The ROC hosted this workshop to facilitate collaborations among those researchers formalizing concepts in RNA, those developing RNA-related software, and those performing genome annotation and standardization. The workshop included three software presentations, extended round-table discussions, and the constitution of two new working groups, the first to address the need for better software integration and the second to discuss standardization and benchmarking of existing RNA annotation pipelines. These working groups have subsequently pursued concrete implementation of actions suggested during the discussion. Further information about the ROC and its activities can be found at  The RNA Ontology Consortium is an international coalition of RNA researchers working to develop controlled vocabularies pertaining to RNA function and based on RNA primary sequence, secondary structure, and tertiary structure. Launched in 2005 with funding from the National Science Foundation, the ROC includes nine working groups addressing the ontology needs of RNA domains ranging from backbone conformation to multiple sequence alignment. The Consortium has published an RNA Ontology (RNAO)  and an RThis workshop furthered the goals of the consortium by making progress on several of the RNA structure mapping concepts elaborated upon during previous ROC meetings in Kona and in Strasbourg. In addition, it brought together members of the ROC with members of the Genomic Standards Consortium (GSC) , an openThe overall structure of the meeting consisted of a working dinner, software presentations, a roundtable discussion on annotation challenges, and assembly of working groups to address these challenges. The major annotation issues that were identified included: 1) How can information that is useful for drawing/representing RNA structures be exchanged among different software programs and incorporated into different genomic and metagenomic web sites for display to end users? 2) How can information about covariance models automatically be used to infer correspondence groups? 3) How can information about structure mapping experiments be captured to facilitate automatic secondary structure inference and propagated and displayed in a way that is useful to end users?Eric Nawrocki provided an introduction to structural RNA homology search using Infernal (INFERence of RNA ALignment) [ignment) . He noteJesse Stombaugh presented a live demonstration of Boulder ALE (ALignment Editor) [ Editor) , a tool  Editor)  inputs fZasha Weinberg introduced R2R, software new application to aid in drawing publication-quality figures for single and consensus RNA structures [ructures . The goaAfter the three software presentations, Day One proceeded with an extensive round-table discussion of the role of the RNAO in annotation pipelines for RNA discovery. While protein (gene) annotation is a relatively mature field, RNA annotation is much less mature. Given this fact, there is an opportunity to implement ontological structure and support from the very beginning of the standardization of the field. The discussion focused on identifying ontological needs for end users who create RNA annotations or integrate them into larger genome resources.In such a scenario, one key use for ontologies would be to identify the provenance of RNA sequences incorporated into annotation pipelines and describe the techniques used to generate these sequences. It was noted that researchers performing RNA annotation on sequences generated for protein-level work (i.e. genome and metagenome sequences) may be unaware of quality issues associated with these sequences. For example, some sequencing facilities pre-filter genomic and metagenomic sequences to remove low-quality sequences before distributing the sequences to researchers. However, the researchers may not be given the parameters and software tools that were used for the pre-filtering step and be unaware that filtering steps had been applied with a potential loss of RNA sequences as a consequence.This caveat also applies to RNA structure information. It was agreed that evidence codes, analogous to those applied to gene annotations, to describe the process by which publicly available RNA secondary and 3D structures were constructed would be extremely useful. In the ideal situation, service(s) would exist that could be queried with a structure identifier and would return both an evidence code summarizing the quality of and backing for the structure model as well as a persistent identifier linking to more detailed metadata. Participants also argued that improving interoperability and comparability of annotation tools and pipelines is a major need. Day Two was therefore structured to create time for two new working groups to form and meet, the first to address the need for better software integration and the second to discuss standardization and benchmarking of existing RNA annotation pipelines.This working group includes the developers of various tools for building, visualizing and manipulating RNA alignments and structure mapping information , as well as the developers of the Single Nucleotide Resolution Nucleic Acid Structure Mapping (SNRNASM) standard for capturing information, describing and reporting single-nucleotide resolution nucleic acid structure mapping assays ,12. The One issue that was identified was that some databases such as Rfam  do not pAs a result of the meeting, the authors of the three packages  agreed to modify their programs to use the extended format. It was also decided to encourage the developers of other software, notably VARNA , to adopConsistent RNAannotation is a major need of both producers of genomic and metagenomic data, and researchers analyzing vast numbers of sequences currently being generated. It was proposed that standardization of such annotations could be brought under the umbrella of the GSC.A clear path forward to standardization of RNA annotation would be to set up an open competition in which existing annotation pipelines perform annotation of RNAs on a gold-standard data set. The pipeline with the best results, i.e. the one that correctly identifies and annotates most known RNAs, would be the ‘gold standard’ for that year, and the results of the competition would be made available to all pipeline developers for use in improving their tools. One point raised was that most current pipelines include significant pre-filtering steps, which are frequently not made public with the pipeline or described in detail. Such a competition could address the question of whether or not these pre-filtering differences meaningfully affect results and, if so, encourage open discussion of these steps.The working group included researchers with access to completed, but currently unpublished genome data that could serve as the gold-standard data set. In addition, members began investigating potential funding opportunities to support the administration, implementation, and publication of the proposed competition.This meeting provided a forum for knowledge exchange and discussion and led to the establishment of two working groups to promote work on software integration and standardization of RNA descriptions , ideally under the auspices of the GSC. We established an extension of the Stockholm format for annotating alignments that is compatible with Infernal, Boulder Ale and R2R. This format, based on that of R2R, will allow people to make 2D diagrams from their alignments, and will be used to improve covariance models in Infernal. Overall, the prospects for improved RNA annotation are bright, and the meeting was extremely productive in bringing together members of previously non-overlapping communities, this time also including several leading members of the GSC. The participants thank the ROC and the NSF for the opportunity to hold this meeting, which all found extremely valuable.


Climate change has emerged as a principal threat to coral reefs, and is expected to exacerbate coral reef degradation caused by more localised stressors. Management of local stressors is widely advocated to bolster coral reef resilience, but the extent to which management of local stressors might affect future trajectories of reef state remains unclear. This is in part because of limited understanding of the cumulative impact of multiple stressors. Models are ideal tools to aid understanding of future reef state under alternative management and climatic scenarios, but to date few have been sufficiently developed to be useful as decision support tools for local management of coral reefs subject to multiple stressors. We used a simulation model of coral reefs to investigate the extent to which the management of local stressors  might influence future reef state under varying climatic scenarios relating to coral bleaching. We parameterised the model for Bolinao, the Philippines, and explored how simulation modelling can be used to provide decision support for local management. We found that management of water quality, and to a lesser extent fishing, can have a significant impact on future reef state, including coral recovery following bleaching-induced mortality. The stressors we examined interacted antagonistically to affect reef state, highlighting the importance of considering the combined impact of multiple stressors rather than considering them individually. Further, by providing explicit guidance for management of Bolinao's reef system, such as which course of management action will most likely to be effective over what time scales and at which sites, we demonstrated the utility of simulation models for supporting management. Aside from providing explicit guidance for management of Bolinao's reef system, our study offers insights which could inform reef management more broadly, as well as general understanding of reef systems. Coral reefs are of immense natural and anthropocentric value. Aside from boasting the highest diversity of all marine ecosystems Given that climate change impacts on coral reefs cannot be mitigated directly, the question arises whether reduction of stressors that originate and can be managed at a local scale  provides a tractable opportunity to increase the potential of coral reefs to cope with inevitable changes in the climate Inherent to assessing the importance of local management action is knowledge of the combined impact of multiple stressors Models are ideal tools for tackling questions regarding cumulative stressors at appropriate spatial and temporal scales, and for supporting environmental decision-making in general, through elucidating the consequences of alternative management actions in the context of global climate change Models have been used widely to aid understanding of the impact of stressors on coral reefs Recent work by Melbourne-Thomas et al. Here we use a simulation model to investigate the extent to which management of local-scale stressors might influence future trajectories of coral reefs under varying climatic scenarios. We use a model originally developed by Fung et al. Thus our study has two distinct aims: first, to ascertain how management of local stressors might affect future trajectories of coral reefs under varying bleaching scenarios. We address this by investigating (1) the cumulative impact of fishing, poor water quality (i.e. nutrification and sedimentation) and bleaching-induced coral mortality on reef systems, and how their interactions manifest over time; and (2) patterns of reef recovery following bleaching. Our second aim is to assess the utility of simulation modelling for providing decision support for local-scale management of coral reef systems subject to multiple stressors, which involves providing guidance on which courses of management are most likely to be effective in different locations of the Bolinao reef system and over what time scales. This assessment also involves identifying the circumstances under which projections of future reef state are likely to be most certain.The University of The Philippines Marine Science Institute (UPMSI) has been given Prior Informed Consent (PIC) by the Mayor of Bolinao, Pangasinan, to undertake the research from which data was drawn for this modelling analysis. The PIC is continually updated by the Deputy Director for the Bolinao Marine Laboratory. Presently the director is Dr. Ronald Villanueva, and previously, during the research cited, the director was Dr. Antonette Juinio-Menez. Since there were no materials transferred outside the country, there was no need for a gratuitous permit for materials transfer from the Bureau of Fisheries and Aquatic Resources (BFAR) of the Department of Agriculture (DA). Since the sites are not part of the National Protected Areas System, the PIC from the Mayor was sufficient. Data from 1988 & 1995 was collected by Dr. Cleto Nanola, under the UPMSI Community Ecology (ComEco) Lab with permission from Dr. Porfirio M. Aliño as the ComEco Lab director. During this period (1988 – 1995), no PIC was required to undertake field observations in non-protected areas such as those cited in the publication. For all studies only visual censuses of fish and benthic communities were conducted; no fauna or flora were collected or manipulated.Ecological dynamics are simulated using a local-scale mean-field model developed by Fung A range of natural and anthropogenic stressors can be applied as external stressor functions (hereafter ‘forcings’), that include nutrification and sedimentation , and fishing and coral bleaching  in the Guiguiwanen Channel since 1995 The Bolinao reefs are highly degraded, with the most significant stressors being poor water quality and fishing The Bolinao reef system provided an ideal case study to examine local-scale management of multiple stressors because: (1) reefs are heavily affected by numerous stressors originating from anthropogenic activities and climate change; (2) marine governance is undertaken at the local-scale; and (3) data were available to parameterise and validate the model.viz. Tomasa, Lucero, Cangaluyan and Malilnep , coral mortality due to bleaching (three levels) and water quality  Simulated future reef states were analysed using multivariate statistical techniques, and by examining model trajectories under these scenarios. Both approaches provide useful complementary insights into the effect of stressors on reef function. We used multivariate analyses to analyse modelled reef states for each site at four points in time selected and variability in reef state (i.e. dispersion), we used permutational analysis of multivariate dispersions (PERMDISP) a posteriori pairwise comparisons to identify the treatment groups that differed. Following the method of Anderson Given that terms in the PERMANOVA can be significant due to differences in mean reef state (i.e. location) only, or differences in reef state −2 and %). 4,999 permutations of the residuals under a reduced model were used to calculate p values for PERMANOVA, PERMDISP and CAP analyses.We used canonical analysis of principal coordinates (CAP) We examined trajectories of reef state over 40 years for six of the 18 scenarios, representing all combinations of the different levels of fishing and water quality under the climatic scenario of decadal bleaching. These scenarios were chosen based on results from the statistical analysis and because fishing and water quality, but not bleaching, can potentially be managed at a local scale. Analysis of the trajectories allowed us to visualise changes in the functional groups driving differences in reef state detected in the PERMANOVA, and to assess whether these changes were meaningful in a management context. Further, trajectory analysis elucidates patterns of recovery following bleaching events under alternative management scenarios. Here we define coral recovery as the rate at which coral cover increases after simulated bleaching. To allow assessment of coral recovery, the magnitude and timing of bleaching events was fixed for each scenario and site for the trajectory analysis. Bleaching events were set to occur 20 and 38 years into the future and to reduce coral cover by 30% and 15% respectively.Modelled reef communities differed significantly under the alternative management and climatic scenarios, in terms of multivariate location (i.e. mean composition of the reef) and, in some cases, dispersion  in both the PERMANOVA and PERMDISP analyses. However, the F×B interaction occurred only where a significant three-way interaction was also evident, and thus will be analysed only in the context of the three-way interaction. In considering results, first we characterise the NS×F and NS×B interactions in terms of reef state only, through examining those terms which were significant in the PERMANOVA but did not differ in dispersion between scenarios (i.e. non-significant PERMDISP results). Second, we investigate NS×F and NS×B interactions terms which were significant in both the PERMANOVA and PERMDISP, which affords some insights into the predictability of future reef condition under alternative management and bleaching scenarios. Finally, we examine the nature of significant NS×F×B interactions, guided by our analysis of the two-way interactions.The precise nature of the NS×B and NS×F interactions differed among scenarios, but in each case were consistent across all sites and time periods. For both interactions, reef state under scenarios in which nutrification-sedimentation was present (at any level of the other stressor), was characterized by high macroalgae cover and low piscivorous and herbivorous biomass , 3. GiveFor the NS×B interaction, while there was a significant distinction between levels of bleaching when this stressor acted alone, when bleaching occurred together with nutrification-sedimentation, there was no difference between the levels of bleaching . This waConversely, for the NS×F interaction, all levels of one factor were significant at each level of the other factor, but the combined effect of both impacts was less than the sum of the individual effects . FollowiPatterns of dispersion differed between the NS×B and NS×F interaction, but in each case were consistent across all sites and time periods. For both kinds of interactions modelled reef state was significantly less variable for scenarios in which nutrification-sedimentation was present , and thiFor the NS×B interaction dispersion of modelled reef state did not differ significantly between levels of bleaching for either level of nutrification-sedimentation . Howevera posteriori pairwise tests, there was a risk of compounding of Type I errors . However, since significant terms were highly significant  and non-significant terms were highly non-significant , there is no ambiguity in the results.Three-way interactions between nutrification-sedimentation, fishing and bleaching, were significant only for Tomasa and Cangaluyan . All of Patterns in dispersion of reef state identified for the two-way interactions were similarly evident for third order interactions. Following the method of Anderson et al. −2 for all sites. For reference, Nañola et al. −2 as ‘very low’ in their report on the status of Philippine coral reefs. Thus fish biomass was too small to be meaningful in a management context, suggesting that regulating fishing will have negligible effect when water quality is poor.Our analysis of reef state trajectories for various fishing and water quality scenarios under decadal bleaching revealed that differences in reef state under various scenarios detected in the PERMANOVA were driven by changes in all functional groups . The dom-2. This is largely because fish recruitment at Tomasa was constrained by low coral cover . While statistical analysis indicated that there was a significant difference between all fishing and water quality scenarios, trajectory analysis showed that these differences are not ecologically meaningful and thus are irrelevant in a management context for Tomasa. Therefore, projected responses to management were strongly non-linear and depended on the initial state of the reef.Under scenarios in which nutrification-sedimentation was not present, the effect of different levels of fishing on reef state was evident for all functional groups at all sites (except Tomasa in regards to fish). Coral cover increased as fishing pressure decreased for all sites; after 40 years coral cover was 15% higher at Lucero, 24% higher at Malilnep, 300% higher at Tomasa and 400% higher at Cangaluyan, under the zero fishing scenario compared with the high impact fishing scenario. While the differences in coral cover between fishing scenarios were highest for Tomasa and Cangaluyan, absolute coral cover was less than 3% at Tomasa and less than 12% at Cangaluyan for all scenarios. Further, at Tomasa, under all scenarios of fishing and water quality, modelled fish biomass was almost identical and was less than 500 kg kmAnalysis of the trajectories for scenarios in which nutrification-sedimentation was absent  highlighted that management of local stressors influenced patterns of recovery after bleaching events, although this was dependent on initial reef state . For allThe increase in coral cover varied between sites (in relative and absolute terms) reflecting initial reef state; i.e. changes in cover was highest at Lucero (which increased by more than 50%), followed by Malilnep and Cangaluyan. The exception to this trend was Tomasa, where the relative increase in coral cover (when fishing was present) was 50% during 2028–2044, although the absolute increase in cover was only 1%. In Tomasa and Cangaluyan during 2028–2044 changes in coral cover – i.e. very small increases in coral cover of 1–2% or maintenance of very low coral cover – were mainly due to external coral recruitment. Thus coral recovered more quickly after bleaching events at those sites which were subject to fewest additional stressors and disturbances, and were least degraded (and thus had higher endogenous recruitment). These results suggest that if coral becomes too degraded, such as at Tomasa, coral will not recover from bleaching-induced mortality irrespective of the absence of other stressors.While reducing the impact of local stressors is widely advocated to bolster coral reef resilience under anticipated future changes in the climate Integral to assessing how management of local-scale stressors might influence future trajectories of reef state is investigating the cumulative impact of those stressors, including whether the effect of multiple stressors is the sum of single-stressor effects (additive), or an amplification (synergism) or a reduction (antagonism) relative to an additive effect The antagonistic interactions between stressors identified in this study may be driven by the asymmetric effects of the three stressors. The individual effect of poor water quality on overall reef state was much greater than the individual effect of the two other stressors, particularly bleaching. This suggests that the interactions between poor water quality and the other two stressors are caused by the dominant impact of nutrification-sedimentation. Antagonistic interactions driven by a dominant stressor have been identified in other studies Management of multiple stressors requires an understanding of how interactions between stressors manifest in different contexts. This is particularly pertinent to coral reef management given that reefs are almost always subject to more than one stressor A critical component of understanding the importance of managing local-scale stressors to conserve coral reefs in the context of future climate change is knowledge of the factors influencing coral recovery post bleaching. We found that simulated coral recovery following bleaching was greatest at sites that were least degraded and subject to the least impact from local stressors. For the most degraded site (i.e. Tomasa), coral did not recover (under high and medium levels of fishing) or showed very little recovery (under no fishing), with a only 1% increase in absolute coral cover over 18 years. This indicates a hysteresis effect, and suggests that if coral cover becomes too depleted recovery is not possible even in the absence of stressors. This is in part because endogenous supply of coral recruits is less for degraded reefs, with a number of studies showing that coral recovery is highly dependent on the extent of remnant coral survival These results support consensual understanding of conditions which foster coral resilience following bleaching-induced coral mortality Our research supports the paradigm that managing local-scale stressors is critical to the persistence of coral reefs in the context of global climate change, a concept that is widely advocated For our study sites at Bolinao, poor water quality emerged as the pre-eminent driver of deterioration of coral reefs, and overrode the effects of fishing and bleaching. The combined effect of excess nutrient and sediment input is well known to degrade real coral reef systems The utility of simulation modelling to environmental management lies in the capacity of models to estimate future ecosystem state under alternative management and climatic scenarios. Through this process, models can greatly aid identification of drivers of degradation and consequently provide guidance on appropriate management actions, where they will most likely to be effective and over what time scales. Given that resources for management are limited and that management is often constrained by human use of marine resources, this information also usefully informs a ‘triage’ approach to management, whereby locations for undertaking management actions are prioritised according to where actions are most likely to be effective given existing limitations such as resources, time and knowledge.We have demonstrated how simulation modelling can be used to support coral reef management at local scales. Our results clearly indicate that reefs in Bolinao would benefit from improving water quality above all other stressors, because even in the absence of fishing and bleaching, the present levels of nutrient enrichment and sediment deposition are likely to lead to loss of corals within a short time scale (within 12 years). Improvement in water quality could be achieved by regulating the aquaculture industry, including reducing the stocking density and number of fish cages. If water quality is improved, our results suggest that under decadal bleaching the present level of fishing can be supported at Malilnep and Lucero, and half of the present level at Cangaluyan, without further decline in coral cover. Conversely, any management intervention at Tomasa, including action to improve water quality, is unlikely to make a meaningful difference to reef health over at least a 40 year time frame. Thus, responses to management are strongly non-linear depending on the state of the reef, and management focused initially on the Lucero and Malilnep will likely yield greatest recovery for the least investment. This example highlights the importance of site-specific information in management.Critical to using models to forecast future reef state under alternative scenarios is consideration of the predictability of those trajectories. Variability in modelled reef state (i.e. dispersion of modelled reef state between model runs) affords insights into the predictability of future reef state. We found reef state more predictable for degraded reefs, especially if the reef was subject to high impact stressors , because these sites converge on a reef state dominated by algae with few fish and coral with low probability of recovery. Note however, that predicting the exact future state of a reef under a given scenario is not our intention, nor is it necessary to achieve good management outcomes. Rather, managers are more interested in broad-scale trends that indicate whether reefs will be broadly healthy or degraded.Further, in considering the utility of simulation modelling to environmental management, it is important to acknowledge the host of other biological and socioeconomic factors which were not included in our modelling but which will greatly influence decision making in regards to management of fisheries, bleaching and water quality in Bolinao and elsewhere. For example, marine reserve design may be contingent upon factors such as the presence of threatened species, distribution of fishing effort by gear type or cultural practices associated with fisheries. Thus, we suggest that simulation modelling be used in combination with other decision support tools such as fisheries stock assessment models Uncertainty is inherent to modelling complex systems Uncertainty is related to our model validation process mainly due to the paucity of empirical observations of reef state in Bolinao in the past. We validated the model for each of the four sites through assessing whether the model could reproduce reef dynamics from 1987–2008 given empirical data on reef state in 1987 and a known series of stressors at these sites ,Text S3.Given imperfect knowledge of both future impacts and how a system will react to these factors Uncertainty is also related to the scenarios under which projections about the future are made. For instance, the frequency of bleaching events assumed in the future scenarios modelled here were predicted using climate models In summary, there are inevitably sources of uncertainty in scenario analysis which translates to uncertainty in the associated projections. In this sense the kind of stochastic simulation model used here is realistic because uncertainty about the future is inevitable and is captured through the model providing a probabilistic spectrum of possible futures for a given scenario. Future scenarios are intended to illustrate the range of possibilities for the future, rather than predict actual trajectories Our analysis of potential reef state under alternative management and climatic scenarios highlights two key principles critical to management of coral reefs in the face of climate change. First, local-scale reef management to improve water quality and prevent overfishing can play a significant role in conserving coral reefs under the expected effects of global climate change. Second, to develop effective management actions it is critical to consider the combined impact of multiple stressors, and potential interactions between them. However, the relative importance of different stressors and how they interact is likely to vary with context, ensuring that generalization and extrapolation of specific findings is difficult. Therefore, managers require decision support tools to identify drivers of degradation for particular reefs, allowing the development of site-specific management. Simulation models, such as the one used here, are ideal tools in this endeavour, and can further support management decisions by providing insights into the efficacy of different management approaches under alternative climate (or other) scenarios. Thus we suggest that simulation modelling offers a critical and much needed opportunity for aiding local-scale management of coral reefs.Figure S1The ecological processes between the seven functional groups in the local-scale mean-field model.(DOCX)Click here for additional data file.Figure S2Historical timeline of stressors to Bolinao's coral reef system during 1987 – 2008.(DOCX)Click here for additional data file.Figure S3Comparison of empirical observations and model trajectories of benthic covers in the four sites at Bolinao from 1987 – 2008.(DOCX)Click here for additional data file.Figure S4Comparison of empirical observations and model trajectories of consumer biomass in the four sites at Bolinao from 1987 – 2008.(DOCX)Click here for additional data file.Figure S5Mean distance-to-centroid in Euclidan space of the 20 predicted community states (i.e. results from Monte Carlo simulations) for each of the 18 scenarios for Cangaluyan after 10 years.(DOCX)Click here for additional data file.Table S1Parameter definitions and values derived for Bolinao, the Philippines.(DOCX)Click here for additional data file.Table S2Exogenous recruitment parameters for consumer groups used for historical trajectories.(DOCX)Click here for additional data file.Table S3Additional forcings (i.e. stressors) used in model validation.(DOCX)Click here for additional data file.Table S4Validation data for the historical reconstruction trajectories for Bolinao.(DOCX)Click here for additional data file.Text S1Model equations.(DOCX)Click here for additional data file.Text S2Additions to the model for Bolinao instantiation.(DOCX)Click here for additional data file.Text S3Model validation.(DOCX)Click here for additional data file.


Overall, choice order depended on the degree of relative preference among alternatives and a peak-first bias, providing evidence for prospective evaluation when choice order is self-generated.Animals typically must make a number of successive choices to achieve a goal: e.g., eating multiple food items before becoming satiated. However, it is unclear whether choosing the best first or saving the best for last represents the best choice strategy to maximize overall reward. Specifically, since outcomes can be evaluated prospectively (with future rewards discounted and more immediate rewards preferred) or retrospectively (with prior rewards discounted and more recent rewards preferred), the conditions under which each are used remains unclear. On the one hand, humans and non-human animals clearly discount future reward, preferring immediate rewards to delayed ones, suggesting prospective evaluation; on the other hand, it has also been shown that a sequence that ends well, i.e., with the best event or item last, is often preferred, suggesting retrospective evaluation. Here we hypothesized that when individuals are allowed to build the sequence themselves they are more likely to evaluate each item individually and therefore build a sequence using prospective evaluation. We examined the relationship between self-generated choice order and preference in rhesus monkeys in two experiments in which the distinctiveness of options were relatively high and low, respectively. We observed a positive linear relationship between choice order and preference among highly distinct options, indicating that the rhesus monkeys chose their preferred food first:  Animals live in the present but benefit from remembering the past and predicting the future. For goal-directed behavior, it has long been appreciated that many animals, including humans, consider future events, but discount their value based on the delay to receiving them, indicating that an immediate reward is more valuable than the same amount of reward in the future peak-end rule in which people judge previous events by averaging the peaks  and the end peak-end bias. Indeed, there are numerous examples in everyday life that appear to reflect this preference: from dessert at the end of meals to the climax of stories or performances normally occurring at the end.In fact, this retrospective evaluation process has been well-documented in people, leading to what has been called a Thus, outcome evaluation appears to be a decreasing function of distance in time from the present to both the future and the past. However, the conditions under which individuals use prospective or retrospective evaluation to guide behavior remain unclear. We hypothesize that the type of evaluation process evoked depends in part on the extent to which a series of events are organized into a unit or episode. When individual events are seen as parts of a larger episode, the evaluation process may be conducted on the episode as a whole once it is completed. In this case, memory is needed, and retrospective evaluation would occur. If, however, the events are seen as separate entities, prospective evaluation would take place, with each event being processed individually in order to maximize immediate reward.first one As a test of this hypothesis, we conducted a study with rhesus monkeys, which have proven to be an excellent primate model of human decision-making; and at the same time help delineate the evolutionary origins of these evaluation processes. It had previously been shown that when receiving a sequence of qualitatively distinct rewards, rhesus monkeys did not exhibit a clear peak-end bias, and if anything, appeared to show a peak-first sequence. Thus, for the current study, we asked, if given a sequence of choices without replacement, in which all items are initially available and an individual must select them in their preferred order, which evaluation process would be used? Subjects who determined the sequence retrospectively should have a peak-end bias and should choose in the opposite order of their preference, saving the best for last, whereas those who chose prospectively, should have a peak-first bias and should choose in the order of their preference, selecting their favorite first and so on. We found that all four monkeys exhibited strong peak-first order preferences, but only when there were large enough differences in preferences among the food items.Following the logic of items being evaluated prospectively if considered individually, we hypothesized that if the monkeys were offered four qualitatively distinctive food items, which they could obtain in any sequence and were allowed to generate the sequence themselves, they would evaluate each item in the sequence individually, leading to prospective evaluation and a preference for a peak-Animal care and use complied with all current laws, regulations, policies, and guidelines of the United States, the United States Department of Agriculture (USDA), the Public Health Service (PHS), and all procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of Dartmouth College.Macaca mulatta) participated in Experiments 1 and 2: Hamlet, Caesar, Puck, and Titus. Hamlet, Puck, and Titus. The average age of the monkeys was 8.75±0.48 (mean ± s.e.m). They were housed in 32×27×68 (width x depth x height) inch cages  in a homeroom with automatically regulated temperature, ventilation, humidity, and lighting . The monkeys were intermittently housed in pairs and individually: at times when they engaged in confrontations, which is normal periodic behavior in young rhesus macaque males of similar size and temperament Four male rhesus macaques , supplemented with fresh fruit and vegetables, as well as various treats that included peanuts, cereal, and dried fruits .The Center for Comparative Medicine and Research (CCMR) at Dartmouth maintains a full-time animal care and veterinary staff that monitors the monkeys’ daily health and well-being. The monkeys were maintained at approximately 95% of their To allow for continued social stimulation, the subjects had direct visual contact with the other monkeys in the colony, the animal care staff, and experimenters. When pair-housed, they had direct physical contact with each other, and also when individually-housed, through a mesh grading divider between their cages. In addition, environmental enrichment included two or more enrichment items in their home cages at all times, daily playing of radio or videos in the room , and regular access to a larger enrichment cage (68×38×72 inch) in an adjacent room. The monkeys were brought to the testing room in custom-made chairs. The chairs were used (a) to minimize disruption in the test subjects’ daily routines, given that they were already acclimated to them from previous experiments; (b) to have precise control over the experimental testing conditions; and (c) to obtain clear, unbiased choice responses, with tray compartments (described below) at fixed positions relative to the monkey on every trial.Each monkey sat across from the experimenter in the chair. The chair loosely restrained the left arm of the monkey while allowing free movement of the right arm. An opaque plastic divider separated the experimenter and the monkey, thus preventing the monkey from seeing the face and upper body of the experimenter. An opening at the bottom of the divider allowed the experimenter to present a transparent plastic food tray to the monkey. The tray contained four separate compartments, which we labeled according to their position relative to the experimenter: left (L), middle left (ML), middle right (MR), and right (R). On a given trial, food was placed on a circular platform in a compartment so that the monkey could clearly see and easily select his choice of food item. Each compartment was covered by a transparent lid, which the monkey had to lift to gain access to a food item. The food items in Experiment 1 consisted of four different types of food, with all being generally yellow in color: shelled peanut halves (PN), yellow BioServ® Fruity Gems (FG), BioServ® banana-flavored dustless precision pellets (PL), and rice krispies®, or krisps (KR). The food items in Experiment 2 consisted of four differently colored  BioServ® Fruity Gems (FG).Both Experiments 1 and 2 consisted of a two-condition interleaved paradigm (described below) that studied ordered choice among four food items without replacement (Condition 1) and binary choice between two food items (Condition 2). Four different types of food  for Experiment 1 and four differently colored fruity gems for Experiment 2 were used. Because the binary choice condition allowed only one selection per trial, and the loss of the item not selected, we reasoned that choice in this condition would reveal preference. In the ordered choice condition without replacement, the monkeys would obtain all four food items regardless of order; and thus, selection order might or might not reflect preference. For example, an individual might select his favorite items first, or save them for last. The experiments, therefore, examined the specific relationship between choice order and preference.The conditions were interleaved in that each test session consisted of 1 trial of Condition 1  and 12 trials of Condition 2 . We interleaved the conditions to make sure choice order and preferences remained stable; and we conducted the ordered choice condition first to provide the monkeys with experience of all four food items before conducting the binary choice preference tests.In Condition 1, the ordered choice condition, a different food item was placed into each of the four compartments . The locTo measure food preferences, in Condition 2 we used the binary choice paradigm, in which two different food items were placed in the two middle containers (ML and MR), with one food item per container . To begiOnce the data was collected, for Condition 1, we calculated the choice rate of each item for a given choice order. The overall individual's choice order with respect to different items was determined by selecting an item that provides the highest choice rate for a given order. For Condition 2, we calculated the winning percentage of each food item, the percentage of winning in the binary choice paradigm. Then we obtained the rank order of individual preference by ranking individual's winning percentages of food items.F  = 408.0, p<0.001 for PN; F  = 148.8, p<0.001 for FG; F  = 7.4, p<0.01 for PL; F  = 7.7, p<0.01 for KR). This indicates that the four monkeys showed a similar choice order for each food item. Individually, all four monkeys exhibited significant choice orders for the different food items across sessions   = 204.0, p<0.001; Caesar: χ2(9)  = 192.0, p<0.001; Puck: χ2(9)  = 110.0, p<0.001; Titus: χ2(9)  = 158.7, p<0.001 ), indicating a stable choice order for each food item across sessions.In Condition 1, we tested the order in which the monkeys chose a food item among the four different food items without replacement. Overall, the monkeys predominantly chose the peanut half , fruity gem , pellet , and rice krisp  for their first, second, third, and fourth choices, respectively . There wF  = 54.8, p<0.001), indicating general food preferences in the monkeys.For Condition 2, we calculated the winning percentage of each food item using a binary choice paradigm that presented all possible combinations of two food items in order to measure preference between food items. 2(3)  = 154.9, p<0.001; Caesar: χ2(3)  = 143.1, p<0.001; Puck: χ2(3)  = 126.0, p<0.001; Titus: χ2(3)  = 120.8, p<0.001). The food preferences were the same for three of the monkeys , and somewhat similar for the other monkey . All four monkeys preferred the peanut half most, then the fruity gem. Overall, there were strong food preferences for all individuals and small individual differences in less-preferred items.Individually, all four monkey showed significant preferences toward an option among the four alternatives in Condition 2 .Because all four monkeys showed the greatest preference for peanut halves and the second-greatest preference for fruity gems it indicates general preferences for these food items. This general preference suggests there are particular factors, such as caloric value, that may underlie preference. Thus, we tested whether the winning percentage was correlated with the caloric values of the food items : peanut half (66.9%); fruity gem (25.5%), pellet (5%), krisp (2.6%). The winning percentage for each food item was indeed significantly correlated with their caloric values   = 287.1, p<0.001), confirming that individual preference among ranks was significantly biased toward the favorite options.Although the individual monkeys exhibited similar preferences among food items, individual preference does not necessarily correspond to group preference . Thus, we defined rank as the order of winning percentage within individuals. Then, we compared the winning percentage with respect to rank to quantify the extent of preference toward an individual's favorite options . There wp<0.001) ; the fourth monkey, Puck, chose his first and second preferred items in order, but then flipped the choice order for the third and fourth preferred items . It is unclear why Puck tended to select the fourth preferred item (pellet) over the third (krisp) when choosing between them in the choice order condition, but selecting the third over the fourth in the binary choice condition. Although the difference in winning percentage between the third and fourth preferred items (17.4%) was less than the overall average difference (29.5%) or Puck’s average difference (27.6%), it, however, was not the smallest difference among the preferences (which was the difference between Caesar’s first and second preferred items: 16%).Comparing the two conditions, we found a direct linear relationship between choice order and the winning percentage of food items  . RegardiOverall, the monkeys exhibited a general peak-first bias, with at least the first and second favorites being selected in order.To help delineate the factors underlying preference and choice order, Experiment 2 tested color, which is considered a salient cue to primates with trichromatic color vision, such as rhesus monkeys F  = 2.9, p = 0.079 for red; F  = 0.27, p = 0.848 for orange; F  = 1.3, p = 0.330 for green; F  = 0.46, p = 0.715 for purple). This indicates that there was no generally preferred order with respect to color among the monkeys. Individually, three of the four monkeys exhibited no significant choice order for the different colors across sessions   = 5.3, p = 0.810; Caesar: χ2(9)  = 12.5, p = 0.188; Titus: χ2(9)  = 13.6, p = 0.138), indicating an unstable choice order with respect to color across sessions. In contrast, one monkey exhibited a significant choice order for color   = 21.0, p<0.013).In Condition 1, we tested the order in which the monkey chose the four differently colored but otherwise identical food items (fruity gems). The monkeys chose green (mean ± s.e.m = 28.1±6.5%) and purple (28.1±11.1%) most for their first choice in a two-way tie, purple most for their second choice (28.1±5.0%), red most for their third choice (35.4±9.2%), and red (28.1±2.8%) and green (28.1±6.1%) most for their fourth choice in a two-way tie . There wF  = 0.713, p = 0.563). Individually, however, all four monkeys had significant color preferences   = 13.3, p<0.01; Caesar: χ2(3)  = 21.6, p<0.001; Puck: χ2(3)  = 12.8, p<0.01; Titus: χ2(3)  = 20.1, p<0.001 )  . The colF  = 31.1, p<0.001), indicating that individual preference was significantly biased toward the favorite colors.Since there were large individual differences in specific color preferences, we defined rank as the order of winning percentage within individuals. Then, we compared the winning percentage with respect to rank to measure the extent of preference toward an individual's favorite option . There wp = 0.25)  .p = 0.61; Caesar: slope  =  5.42, Adj. R-Square  =  –0.21, p = 0.56; Puck: slope  =  6.94, Adj. R-Square  =  0.32, p = 0.26; Titus: slope  =  6.12, Adj. R-Square  =  0.20, p = 0.32). Thus, the monkeys did not exhibit an order preference with respect to color.Examining choice order in relation to specific color preferences individually, none of the monkeys exhibited a significant tendency to choose the most preferred item   = 14.7, p<0.001). Thus, although significant preferences were found among the food items in both experiments, the relative preference among options was significantly higher in Experiment 1 than in Experiment 2.Finally, to compare the relative preference among the choice options in Experiments 1 and 2, we calculated the average difference among winning percentages for each monkey in Experiment 1  and compared them to the same in Experiment 2 . The average difference in relative preference among the food items was significantly different between the two experiments  across the food items in Experiment 1 compared to Experiment 2. Thus, the relationship between relative preference and behavior—in this case choice order—is complex and apparently nonlinear in rhesus macaques. It will be important for future work to further characterize this nonlinear functional relationship to determine when and how relative differences become sufficiently salient to influence choice order However, when the relative differences were sufficiently salient, choice order was clear: when allowed to take four food items in any sequence, the monkeys chose the food items in order of preference, starting with their favorite first. In a study in which one choice response was followed by a sequence of outcomes , rhesus monkey order preferences were less clear, although if anything, they also tended to exhibit a peak-first preference In contrast, humans appear to evaluate sequences retrospectively, often exhibiting a peak-end preference Thus far, then, humans appear to exhibit a peak-end preference for outcome sequences, considering them as an episode, whereas rhesus monkeys, if anything, appear to exhibit the opposite. Whether this difference is due to species or methodological differences, however, remains unclear. For example, the human studies often use sequences composed of quantitative differences, which might be more conducive to sequence detection and formation; whereas, the rhesus study used qualitatively distinct food items. It will therefore be important to compare humans and non-human animals with more similar testing conditions, and in particular, more comparable items, which may influence sequence formation and the type of evaluation process used.If prospective versus retrospective evaluation is at least partly based on the extent individual events are combined into sequences, one might expect self-generated sequences to promote the evaluation of each choice outcome individually, which could explain why the current results from self-generated sequences more strongly revealed a peak-first preference as opposed to the experimenter-generated sequences in the Xu et al. (2011) study. It is therefore important to ask what order humans would produce if they generated the sequences themselves. Recently, Jeong et al. (under revision) conducted such an experiment with pieces of sushi. When choosing sushi pieces without replacement, two general populations of people emerged: some people selected their preferred items first, while others saved them for last. Different subpopulations of people with opposite preferences suggest that in addition to contextual factors, individual traits may also influence the preferred sequence order. A critical individual factor might be the degree of impulsivity versus self-control, given that there are clear individual differences among humans on this trait Comparing our results with those in the sushi experiment, our rhesus monkeys match the group preferring the peak-first sushi order. Indeed, evidence points to rhesus monkeys showing relative impulsivity, compared, for example, to great apes. When required to wait for a food item, rhesus monkeys rarely exceeded a 30-second delay, whereas chimpanzees and an orangutan waited roughly six times longer However, it has also been argued that the apparent riskiness of rhesus monkeys may be context driven Future work will need to further delineate the environmental conditions and individual factors determining the use of prospective versus retrospective evaluation. In general, goal-directed behavior involves the generation, maintenance and seeking of goals, which appears to promote prospective evaluation In more stable environments, however, sequences of events, actions and/or outcomes are more reliable, and linking (or ‘chunking’) them together into a sequence can provide efficient hierarchical memory storage and heightened predictability Finally, even though humans appear to have a peak-end bias for a series of events, including, e.g., personal experiences, performances, and stories, it does not necessarily mean that if given the opportunity to build the sequence themselves, they would do so. Here we demonstrated that rhesus monkeys showed a peak-first preference in self-generated choice order. Whether self-generated sequencing imposes a heavier burden on self-control and/or sequence formation is yet to be determined. To be sure, it is likely that different underlying processes, such as prospective and retrospective evaluation, impulsivity and self-control, and the ability to detect and formulate sequences, interact to determine our preferences, and more work including comparative analyses is necessary to identify the nature of these underlying processes.


Understanding the nuances of RNA structure as they pertain to biological function remains a formidable challenge for retrovirus research and development of RNA-based therapeutics, an area of particular importance with respect to combating HIV infection. Although a variety of chemical and enzymatic RNA probing techniques have been successfully employed for more than 30 years, they primarily interrogate small (100–500 nt) RNAs that have been removed from their biological context, potentially eliminating long-range tertiary interactions (such as kissing loops and pseudoknots) that may play a critical regulatory role. Selective 2′ hydroxyl acylation analyzed by primer extension (SHAPE), pioneered recently by Merino and colleagues, represents a facile, user-friendly technology capable of interrogating RNA structure with a single reagent and, combined with automated capillary electrophoresis, can analyze an entire 10,000-nucleotide RNA genome in a matter of weeks. Despite these obvious advantages, SHAPE essentially provides a nucleotide “connectivity map,” conversion of which into a 3-D structure requires a variety of complementary approaches. This paper summarizes contributions from SHAPE towards our understanding of the structure of retroviral genomes, modifications to which technology that have been developed to address some of its limitations, and future challenges. Cis-acting sequences within the (+) strand RNA genomes of retroviruses and long terminal repeat (LTR) containing retrotransposons control several critical events in their life cycle, including transcription [2+ [in vitro transcription and labeled with 32P, eliminating any positional context, that is, regulatory roles that might be mediated by long-range, tertiary interactions. Although this challenge has in part been addressed by Paillart et al. via ex vivo footprinting of virion-associated RNA with dimethyl sulfate [in vitro and ex virio, and with fewer base-specific reagents, would clearly be advantageous.cription , translacription , dimerizcription , packagicription , RNA expcription , and DNAcription . Developcription  and nuclcription ) or chemcription , 10, andtion [2+ ). While tion [2+ –23, the  sulfate , a more in vitro and ex vivo, these studies have also provided important information on HIV genome organization and the role played by chaperone proteins. Finally, the recent advent of the self-inactivating electrophile benzoyl cyanide (BCN) [ Selective 2′ hydroxyl acylation analyzed by primer extension (SHAPE), reported in 2005 by Merino and colleagues , has emede (BCN)  opens th Despite these benefits, it should be borne in mind that SHAPE effectively provides a secondary structure nucleotide “connectivity” profile; that is, it does not report directly on long-distance tertiary interactions such as kissing loops and pseudoknots and is best used in conjunction with other solution techniques, such as X-ray crystallography, NMR spectroscopy, and small angle X-ray scattering in order to generate an accurate 3-D model. Where possible, combining structural data with a genetic analysis, via construction of disruptive and complementary mutations, should be seen as an important complement. In this communication, we have reviewed the basic SHAPE methodology and its application to understanding the structure of regulatory elements of both retroviral and retrotransposon genomes. Modifications to the probing technology which have allowed us to (i) investigate tertiary interactions important for regulating nucleocytoplasmic RNA transport and (ii) combine chemical modification with tandem mass spectrometry to understand conformational dynamics of RNA/DNA hybrids containing polypurine tract (PPT) primers of (+) strand DNA synthesis, are presented. Finally, future challenges of SHAPE, including increasing sensitivity where the amount of biological material is limiting, and studying interconverting RNA structures, are also discussed.O-adduct [32P allows primer extension products of 50–300 nt to be fractionated by conventional denaturing polyacrylamide gel electrophoresis, while multiplexing with fluorescently-labeled primers and automated capillary electrophoresis permits resolution of 500–750 nt in a single electropherogram  strand, DNA-dependent DNA synthesis from the polypurine tract (PPT) RNA primer. Although we have gleaned important information on PPT function using mutants of HIV RT –37 and tSaccaromyces cerevisiae LTR-retrotransposon Ty3 was insensitive to NMIA, acylation of ribonucleotide +1G was observed. These results were in agreement with NMR data [4 footprinting, which differentiates between thymines in a single-stranded and duplex configuration [The utility of our approach , designaNMR data , suggestguration , SHA-MS MusD (MTE) revealed a complex structure containing a combination of a kissing loop and a pseudoknot [Originally defined as an intermolecular interactions that mediate HIV-1 RNA genome dimerization , kissingeudoknot . Such tein vivo, where MusD-dependent nucleocytoplasmic RNA transport was abrogated and restored by disruptive and compensatory kissing loop mutations, respectively. The structure of the MusD pseudoknot was likewise confirmed by ai-SHAPE, while a genetic analysis indicated that the ability to assume a pseudoknot configuration was a more critical determinant of function than absolute nucleotide sequence.This strategy involves hybridization of short (5–10 nts) oligonucleotides to the proposed RNA duplex and determining whether this induces enhanced NMIA reactivity of the displaced strand. In view of their length, antisense oligonucleotides were constructed containing 2′-O-methyl and locked nucleic acid substitutions, both of which have been shown to improve duplex stability. Such interfering oligonucleotides are invasive inasmuch that they will hybridize to their partner sequence in an RNA that has already adopted its 3D structure. When applied to the MusD MTE, an interfering octanucleotide hybridized to internal loop 8 (IL8) stimulated NMIA reactivity at several positions in its kissing partner, loop 3  structure occludes the DIS, while the gag initiation codon is positioned within a short stem-loop, the stability of which would facilitate translation over dimerization and packaging. In the MSL, the tRNA primer binding site is also inaccessible. Support for interconverting structures of the 5′ UTR was provided by the observation that the FIV mutant AN14, demonstrated in vivo to have impaired packaging [in vitro, while dimerization was enhanced when the RNA was stabilized in the LRI form ['-bisacryloylcystamine (BAC) or N,N-diallyltartardiamide (DATD) would allow solubilization and recovery of nucleic acid for subsequent cDNA synthesis. Should ribose acylation alter RNA conformation, in-gel probing directly following fractionation by nondenaturing electrophoresis is an alternative strategy. The long range interaction (LRI) model, originally proposed by Kenyon et al. , exposesackaging , exhibitLRI form . Though Asp tertiary structure [Understanding RNA structure-function relationship requires accurate three-dimensional structure modeling methods. At present, there is a substantial gap in obtaining high-throughput 3D information for RNA molecules larger than 150 nts. The techniques frequently used to obtain atomic resolution of RNAs, such as NMR spectroscopy and X-ray crystallography, have restrictions that preclude structural analysis. In NMR spectroscopy, the excited signal from individual atomic nuclei becomes congested and difficult to analyze with the increasing size of RNA molecule. Even though X-ray crystallography does not suffer from size limitations, obtaining crystals for flexible and diverse RNA structures represents a great challenge. These difficulties however, are now being addressed by combining SHAPE with methidiumpropyl-EDTA- (MPE-) directed through-space hydroxyl radical cleavage, as outlined schematically in tructure .Asp). However, synthesis of MPE has been reported, and this strategy opens the intriguing possibility of developing “molecular rulers” by introducing linkers of different length between the intercalating and hydroxyl radical generating moieties.MPE is a methidium intercalator moiety tethered to EDTA that preferentially intercalates at G-C rich helices in RNA at sites adjacent to a single nucleotide bulge. The intercalated MPE occupies roughly the same space as a single base pair and is oriented in the motif such that the EDTA moiety points toward the bulge. Upon addition of Fe(II) and a reducing agent, ferrous ion binds the EDTA and generates short-lived hydroxyl radicals that cleave proximal regions of the RNA backbone . The MPEgag-pol ribosomal frameshift signal, which constituted one component of a three-helix structure (P1-P2-P3). The slippery sequence forms one of the three helices (P2), while two others (P1 and P3) are stabilized by an anchoring stem with two bulges. Additional RNA elements were identified in protein-coding regions of the genome, from which it has been tentatively postulated that RNA structure constitutes an additional organizational level of the genetic code. Since many proteins appear to fold co-translationally, highly structured RNA might induce pausing of the translational machinery, promoting protein folding in a more native-like conformation. In contrast, highly unstructured regions were observed in hypervariable regions of the HIV-1 genome, which have important roles in viral host evasion. These unstructured regions were shown as separated from the rest of the genome by stable helices that have been proposed to function as structural “insulators.”Most structural analyses have historically targeted small RNA motifs (<500 nt) in artificial contexts and, in the absence of complementary genetic and phylogenetic data, may not accurately relate their structures to the biology of the larger RNAs from which they were derived. In contrast, SHAPE provides an unprecedented opportunity to view an entire RNA molecule, giving the researcher the opportunity to connect simple elements to the components of larger RNA motifs. This concept has recently been exemplified through the application of SHAPE to decode the structure of the entire HIV-1 genome (~9750 nucleotides) at single-nucleotide resolution . This sein vivo, (ii) ex vivo, where genomic RNA had been gently deproteinized, (iii) in vivo, but where important interactions between the nucleocapsid protein (NC) and genomic RNA had been compromised by covalent modification with aldrithiol-2  than coding regions. A comparison of acylation profiles for the in vivo state with those following covalent modification by AT-2 defined several high affinity NC recognition sites, consistent with the role of this critical RNA chaperone in governing packaging of viral RNA. All NC binding sites were characterized by a G-rich single-stranded sequence flanked by stable helices. Additionally, RNA motifs where NC increases local flexibility were also identified, comprising single-stranded A/U-rich motifs adjacent to a duplex in which the first base pair includes a guanosine nucleotide. Collectively, this genome-probing approach suggests that local protein interactions can be organized by the long-range architecture of RNA. Although a limited region of the genome of the formerly known gammaretrovirus xenotropic murine leukemia virus related virus (XMRV) was examined using this strategy, it yielded similar conclusions on high affinity NC binding sites [The versatility of SHAPE extends to studying viral RNA not only in the context of the intact genome, but also at different biological states, providing information with respect to RNA conformational changes underlying different stages of viral life cycle. As an example, Wilkinson et al.  have pro-2 (AT-2 ), and  cDNA ligation to incorporate the second Illumina adapter. Single-stranded cDNA ligation is achieved using a thermostable ligase  and a blocking group on the 3′ end of the adapter to prevent concatemerization [Initially, input RNA templates are bar-coded with a unique sequence. Such barcodes comprise tetranucleotide sequences that are placed in the 3′ structural cassette and introduced prior to rization . FinallyWhen compared to conventional SHAPE, SHAPE-Seq permits rapid, fully-automated analysis and eliminates the necessity for manual, time-consuming data manipulations associated with quantification of fluorescently-labeled cDNAs by capillary electrophoresis. By ligating single-stranded cDNA products with 5′ adapters followed by PCR-amplification, with minute amounts of RNA needed to generate the reactivity spectrum of a given RNA, SHAPE-Seq represents a more generally-applicable and sensitive technique studying RNA samples that are limiting, from a biohazardous source, or both. For example, it was shown for the RNase P specificity domain that with as little as 0.1 pmol of input RNA, SHAPE-Seq reactivities of over 800 bar-coded RNA species could be inferred . SHAPE-Sin vivo analysis of a short portion of the formerly known XMRV genome. In contrast to earlier in vivo studies that required 1–3 pmole of input RNA, acylation profiles could be obtained with as little as 50 fmole aliquots of genomic RNA. As might be predicted, structural features of the XMRV leader RNA were similar to the extensively-studied counterpart Moloney murine leukemia virus, although binding sites unique to the XMRV nucleocapsid protein were proposed. More importantly, this study, which required in-house construction of a dedicated two-color capillary electrophoresis instrument, opens the exciting prospect of future functional studies on low abundance RNAs of clinical significance.Using a two-color automated capillary electrophoresis with subfemtomole sensitivity, Grohman et al.  have rec2+ cleavage, ai-SHAPE and threading intercalators) and biophysical approaches . The benefits of capillary electrophoresis-based high throughput SHAPE must also be balanced by the demand this makers on the number of fluorescent oligonucleotide primers required for multiplexing, and the necessity for expensive instrumentation, features that also hold for femtomole SHAPE and SHAPE-Seq. Moreover, Kladwang and coworkers [Rather than giving an exhaustive review of projects that have made use of SHAPE, which have included structures of wild type and mutant variants of the HIV-1 Rev response element , NC bindoworkers  compared


This affords 3′ transcript degradation and consequent Pol II termination. In effect, R-loops formed over G-rich pause sites, followed by their resolution by senataxin, are key steps in the termination process.We present a molecular dissection of pause site-dependent transcriptional termination for mammalian RNA polymerase II (Pol II)-transcribed genes. We show that nascent transcripts form RNA/DNA hybrid structures (R-loops) behind elongating Pol II and are especially prevalent over G-rich pause sites positioned downstream of gene poly(A) signals. Senataxin, a helicase protein associated with AOA2/ALS4 neurodegenerative disorders, acts to resolve these R-loop structures and by so doing allows access of the 5 ► Senataxin is required for pause-dependent Pol II termination in human genes ► R-loops accumulate over G-rich pause regions and are required for termination ► R-loops dependent on transcription, functional poly(A) signal, and pause elements ► Senataxin resolves R-loops to allow Xrn2 to mediate Pol II release from the gene All steps in gene expression are thought to be interconnected and co-ordinately regulated . TranscrS. cerevisiae  and amyotrophic lateral sclerosis type 4 (ALS4) were mapped to the senataxin gene (SETX) (reviewed in We describe here the role of senataxin in the regulation of gene transcription. We demonstrate that senataxin depletion in HeLa cells causes an increase in readthrough RNA and Pol II density downstream of the poly(A) site, implying an involvement in transcriptional termination. Using DNA immunoprecipitation (DIP) analysis on endogenous genes and transfected constructs, we demonstrate that RNA/DNA hybrid structures (R-loops) are formed across transcription units by interaction of the nascent transcript with the ssDNA template behind the elongating Pol II. R-loops are dependent on transcription, functional poly(A) signals, and termination elements. Overexpression of RNase H1 in vivo acts to resolve R-loops and causes transcriptional readthrough. Upon senataxin depletion, R-loops are stabilized downstream of the poly(A) signal, preventing Xrn2 recruitment, nascent RNA degradation, and efficient termination. We predict that senataxin acts to resolve R-loops in pause-dependent transcription termination.S. cerevisiae has demonstrated that Sen1 is required to promote Pol I and Pol II transcriptional termination (trans-activator. Both the β-actin pause element and MAZ4 G-rich pause sequences have been previously shown to promote poly(A) site- and Xrn2-dependent transcriptional termination  on nuclear and cytoplasmic RNA harvested from mock-treated and senataxin- siRNA1-treated cells, detecting nascent transcripts that readthrough the normal termination signals . In thesThe termination efficiency of β-globin/β-actin and β-globin/MAZ4 constructs in senataxin-deficient cells was also assessed using quantitative RT-PCR analysis. In this experiment, we measured the amount of readthrough transcript A and the amount of correctly initiated transcript B .The majority of senataxin mutations observed in AOA2/ALS4 patients are detected within the helicase or N-terminal protein interaction domains, suggesting their importance in senataxin function. Furthermore, the function of yeast Sen1 in transcription is likely to be associated with Sen1 RNA/DNA helicase activity, which acts to resolve RNA/DNA hybrids formed between nascent RNA and template DNA strand behind elongating Pol II .trans-activator TAT and observed a reduction of R-loop signal over the coding regions of the gene , which relies on the immunoprecipitation of noncrosslinked purified RNA/DNA hybrids, using S9.6 antibody, which recognizes RNA/DNA duplexes . We democis mutations affecting transcriptional termination of β-globin/MAZ4 construct can affect the R-loop distribution profile. Here, we used constructs containing either mutated poly(A) signal (ΔpA) or mutant MAZ sequences (mMAZ4), which do not promote transcriptional pausing. Both of these mutations cause transcriptional termination defects  C. Here, The above experiments rely on analysis of gene constructs with different termination sequences. To validate these data using an endogenous gene system (in native chromatin), we also investigated the role of senataxin in termination of the endogenous β-actin gene in HeLa cells. In contrast to transiently transfected plasmids, transcriptional profiles of endogenous genes can be reproducibly analyzed by chromatin immunoprecipitation (ChIP) experiments, employing a number of PCR primers located over the coding and 3′-flanking regions. Previously, we have mapped a 3′-flanking pause element involved in transcriptional termination of the human β-actin gene . As showTo further substantiate a role for senataxin in transcriptional initiation/elongation and termination, we employed NRO using Br-UTP as the labeled nucleotide . Br-UTP S. cerevisiae have shown that Sen1 is required to promote Pol I and Pol II transcriptional termination  and reverse transcribed with SuperScript III Reverse Transcriptase (Invitrogen), using gene-specific reverse primers . SplicedChIP was carried out using anti-senataxin , anti-Pol II (H-224) , and anti-Xrn2  antibodiDIP was performed without a crosslinking step, following the ChIP protocol with some modifications. After the nuclear lysis reaction, extracts were incubated with 30 μg of proteinase K (Roche) at 55°C for 3 hr, and genomic DNA was isolated. Following sonication, DIP analysis was carried out using an antibody that recognized RNA/DNA hybrids, purified from S9.6 hybridoma cell lines . The imm


At the beginning of the last decade, it was believed that animal and plant genomes encoded, on average, 30,000 genes, surrounded by junk non-coding sequences. The dogma “from DNA to RNA to proteins” assigned RNA the role of transducer of genetic information into proteins. Large-scale studies have demonstrated that non-protein-coding RNAs are a massive output of transcription and can be regarded as regulatory RNAs. Following this new understanding, DNA is a static element of genetic information, a hardware, while RNA, in the form of multiple species of short and long non-coding transcripts constitutes the active information, the software  in plants include microRNAs, siRNAs , tasi-RNA (trans-acting siRNAs), and nat-siRNAs . The presence of methylated 3′ OH ends caused an under-representation of sRNAs in the analysis of RNA composition in plants, producing a bias in RNAs that were sequenced using tag sequencing and next-generation sequencing methods.Today, sRNAs include an expanding number of 20–40 nt RNAs that function in the regulation of gene expression by affecting mRNA decay and translational inhibition, or lead to DNA methylation and gene silencing. They generally involve double-stranded RNA or stem loops and imply transcriptional or post-transcriptional gene silencing (PTGS). Gene silencing in plants can be mediated by siRNA, microRNA, tasi-RNAs, and nat-siRNAs. These RNAs act as single-stranded filaments incorporated into the effector complexes  containing one of the Argonaute enzymes degrading the target RNA complementary to sRNA Bartel, . DNA-depIn higher plants, a number of physiological processes are regulated by the transfer of signaling molecules through the phloem. This phloem-mediated remote-control system provides specific and efficient regulation to fine-tune many plant developmental programs. In addition to hormones, proteins, and small peptides, phloem is rich in RNA-binding proteins that function as chaperones for systemic RNA movement.1, supported the production of the first Arabidopsis non-coding RNA macroarrays, that characterized their regulation in different tissues and in plants subjected to environmental stresses  was used before the advent of next-generation sequencing as a high-throughput method to identify short transcripts devoid of poly-A tails, as microRNAs and sRNAs , miR398 (copper), and miR399 (phosphate), were increased in phloem sap during the growth of plants under the respective starvation conditions  mRNA . This was shown using non-conventional approaches that exploit virus-induced RNA silencing and meristem exclusion of virus infection (Li et al., FT mRNA structure and from other RNA sequences, it was shown that the tertiary structure is important to define their function (Zhong et al., Several messenger RNAs travel a long-distance in the phloem, such as 2. Several new-omics layers such as the interactome, epigenome, peptidome, and hormonome are taking the lead that will open new perspectives on the complex regulation of communication between plant tissues and in signaling at a distance.Omics and bioinformatics are essential to understanding the molecular systems that underlie various plant functions. Recent sequencing technologies have revitalized sequencing approaches in genomics and have produced opportunities for various emerging analytical applications. A new EU FP7 project now started, AB-Stress, will study the epigenetic regulation and the stress induced sRNA world in legumes during biotic and abiotic stresses


Trichomonas vaginalis is a sexually transmitted flagellated protist parasite responsible for trichomoniasis. This parasite is dependent on high levels of iron, favoring its growth and multiplication. Iron also differentially regulates some trichomonad virulence properties by unknown mechanisms. However, there is evidence to support the existence of gene regulatory mechanisms at the transcriptional and posttranscriptional levels that are mediated by iron concentration in T. vaginalis. Thus, the goal of this study was to identify an RNA-binding protein in T. vaginalis that interacts with the tvcp4 RNA stem-loop structure, which may participate in a posttranscriptional iron regulatory mechanism mediated by RNA-protein interactions. We performed RNA electrophoretic mobility shift assay (REMSA) and supershift, UV cross-linking, Northwestern blot, and western blot (WB) assays using cytoplasmic protein extracts from T. vaginalis with the tvcp4 RNA hairpin structure as a probe. We identified a 135-kDa protein isolated by the UV cross-linking assays as α-actinin 3 (TvACTN3) by MALDI-TOF-MS that was confirmed by LS-MS/MS and de novo sequencing. TvACTN3 is a cytoplasmic protein that specifically binds to hairpin RNA structures from trichomonads and humans when the parasites are grown under iron-depleted conditions. Thus, TvACTN3 could participate in the regulation of gene expression by iron in T. vaginalis through a parallel posttranscriptional mechanism similar to that of the IRE/IRP system. Cellular iron is an essential cofactor for many biochemical activities, including oxygen transport, cellular respiration, and DNA synthesis. Thus, iron deficiency can cause cell growth arrest and death. However, iron overload is also potentially toxic; under aerobic conditions, it catalyzes the formation of reactive oxygen species and generates highly reactive radicals through the Fenton reaction . The duaIn vertebrates, cellular iron homeostasis is maintained by the coordinated expression of proteins involved in iron uptake, storage, utilization, and export, which are regulated at the posttranscriptional level. This mechanism is based on the interactions of cytoplasmic iron regulatory proteins (IRPs) with conserved RNA stem-loop structures known as iron-responsive elements (IREs), which are located in the untranslated regions (UTRs) of specific mRNAs –7, underα, β-APP, and α-synuclein, which control iron storage, erythroid iron utilization, energy homeostasis, iron efflux, hypoxic responses, and neurological pathways. Conversely, the IRE/IRP interaction in the 3′-UTR stabilizes mRNAs encoding TfR1, DMT1, Cdc14A, and MRCKα, which are involved in iron uptake, iron transport, the cell cycle, and cytoskeletal remodeling  UTP  was included in the transcription reaction. The IRE-fer transcript encoding the canonical IRE was used as the positive control where indicated. An unrelated RNA transcript was used as a negative control  coupled to peroxidase) (Bio-Rad) at a 1 : 3,000 dilution in 10% fat-free milk in PBS-T for 2 h at 25°C, washed five times with PBS-T, and developed with 4-chloro-1-naphthol (Bio-Rad) or by chemiluminescence using a SuperSignal West Pico Kit (Pierce). The corresponding PI rabbit or mouse serum was used as a negative control. These experiments were independently performed at least three times and yielded similar results.After electrophoresis, recombinant TvACTN3r, DIr, DIIr, or DIIIr, or cytoplasmic extracts of μg/mL yeast tRNA (Sigma) in EB (EBY) for 18 h at 4°C, and washed three times with EBY at 25°C. The NC membranes were incubated for 18 h at 4°C with EBY containing 10–15 ng/mL radiolabeled RNA; washed twice with EB; washed another two times with B buffer (10 mM Tris-HCl [pH 7.5] and 50 mM NaCl); and blocked again for 1 h at 37°C with STMT buffer  containing 3% BSA. After extensive washing with EB, the NC membranes were air-dried and exposed for autoradiography. These experiments were performed independently at least three times, with similar results.The Northwestern blot assay was performed by combining previously described methods , 24 withT. vaginalis genome project database (http://www.trichdb.org/). To identify functional RNA-binding domains, we analyzed the deduced protein sequence of TvACTN3 using the SMART (http://smart.embl-heidelberg.de/)  . These α-actinin proteins have different sizes  and different identities compared with TvACTN3 . The most conserved region among them is the N-terminal corresponding to the actin-binding domain. The most divergent region is the central domain that contains the spectrin repeats .In this figure we show a multiple alignment of the amino acid deduced sequence of the five actinin genes found in the Click here for additional data file.


Peromyscus maniculatus), the natural reservoir of Sin Nombre hantavirus, have shown that viral RNA can be transiently detected well past the early acute infection stage, but only in the minority of infected mice. Here, using a non-degenerate RT-PCR assay optimized for SNV strains known to circulate in Montana, USA, we show that viral RNA can be repeatedly detected on a monthly basis in up to 75% of antibody positive deer mice for periods up to 3–6 months. More importantly, our data show that antibody positive male deer mice are more than twice as likely to have detectable SNV RNA in their blood as antibody positive females, suggesting that SNV-infected male deer mice are more likely to shed virus and for longer periods of time. Hantaviruses are widespread emergent zoonotic agents that cause unapparent or limited disease in their rodent hosts, yet cause acute, often fatal pulmonary or renal infections in humans. Previous laboratory experiments with rodent reservoir hosts indicate that hantaviruses can be cleared from host blood early in the infection cycle, while sequestered long term in various host organs. Field studies of North American deer mice ( Bunyaviridae, genus Hantavirus) are negative-stranded RNA viruses that occur across Europe, Asia, and the Americas and are hosted by rodents, insectivores, and possibly bats [Peromyscus maniculatus) demonstrated SNV viral antigen in tissues and viral RNA in blood for up to 217 days post-inoculation [P. maniculatus in outdoor enclosures, in which viral RNA was consistently detected in weekly blood samples up to 60 days PI using a newly developed and highly sensitive real-time RT-PCR assay that targeted a conserved region of the genome [Hantaviruses  to Ab+ than females [Rattus norvegicus) system, experimental infection studies of SNV in deer mice have thus far not reported similar findings after examining tissue, saliva, and excreta of infected mice [P. maniculatus are more likely to have circulating RNA than SNV Ab+ females; a finding that might provide an additional explanation for why SNV transmission in the wild appears male dominated.In many naturally occurring hantavirus rodent reservoir host systems, adult males are more likely to be antibody positive (Ab+) than females ,11,12, a females . This ob females ,14,15,16 females , they ar females . While dted mice ,19. Furtted mice ,8. As a et al., [et al., [Blood samples from known SNV Ab+ mice were analyzed using two RT-PCR assays, each optimized for S and M genomic segments of SNV strains known to circulate in Montana . Of 78 STo determine if the increased levels of detection we found were due to more sensitive methods rather than a temporal spike in virus prevalence during the 2011–2012 collection period, we tested 20 archived SNV antibody-positive samples collected from the same region in 2007 and 2009. These archived samples were originally analyzed using the RNA extraction method [P. maniculatus to clear SNV virus from the bloodstream. As shown in versus 29% (12/42) for females. By sex, 86% of SNV Ab+ males have detectable viral RNA in their blood (30/35) versus 57% (12/21) for females  = 4.66, p = 0.03083). Further, in persistently ‘viremic’ animals , 90% were males (9/10) versus only 10% for females (1/10), and by sex, 100% (9/9) of Ab+ males were viremic versus 25% (1/4) for females. For those mice RNA+ for three or more consecutive months, 100% (4/4) were males , and found the vast majority, 77%, to be RNA+ each time . Extendire males , includiUsing geographic region-specific RT-PCR assays to screen for viral RNA in blood samples of SNV Ab+ North American deer mice, we found 75% to be RNA+ with some mice showing evidence of sustained circulating RNA for 4–6 months. These data demonstrate a much higher prevalence of circulating RNA than in previous SNV studies in Montana and Canada where 19%–47% of mice were RNA-positive . Experiments in at least one other New World hantavirus-host system showed that viral RNA levels detected in blood mimicked patterns of infectious virus in the blood and tissues, including the salivary glands . Microtus pennsyvlanicus. At these sites, deer mice often share habitat and burrows with Microtus spp., and inter-species virus spillover has been documented in other deer mouse-SNV systems [Twenty-three percent of the Ab+ mice were negative for SNV RNA. This may be explained by the fact that 80% of the RNA-negative (RNA-) samples were from large adults 23–27 g), raising the possibility that these individuals were infected in a previous season but not captured and tested, a likely consequence of less frequent monthly recapture studies. Alternatively, it is known that the antibody test for SNV is highly cross-reactive with other North American hantaviruses, and therefore we cannot rule out that some of the Ab+ but RNA- mice were infected with other hantaviruses such as Prospect Hill virus, hosted by meadow voles,  systems . Finally systems ,23. Depe–27 g, ra systems . et al. [et al. [et al. [We obtained blood samples from an ongoing 18-year monthly longitudinal mark-recapture study at three sites in Montana , and most closely matched those from mice sampled previously from the same region.We found evidence that males are more likely to be circulating RNA in their blood as compared to females. Future research should explore the relationship of the immunological, physiological, and ecological factors that can influence the pattern of infection, including the role of testosterone and estrogen and their relationship to hantavirus transmission-related behaviors and host immunology and pathology in deer mice. Also, further study should focus on exploring viral RNA and antibody levels, as well as other related immune system components, in response to natural SNV infection in males and females to understand the relevant roles of each sex in the maintenance of SNV transmission in the wild.


Hepatitis C virus (HCV) RNA initiates its replication on a detergent-resistant membrane structure derived from the endoplasmic reticulum (ER) in the HCV replicon cells. By performing a pulse-chase study of BrU-labeled HCV RNA, we found that the newly-synthesized HCV RNA traveled along the anterograde-membrane traffic and moved away from the ER. Presumably, the RNA moved to the site of translation or virion assembly in the later steps of viral life cycle. In this study, we further addressed how HCV RNA translation was regulated by HCV RNA trafficking. When the movement of HCV RNA from the site of RNA synthesis to the Golgi complex was blocked by nocodazole, an inhibitor of ER-Golgi transport, HCV protein translation was surprisingly enhanced, suggesting that the translation of viral proteins occurred near the site of RNA synthesis. We also found that the translation of HCV proteins was dependent on active RNA synthesis: inhibition of viral RNA synthesis by an NS5B inhibitor resulted in decreased HCV viral protein synthesis even when the total amount of intracellular HCV RNA remained unchanged. Furthermore, the translation activity of the replication-defective HCV replicons or viral RNA with an NS5B mutation was greatly reduced as compared to that of the corresponding wildtype RNA. By performing live cell labeling of newly synthesized HCV RNA and proteins, we further showed that the newly synthesized HCV proteins colocalized with the newly synthesized viral RNA, suggesting that HCV RNA replication and protein translation take place at or near the same site. Our findings together indicate that the translation of HCV RNA is coupled to RNA replication and that the both processes may occur at the same subcellular membrane compartments, which we term the replicasome. Hepatitis C virus (HCV) is a positive-sense RNA virus that is estimated to chronically infect as many as 3% of the world's population. As a member of Flaviviridae, HCV is an enveloped virus with a single, positive-stranded RNA around 9.6 kb in length Using the HCV subgenomic replicon system as well as infectious virus system, many host factors have been identified to be involved in HCV RNA replication, including the human homologue of the 33-kDa vesicle-associated membrane protein-associated protein (hVAP-33) The balance between viral RNA transcription and translation is critical for the replication of positive-stranded RNA viruses, since the same RNA is used both for translation and as the template for negative-strand RNA synthesis. Transcription of poliovirus has been reported to be dependent on the translational activity of the viral RNA in vivo. Finally, we found that at least most of the newly synthesized HCV proteins colocalized with the newly synthesized viral proteins. These findings together thus indicate that HCV replication and translation are coupled, in the sense that replication of viral RNA is linked to translation of viral RNA in situ.In this study, we observed that HCV RNA exit from the site of RNA synthesis to the Golgi complex, a process that can be blocked by nocodazole, an inhibitor of the ER-Golgi transport pathway. Surprisingly, HCV protein translation was enhanced when HCV RNA movement was blocked, suggesting that the translation of viral proteins occurred near the site of RNA synthesis. We also found that the translation of HCV proteins was dependent on active RNA synthesis: inhibition of RNA synthesis resulted in decreased HCV viral protein synthesis before there was significant decrease in the total amount of HCV RNA, and that the replication-defective HCV RNA could not be translated efficiently Huh7 or Huh7.5 cells were grown at 37°C in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and nonessential amino acids. Huh7 cells were obtained from Dr. Sato's lab in vitro transcription, the plasmids were digested by Xba I and Mungbean nuclease and gel-purified. For electroporation, Huh7 or Huh7.5 cells were trypsinized, washed and resuspended in serum-free DMEM. HCV replicon RNA or JFH full-length RNA were transcribed in vitro by T7 MegaScript (Ambion). A total of 6 to 10 µg of RNA and 107 Huh7 cells were mixed and incubated on ice for 5 minutes and subjected to an electric pulse at 975 µF and 220 V. Cells were immediately transferred to 8 ml of DMEM containing 10% FBS for incubation.HCV JFH1 and JFH-GND constructs were obtained from Dr. Wakita’s lab  4 cells per well. Two days after seeding, cells were incubated with actinomycin D (10 µg/ml) for 1 hour to inhibit cellular RNA synthesis, and in some experiments, also with 20 nM of hippuristanol Labeling of de novo-synthesized viral RNA, immunofluorescence staining and confocal microscopy were modified from the previously described methods G TGT CGT GCA GCC TCC A 3′ and 5′ CAC TCG CAA GCA CCC TAT CA 3′) of the HCV 1b sequence CCC GCA AGA CTG CTA GCC GAG TAG TGT TGG (TAMRA) 3′] spanning these two regions. The RT step was performed at 60°C for 50 min, followed by 1 min at 50°C. The amplification condition was as follows: 95°C for 5 min and 50 cycles of denaturation at 94°C for 15 sec, annealing at 55°C for 10 sec, and extension at 69°C for 1 min.To determine the quantity of RNA by real-time PCR, a single-tube reaction was performed by using the TaqMan EZ RT-PCR Core Reagents (Applied Biosystems). Duplicate reactions for RNA standards and the samples were performed in 20-µl volume using 1 µl of HCV RNA, primers from HCV 5′ non-coding region (5′ GA1–106 copies per reaction) of HCV genotype 1b transcript. The same software was used to calculate the coefficients of regression. Values were normalized to that of GAPDH (Applied Biosystems). Each test was done in triplicate and averages were obtained.Using the ABI Prism 7900 program, standard curves of the assays were obtained automatically by plotting the three hold values against each standard dilution of known concentration , ERGIC (ERGIC53), and Golgi apparatus (GS27), respectively . Consist3H-uridine for 30 minutes and chased for 30 minutes to 3 hours. The labeled cell lysates were separated into ER and Golgi fractions by ultracentrifugation. Immunoblotting studies showed that the ER and the Golgi apparatus were efficiently separated by this procedure  and HCV NS3 proteins, both of which are encoded from the HCV replicon RNA but under the control of separate IRES elements. We found that after a 4-hour nocodazole treatment, the total amount of the labeled NS3 protein, as detected by anti-NS3 or HCV patients’ sera, was significantly increased , indicating that the open reading frames in these RNAs are intact. The wild-type HCV replicon (Rep) and its GND mutant (Rep*GND) RNAs were then transfected into Huh7 cells, and the transfected cells were metabolically labeled with 35S-Met for 14 hours to detect protein syntheses by immunoprecipitation with anti-NS3 or -NPT antibodies. The result showed that, the GND mutant yielded very little NS3 as compared with the corresponding wildtype replicon RNA . Immediately after transfection into Huh7 cells, both luciferase activities were equivalent initially; however, the FFLuc/RLuc ratio increased over time (We further used a replication-defective replicon RNA (GND mutation in the NS5B region) to assess if active RNA replication of HCV is required for HCV protein translation. A separate experiment using an icon RNA . The amoicon RNA . This reicon RNA . Both cover time . The revver time . We obsever time , at whic35S-Met during the 0–8, 8–16, or 16–24 hours post-transfection, and followed by immunoprecipitation. The amounts of these two RNAs at 24 hours post-transfection were almost the same ; at 2 days after infection, the cells were labeled with Cy5-UTP and BODIPY-FL-lys-tRNA in the presence of hippuristanol and actinomycin D, which blocked eIF4A-dependent translation and host RNA transcription, respectively The mechanisms of replication and translation of HCV RNA have been extensively studied in the past few years. However, the exact subcellular localization of HCV RNA replication and translation is still unclear. Evidence has previously been presented that HCV RNA replication occurs on the detergent-resistant membrane (DRM) possibly derived from the ER cis Although coupling of translation and RNA replication has been reported for many RNA viruses These findings raised an important issue, namely, how the initial viral translation is carried out, since, as a positive-strand RNA virus, the very initial round of translation from the incoming HCV viral genome has to take place before viral RNA replication can occur. Conceivably, the free viral RNA genome generated by uncoating of the incoming virion in the endosome  can associate with ribosomes on the rough ER and be translated in an RNA replication-independent manner. Such translation is likely of low efficiency, but is sufficient to support first round of HCV RNA translation. These initial viral protein products and RNAs will then be encased into the membranous replication complex and become part of the replication-translation machinery. The latter process will then become the main mechanism of HCV replication-translation.In summary, we propose the following pathway for HCV RNA replication and translation . PreviouFigure S1Benzothiadiazine treatment in Huh-N1b replicon cells. Huh7 and Huh-N1b cells pre-treated with actinomycin D, Benzothiadiazine (NS5B inhibitor), or combination of both for 4 hours, and then labeled with 3H-Uridine (A) or BrUTP (B). RNA synthesis was detected by RNA precipitation followed by scintillation counting (A), by immunofluorescence staining (B), or by real-time RT-PCR . Autoradiography of the H3-uridine-labeled RNA was also performed . The relative amounts of intracellular HCV RNA after the various treatments are shown in . Benzothiadiazine specifically inhibited viral transcription, but did not affect the intracellular replicon RNA levels under this condition (C). (D), long-term Benzothiadiazine treatment (over 2 days) significantly decreased the replicon RNA levels.(TIF)Click here for additional data file.Figure S2Preferential translation of JFH1 wildtype over the GND mutant HCV RNA. (A) A time-course study of HCV NS protein translation in cells transfected with JFH1 or its GND mutant. The cells were labeled with S35-methionine from 0–8, 8–16 and 16–24 hours posttransfection and followed by immunoprecipitation with anti-NS3 or HCV patient serum. NS3 and NS5A were detected by immunoprecipitation and separated by SDS-PAGE. (B) The corresponding intracellular HCV RNA levels in (A). The intracellular RNA levels at 24 hour post-transfection were determined by real-time RT-PCR.(TIF)Click here for additional data file.