling. The time-consuming nature of these surveys prevented repeating them at the deeper site (17 m) where the required bottom time on scuba was restricted by safe diving practices. In the back reef, juvenile Pocillopora spp. in 2010 were counted in the photoquadrats recorded at LTER 1 and 2, and ∼5 y later (January 2015) the density of juvenile colonies was quantified using quadrats (0.5 × 0.5 m) positioned randomly at three back reef sites near LTER 1 and 2. Finally, the density of Pocillopora spp. colonies (regardless of size) was evaluated in 2010 , 2014 , and January 2015 (back reef). Analyses of Pocillopora spp. populations in the back reef were conducted in January 2015 (rather than using photographs recorded in April 2014), because in situ counts provide better resolution of small corals than photographs, and we reasoned this was an acceptable trade-off against the ∼10 month discrepancy in sampling date relative to analyses of the outer reef community (in April/May 2014). For the outer reef in 2010 and 2014, and the back reef in 2010, colony densities (no. 1/4 m−2) were evaluated using the photoquadrats described above. For the back reef in January 2015, Pocillopora spp. densities were evaluated using quadrats (0.5 × 0.5 m) randomly placed at the same three sites where densities of juvenile Pocillopora spp. were measured.To gain further insight into the demographic processes mediating population growth of spawning Pocillopora spp. on the north shore of Moorea was estimated. Several reasonable assumptions were necessary to achieve these objectives. First, the abundance of Pocillopora spp. recruits along the shore was estimated from the recruits on settlement tiles, which assumes that the density of recruits on tiles and on natural reef surfaces is similar. While this is unlikely to be true (Pocillopora spp. recruits on tiles at each site (LTER 1 and 2) and depth was averaged between sites and depths. The densities at each sampling within a year (January/February and August/September) then were summed to estimate annual recruitment . This value was scaled proportionately to the area of hard bottom on the outer reef (∼3.15 km2), and calculations were completed for 2010 and 2014. For the back reef, the mean density of Pocillopora spp. recruits on tiles at each site (LTER 1 and 2) was averaged between sites, and the densities at each sampling within a year (January/February and August/September) summed to estimate annual recruitment . This value was scaled proportionately to the area of hard bottom in the back reef (∼4.58 km2), and calculations were completed for 2010 and 2014.Using the empirical data described above, the population size, fecundity, and larval production of  be true , settlem be true . Moreove be true , includiPocillopora spp. on the north shore was estimated from the number of colonies ≤ 4 cm diameter in quadrats. For the outer reef, the mean number of juvenile colonies (number 1/4 m−2) scored in situ during April/May at 10 m depth at LTER 1 and 2 was averaged between sites, and scaled proportionately to the area of hard bottom in the outer reef to 17-m depth. This approach assumes that the density of juvenile colonies is similar among depths, which is reasonable given the similarity of Pocillopora spp. recruitment at 10-m and 17-m depth . The abundance of juvenile corals was calculated as described above for 2010 and 2014. For the back reef, in 2010 the mean number of juvenile colonies at LTER 1 and 2 was determined in April/May using photoquadrats, and the mean values by site were averaged (number 1/4 m−2), and scaled proportionately to the area of the hard bottom in the back reef. To estimate densities of juvenile corals in the back reef in April/May 2014 , in situ surveys were conducted during the next field expedition in January 2015. In situ surveys conducted ∼10 months after the outer reef sampling were used for this purpose, as they provide better resolution of small corals than photographs, and because anecdotal field observations suggested the abundance of juvenile corals broadly were similar in April/May 2014 and in January 2015. Densities of juvenile colonies were averaged by site (number 1/4 m−2) and scaled proportionately to the area of the hard bottom in the back reef.Second, the abundance of juvenile Pocillopora spp. (regardless of size) was estimated as the number of colonies along the north shore. For the outer reef, the mean number of colonies (number 1/4  m−2) at LTER 1 and 2 was averaged between sites and depths, and scaled proportionately to the area of hard bottom in the outer reef to 17-m depth. This calculation was completed separately for 2010 and 2014. For the back reef, the mean number of colonies at each site was averaged within each year (number 1/4 m−2), and scaled proportionately to the area of the hard bottom in the back reef.Third, the population size of Pocillopora spp. were difficult to generate, as none of the critical pieces of information necessary for this purpose were available for spawning members of this genus in Moorea. The estimation process focused on spawning Pocillopora spp., which dominate in Moorea, and was accomplished by estimating the number of sexually mature colonies, fecundity (eggs polyp−1), and fertilization success. The size of sexual maturity of Pocillopora spp. in Moorea is known only for the uncommon brooder P. damicornis (Combosch & Vollmer 2013) and, therefore, this size for spawning taxa was taken as 14-cm diameter, which is the size of sexual maturity in Hawaii for the congeneric spawner P. meandrina  between tissue area  and colony diameter  for P. verrucosa collected from 10 m depth in Moorea in April 2014. This calibration was prepared for a separate study, and used wax-dipping  of spawning Pocillopora spp. was estimated from three studies reporting 7,300 eggs cm−2 y−1 ). The mean fecundity from these studies ) was converted to area-normalized fecundity assuming Pocillopora spp. have 66 polyps cm−2 , 63 egg Red Sea ) and 114yps cm−2 , and thePocillopora spp. in Moorea, therefore we assumed that eggs were fertilized at a rate similar to that reported in two studies of spawning corals—n = 3 in Fig. 6 for Montipora digitata) and n = 12 in Fig. 9 for Orbicella spp.)—which together indicate a mean fertilization success of 15 ± 21% (±SD).Nothing is known about the fertilization success of spawning n = 20,000) was applied to the calculations, assuming fecundity and fertilization were normally distributed with SD values as reported above. Monte Carlo sampling was conducted using Caladis software   . The valdis.org)  in R sofdis.org) , and norPocillopora spp. (15% cover). At 17-m depth, coral cover was higher (47% cover), and 28% was Pocillopora spp. (13% cover). Five years later, predation by Acanthaster planci and damage by Cyclone Oli had reduced mean coral cover to 2% at 10-m depth, and 1% at 17-m depth, and Pocillopora spp. virtually was absent (cover < 0.2% at both depths). The recovery of coral community structure was well underway 4 y later (2014), when mean coral cover had risen to 27% at 10-m depth, and 8% at 17-m depth, the majority of which (>53%) was Pocillopora spp. at 10-m depth (18% cover) and 17-m depth (4% cover). A different pattern was observed in the back reef, where mean coral cover declined from 20% in 2010 to 10% in 2014, and Pocillopora spp. was a minor component of this cover. In 2010, mean Pocillopora spp. cover was only 0.2% in 2010 and 0.1% in 2014 . Together, summed pocilloporid densities in January and September 2010 were 3.58 recruits 225 cm−2 at 10-m depth, 2.99 recruits 225 cm−2 at 17-m depth, and 3.29 recruits 225 cm−2 averaged across depths. Assuming these densities apply to the outer reef along the north shore, recruitment in 2010 brought 4.27 × 108 recruits to the outer reef. Likewise, pocilloporid recruitment at the three back reef sites in 2010 was 0.27 recruits 225 cm−2, 0.80 recruits 225 cm−2 and 0.07 recruits 225 cm−2, so that overall recruitment (averaged among sites) was 0.38 recruits 225 cm−2. Assuming this density applies to all hard surfaces, pocilloporid recruitment in 2010 brought 7.74 × 107 recruits to the back reef. Pocilloporid recruitment in the back reef and outer reef in 2010, therefore, brought 5.05 ×108 recruits to the whole reef . Four years later (2014), densities of recruits on tiles on the outer reef were depressed 71% compared to 2010, with 0.88 recruits 225 cm−2 , but densities on tiles in the back reef were elevated 23% compared to 2010, with 0.47 recruits 225 cm−2 . Scaling these densities for pocilloporids to the north shore as described above for 2010, indicates that 2014 brought 9.51 ×107 recruits to the back reef, 1.23 × 108 recruits to the outer reef .Following Cyclone Oli in February 2010, recruitment of pocilloporids to tiles on the outer reef was evaluated in September 2010, when overall recruitment (averaged between sites) was 3.37 recruits 225 cm-m depth –2E. Pociter reef , and 2.1Pocillopora spp. at 10-m depth on the outer reef were censused in April 2010, densities were low  densities of juvenile Pocillopora spp. were higher, with a 19-fold increase in the outer reef to 4.75 colonies 0.25 m−2 (averaged between sites), and by January 2015, there was a 24-fold increase in the back reef to 0.60 colonies 0.25 m−2 (averaged among sites). These values suggest that in April 2014 there were 5.98 ×107juvenile Pocillopora spp. on the outer reef, and in January 2015 there were 1.10 ×107juvenile Pocillopora spp. in the back reef  greatly varied between 2010 and 2014 (Pocillopora spp. colonies was 0.01 colonies 0.25 m−2 in the outer reef (averaged between sites and depth), and 0.10 colonies 0.25 m−2 in the back reef (averaged between sites), but 4 y later increases in these mean values were large. By 2014 (and relative to 2010), the mean density of Pocillopora spp. on the outer reef had increased 1,210-fold to 9.08 colonies 0.25 m−2, and by January 2015, in the back reef it had increased 8-fold to 0.80 colonies 0.25 m−2. These densities suggest that in 2010, there were 9.45 × 104Pocillopora spp. colonies on the outer reef of the north shore, with an additional 1.83 × 106 in the back reef. These numbers rose to 1.78 × 108 on the outer reef by April 2014, and to 1.47 × 107 in the back reef by January 2015 . Assuming sexual maturity of spawning Pocillopora spp. is obtained at ≥14-cm diameter, in 2010, 21% (n = 19 colonies in the quadrats censused) of Pocillopora spp. on the outer reef was sexually mature, as were 50% of the colonies in the back reef (n = 2 colonies in the quadrats censused). By 2014, these numbers had changed to 0% on the outer reef , and 10% in the back reef (n = 191 colonies in the quadrats censused). The mean diameter of sexually mature colonies in the back reef was 19.1 cm (n = 4) in 2010, and 22.3 cm (n = 19) in 2014, and in the outer reef it was 17.1 cm (n = 1) in 2010. Using an empirical relationship between diameter and tissue area for P. verrucosa in Moorea, and a mean fecundity of 6,327 eggs polyp−1 y−1, these sexually mature colonies would each release 1.44 ×107eggs cm−2, 2.07 × 107 eggs y−1, and 1.12 ×107 eggs y−1, respectively. Based on the proportion of the Pocillopora spp. in the population that was sexually mature , Pocillopora spp. on the outer reef of the north shore probably released 5.29 × 1011 eggs in 2010, and no eggs in 2014, and in the back reef, they probably released 5.23 × 1011 eggs in 2010, and 3.03 × 1013 eggs in 2014. If 15% of these eggs are fertilized in the seawater, on the outer reef they would produce 7.89 × 1010 zygotes in 2010, and no zygotes in 2014, and in the back reef, 8.30 × 1011 zygotes in 2010, and 4.54 × 1011 zygotes in 2010. Without information on the proportion of zygotes that develop into competent larvae, the number of zygotes was taken as the best estimate of larval production for the study populations , and in the back reef, 8.31 × 1011 ± 2.06 × 1011 larvae in 2010, and 4.54 × 1012 ± 1.13 × 1012 in 2014. The frequency distributions of estimated larval production  were positively skewed , but leptokurtic  larval productions in the outer reef of ∼7.89 ×10Pocillopora spp. in Moorea to support local recruitment and population growth, but they do not replace genetic tools . Moreover, our calculations suggest that Pocillopora spp. on the north shore had the capacity to export larvae from this shore, even while the outer reef populations were devastated, or in an early recovery stage, and before most colonies of mass spawning members of this genus were sexually mature. Implicit in this conclusion is that Pocillopora spp. in the back reef, as well as any large Pocillopora spp. colonies that survived the disturbances on the outer reef, produced the larvae necessary to support the ongoing recovery of Pocillopora spp. on the outer reef (Our calculations estimate the capacity of ls e.g.,  for quanter reef . Althougter reef .Pocillopora (P. verrucosa among Pocillopora spp. on the outer reef (Pocillopora spp. larvae recruit locally, or that distantly-produced Pocillopora spp. larvae support local recruitment. Both possibilities are supported by different genetic studies of Pocillopora spp. in the Society Islands, with one study using samples collected between 2001 and 2003 highlighting connectivity between populations of P. meandrina on Moorea and Tahiti (Pocillopora spp. in Moorea and Tetiaroa represent unique samplings of genetically discrete larval assemblages (Montipora capitata in the Hawaiian archipelago, and found >90% self-recruitment, even though pelagic larval duration of this species can exceed 200 d.Our approach has limitations consistent with a first order approximation, but nonetheless, it represents a reasonable summation of available data to realize emergent properties. One important focus of these limitations is uncertainty arising from the complexity of poorly-resolved species within the genus illopora , and theillopora  with impter reef . Regardlter reef , a key ld Tahiti , and anoemblages . The resemblages . Clearlyemblages , but regemblages . In thisPocillopora spp. in Moorea, our estimates of fecundity and fertilization success have the greatest uncertainty, because our calculations rely on values from studies conducted in different locations and at various times. Monte-Carlo simulations using variation about these values provide an indication of the implications of this uncertainty. As expected, these simulations reveal variation in the estimates of larval production by Pocillopora spp. in Moorea, but the leptokurtic distribution of these values suggest that variation in fecundity and fertilization success do not greatly affect the present conclusions regarding the capacity of Pocillopora spp. to self-recruit.Of the demographic properties described for Pocillopora spp. recruitment on the north shore of Moorea following recent catastrophic disturbances suggests that Pocillopora spp. larvae saturate the outer reef and drive negative density-association recruitment (Pocillopora spp. may be capable of producing enough larvae to support self-seeding, at least when produced in conjunction with conditions favoring retention, settlement, and post-settlement success. Future studies now are required to test this hypothesis in an inferential framework.The history of investigating connectivity among coral populations has changed from favoring self-seeding and short dispersal , to wideruitment . This em


Various steps in the RNA world required cooperation. Why did life’s first inhabitants, from polymerases to synthetases, cooperate? We develop kin selection models of the RNA world to answer these questions. We develop a very simple model of RNA cooperation and then elaborate it to model three relevant issues in RNA biology: (1) whether cooperative RNAs receive the benefits of cooperation; (2) the scale of competition in RNA populations; and (3) explicit replicator diffusion and survival. We show: (1) that RNAs are likely to express partial cooperation; (2) that RNAs will need mechanisms for overcoming local competition; and (3) in a specific example of RNA cooperation, persistence after replication and offspring diffusion allow for cooperation to overcome competition. More generally, we show how kin selection can unify previously disparate answers to the question of RNA world cooperation. Life very likely began as simple replicating RNA molecules ,2,3. TheHowever, in addition to posing biochemical problems, many of the key steps in the evolution of the RNA world are also problems of cooperation ,5,6,7. FThe links between these different suggested solutions for the problem of cooperation in the RNA world are not clear. Are they different explanations, or can we, instead, identify an overarching framework that links them all? It is useful here to make a comparison of the literature on the evolution of cooperation in higher organisms, ranging from animals to bacteria. Over the last 50+ years, work in this area of ‘social evolution’ has produced relatively unified theoretical and empirical literature that can explain cooperation across the tree of life ,19,20. OKin selection requires that cooperation be directed towards relatives. While this can involve mechanisms to discriminate kin from non-kin, it can also work via limited dispersal keeping relatives together. For example, when bacteria grow clonally, a cell will be tend to be surrounded by genetically identical cells, facilitating cooperation. This could potentially be important in the RNA world if limited diffusion keeps copies of the same molecule together . Indeed,Our aim is to test the utility of using existing kin selection methodologies to explain cooperation in the RNA world. We use a game theory approach to determine under what conditions cooperation would be evolutionarily stable, and hence be favoured by natural selection. This approach is deliberately simple, abstracting away biological details, to focus on key parameters that are likely to be important across different systems. We start with the simplest possible model, and then elaborate by adding in details that could have been important in the RNA world. Our more specific questions are: (1) Why would cooperation have been favoured in the RNA world? (2) How will different RNA biochemistries influence the evolution of cooperation? (3) Can we gain anything from applying the kin selection approach to the RNA world?We start by developing a simple model of cooperation in RNA molecules using standard kin selection techniques . We delix and that the average phenotype of the social partners it is interacting with is y. The phenotype could represent the likelihood (bounded between zero and 1) or amount (unbounded) of cooperation. For example, x could be the probability, between 0 and 1, of becoming a cooperative enzyme. A simple way to model the costs and benefits of cooperation is to assume that our focal RNA molecule has a baseline replication rate of C), and increased by some function of the level of cooperation in the local group of RNA molecules (B). The replication rate, w, can then be expressed as:In this case, the replication rate, or fitness, of an RNA molecule will be a function of its own level of cooperation  and the local level of cooperation amongst the RNA molecules it is interacting with (the group benefit). We assume that the focal RNA molecule has phenotype C as the cost of the cooperative trait and B as the benefit of the trait. Equation  showed that, assuming weak selection, this happens when the derivative of fitness with respect to genotype is zero :(2)dwdgR is genetic relatedness, which is a measure of genetic similarity ).).a=0), ta increases), the condition for cooperation to evolve becomes more stringent. When competition (a) is high, the competitive effects of the extra offspring copies produced by, e.g., cooperative enzyme activity, are experienced locally. This means that in addition to benefiting from cooperation, relatives suffer increased competition from cooperation, making it harder for cooperation to evolve  different forms of RNA dispersal, other than simple limited diffusion, which could decrease local competition; or (2) other features of RNA biology that might achieve the same effect. We provide an example of the latter in the next section.The above analysis also links RNA world with the wider literature on the influence of local competition. Previous work on bacteria and insects has demonstrated how local competition can reduce selection for cooperation ,49,50. FR is independent from the other model parameters. In reality this is unlikely to be the case. For example, limited diffusion leads to both local competition and higher relatedness, and so a and r should be positively correlated [In our analysis above we implicitly assumed that rrelated ,35,54,55R and model parameters (‘open’ models) [N. Island models are standard in evolutionary biology, but distinct from the lattice approach often used in RNA models, in that we do not explicitly track distance. While this island model approach is taken for analytical tractability, it has been shown to give qualitatively similar results to explicit lattice structures [We can allow for this by modelling an explicit RNA life history, the parameters of which can then be used to calculate an estimate of relatedness in system. Incorporating specific life history parameters and using them to calculate relatedness in the model is called ‘closing the model’. This is an alternative to our previous models, which assumed independence between  models) ,56. We dructures .We extend the model of cooperation in Equation  by explik, survives into the next generation, and the remaining d, of offspring copies diffuses elsewhere, while the other x, the average phenotype of the other individuals on the patch, y, and as the whole-group average , Z , limited diffusion (lower d), and smaller local group size .Cooperation is favoured by higher parent survival , rather than being an open parameter. The benefit of this approach is that it reveals the exact relationship between model parameters and cooperation. The downside is that it required being explicit about the life history and population structure of the RNA molecules. In situations in which we know these details this approach will be useful. Otherwise it may be useful to keep the model open, and subsume unknown population processes in R) . GenetiEquation . We haveWe elaborated on our most simple model by incorporating specific biological details that might be especially relevant in RNA world. We examined whether cooperative RNAs can receive the benefits of cooperation ( Equation ). We expWe have made a number of assumptions that are standard in kin selection analyses, and it is worth questioning their validity for the RNA world. One issue is that we have assumed that all phenotypes in the strategy set . We do Finally, the kin selection approach allows us make conceptual links to other taxa, unifying our evolutionary explanations across the tree of life. The tools we have used here are the same as those used to study cooperation in bacteria, birds, and mammals ,61, and


In this study, Eaton et al. use a new gene-editing approach to delineate the mechanism by which RNA polymerase II terminates. They generated human cell lines from which the 5′-to-3′ exoribonuclease Xrn2 or the poly(A) signal endoribonuclease CPSF73 can be rapidly controlled and show that efficient termination on most protein-coding genes involves CPSF73-mediated RNA cleavage and cotranscriptional degradation of polymerase-associated RNA by Xrn2. Termination is a ubiquitous phase in every transcription cycle but is incompletely understood and a subject of debate. We used gene editing as a new approach to address its mechanism through engineered conditional depletion of the 5′ → 3′ exonuclease Xrn2 or the polyadenylation signal (PAS) endonuclease CPSF73 (cleavage and polyadenylation specificity factor 73). The ability to rapidly control Xrn2 reveals a clear and general role for it in cotranscriptional degradation of 3′ flanking region RNA and transcriptional termination. This defect is characterized genome-wide at high resolution using mammalian native elongating transcript sequencing (mNET-seq). An Xrn2 effect on termination requires prior RNA cleavage, and we provide evidence for this by showing that catalytically inactive CPSF73 cannot restore termination to cells lacking functional CPSF73. Notably, Xrn2 plays no significant role in either Histone or small nuclear RNA (snRNA) gene termination even though both RNA classes undergo 3′ end cleavage. In sum, efficient termination on most protein-coding genes involves CPSF73-mediated RNA cleavage and cotranscriptional degradation of polymerase-associated RNA by Xrn2. However, as CPSF73 loss caused more extensive readthrough transcription than Xrn2 elimination, it likely plays a more underpinning role in termination. Transcriptional termination can be defined as the cessation of RNA polymerization and dissolution of the ternary complex of RNA polymerase II (Pol II), DNA, and RNA. Termination is a biologically important process, as it prevents transcriptional interference of genes and ensures that polymerases are available for new rounds of gene expression. Despite the fact that all transcription ends this way, it is perhaps the least understood phase in the cycle. A polyadenylation signal (PAS) is a prerequisite for termination, and mutations within it were shown decades ago to cause extended transcriptional readthrough . Two modEarly support for the torpedo model came from observations that depletion of the nuclear 5′ → 3′ exonuclease Xrn2 caused termination defects on transfected plasmids . Its homCaenorhabditis elegans, Xrn2 depletion does not affect termination on the majority of protein-coding genes, suggesting that the torpedo mechanism is less widely used in that organism . Further RNA analyses performed throughout this study also showed that RNA degradation functions are virtually unimpaired in XRN2-AID cells.Western blotting confirmed homozygous targeting in two selected positive clones, shown by the higher-molecular-weight Xrn2 and the absence of any signal at the size expected for native Xrn2 D. It is XRN2-AID cells completely prevented cell colony formation, showing that Xrn2 is an essential protein .To test Xrn2-AID depletion, Western blotting was performed over a time course of auxin addition E. Xrn2-AMYC and ACTB using quantitative RT–PCR (qRT–PCR). RNA was isolated over the same time course as for the Western blot in ACTB . Importantly, 3′ flanking region RNA was stabilized only in the combined presence of the AID tag, Tir1, and auxin, showing that no individual factor indirectly causes the effect . These findings are unlikely to result from secondary effects due to the speed of Xrn2-AID depletion, especially by comparison with RNAi, with the near-complete elimination of Xrn2-AID revealing function without overexpression of the inactive protein.Next, we tested the effect of Xrn2 loss on PAS cleavage and the stability of 3′ flanking region RNA from XRN2-AID cells treated with auxin or untreated. We also performed this analysis on a HCT116 cell line that was unmodified at XRN2 and grown in the absence of auxin. Analysis of individual gene tracks confirmed the effect on MYC and ACTB, where an enhanced signal beyond their PASs was observed upon Xrn2-AID elimination  precursors , showing other ways of Xrn2 substrate generation.We then sought to test the generality of the effects seen on Xrn2-AID loss using nuclear RNA sequencing (RNA-seq) carried out on mination B. Furthend RPL30 C. XRN2-AXRN2-AID cells . We note that Xrn2-AID loss is associated with a slight reduction in reads upstream of the PAS, potentially reflecting mildly reduced gene expression that might be caused by Pol II recycling defects. Finally, closer analysis of the TES (PAS) region showed that read counts at this position are similar in all samples . This again suggests that major PAS cleavage defects are not widespread following Xrn2 loss, which is consistent with the analysis of MYC and ACTB shown above.Metagene plots were then generated for protein-coding genes that were separated from any reads within 3 kb of their transcription start site (TSS) and 7 kb of the PAS (denoted as TES [transcript end site]). This left 4701 genes for analysis and revealed a clear enhancement of 3′ flanking region RNA upon auxin treatment of ID cells D. Xrn2-AXRN2-AID cells treated with auxin or untreated and analyzed it by qRT–PCR  analysis in the presence of 4-thio UTP (4sUTP). In this experiment, transcriptionally engaged Pol II was allowed to run on and label the 3′ ends of nascent transcripts in vitro. These were purified via linkage of biotin onto 4sUTP followed by streptavidin capture  and subjected to qRT–PCR to analyze UCPA and 3′ flanking region transcripts from MYC . Finally, stable integration of wild-type or catalytically inactive (D235A) XRN2 into XRN2-AID cells demonstrated that both RNA degradation and termination defects caused by Xrn2-AID elimination are completely rescued by wild-type Xrn2 but not by D235A . The Xrn2 effects on transcriptional termination therefore require its exoribonuclease function.As a second measure of cotranscriptional degradation, we isolated nuclei from control or auxin-treated from MYC G. This eNext, we precisely interrogated the global function of Xrn2 in transcriptional termination using mNET-seq . In thisMYC and RPL30 mNET-seq profiles are shown in ACTB in Supplemental Fig. 5A). In cells not treated with auxin, termination occurs downstream from the PAS, where the mNET-seq signal reaches background. When Xrn2 is eliminated, a clear termination defect is observed, and, due to the high resolution of mNET-seq, it is possible to visualize two manifestations of this. First, where flanking region signal is detected in control cells, it is frequently elevated over the same positions in cells lacking Xrn2. This can be seen in the MYC and RPL30 examples in RPL30 gene plot in Supplemental Fig. 5B). Interestingly, some genes were especially sensitive to Xrn2 elimination and showed more extensive readthrough than the genome-wide trend—as exemplified by TBL1XR1 in TBL1XR1 .We next addressed the generality of Xrn2 function in termination by generating metagene plots from control and auxin-treated cells. We analyzed expressed genes separated from upstream and downstream reads by at least 1 and 15 kb, respectively, which revealed a general transcriptional termination defect upon loss of Xrn2 C. IntereMIR17HG and MIR31HG . snRNAs also undergo 3′ end cleavage by the integrator complex, and this may also precede Xrn2 activity . As such, 3′ end cleavage is not always sufficient to promote an Xrn2-dependent termination process.Although not polyadenylated, Histone RNAs also use CPSF for 3′ end formation, which could provide an entry site for Xrn2 , and we activity . HoweverEscherichia coli DHFR-based degron using the system used for Xrn2-AID  was performed in and ACTB C,D. In band ACTB . DespiteCPSF73-DHFR cells ±TMP, on XRN2-AID cells, and on D235A XRN2-AID cells +auxin . Pol II occupancy over extended readthrough regions of MYC and ACTB was plotted relative to the signal from upstream of the PAS. For both genes, CPSF73 depletion resulted in greater signals over extended positions than elimination of Xrn2 function, suggesting that termination is more adversely effected by loss of CPSF73. qRT–PCR analysis of readthrough RNA over the same positions confirmed this result . Inhibition of CPSF30 function by influenza NS1A protein .We next tested whether CPSF73 and Xrn2 produced differential effects on readthrough transcription. For this, Pol II ChIP was compared for s +auxin A,B. D235 protein  also cauD235A cells represent the scenario most lacking in 5′ → 3′ degradation of RNA, the smaller effect on termination relative to CPSF73 loss may be due to incomplete Xrn2 depletion or other 5′ → 3′ nucleases acting in its absence. To address this, we analyzed the turnover rate of 3′ flanking region transcripts from MYC and ACTB in more detail. A time course was used in XRN2-AID cells treated with auxin or untreated and in D235A cells treated with auxin following transcriptional inhibition by actinomycin D (Act D)  C. In XRND235A cells could be by alternative 5′ → 3′ exonucleases or from the 3′ end by the exosome. To address this, we treated D235A cells with control or human Rrp40 (hRrp40)-specific siRNAs before auxin addition . The same experiment was performed on XRN2-AID cells not treated with auxin to determine any exclusive effects of hRrp40 depletion. We first tested the effects of these conditions on the levels of MYC and ACTB 3′ flanking region RNA. hRrp40 depletion alone gave no substantial effect, whereas auxin treatment of D235A cells gave the expected strong accumulation. When hRrp40 was depleted from D235A cells treated with auxin, there was an accumulation of 3′ flanking region RNA above what was seen upon manipulation of Xrn2 that was most marked for MYC transcripts. Therefore, the exosome contributes to readthrough RNA degradation in the absence of Xrn2 function. The level of UCPA transcripts was similar under each of these conditions, arguing that PAS cleavage is unaffected .The degradation of 3′ flanking region RNA in auxin-treated XRN2-AID sample treated with control siRNA, Act D treatment caused depletion of ACTB and MYC flanking transcripts as expected, and hRrp40 depletion gave a similar result. In auxin-treated D235A cells, ∼40%–60% of 3′ flanking region RNA was again degraded in the absence of Xrn2 function. Importantly, hRrp40 depletion from D235A cells grown in auxin essentially blocked degradation, as the level of RNA recovered after transcription inhibition was similar to before Act D addition. This shows that the exosome rather than other 5′ → 3′ exonucleases is responsible for the degradation of RNA that occurs in the absence of functional Xrn2. As such, auxin treatment of D235A cells effectively blocks degradation of 3′ flanking region transcripts from their 5′ ends. A similar result was obtained when transcription was inhibited using flavopiridol . Act D time course analysis also revealed that CPSF73 elimination prevented turnover of 3′ flanking region RNA, arguing that PAS cleavage is necessary to promote their degradation . These data argue that the differential effect of Xrn2 and CPSF73 on transcriptional termination is unlikely to be due to an incomplete block of 5′ → 3′ degradation when Xrn2 is manipulated. As such, they support the existence of additional termination mechanisms that occur in the absence of 5′ → 3′ degradation.Next, the impact of the exosome on degradation of 3′ flanking RNAs was assessed after 20 min of Act D treatment E. In theCPSF73 or a point-mutated derivative (H73A) shown previously to have diminished nuclease activity  of CPSF-DHFR via 10 h of TMP withdrawal. Chromatin-associated RNA was then isolated to study termination via the extent of nascent RNA transcription, which was assayed by qRT–PCR for MYC and ACTB genes . These observations may be of importance beyond Xrn2, as they suggest that a degron-based approach can yield a fuller repertoire of functions for some proteins than RNAi alone.Previous reports have reached different conclusions on the role of Xrn2 in termination. Originally, RNAi of Xrn2 caused a termination defect on transfected β-globin plasmids, while a subsequent global analysis found no genome-wide function for Xrn2 in termination at gene 3′ ends using mNET-seq . An explAnother finding in our study is that termination is not readily observed in the absence of CPSF73 activity, suggesting that PAS cleavage is required. In vitro experiments suggest that PAS cleavage is not absolutely required for termination . HoweverMIR17HG transcription to enter the downstream GPC5 gene, whereas transcription terminates before this point following Xrn2 loss , CPSF73 , CPSF73 for XRN2 or CPSF73 was confirmed by Sanger sequencing. An optimized sleeping beauty transposon system . For CRISPR, 1 µg of guide RNA plasmid and 1 µg of each repair plasmid were transfected into six-well dishes. Twenty-four hours later, culture medium was changed, and, a further 24 h later, cells were split into a 100-mm dish containing 800 µg/mL neomycin and 150 µg/mL hygromycin. After ∼10 d of selection, single colonies were transferred to a 24-well plate and screened by PCR or Western blotting. The presence of repair cassettes at n system  was usedTri reagent (Sigma) was used to isolate total RNA following the manufacturers’ guidelines, and RNA was treated with Turbo DNase (Life Technologies) for 1 h. In all cases, reverse transcription of 1 µg of RNA was primed with random hexamers using Protoscript II (New England Biolabs). qPCR was performed using Brilliant III (Agilent Technologies) in a Qiagen Rotorgene instrument. Comparative quantitation was used to establish fold effects.g for 5 min) and resuspended in 400 µL of RIPA buffer . Samples were sonicated in a Bioruptor sonicator (30 sec on and 30 sec off) 10 times on high setting. Tubes were spun at 13,000 rpm for 10 min. Supernatant was then split into two and added to 30 µL of Dynabeads (Life Technologies) that had been incubated for 2 h with 3 µg of antibody or, as a control, mock-treated. Ten percent of the supernatant was kept for input. Immunoprecipitation was for 2–14 h at 4°C, and beads were then washed twice in RIPA, three times in high-salt wash buffer , and once in RIPA. Samples were eluted (0.1 M NaHCO3 + 1% SDS), and cross-links were reversed overnight at 65°C. DNA was purified by phenol chloroform extraction and ethanol precipitation. Samples were generally resuspended in 100 µL of water, with 1 µL used per PCR reaction. For RNA immunoprecipitation, cross-links were reversed for 45 min at 65°C. RNA was purified by phenol chloroform extraction and ethanol precipitation followed by DNase treatment and reverse transcription.For ChIP, one 100-mm dish of cells was cross-linked for 10 min in 0.5% formaldehyde, and cross-links were quenched in 125 mM glycine for 5 min. Cells were collected . This was underlayered with HLB + 10% sucrose and spun at 500g for 5 min. Nuclei were resuspended in 100 μL of NUN1 . One milliliter of NUN2  was added before incubation for 15 min on ice with regular vortexing. Chromatin pellets were isolated by centrifugation at 13,000 rpm in a benchtop centrifuge, and RNA was isolated using Trizol.Nuclei were isolated from cells by resuspending cell pellets from a 100-mm dish in hypotonic lysis buffer . This was supplemented with rA, C, and G together with 4sUTP . Following incubation for 15 min at 30°C, RNA was isolated, and biotin linkage and capture were performed as described in Nuclei were isolated as for chromatin-associated RNA. These were resuspended in an equal volume of 2× transcription buffer  according to the user manual. Libraries were prepared using TruSeq stranded total RNA library preparation kit (Illumina) according to the manual and purified using Ampure XP beads (Beckman Coulter). Libraries were screened for fragment size and concentration by Tapestation D1000 (Agilent) and sequenced using HiSeq 2500 (Illumina).http://www.R-project.org). Normalized gene coverage plots were visualized using the Integrated Genome Viewer suite (Raw single-end 50-base-pair (bp) reads were screened for sequencing quality using FASTQC, adapter sequences were removed using Trim Galore (wrapper for Cutadapt), and trimmed reads <20 bp were discarded. Reads were aligned to the GRCh38 human genome using Hisat2  with spler suite .XRN2-AID cells were treated for 2 h with auxin or left untreated. NEBNext small RNA libraries were sequenced using HiSeq 2500 (Illumina). Raw 50-bp paired-end sequences had adapter sequences removed using Trim Galore, and resultant reads with a quality of <20 and fragment size of <19 bp were discarded. Reads were aligned using HiSat2 against GRCh38 (Ensemble) with known splice site annotation (Gencode), and concordantly mapped read pairs were selected (A detailed description of the mNET-seq protocol can be found in the study by selected .The mNET-seq traces used single-nucleotide resolution BAM files corresponding to the 3′ end of the RNA fragment . For met


This review will explore these recent studies and how they broaden our understanding of the functions and regulation of ADAR-mediated RNA editing.One of the most prevalent forms of post-transcritpional RNA modification is the conversion of adenosine nucleosides to inosine (A-to-I), mediated by the ADAR family of enzymes. The functional requirement and regulatory landscape for the majority of A-to-I editing events are, at present, uncertain. Recent studies have identified key in vivo functions of ADAR enzymes, informing our understanding of the biological importance of A-to-I editing. Large-scale studies have revealed how editing is regulated both in  The post-transcriptional modification of RNA is a key process controlling the output of the genome, shaping the transcriptional landscape and ultimately cellular and organismal fate. Many types of RNA regulation have been identified, from differential splicing and isoform usage through to disctinct classes of chemical modification . There aXenopus eggs [Gria2) [A-to-I editing was initially identified as an activity causing the unwinding of transfected RNA duplexes in pus eggs , 7. It wpus eggs , 9. This [Gria2) . In this [Gria2) –14.Alu and retrotransposons, is assessed due to the high level of sequence similarity present in these regions.A-to-I editing is performed by the adenosine deaminase acting on RNA (ADAR) family of proteins –18. ADAROur knowledge of editing was largely confined to a select few well-studied targets, until the parallel advances in computational methods and sequencing approaches that generate significantly greater transcriptome coverage converged to allow the accurate identification of editing in many different species. Since the identification of this modification and with the relatively recent advances in sequencing methods, the number of known sites that can be subjected to A-to-I editing has grown exponentially, with current estimates of up to 100 million possible editing events in the human genome , 26.Gria2 to less than 0.1%, with the majority of editing occurring at a frequency of less than 20% when assessed genome-wide [Two primary types of A-to-I editing have been defined. The first is site-selective editing , 27–29. ome-wide . An adenome-wide . There aome-wide , 32–34. ome-wide . Despiteome-wide , 35. Theome-wide , 32, 33.Alu elements and other types of repetitive regions [The second and distinctive type of A-to-I editing is hyper-editing , 37, whi regions , 35, 43. regions , 44–47.The initial identification of A-to-I editing sites was largely based on serendipitous discoveries stemming from the detailed assessment of a single transcript , 48. EviMethodologies to systematically map A-to-I editing have primarily utilized the in silico analysis of expressed sequence tag databases or, more recently, the analysis of large RNA-seq datasets , 53–55. Intuitively, RNA editing sites can be identified by finding genetic variants  present in the RNA-seq data but absent in the matched whole genome sequence from the same individual or species , 64. MorADAR), ADAR2 (ADARB1), and ADAR3 (ADARB2); Drosophila melanogaster has a single Adar ; anectively ). Each Aectively , 73. Mamectively , 75. Unlectively , 74. Simactivity .The expression of each of the ADARs varies across development and tissues in mammals . ADAR1 iC. elegans deletion of adr-1 or adr-2 resulted in defects in chemotaxis [rde-1 and rde-4, implicating an interaction between RNA editing and RNAi pathways [adr-2-deficient C. elegans has been determined to be an editing-dependent effect [clec-41, a predicted C-type lectin protein, was dependent upon editing by ADR-2. In ADR-2-deficient cells, the expression of clec-41 was significantly reduced. When clec-41 expression was restored in adr-2-deficient neural cells, the chemotactic defect could be rescued, providing direct evidence that neuronal/chemotactic phenotypes of adr-2 mutants can be attributed to altered gene expression of an edited transcript [The phenotypes associated with loss of function of ADARs differ between species. In emotaxis , phenotypathways . Very ret effect . The noranscript .Drosophila resulted in behavioral and locomotion abnormalities with brain lesions upon aging [Drosophila which have defects in sleep patterns [Adar2–/–- mice [C. elegans and Drosophila, the germline deletion of ADARs is compatibile with life and the mutants are viable but phenotypic [Drosophila Adar null mutants, but that mammalian ADAR1 could not [Deletion of the single ADAR in on aging , 84, 85.patterns , with ev/–- mice . In bothenotypic . Phylogeould not . This reAdar2 resulted in the fully penetrant development of postnatal seizures that ultimately result in death by 20–25 days of age [Gria2 gene, mimicking constitutive editing at this site [Adar2–/–Gria2R/R animals have a normal lifespan, are fertile, but have some subtle phenotypes that were revealed by broad-based phenotyping [In mice, deletion of s of age . This phhis site , 13. Thenotyping . This elAdar2–/–- phenotype, the deletion of Adar1 , th150–/–- ), or theeficient ) resulteeficient . Using gand MAVS , 78, 92. of MAVS . It is nAdar2–/– animals, with the profound rescue of the phenotype in these mice by the Gria2R/R allele demonstrating that the physiologically most important function of ADAR2 is A-to-I editing. The Adar2–/–Gria2R/R animals do have additional subtle phenotypes that were revealed after a comprehensive phenotypic analysis and testing suggesting that there are specific requirements for ADAR2 outside of Gria2 editing; however, whether these reflect the lack of editing of specific substrates or editing-independent functions is not clear [Adar1–/–Mavs–/– and Adar1p150–/–Mavs–/– rescued mice that survived past 10 days of age had developmental defects in the kidney, small intestine, and lymph node and a failure of B lymphopoiesis [Adar1E861A) demonstrated highly comparable phenotypes in both a germline-deficient or acute adult somatic deletion model to ADAR1 null alleles [A question that has not been definitively resolved is the extent to which the phenotypes seen in the different mutant mouse models are due to editing-dependent or editing-independent functions. This is reasonably clear for the ot clear . In the ot clear –100, affot clear , alternaot clear , and altot clear , 104 andot clear . In vivoopoiesis . In cont alleles , 41, 106C. elegans and mammalian models that ADAR3 may act to reduce the availability of substrates for ADAR1 or ADAR2, resulting in a net overall inhibitory effect on editing levels [Adar1 and Adar2 mutants have been reported for Adar3–/– animals to date. Therefore, it is the combination of expression patterns of the different ADAR isoforms that can determine the nature and extent of editing in a given cell and tissue, with ADAR3 providing a counterpoint to ADAR1 and ADAR2 [No editing activity has been demonstrated by ADAR3. The role of mammalian ADAR3 is less clear, but data are accumulating from both g levels , 75, 76.These genetics studies have refined our understanding of the functions of A-to-I editing and of the individual roles that ADAR isoforms fulfill in vivo: ADAR2 is key to site-selective editing, especially in the CNS, whereas ADAR1-mediated editing has an essential role in the prevention of activation of the cytosolic dsRNA innate immune sensing system by endogenous RNA  surrounding the editing site guide the preference and selectivity of ADARs. ADAR has a preferred sequence motif neighboring the targeted adenosine, in particular the 5′ and 3′ nearest neighboring positions to the editing site, with the depletion and enrichment of G upstream and downstream of the editing site, respectively [cis regulatory code of RNA editing.How ADARs target a specific A-to-I RNA editing site is a long-standing question that is not well addressed. Both the primary sequence and secondary structure  associated with differences in RNA editing [cis regulatory code of RNA editing. Consistent with these results, an assessment of editing across 21 diverse organisms concluded that editing is enriched in regions of putative double-strandedness and is relatively rare in coding regions [We and others recently applied a quantitative trait locus (QTL) mapping approach to identify genetic variants associated with variability in RNA editing –118. Wit editing . We demo editing . While t regions . This an regions , 56, 60.An additional finding from the analysis of the crystal structures of the human ADAR2 deaminase domain bound to an RNA substrate was that differences between the ADAR proteins themselves may affect substrate specificity . It was trans regulators involved in the RNA editing machinery. In Drosophila, the fragile X protein FMR1 biochemically and genetically interacts with ADAR to influence editing levels [Drosophila, underscoring the need for additional efforts to identify editing regulators with broader effects.Beyond ADAR editing enzymes themselves only a handful of proteins have been identified that modulate RNA editing, despite speculation about the existence of additional g levels , the RNAg levels , and theg levels . HoweverIn mammals, two proteins are known to regulate ADAR2’s global activity through post-translational modifications. Pin1 promotes editing by binding ADAR2 in a phosphorylation-dependent manner, while WWP2 decreases editing by targeting ADAR2 for ubiquitination . By takiADAR2 have not been reported to be associated with human disease. In contrast, mutations of ADAR are associated with the human diseases dyschromatosis symmetrica hereditaria (DSH) [ADAR mutations have been reported in DSH and are associated with altered pigmentation (areas of hypo- and hyperpigmentation) on the face and dorsal aspects of the extremities that first appear in infancy/early childhood. This condition is not fatal and the symptoms appear to be largely restricted to the skin.The available data suggest a more pronounced separation of biological function between ADAR1 and ADAR2 than was previously expected. Mutations in ia (DSH) , 126 andia (DSH) –129. OveADAR mutations as one of the genetic causes of AGS [ADAR mutations, develop a severe neurodevelopmental disorder characterized by intracranial calcifications and motor disorders, and have evidence of an activated innate immune/interferon response (“interferonopathy”) in their peripheral blood, consistent with the results from murine mutants [TREX1, RNASEH2B, RNASEH2C, RNASEH2A, SAMHD1) and those regulating cytosolic RNA metabolism (ADAR and IFIH1) [ADAR are seen in affected patients and are predicted to be significantly more detrimental to the RNA editing/interacting potential of the mutant proteins. Interestingly, despite the significantly different numbers of repetitive elements between the species (Alu repeats are primate restricted), the transcriptional response to ADAR1 deficiency is conserved between mouse and human, as is the specific requirement for MDA5 in this response [ADAR2 or ADAR3 have not yet been clearly described or associated with human disease.More recently, and contrasting with the phenotypes of DSH, Crow, Rice, and colleagues identified biallelic s of AGS . AGS has mutants . Mutatiod IFIH1) , 131. Inresponse . These rADAR.A range of different human diseases are associated with altered editing and ADAR activity. In these cases, the direct mutation of the ADAR genes does not cause this association, as is seen in AGS. There is a significant body of work demonstrating reductions in editing, principally ascribed to ADAR2, in a range of neuronal and CNS disorders, including Alzheimer’s disease and amyotrophic lateral sclerosis –136. In ADAR gene resides, and the activation of interferon/innate immune sensing responses in tumors leading to an increase in ADAR1 expression. The biological consequences of increased ADAR1 and an increased level of overall editing in tumors is only beginning to be explored. In some specific examples, such as in melanoma, reduced editing efficiency has been proposed to be important in the pathogenesis of these tumors [Our appreciation of the extent and characteristics of A-to-I editing have rapidly expanded, paralleling the technological advancements in sequencing methods. This has been particularly informative in the context of cancer, where large datasets from diverse human cancers have been harnessed to identify links between altered A-to-I editing levels and a range of different cancer types. Initial reports described changes, generally reductions, of ADAR2-mediated editing at selected targets in tumors of the CNS such as glioblastoma and astrocytoma , 138. Ree tumors , 147, alcis regulation of RNA sequence and structure surrounding the edited adenosine and, to a lesser extent, the trans regulation of ADAR protein activity/levels by other cellular proteins. At the cellular level, how altered A-to-I editing, both increased and decreased, impacts cell fate is only beginning to be explored. This is particularly relevant in disease contexts, where evidence has solidified that there is altered activity of ADAR proteins. In inherited disorders such as AGS the loss/reduction of ADAR1 activity has a profound impact on normal functioning and is ultimately lethal. In cancer, where elevated ADAR1 expression and activity have been frequently reported, it remains to be shown if these reflect a function in driving tumor initiation and maintenance or reflect the physiological function of ADAR1, to edit endogenous dsRNA to prevent activation of the innate immune system. Many of the tools developed to allow our present understanding of the physiological roles of ADARs can be applied to understand these pathogenic roles.Our understanding of the landscape of A-to-I editing has rapidly expanded over the past decade. The efforts of many investigators have enabled us to catalogue editing across the transcriptomes of many species. The ability to identify editing with high confidence at the genome scale has enabled a better understanding of how editing contributes to genome diversity in a range of contexts: evolutionarily, developmentally, and pathogenically. Paralleling the identification of A-to-I editing events, studies using genetically modified organisms have greatly enhanced our understanding of the in vivo roles and functions of ADARs. These studies have established that ADAR1 serves a unique function in the regulation of the innate immune response to self-RNA, while ADAR2 principally contributes to editing in a more site-selective manner, and ADAR3 competes with ADAR1/2 for substrates, but does not edit them directly. Further studies have broadened our understanding of factors contributing to A-to-I editing efficiency of a given substrate, principally the N(6)-methyladenosine (m6A) is the most frequent internal modification of mRNA [6A and those of A-to-I editing, including roles in the viral life cycle [6A and A-to-I editing interact and alter the fate of the targeted RNA transcripts is at present unclear [Modifications of RNA, outside of A-to-I editing, are increasingly being defined as key regulators of transcriptional output and more than 100 distinct types of modifications have been identified to date . This ra of mRNA . There afe cycle , 150 andfe cycle –153 and fe cycle –156. Giv unclear . It may


Oyster reefs provide myriad ecosystem services, including water quality improvement, fisheries and other faunal support, shoreline protection from erosion and storm surge, and economic productivity. However, their role in directing flow during non-storm conditions has been largely neglected. In regions where oyster reefs form near the mouth of estuarine rivers, they likely alter ocean-estuary exchange by acting as fresh water “dams”. We hypothesize that these reefs have the potential to detain fresh water and influence salinity over extensive areas, thus providing a “keystone” ecosystem service by supporting estuarine functions that rely on the maintenance of estuarine  conditions in the near-shore environment. In this work, we investigated the effects of shore-parallel reefs on estuarine salinity using field data and hydrodynamic modeling in a degraded reef complex in the northeastern Gulf of Mexico. Results suggested that freshwater detention by long linear chains of oyster reefs plays an important role in modulating salinities, not only in the oysters’ local environment, but over extensive estuarine areas (tens of square kilometers). Field data confirmed the presence of salinity differences between landward and seaward sides of the reef, with long-term mean salinity differences of >30% between sides. Modeled results expanded experimental findings by illustrating how oyster reefs affect the lateral and offshore extent of freshwater influence. In general, the effects of simulated reefs were most pronounced when they were highest in elevation, without gaps, and when riverine discharge was low. Taken together, these results describe a poorly documented ecosystem service provided by oyster reefs; provide an estimate of the magnitude and spatial extent of this service; and offer quantitative information to help guide future oyster reef restoration. Oyster reefs provide myriad ecosystem services, including water quality improvement, fisheries and other faunal support, shoreline protection from storm surge, and economic productivity –3. OysteGiven their central role in supporting coastal resilience  and provIn many regions, oyster reefs form linear structures parallel to the coast and/or across the path of estuarine and riverine outflows , potenti2 in the coastal counties that make up the Big Bend region, compared to a statewide average of >100 [In the Big Bend region of Florida (USA), oyster reefs form km-long structures that are nearly parallel to the coast and lie wholly or partially across the mouths of the Suwannee and smaller coastal rivers and creeks . While e of >100 ), littleMultiple hypotheses exist to explain reef loss in the region, however recent evidence  suggestsThe expected effect of freshwater detention by shore-parallel oyster reefs is the maintenance of consistently lower salinities on the landward sides of reefs relative to seaward sides. To test this expectation, six monitoring stations were established to measure salinity differences between seaward (SEA) and landward (LAND) sides of the Lone Cabbage Reef, located in the Suwannee Sound near Suwannee, FL . ROMS is a 3-D, free-surface, terrain-following numerical model that solves finite-difference approximations of the Reynolds-Averaged Navier-Stokes (RANS) equations using the hydrostatic and Boussinesq assumptions , 19 withSimulations were performed in two steps. The first step was to consider a morphology that closely mimicked the current geometry of the Lone Cabbage Reef. The aim of this simulation was to verify that the simplified model domain captured essential features of the overall physical behavior of the Suwannee Sound and Long Cabbage Reef, rather than explicitly matching observations. After this verification, the second step was to explore ten idealized reef configurations that spanned a range of reef lengths, widths, heights, and inlet spacing . In bothx (east) and y (north) directions, and flow structure was solved using 10 terrain-following, equidistant vertical layers. The salinity and temperature at the southern boundary were considered constant . River salinity and temperature were assumed to be 0 g/kg and 12°C, respectively. Newman boundary conditions were applied at the eastern and western boundaries. Bed friction was computed assuming the “law of the wall.” Bed-roughness lengths were chosen as 0.02 cm over sandy areas and 0.78 cm over the oyster reefs, according to [2/s, based on Madsen et al. [3/s at the northern boundary. The tidal wave was defined by the four most energetic constituents in the region . The amplitudes and phases were based on measurements at the nearest tidal gauge . The model was run for a period of 2 months for each value of freshwater discharge.Model cells were 50 m in the rding to . Eddy vin et al. . To simuSalinity at all sites was highly variable over seasons and tidal cycles, varying between ca. 0 and 27 g/kg and was generally inversely correlated with Suwannee River discharge. Tide-filtered mean daily salinity  was loweTide-filtered, mean daily salinity modeled in Run 2  was lowest closest to the river mouth and increased with distance , consistNext, different reef geometries were considered to ascertain the role of reef length, height, and width, as well as the width of the inlet openings . Reef leWe used field observations and numerical modeling to explore the role of oyster reefs in detaining fresh water from rivers and modifying salinities in the coastal environment. Results provide evidence that freshwater detention by oyster reefs plays a key role in modulating salinities not only in the oysters’ local environment, but over extensive coastal areas (tens of square kilometers) as a function of reef morphology. These findings have relevant implications for coastal freshwater management, oyster reef restoration, estuarine eco-hydrological modeling, and the appropriate valuation of ecosystem services provided by ecosystem engineers.Field measurements (Section 3.1) confirmed that salinity differences exist between landward and seaward sides of the Lone Cabbage Reef over short distances (<50 m) and over both tidal and seasonal time scales . These eNumerical results (Section 3.2) expanded these observational findings by illustrating how reefs affect the lateral and offshore extent of freshwater influence. In general, freshwater detention was most evident when the reef extended completely across the sound . If the Model results also suggest that the capacity for freshwater detention within the sound increases when the reef is highest (echoing the results above), without inlets, and when discharge is low  . The preThe coupling of reefs with salinities provide a potential feedback loop in which oyster reef degradation allows more saltwater intrusion during low freshwater flow events, leading to further oyster mortality and reef degradation. The strength and rates of these feedbacks depend on interactions between the rate of change of oyster reef configuration  and the salinity regime. These reciprocal feedbacks may dictate the potential for critical transitions  between Understanding when and where these critical transitions and stable equilibria occur will be essential for guiding successful oyster reef management and restoration and represents an important next step for advancing this work. A robust understanding of the feedbacks among freshwater flow, reef morphology, and estuarine salinity can be leveraged via active restoration to make coastal environments more resilient to sea level rise and episodic declines in freshwater discharge, particularly since oyster reefs have been shown to grow vertically at rates far in excess of sea-level rise . AdditioFinally, oyster reefs have been shown to provide a number of important ecosystem services, including storm surge abatement, water filtration, sequestration of nutrients, stabilization of sediments, and creation of nursery habitat for fishes and invertebrates , 29, 30.S1 Dataset15-minute salinity, temperature, and water level data from the six monitoring stations shown in (XLSX)Click here for additional data file.


Flavivirus comprises a large number of small, positive-sense single-stranded, RNA viruses able to replicate in the cytoplasm of certain arthropod and/or vertebrate host cells. The genus, which has some 70 member species, includes a number of emerging and re-emerging pathogens responsible for outbreaks of human disease around the world, such as the West Nile, dengue, Zika, yellow fever, Japanese encephalitis, St. Louis encephalitis, and tick-borne encephalitis viruses. Like other RNA viruses, flaviviruses have a compact RNA genome that efficiently stores all the information required for the completion of the infectious cycle. The efficiency of this storage system is attributable to supracoding elements, i.e., discrete, structural units with essential functions. This information storage system overlaps and complements the protein coding sequence and is highly conserved across the genus. It therefore offers interesting potential targets for novel therapeutic strategies. This review summarizes our knowledge of the features of flavivirus genome functional RNA domains. It also provides a brief overview of the main achievements reported in the design of antiviral nucleic acid-based drugs targeting functional genomic RNA elements.The genus  The great plasticity of RNA virus genomes allows them to perform different functions during the infectious cycle, helping viral populations adapt to novel molecular and cellular contexts, and to escape host defenses. It also contributes toward the development of resistance to antiviral drugs. These feats are achieved by the genome preserving a degree of variability while avoiding challenges to viral fitness. Genome variability can become a threat to viral survival if it reaches the error catastrophe limit , but RNAFlavivirus spp. (from now on flaviviruses) belong to the family Flaviviridae. They are small (40–65 nm diameter), enveloped  viruses with a positive single-stranded RNA genome. The genus includes important human pathogens responsible for ongoing/recurrent outbreaks of disease in areas where such diseases are not traditionally endemic; West Nile virus (WNV), dengue virus  and Zika virus (ZIKV), for example, are all dramatically expanding their original geographic distribution. Other well-known flaviviruses include the causal agents of yellow fever (YFV), Japanese encephalitis (JEV), St. Louis encephalitis (SLEV), Murray Valley encephalitis (MVEV), or tick-borne encephalitis (TBEV) among of over 70 flaviviruses that have been identified. Some authors believe there could be over 2,000 left to discover (discover .Table 1). These clusters can be further divided into clades and species. The members of the MBFV and TBFV clusters replicate in vertebrates and arthropods, while the NKV flaviviruses can be subdivided into two clades infecting solely bats or rodents, with no arthropod vector involved in the infective cycle. A fourth cluster, gathers together the insect-specific flaviviruses (ISFV), has recently been defined and characterized (Aedes spp. and Culex spp.). ISFVs do not infect any vertebrate host (Table 1). Finally, Tamana bat virus (TABV), which infects exclusively mammalian cells, shows no serological relationship with any other flavivirus, and has only very distant phylogenetic relationships with them. Its taxonomic position, therefore, is not well defined (Most flaviviruses are transmitted to vertebrate hosts by the bite of haematophagous arthropods (thus classifying them within the heterogeneous group of arboviruses). Flaviviruses have traditionally been assigned to one of three clusters according to their arthropod vectors : mosquitcterized . It is t defined .1 and the Centers for Disease Control2 therefore both cite flaviviruses as a global health threat.Certainly, flaviviruses pose health problems for humans (and some other vertebrates) that may be associated with enormous social and economic costs. Over the last decade, the number of outbreaks of flavivirus-induced disease has increased all over the world. The main causes include the geographic expansion of their mosquito vectors, and increasing human travel to the areas of highest infection risk. They cause non-specific symptoms in the initial phase of infection in humans, which hinders their control, and as for other RNA viruses, no efficient therapeutic or immunoprophylactic strategies have been developed. The World Health OrganizationThe functional importance of the highly conserved structural genomic RNA domains in different RNA viruses  renders Figure 1A). This then reaches the surface of the endoplasmic reticulum (ER) where the molecular environment that allows the viral cycle to proceed is created, while preventing interferon response signaling .During the early phase of the flavivirus cycle, the viral genome is mainly employed as mRNA in viral protein synthesis. The initiation of translation occurs in a cap-dependent manner. In DENV, the process starts with the binding of the eukaryotic initiation factor 4E (eIF4E) to the 5′cap, and the further recruitment of eIF4G and eIF4A . This riOnce viral proteins levels are appropriate, the ER membrane undergoes structural rearrangements that promote the formation of replication complexes . In addiIt has been recently shown that flaviviruses suppress host protein synthesis in human cells early post infection (host translation shutoff) while viral RNA translation is maintained . This stFigure 1A). Finally, the infectious particles are released by exocytosis at 8–10 h post infection (hpi). Peak extracellular virus titres are usually observed at 18–24 hpi  at the ER . The res8–24 hpi .7GpppAmp) . The flanking UTRs are defined by discrete, functionally active structural RNA elements that play important roles in the viral cycle. These can be divided into essential partners in the infection process  and other elements not essential for viral RNA propagation but which help to regulate the processes involved. The functional RNA elements of all flaviviruses appear as highly conserved, complex folding regions, despite the lack of extensive sequence conservation across the Flavivirus genus   but it lGpppAmp) . The RNAGpppAmp) . It servGpppAmp)  to yieldGpppAmp)  . The 5′UTR is relatively short in comparison with that of the IRES-dependent members of the Flaviviridae family. Different isolates of the same flavivirus show strong sequence conservation, and significant identity is observed among members of the same flavivirus group. Less nucleotide conservation is seen among members of different groups .Various functional RNA elements have been identified in the 100 nt-long 5′UTR and the 5′ end of the coding sequence of the flavivirus genome  . The SLA architecture is recognized by viral RNA polymerase (NS5), an RNA-dependent-RNA polymerase (RdRp) involved in viral replication  that emerges from it. The size of the essential SSL stem and the sequence of its loop vary across flaviviruses  . An oligo(U) tract providing an at-least-10 nt spacer between the two stem-loop structures has been observed in DENV .In most flaviviruses, including DENV and WNV, a second, smaller stem-loop (SLB) is present downstream of SLA that shows a certain variability in its size and shape . The SLB in DENV . In WNV,Figure 2). It was first identified in DENV, and despite the reduced conservation of its sequence it was predicted to be preserved in the mosquito- and TBFV flaviviruses  – was first described in the DENV genome. It was shown to modulate the DENV life cycle in mammalian and mosquito cells, likely acting during a post-RNA synthesis stage and possibly regulating viral assembly , which show different degrees of sequence and structure conservation across members of the genus, with the 3′ extreme region – known as small hairpin 3′ stem-loop (sHP-3′SL) – the most conserved of all. A defining feature within the 3′UTR is the presence of duplications of structural cassettes. These are composed of various structural elements in MBFV and TBFV, but not in ISFV or NKV flaviviruses. Compelling experimental evidence indicates each duplicated cassette to play a different role in viral replication. An association between the duplication of structural elements and the capacity of the genome to replicate in mammalian and arthropod hosts has been established (for review see The 3′ end of the genome terminates in a 700 nt-long untranslated region (3′UTR) that lacks a poly(A) tail. It ends in a conserved CUcleotide  in MBFV cleotide . The 3′Ucleotide . Its strFigures 3, 4); in YFV (Table 1), the hypervariable region folds into an AU-rich stem-loop (SL-I) followed by a highly conserved branched element (SL-II) immediately preceded by a short conserved hairpin (RCS3) . This structural unit (SL-I•SL-II•RCS3) is repeated to yield the SL-III, SL-IV, and CS3 elements . Deletion and sequence mutation analyses of SL-I and II, and of the motifs RCS3 and CS3, have revealed their roles as regulatory replication elements . The formation of this PK is critical for infectivity (Domain I is located just downstream of the translation stop codon. In most flaviviruses it appears as a hypervariable sequence followed by two conserved stem-loop domains (SL-I and -II) similar in sequence and structure   . In the DENV and JEV groups (Table 1), it contains a sequence motif duplicated in tandem (RCS2 and CS2) that forms an essential component of the respective functional dumbbell structures 5′DB and 3′DB  . This architecture is functionally important for viral replication (Domain II is moderately conserved and in MBFV and NKV flaviviruses contains a characteristic structure known as a dumbbell (DB); this is involved in the formation of a PK structural element (igure 3) . YFVs coigure 3) . This stlication , translalication  and infelication . NKV flalication .Figures 3, 4). The presence of both has been confirmed by chemical probing  has been shown indispensable for virus replication in DENV . In addition, all flaviviruses show a bulge in the upper portion of the 3′SL stem . This bulge induces a bend in the duplex, which might be required for NS5 protein recognition. The functions of sHP and 3′SL have been studied in depth, and are essential for viral replication  and 3′SL . This resistance to Xrn1 activity is dependent on specific residues; these have been elucidated for WNV , which is essential for sfRNA generation. In the DENV and JEV groups (Table 1), this structure has been located within the SL-I and SL-II of Domain I of the 3′UTR, respectively , while in YFV the single SL (SL-E) provides the stalling point . In MBFVs, it has been reported that the abrogation of PK1 leads to the production of shorter species of sfRNAs derived from the Xrn1 stalling at the downstream pseudoknot structures PK2  and/or PK3 [5′DB in JEV and DENV and DB in YFV ]  ranging from 300 to 500 nt-long accumulate in the cytoplasm . These m for WNV , YFV ] . Consecugure 4)] . Thus, rgure 4)] .From a functional point of view, full-length sfRNAs play important roles in regulating the switch between translation and replication during the infectious cycle . They prThe acquisition of a circular conformation in viral RNA genomes is a successful strategy that provides important advantages in the completion of the infective cycle. First of all, it efficiently ensures the propagation of undamaged, full-length genomes . FurtherFigure 4). Such interactions have been probed by psoralen/UV crosslinking assays . These include:In MBFV, at least three pairs of sequence motifs have been shown to participate in the cyclization process ((i)Figure 3) just upstream of the sHP domain at the 3′ terminus of the genome. It contains an 8 nt-long stretch conserved across the MBFVs. The 3′CYC perfectly matches its complementary partner in the cHP domain at the extreme 5′ end (5′CYC) . The 5′–3′CYC interaction  must be preserved for efficient virus replication  – is included in the conserved sequence CS1 ( (5′CYC)  Figures 4). It has been suggested that switching from the formation of the stem to the long-distance interaction with the 5′UAR releases the 3′ terminus of the viral genome for recognition by the flaviviral RNA polymerase (NS5) during the initiation of the minus-strand RNA synthesis . The UAR pair, which involves residues upstream of the AUG start codon at the 5′ end of the viral genome, 5′UAR, and a complementary sequence located within the basal portion of the stem in the 3′SL element, 3′UAR  Table 1), two DAR motifs have been described – 5′DAR I and 5′DAR II – within the stem and the base of the SLB domain, which interact, respectively, with 3′DARI and 3′DARII . During the initiation of minus-strand RNA synthesis, NS5 first recognizes the SLA element and the 5′DARII in the context of a circular RNA, and interacts with 3′DARI and II, probably leading to the initiation of viral replication . These findings suggest a role for protein recruitment in DAR interactions and the subsequent genome cyclization process. The DAR sequences motifs. In the DENV group, a single sequence motif 5′DAR within the linker between the SLB and cHP stems (at the 5′ end of the genome) interacts with the corresponding complementary 3′DAR sequence (included in the CS1 sequence) within the sHP stem at the genome 3′ terminus. In the JEV group  as potential anti-WNV drugs has been reported . Two oliCellular RNA interference (RNAi) has also been widely examined in recent years as a means of generating novel antiviral RNA molecules. This strategy is based on the design of short, double-stranded RNA molecules , which are loaded into the RNA-induced silencing complex (RISC). The sense strand of the duplex then guides the complex to the target region, where it base-pairs fully to induce degradation of the target RNA molecule. As antisense oligonucleotides, siRNAs can be chemically produced or endogenously synthesized from appropriate expression vectors. Numerous authors have reported the use of siRNAs – both in cell culture and in infected mice – against the coding region of the WNV genome  and the The use of RNA or DNA aptamers  represents another promising strategy for developing antiviral agents against flaviviruses. They also provide an interesting means of developing molecular tools for deciphering the functional role of genomic structural elements, and therefore the identification of potential therapeutic targets. This has already been shown for other, closely related viruses such as HCV  as well The successful clinical use of any of the above strategies is conditioned by the appearance of resistant mutants. In fact, WNV particles resistant to PMOs targeting the conserved 3′UAR sequence motif have already been isolated . They coFlavivirus, provide alternative and complementary potential targets to the viral proteins for novel antiviral compounds. Advances made in the field of nucleic acid synthesis have provided excellent candidate molecules for fighting RNA viruses by interfering with the essential functions performed by their genomic functional domains. Different pharmaceutical companies are now investigating the potential of nucleic acid therapeutic strategies, assessing long-term antiviral responses and trying to minimize secondary effects.The acquisition of compact genomes was an important evolutionary achievement of RNA viruses; these genomes can store all the information required for the completion of the infectious cycle in reduced packages. This is possible due to the existence of a supracoding system beyond the nucleotide sequence, defined by discrete, folded domains, and higher-order structures. These elements operate both alone and in combination to create complex networks of contacts that regulate multiple steps of the viral cycle, and to recruit host and viral factors. Understanding how host–virus interactions shape viral evolution will help to elucidate the factors that govern the emergence of new viruses and the expansion of already known RNA viral pathogens. The lack of technics or experimental approaches to determine the RNA structure and to analyze the kinetics of RNA–RNA interactions in cell culture, together with the lack of experimental strategies to specifically interfere with the folding of the RNA genomic elements, represent an important limitation for understanding their function in the viral cycle. Importantly, the phylogenetic conservation of the genomic RNA structural domains and their interactions across members of All authors participated in writing the manuscript (led by CR-L and AB-H), commented upon, and approved its final version. AF-S and PR-M prepared the figures.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.