Animals often use public information for mate-choice decisions by observing conspecifics as they choose their mates and then copying this witnessed decision. When the copier, however, is detected by the choosing individual, the latter often alters its behavior and spends more time with the previously non-preferred mate. This behavioral change is called the audience effect. The deception hypothesis states that the choosing individual changes its behavior to distract the audience from the preferred mate. The deception hypothesis, however, only applies if the audience indeed copies the pretended mate choice of the observed individual. So far, this necessary prerequisite has never been tested. We investigated in Atlantic molly males and females whether, first, focal fish show an audience effect, i.e., alter their mate choices in the presence of an audience fish, and second, whether audience fish copy the mate choice of the focal fish they had just witnessed. We found evidence that male and female Atlantic mollies copy the pretended mate choice of same-sex focal fish. Therefore, a necessary requirement of the deception hypothesis is fulfilled. Our results show that public information use in the context of mate choice can be costly. The evolution of mate choice is one of the most fascinating topics in biology. Since Darwin 1871 ,3,4,5,6,One form of using public information in the context of mate choice is mate-choice copying (hereafter MCC) ,23. MalePoecilia reticulata [Poecilia latipinna [Poecilia mexicana [Limia nigrofasciata [Oryzias latipes [Gasterosteus aculeatus [Amblyglyphidodon leucogaster [Symphodus ocellatus [Syngnathus typhle [So far, MCC has been found in several taxa like mammals ,45,46,47ticulata ,62,63,64atipinna ,67,68,69fasciata , in the  latipes ,72; and culeatus , the whicogaster , the ocecellatus , and thes typhle . Besidess typhle ,74,75. As typhle ,21,64,66Betta splendens [G. aculeatus [P. reticulata [P. latipinna [P. mexicana [When animals observe other conspecifics during mate choice, it is possible that the observing individual (the audience) is detected by the interacting conspecifics. The presence of an audience can lead to a change in behavior of the observed individuals. This phenomenon is called audience effect. The audience effect is a wide-spread phenomenon and has been studied in various contexts  show an audience effect, by altering their initial mate-choice decision when a same sex audience is present, and (2) whether the audience fish that had just witnessed the mate choice of the focal fish copied that mate-choice decision. Thus, we combined the typical setup to test the audience effect with a MCC experiment. We used the Atlantic molly as a model species because males and females copy the mate choice of others ,103, andWe predicted that focal fish would at least decrease their preference for their initially preferred mate in the presence of an audience fish and that the audience fish which had witnessed the altered mate choice would copy that mate choice. Our results provide support for the deception hypothesis in the context of the audience effect in mate choice. 3), each with approximately 30 individuals, under a 14:10 h light-dark-cycle and a constant temperature of 26 ± 1 °C. For more detailed information, see Artemia spec. One week before we started the experiments, we separated fish by sex and housed them under the same condition in three large tanks, two for the females and one for the males. During the testing period, fish used in experiments were separated in individual tanks (40 × 25 × 41 cm3) and kept under the same light regime and nutrition condition as in the home tanks. For the performed experiments, we used focal fish, audience fish, and stimulus fish. Focal fish and audience fish were of same sex within each experiment and could choose between two stimulus fish of the opposite sex successively. The audience fish was presented during the second mate-choice test of the focal fish. After finishing the experiments, we released all fish back into their home tanks. Atlantic mollies are small neotropical fish inhabiting fresh and brackish water . They arWe performed male and female mate-choice experiments and controls in the same manner. We conducted single mate-choice experiments to determine the initial mate choice of audience fish, social mate-choice experiments to investigate the audience effect on focal fish and the MCC response of audience fish see , and conAll experiments and controls were performed in a large test tank (100 cm × 50 cm × 40 cm) and two small stimulus tanks (20 cm × 25 cm × 40 cm) each adjacent to each end of the large tank see . All tanThe single mate-choice experiment was conducted one day before the social mate-choice experiment (see below). In the single mate-choice experiment, the fish later used as the audience fish in the social mate-choice experiment could choose between two stimulus fish of the opposite sex without any other conspecific present. First, we inserted opaque screens between the test tank and the two smaller tanks for stimulus fish. The audience fish was placed into the test tank and two stimulus fish (different in body length of at least 5 mm) were placed into the stimulus tanks one at each side of the test tank. The side on which the larger stimulus fish was presented was randomized. Each fish could explore and acclimatize to the new environment. After 20 min, the audience fish was gently placed into a clear Plexiglas cylinder (11 cm diameter) in the middle of the test tank, and the opaque screens were removed. During this period, the audience fish was able to observe both stimulus fish for 10 min see a. After We measured the absolute association time (in seconds) an audience fish spent within each mate-choice zone by adding the time spent in the same mate-choice zone in both 10 min trials of the single mate-choice test. The audience fish was considered to prefer a particular stimulus fish if it spent more than 50% of the total association time within the mate-choice zone in front of that particular stimulus fish during the two 10 min mate-choice trials. This is a commonly used criterion to decide which of two stimuli was chosen . AssociaAn audience fish was considered to be side biased if it spent more than 90% of the total time in the same mate-choice zone in both 10 min trials although stimulus fish had been switched. Those audience fish were rejected from the analysis. This criterion has been used in several other studies with Poeciliids and other fish species ,113,114.After finishing a single mate-choice experiment, we started with the social mate-choice experiment combining the audience effect and MCC. The social mate-choice experiment consisted of two phases; Phase 1 including the first and Phase 2 the second mate-choice test of the focal fish, and Phase 3 including a mate-choice test of the audience fish. The second mate choice test of the focal fish was performed in the presence of the audience fish see b. We wouPhase 1: 1. Mate-Choice Test of the Focal FishThe first mate-choice test of the focal fish was performed without an audience fish and served to determine the initial mate choice of the focal fish. It was performed in the same manner as the single mate-choice test described in detail above . Phase 2: 2. Mate-Choice Test of the Focal FishAfter this first mate-choice test, we immediately started the second mate-choice test and tested whether the focal fish would change its mate-choice decision when the audience fish is present. Thus, we tested whether male and female Atlantic mollies showed an audience effect.First, we inserted the opaque screens between the test tank and the stimulus tanks, placed the focal fish back into the Plexiglas cylinder for 5 min and placed an audience fish of the same sex and of similar body length as the focal fish into the large test tank so that the audience fish could explore the tank. After 5 min the audience fish was then gently placed into another clear Plexiglas cylinder (11 cm diameter) next to the focal fish. We removed the opaque screens and released the focal fish into the test tank and started the mate-choice test within Phase 2. We recorded the time the focal fish spent within the mate-choice zones in front of the same two stimulus fish as in the first mate-choice test, for two 10 min trials, including switching the stimulus fish after the first 10 min trial. We decided to present the audience fish always in the mate-choice test within Phase 2, because we needed to measure first the initial mate-choice decision of the focal fish when no conspecific was present to be able to compare its initial mate choice to the mate choice in the presence of an audience. We controlled for mate-choice consistency in focal fish (Control 1 and 2).Phase 3: Mate-Choice Test of the Audience FishAfter finishing the mate-choice test in Phase 2 , we immeControl 1—Mate Choice Consistency in Focal Fish When no Audience was PresentHere, we investigated whether focal fish choose consistently in the mate-choice test in Phase 1 and 2 of the social mate-choice experiment when no audience was present in the Phase 2. We performed this control in the same setup under the same conditions and same procedures as the social mate choice experiment. The only difference was that the second clear Plexiglas cylinder in Phase 2 contained no audience fish. Control 2—Mate Choice Consistency in Focal Fish When the Audience Fish was Present but not Visible In this control we investigated whether focal fish chose consistently in the mate-choice test in Phase 1 and 2 when an audience fish was presented in Phase 2 but inside an opaque cylinder and thus not visible to the focal fish. We performed this control in the same setup under the same conditions and procedures as the social mate-choice experiment with one exception. We placed the audience fish directly into the opaque cylinder. It was not allowed to explore the tank. Control 3—Mate Choice Consistency in Audience Fish When no Public Information was AvailableFor this control we used data collected in Control 2 to test whether audience fish chose consistently and preferred the same mate-type  when they had no opportunity to copy the mate choice of the focal fish while they were inside an opaque cylinder during the mate-choice test in Phase 2. For this we compared the mate-choice decision of audience fish in Control 2 with their initial mate-choice decision in the single mate-choice experiment.After each single mate-choice experiment, each social mate-choice experiment, and after each control we measured body lengths of all fish used in a test as the standard body length, from the tip of the snout to the end of the caudal peduncle in males and females. Stimulus fish used in a test always differed in body length by about 5 mm. Focal fish and audience fish used within a test were matched for body length. Standard body lengths of all used fish are given in Each male was used only once as a focal male in the social mate-choice experiment. Each audience male was used once in the single mate-choice experiment and afterwards once in the social mate-choice experiment. Stimulus females were used twice, but with the second time always in combination with another female. In each control we used new focal males and audience males. In all experiments and controls, focal males and audience males used together in a test were matched for their initial mate choice, i.e., a mate choice for either larger or smaller females. Thus, we used an audience male with a preference for larger females in a social mate-choice test with a focal male expressing an initial preference for larger females as well. We tested 20 focal males and 29 audience males. We could use 20 focal males and had to exclude nine audience males because of side preferences or a mismatch in mate preferences. In Control 1, we tested 20 focal males. In control 2, we tested 21 focal males and used 20 audience males. One focal male had to be excluded because of side preferences. For Control 3, we used data from 20 audience males used in Control 2. Each female was used only once as a focal female in the social mate-choice experiment. Each audience female was used once in the single mate-choice experiment and afterwards once in the social mate-choice experiment. Stimulus males were used twice but always the second time in combination with another male. In each control, we used new focal females and audience females, where required. In all experiments and controls, focal females and audience females used together in a test were matched for their initial mate choice, i.e., a mate choice for either a larger or smaller male. Thus, we used an audience female with a preference for larger males in a social mate-choice experiment with a focal female expressing an initial preference for larger males as well. We tested 26 audience females and 26 focal females. Twelve females had to be excluded because of side preferences or a mismatch in mate preferences. We could use 20 audience females and 20 focal females. In Control 1, we tested 23 focal females and excluded 3 because of side preferences, leaving 20 focal females. In Control 2, we tested 25 focal females and used 25 audience females. Ten females had to be excluded because of side preferences and mismatch in initial mate choice, leaving 20 focal females and 20 audience females. For Control 3, we used data of the 20 audience females tested in Control 2.The performed experiments and handling of the fish were in line with the German Animal Welfare Act (Deutsches Tierschutzgesetz) and approved by the internal animal protection commissioner Dr. Urs Gießelmann, University of Siegen, and the national Veterinary Authority . All analyses were conducted with the R software 3.4.0 . The mate-choice scores used in the linear model (LM) analysis were calculated as −. For focal fish, the first and second mate-choice test correspond to Phases 1 and 2 of the social mate-choice experiment, respectively b. For aup-values, by dropping out non-significant effects, starting with the highest order interaction. We used Akaike Information Criteria [p = 0.902; focal fish LM: df = 1, F = 1.370, p = 0.244), size difference between audience and focal fish , size of stimulus fish  nor size difference between stimulus fish .The starting model included treatment with two levels (informed/uninformed), the type of fish , and sex  as fixed effects. Informed in this context indicates that fish were able to gather public information (corresponding to the mate-choice experiments) while uninformed fish had no opportunity to observe conspecifics during mate choice (corresponding to the controls). All starting models included interactions between fixed effects. We applied a backward selection method using Criteria  to deterCriteria . With tht-test  focal and audience fish spent in front of one stimulus fish divided by the total time spent in both mate-choice zones. Thus, we obtained a score for the preferred and the non-preferred stimulus fish of each focal and audience fish tested in single mate-choice experiments, in social mate-choice experiments, and in all controls. Here, the predictions were that focal fish should decrease their preference for their initially preferred mate in the presence of an audience fish and that the audience fish should copy that mate choice. To test this, we compared the mate-choice scores for preferred and non-preferred stimuli used within and between tests using Wilcoxon matched-pairs tests, and we compared mate-choice scores for preferred stimulus fish between focal and audience fish using Mann-Whitney U tests. We used non-parametric tests because in this case the normality assumption was rejected for this mate-choice score. To control for body size differences, we tested whether fish used within a test differed in standard body length using a paired p = 0.08) and a significant interaction between type of fish  and sex  . For further analysis, we split the data set by the type of fish and analyzed focal and audience fish separately. For focal fish, we found a significant effect of the sex  and treatment  on the mate-choice score. If focal fish were informed, thus aware of the presence of an audience, this had no effect on males , but did have an effect on female focal fish . In audience fish, neither sex  nor treatment  had an effect on the mate-choice score. It is worth, however, to have a closer look at the mate-choice decision of focal and audience fish in the different situations. In the following, we present the analysis with the scores for preferred and non-preferred stimuli within the mate-choice tests of the social mate-choice experiments and controls. The starting model of variables explaining the first mate-choice score included three main effects (i) treatment (informed/uninformed), (ii) type of fish , and (iii) sex , plus all possible interactions. We found a trend for treatment . Only 11 of 20 focal males spent more time in front of larger females. Thus, focal males altered their mate-choice decision in the presence of an audience male, and therefore, showed an audience effect.Focal males showed a clear preference for one of the two females in Phase 1 . Only 13 of 20 audience males spent more time in front of larger females. Furthermore, they spent the same amount of time in front of the preferred female as the focal fish . Thus, audience males copied the altered mate choice of focal males.Audience males showed a clear preference for one of the two females in the single mate-choice test . In the mate-choice test of Phase 1, 16 of 20 focal males spent more time with larger females and in Phase 2, 13 of 20 focal males spent more time with larger females.Focal males showed a clear preference for one of the two females in the mate-choice test of Phase 1 . Thus, focal males did not choose consistently when an audience male was present but not visible to focal males.Focal males showed a clear preference for one of the two females in the mate-choice test of phase 1 . Thus, audience males chose consistently between female phenotypes when they had no opportunity to copy. Audience males spent more time in front of a specific female phenotype in the single mate-choice experiment . Now 16 of 20 audience females spent more time with larger males. Furthermore, they spent the same amount of time in front of the preferred male as the focal female in the mate-choice test before . Thus, after observing the focal females’ mate choice, audience females changed their mate-choice decision and copied the altered mate choice of the focal female. Audience females showed a clear preference for one of the two males in the single mate-choice test . Twelve of 19 focal females spent more time with larger males in Phase 1 and in Phase 2. Thus, focal females chose consistently when no audience female was present. Focal females showed a preference for one of the two male phenotypes in the mate-choice test of Phase 1 . In phase 1, 15 of 20 focal females spent more time with larger males, and in Phase 2, 16 of 20 focal females preferred larger males.When audience females were present inside an opaque cylinder and thus not visible to the focal female in the mate-choice test of Phase 2, focal females spent significantly more time with a specific male phenotype in the mate-choice test in Phase 1 . Fifteen of 20 audience females spent more time with larger males in Phase 1 and 13 females in Phase 2. Thus, audience females chose consistently between male phenotypes when they had no opportunity to copy.Audience females preferred a specific male phenotype in the single mate-choice test  and sex. For focal fish, we found a significant effect of the sex and treatment on the mate-choice decision. If focal fish were aware of the presence of an audience fish, this had no effect on males, probably due to the inconsistency in control 2, but we found a clear effect in female focal fish. In audience fish, neither sex nor treatment had an effect on the mate-choice decision. Thus, our results were not always clear-cut, but this might be due to the complex test situation and due to the fact that mate-choice behavior including public information is very sophisticated and many factors affected the behavior of the fish during the experiments, like familiarity with stimulus fish between tests and decrease of interest to stimulus fish. Nevertheless, focal fish clearly altered their mate-choice decision when an audience was present, and audience fish copied the altered mate choice of the focal fish, and thus exhibited a different mate choice than without observing the mate choice of the focal fish. To our knowledge, this is the first experimental evidence supporting the deception hypothesis in the context of the audience effect in mate choice. Makowicz et al.  showed iP. mexicana females have genetically based mate preference for larger males [It is known that er males , and simer males . Howeverer males , AtlantiHirundo rustica used deceptive alarm calls when their mates left the nest vicinity during their fertile period, to cheat surrounding males and to lower the risk of extra-pair copulations [Deception is quite common in the animal kingdom and is described in various contexts such as in the case of stealing food ,120,121 ulations . The casulations . In an eulations  could fuBierbach et al.  showed iThe deception hypothesis in the mate-choice context suggests that males deceive other males to reduce risk of sperm competition for the deceiver ,100,101.Castellano et al.  exploredAn alternative to the deception hypothesis explaining the audience effect in the context of mate choice is the ‘split-attention hypothesis’ . The ‘spAnother alternative to the deception hypothesis explaining the audience effect in the context of male mate choice might be male competition for mates. Here, focal males alter their mate choice to adjust their reproductive success to the currently perceived social competitive context ,129. In For testing our prediction, it was absolutely necessary that we always tested first a focal fish without an audience and secondly with an audience. This, however, is in conflict with an order effect. It could be that in general same fish behave differently in the second mate-choice test than in the first one independently of the choice situation. The Controls 1 and 3 in male experiments and the Controls 1–3 in female experiments provide evidence that in general males and females chose similarly in two subsequent tests without any change in test situation.Results from our controls showed that focal males and focal females chose consistently when no same sex audience fish was present during their mate choice (Control 1), and that audience fish chose consistently when they had not witnessed the mate choice of focal fish (Control 3). Interestingly focal males altered their mate-choice decision in Control 2, when an audience male was inside an opaque cylinder, and thus present but not visible to the focal male. We assumed that the focal male was probably able to perceive olfactory cues of the audience male in the opaque cylinder. In general, males use chemical cues to test whether females are receptive by nipping at the females’ genital opening . FurtherWe tested the deception hypothesis in the context of the audience effect in mate choice, and found that both male and female Atlantic mollies alter their mate choice in the presence of a same sex audience fish and mislead the audience fish who copied the altered mate choice. Our study shows how complex the social information network in the Atlantic molly can be and that both sexes use public information for their mate-choice decisions. To our knowledge, this is the first experimental evidence supporting the deception hypothesis in the context of the audience effect in mate choice. Thus, the audience effect can be an adaptive strategy to lead a competitor away from the preferred mate in both sexes. Further experiments are necessary to explore how, when, and why fish use public information in mate choice, and how the use of public information affects sexual selection.


Value-based decision making relies on distributed neural systems that weigh the benefits of actions against the cost required to obtain a given outcome. Perturbations of these systems are thought to underlie abnormalities in action selection seen across many neuropsychiatric disorders. Genetic tools in mice provide a promising opportunity to explore the cellular components of these systems and their molecular foundations. However, few tasks have been designed that robustly characterize how individual mice integrate differential reward benefits and cost in their selection of actions. Here we present a forced-choice, two-alternative task in which each option is associated with a specific reward outcome, and unique operant contingency. We employed global and individual trial measures to assess the choice patterns and behavioral flexibility of mice in response to differing “choice benefits” (modeled as varying reward magnitude ratios) and different modalities of “choice cost” (modeled as either increasing repetitive motor output to obtain reward or increased delay to reward delivery). We demonstrate that (1) mouse choice is highly sensitive to the relative benefit of outcomes; (2) choice costs are heavily discounted in environments with large discrepancies in relative reward; (3) divergent cost modalities are differentially integrated into action selection; (4) individual mouse sensitivity to reward benefit is correlated with sensitivity to reward costs. These paradigms reveal stable individual animal differences in value-based action selection, thereby providing a foundation for interrogating the neural circuit and molecular pathophysiology of goal-directed dysfunction. Novel protocol to reveal stable “trait-like” measures of value-based choice in mice.Mice integrate relative rewards and costs associated with obtaining those rewards in selecting future actions.Reward costs are heavily discounted in the context of large discrepancies in outcome benefit.Both increased operant scheduling and temporal delay to reward decrease the relative value of choice alternatives, but do so in divergent manners.Sensitivity to reward benefit is correlated with sensitivity to cost at the individual animal level.In order to make optimal choices in a complex world, individuals must be sensitive to the costs and benefits of particular actions and integrate those components to holistically control motor output  were used in this study. Animals were housed in cages of at least four, with All experiments were conducted inside a modular chamber with dimensions 8.5 × 7.12 × 5 inches (W × D × H) . Each chamber contained a modified reward magazine through which liquid reward was pumped directly into a custom-made receptacle. On either side of the magazine were retractable levers which had to be fully depressed to register choices. A light in the magazine turned on to indicate the beginning of each trial, after which animals were required to make a sustained (200 ms) magazine head entry to initiate the choice period. The choice period was marked by the extension of levers on either side of the reward magazine, illumination of lights immediately above the protracted levers, and extinction of the magazine light. Mice then had an x-sec temporal window (contingent on current protocol) to register choice via lever press, after which the lever retracted and the trial was considered an omission. Following successful choice selection, the levers were retracted and a variable volume of liquid reward  was delivered via the center magazine, which had its light turned on for the duration of the reward period. Reward volumes were determined by variable activation time of single-speed syringe pumps . Mice were allowed 5 s for reward consumption after which all box lights were inactive for a 1 s inter-trial interval prior to next trial start. All magazine entries (detected by interruption of infrared beam) and lever presses were recorded by MedAssociates software (MedPC-IV). Data were exported to Microsoft Excel via MedAssociates software (MED-PC to Excel).rew = 1.0). Sessions lasted 45 min with no trial number limits. After nine sessions, mice that had completed 2 consecutive days of >150 trials or 1 day >200 trials progressed to the serial reversal task. If mice missed this deadline, they were again assessed after sessions 12 and 15. Mice that failed to meet these criteria by session 15 were excluded from the study (n = 3).In the first stage of training, animals were habituated to behavioral boxes for 10 min., followed by a program that delivered 10 μL of reward every minute for 40 min. via the magazine port. Reward delivery was not contingent on mouse choice. Upon reward delivery, the magazine light turned on for 10 s to cue the mouse to reward, followed by a 50 s inter-trial interval. After 3 days in this introductory program, mice learned a lever press-reward contingency. Trials were initiated as described previously. During the choice phase, 1 of 2 levers were protracted, at random, on each trial. Mice had a 10 s temporal window to register their choice via lever press, otherwise the lever retracted and the trial was considered an omission. If animals registered a selection within the given choice time, 10 μL of reward was delivered  were equally applied to both levers. These contingencies were held constant for the duration of a session. Following choice selection, both levers retracted and the 5 s reward phase initiated.Animals that met the criteria for acquisition of the action-outcome contingency progressed to a forced-choice two-alternative serial reversal paradigm. Trials began as in the previous protocol, with illumination of the magazine light. Again, mice initiated trials with a 200-ms sustained magazine entry, which led to the choice period. Mice then had a 5 s temporal window to register their choice via lever press, otherwise the lever retracted and the trial was considered an omission. On every trial, both levers were presented. Reward volumes were varied according to experiment and reward probabilities .R Studio (3.3.1)  and Pr(B) refer to the probability that mice stay on the choice alternative producing the larger volume outcome (A) and the smaller volume outcome (B), respectively, on the TAdaptability Index was calculated as:ith contingency switch in an individual session and where Relative Initiation Latency was calculated as:Large and LatInitSmall refer to the average latency to initiate trials following large reward and small reward outcomes, respectively, in an individual session.where LatInitThe extent to which the application of reward costs affected the choice distribution of individual animals was calculated as:RAVCost and RAVNoCost refer to the relative action values for animals in reward contingencies with and without cost considerations, respectively. The magnitude of values represent the degree to which costs decrease  or increase  the relative value of choice alternatives to which costs have been applied. The relative sensitivity of animals to particular cost modalities was calculated as the z-score of this value.where n = 5 trials) and their resulting outcomes:A logistic regression model was used to model current choice as a function of past actions (R(i) is the probability of choosing the right-sided alternative on the ith, current, trial. LR(i-p), SR(i-p), and NR(i-p) refer to the outcomes of the pth previous trial. LR(i-p) is defined such that LR(i-p) = +1 if an animal received a large reward on the pth previous trial resulting from a right press, a −1 if an animal received a large reward on the pth previous trial resulting from a left press and 0 if the animal did not receive a large reward. SR(i-p) and NR(i-p) are defined similarly for trials that resulted in small reward and no reward outcomes, respectively. Together these variables account for lateralization of past choices and the resultant outcomes. This method assumes equivalent reinforcement from outcomes regardless of the lateralization of choice. Regression coefficients were fit to individual mouse data using the glm function in R with the binomial error distribution family. Coefficient values for individual mice were averaged to generate the plots and perform the analysis observed in Figure 4.where We fit an adapted Q-Learning Reinforcement Model with four parameters to our behavioral data , is used to update the value of subsequent actions. In this context, α determines the extent to which new information about the state-action pairing alters subsequent behavior. In keeping with standard Q-learning models, values for the unchosen alternative were not updated. In order to model the choice behavior of mice based on these action values, we implemented a softmax decision function to convert values into action probabilities. The operator computed the probability of choosing an alternative A on trial t as:where Ct−1, where Ct−1 is an indicator variable that denotes whether the animal selected alternative A on the previous trial . κ represents the extent to which the animal's previous choice influences its subsequent choice irrespective of outcome. The constant value, c, captures any existing preference for a particular choice alternative. In order to fit this model to our choice data we performed a maximum likelihood fit using function minimization routines of the negative log likelihood of models comprised of different combinations of our three parameters  in MATLAB  are included in All data were initially tested with appropriate repeated measure ANOVA (Prism 7.0). Main effect and interaction terms are described both within figures and accompanying figure legends. Results of relevant rew = 1.0, 0.7, and 0.4 for different cohorts of mice throughout training). The number of trials performed between contingency switches (Block Length) as well as the overall probability that mice chose the rewarded outcome [Pr(Large Reward)] served as global measures of choice efficiency in this task  on future choice (rew = 0.4) driving less robust control of current behavior. Given that the outcome of the T−1 trial largely determined future choice for higher rates of reinforcement , we focused primarily on this trial for analyses of behavior.To understand the changes in action selection underlying this increased behavioral efficiency, we explored how reward differentially shaped behavior in early and late periods of training. When first presented with a choice between levers following rewarded outcomes, mice exhibited random choice preference. Upon further training, these distributions became biased toward choices that produced proximal rewarded outcomes . To furte choice , bottom.rew = 0.7  to perform this analysis. We found that together, the relative action value and adaptability index explained 82% of the variability in individual session performance  choose the higher value option and (b) flexibly alter behavioral responding at contingency changes. To better characterize how different outcome values alter mouse choice patterns, we systematically compared the probability that animals returned to a choice after receiving a reward   , so we f outcome . The rel outcome . While w outcome . We also outcome . Taken trew = 0.7 (rew = 0.4). As demonstrated previously, the rate at which animals selected the large reward outcome in this task was influenced not just by the ability of the animals to discriminate between relative benefits of the lever alternatives, but also by their flexibility at contingency switches. Here we note that adaptability is modulated by differing value contingencies . Here, the learning rate (α) provides a measure of how strongly new reward information modifies values for a specific action. To characterize the extent to which action values influenced choice behavior, we utilized a softmax decision function to solve for the inverse temperature parameter (β). We added additional terms to capture the propensity of mice to repeat actions irrespective of previous outcome, κ, and a constant measure of bias, c, for one alternative over another. We fit this model to our data and observed that it predicted actual mouse choice behavior with high accuracy  in most reward environments tested, suggesting a high gain for proximal reward history—a finding supported by our regression model of choice behavior , 1F. FurWhile manipulations of relative reward contingencies show consistent population effects on outcome valuation and behavioral flexibility, we sought to characterize whether these metrics exhibited stable patterns across multiple sessions and contingencies. For each session, we extracted the relative action value and adaptability index and z-scored these values relative to the population performing the same day of a given operant contingency. This allowed us to estimate the individual value sensitivity and behavioral flexibility of mice relative to the population for each session. We observed a significant correlation of relative action value and adaptability between the first day and subsequent sessions (averaged metrics from days 2 to 3) within the same reward contingency . Next, wrew = 1), with the higher response schedule exclusively paired to the large reward alternative. Interestingly, global task measures  demonstrated that disparities in effort schedule did not significantly alter the performance of mice in the task when the relative reward difference was sufficiently large . An analysis of global performance demonstrated main effects of relative reward ratio and temporal delay on Pr(Large Reward), and a significant interaction between these variables for block length  and applied these delays exclusively to large reward alternatives in any given block . We agaik length .While the global effects of delay largely mirror the effects of effort , we founFor further insight into any systematic relationships between how animals process benefit and cost, we analyzed individual mouse values for benefit sensitivity, with and without relative cost considerations. Our previous data demonstrated trait-like patterns of relative reward bias, reflecting an underlying distribution of reward sensitivities . We hypoThe development of quantitative behavioral assays in mice that probe core features of value-based action selection is an important step toward understanding the neural substrates underlying economic decision-making. These circuits are of critical behavioral relevance, regulating how animals select actions based on the assigned values of available options, a fundamental organizing principle for how organisms interact with their environment . Overall, these data suggest that the changing choice patterns observed during relative reward experiments are not likely due to fundamental changes in how mice approach the task but instead to the nuanced dynamics of the action values themselves. If true, this behavioral paradigm should provide an ideal testing ground to examine the cellular integration of outcome cost and benefit on action values during decision making.To complement our regression-based behavioral analysis, we submitted our data to standard Q-learning models  Katahir. This apin vivo physiological recordings of corresponding neural activity.To further establish the validity of these measures and their stability within animal, we examined their cross-session and cross-contingency consistency. We reasoned that value processing likely represents a behavioral trait—a temporally stable behavioral pattern unique to each subject. In accordance with this, we observe significant within-animal consistency across 3 days of repeated contingencies, for both relative action value and adaptability . As a fuWithin this quantitative framework of mouse behavior, we sought to answer a fundamental neuroeconomic question: How do the benefits and costs of rewarded outcomes shape mouse behavior? Characterizing how diverse features of mouse behavior are modulated by value is a critical step in elucidating neural circuits with specific reward processing function. We observed that mice differentially altered their choice patterns in response to the relative magnitude of the previously rewarded outcome—with more extreme distributions for outcomes of higher benefit . FurtherWe then associated two cost modalities exclusively with high-benefit options in environments with large and small disparities in reward, to assess the integration of reward costs in decision making. We demonstrate that in environments with large differences in reward, costs associated with selecting those outcomes are heavily discounted. Surprisingly, while increased operant scheduling and temporal delay both decrease the relative value of choice alternatives, we found that these two cost modalities differentially altered the relative value of previous outcomes, with effort increasing stay behavior on the small reward and delay reducing stay behavior on the large reward at the population scale compare . This fiIn summary, these findings demonstrate the sensitivity of our behavioral assay to decision-making strategies adopted by mice during economic choice, while revealing stable, mouse-intrinsic differences in value-based action selection. An important direction for future work will be to characterize the local circuits governing the distinct behavioral features described here. For example, the orbitofrontal cortex is intimately involved in value representations central to efficient performance of this task (Schoenbaum et al., We believe that the reliability and robust quantitative nature of these paradigms makes them well-suited to investigating the complex issue of how reward processing is altered by environmental and genetic factors. We uncovered a substantial amount of between-animal variability for value processing, perhaps surprising given the genetic homogeneity of the mouse strain used. We believe this observation is both unsurprising and fascinating. In our view, genetically encoded information provides a basic blueprint for the assembly and maintenance of neural circuits. However, quasi-stochastic processes such as axonal targeting and sub-cellular synapse localization are then superimposed on this basic plan, generating diversity within circuits, and behavior. These differences can be further amplified by existing social hierarchies and other experiences in the home cage leading up to testing (Greenberg et al., OA performed operant experiments, analyzed data, generated analysis code, and wrote the manuscript. MPF performed operant experiments. MVF conceived the project, analyzed data, and wrote the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Schizosaccharomyces pombe that eliminates meiotic transcripts during normal vegetative growth. Mmi1 contains a YTH domain that binds specific RNA sequences, targeting mRNAs for degradation. The YTH domain of Mmi1 uses a noncanonical RNA-binding surface that includes contacts outside the conserved fold. Here, we report that an N-terminal extension that is proximal to the YTH domain enhances RNA binding. Using X-ray crystallography, NMR, and biophysical methods, we show that this low-complexity region becomes more ordered upon RNA binding. This enhances the affinity of the interaction of the Mmi1 YTH domain with specific RNAs by reducing the dissociation rate of the Mmi1–RNA complex. We propose that the low-complexity region influences RNA binding indirectly by reducing dynamic motions of the RNA-binding groove and stabilizing a conformation of the YTH domain that binds to RNA with high affinity. Taken together, our work reveals how a low-complexity region proximal to a conserved folded domain can adopt an ordered structure to aid nucleic acid binding.Mmi1 is an essential RNA-binding protein in the fission yeast  Schizosaccharomyces pombe, Mmi1 (meiotic mRNA interceptor 1) is essential for viability and represses the expression of transcripts required for entry into meiosis during normal vegetative growth RNA-binding proteins regulate gene expression and can elicit profound phenotypic effects. In meiRNA. meiRNA contains DSR elements that are thought to act as a molecular sponge to sequester Mmi1 from its targets  that are present in multiple copies within DSRs (N6-methyladenosine (m6A)  –17. Cryse (m6A) ––20. Surp (m6A) ––, 22.A). This is consistent with a large proportion of RNA-binding proteins (up to one-third) containing substantial intrinsically disordered regions (IDRs), compared with the remainder of the proteome  with no predicted secondary structure A. This iproteome . IDRs caproteome , 25. We proteome . The remHere, we show that a low-complexity region proximal to the Mmi1 YTH domain is required for stable RNA binding. Biophysical and structural analyses show that this region undergoes dynamic changes on RNA binding and decreases the off-rate for DSR RNA substantially. Thus, a low-complexity region proximal to a folded RNA-binding domain can strongly influence interactions with RNA substrates.rec8 3′-UTR . Full-length Mmi1 could only be expressed in small quantities and was difficult to purify to homogeneity. Thus, we tested binding with an Mmi1 YTH domain construct encompassing the RNA-binding domain, plus an additional 20 N-terminal amino acids with no predicted secondary structures that are nevertheless ordered in a published crystal structure and contact RNA  . It is likely that the YTH protein binds RNA but dissociates during electrophoresis, i.e. the off-rate is too fast to observe complex formation by EMSA.To investigate the interaction between the Mmi1 YTH domain and RNA, we performed electrophoretic mobility shift assays (EMSAs) with a fluorescently-labeled RNA substrate composed of a DSR sequence from the rec8DSR, A. Full-lFig. 1A) . AlthougA). Residues 282–326 are largely hydrophilic and include 10 serines, a lysine/arginine-rich stretch (residues 301–309), and three prolines (residues 313–315). Purified Mmi1(282–488) behaved well in solution: nano-differential scanning fluorimetry (nano-DSF) and circular dichroism (CD) spectroscopy data were consistent with a folded YTH domain . Furthermore, size-exclusion chromatography coupled to multiangle light scattering (SEC-MALS) showed that it was monomeric and monodisperse in solution . When we analyzed RNA binding by EMSA using this construct, a stable RNA–protein complex was visualized as a better-defined band on the gel . Therefore, the upstream sequence proximal to the YTH domain likely increases the binding affinity of the YTH domain for DSR RNA. We termed this 45-amino acid segment the “upstream stabilization region” (USR).To determine whether additional N-terminal sequence elements are involved in RNA binding and stabilization of the complex, we used a longer construct, including residues 282–488 A. ResiduDK) of YTH and USR–YTH proteins for RNA, we used fluorescence polarization with a short 15-mer DSR-containing substrate (shortDSR). Longer RNAs exhibit secondary binding events at higher concentrations, observed as a supershifted band in the EMSA . We used increasing protein concentrations and buffer containing either 50 or 150 mm NaCl and measured the change in fluorescence polarization of the labeled RNA. The resulting equilibrium binding curves were fitted with a quadratic single-site binding curve. This revealed that at 150 mm NaCl, the binding affinity (DK) of YTH was 154 ± 85 nm . In comparison, an earlier study reported a binding affinity of 440 nm for Mmi1 residues 322–488, and 390 nm for residues 316–499, with a 10-mer RNA using isothermal titration calorimetry . A similar trend was observed at lower salt . Even though ∼50 residues have low sequence complexity, there was no evidence of substantial random coil regions, which would typically manifest as relatively sharp peaks at 8–8.5 ppm in the proton dimension. Thus, the USR may have some order.Because the USR has no predicted secondary structure, we used NMR spectroscopy to investigate its properties in solution. We recorded tructure A. Even t13C-15N–labeled protein were incomplete and had low signal intensities. This suggested either faster overall relaxation as a result of a large hydrodynamic radius or line broadening caused by conformational averaging. Overall relaxation behavior improved with deuteration of nonlabile carbon-attached protons; however, samples obtained from expression in deuterated media suffered from a lack of “back-exchange” for residues later identified to be from the β-sheet. These slowly-exchanging amide protons yielded signals only after incubation of the sample with a chaotropic agent (4 m urea) followed by dialysis to remove urea. Taken together, this indicates a highly dynamic domain with a rigid core consisting of the β-sheet and more flexible regions where only broad or no signals were observed. In total, only ∼60% of residues were assignable. This included only 14 confirmed residues in the low-complexity region, suggesting that the remainder of the signals from this region are missing due to line broadening; there were ∼50 fewer cross-peaks than expected, even in the BEST-TROSY experiment with nondeuterated protein. The lack of narrow, intense signals attributable to unstructured regions due to quasi-individual mobility of residues means the results obtained here suggest a sampling of interconverting conformations on a micro- to millisecond time scale.We next recorded three-dimensional spectra for backbone assignment of USR–YTH. Spectra of We compared the assigned USR–YTH spectrum with an unassigned YTH spectrum using nearest neighbor minimal chemical shift perturbation maps . These mIn a previous crystal structure of the Mmi1 YTH domain (residues 322–488), most of the USR was not included (Table S1) and built an atomic model for residues 315–488 and all seven nucleotides (Table S1).We were not successful in crystallizing the USR–YTH construct itself, but removal of an additional 19 residues from the N terminus allowed us to crystallize Mmi1 residues 301–488 bound to a 7-mer DSR RNA (UUAAACC). We determined its X-ray crystal structure to 1.4 Å resolution . This results in a network of hydrophobic interactions involving residues Leu-316, Phe-318, and His-324 of the USR, Met-334 and Ile-335 from the N-clamp, and Met-465 and Arg-471 from the YTH domain . Interestingly, the low-complexity N-clamp region adopted a similar conformation in two structures of Mmi1 determined in the absence of RNA . This could stabilize the conformation of the low-complexity region, even in the absence of RNA. Furthermore, structural alignments of all known Mmi1 YTH domain structures show that the core fold is highly similar (r.m.s.d. <0.5 Å), but the N- and C-clamps are more divergent . This observation is also consistent with higher relative B-factors in these regions . In summary, our structure shows that part of the low-complexity USR of Mmi1 (residues 315–326) lacks secondary structure elements but adopts a defined fold on RNA binding, forming contacts with the YTH domain.The core YTH domain of our structure of Mmi1-RNA is similar to apo-Mmi1 and to previously reported structures , 22 withFig. 415N-1H spectrum showed that almost all residues were represented by a cross-peak.Because we were unable to crystallize the full-length USR–YTH construct and the USR may adopt more than one conformational state, we further studied RNA binding using NMR. Again, we performed backbone experiments on USR–YTH, this time bound to unlabeled 19-mer DSR-containing RNA. We could assign ∼70% of the residues in the RNA-bound form. In contrast to the unbound protein, a peak count of the A). When these chemical shift perturbations are mapped onto the crystal structure, few changes are found on the RNA-binding central β-sheet, but instead they are located mainly in the N- and C-terminal regions (B). Surprisingly, 31 additional amide peaks of the low-complexity region could be assigned in the presence of RNA . Some of these correspond to residues not observed in the crystal structure. Thus, in the presence of RNA, it is likely that the conformation of the USR and N-clamp become even more constrained, reducing the line broadening in NMR spectra and thereby leading to the appearance of additional protein amide peaks.Next, to map residues whose chemical environments change on RNA binding, we compared the assigned NMR amide group chemical shifts in the spectra of USR–YTH in the presence and absence of RNA. We observed some large-scale shifts and the appearance of new peaks on RNA binding A. When t regions B. SurpriB, asterisk), suggesting that, in the presence of RNA, this region may adopt multiple conformations or is contacted transiently by the USR. Examination of relative B-factors from our crystal structures suggests that the final C-terminal helix is relatively rigid when bound to RNA . The N terminus of the modeled density in our crystal structure is in a position such that residues further upstream could extend toward the C-clamp (A), consistent with transient interactions with the USR, resulting in the line broadening seen by NMR.RNA binding to USR–YTH also leads to loss of amide signals corresponding to the C-terminal helix of Mmi1  . There are several changes in the USR–YTH spectrum, clustered at the N and C termini. For example, the chemical environments of Asp-467 and Arg-471 at the top of the C-terminal helix change. These residues contact the N-clamp and USR, respectively (B). In addition, a cluster of residues within the N-clamp, including Asn-344, Ile-346, and Ser-347, are in a different chemical environment, possibly because of increased flexibility of this region in the absence of the USR.We also used minimal chemical shift perturbation maps to compare the assigned USR–YTH/RNA spectra to unassigned spectra of YTH bound to RNA to examine how the USR influences binding , A and Bectively B. In add1H-15N heteronuclear NOE (hetNOE) experiments that are sensitive to motion on the picosecond time scale. When a residue is part of a rigid secondary structure element, such as a β-sheet, the hetNOE value is ∼0.8. Residues from more flexible parts of the protein have lower hetNOE values, often reaching negative values at highly flexible N and C termini. The hetNOE experiments show that the N- and C-terminal regions of USR–YTH become more ordered on RNA binding (C). Specifically, the N-clamp, the USR, and residues 485–488 are flexible or unassigned in the absence of RNA but have enhanced rigidity in the presence of RNA, confirming our indirect observation that the addition of RNA stabilizes the dynamic nature of the protein resulting in more residue peaks being present in the NMR data. In addition, the first β-strand of the core YTH domain is exchange-broadened in the absence but not in the presence of RNA, suggestive of enhanced rigidity upon binding. Together, our observations show that the USR influences the chemical environment of both the N- and C-clamps to enhance RNA binding.These data are consistent with the USR contributing to the interaction with RNA via stabilizing the conformation and reducing the flexibility of the N-clamp in solution. The transient nature of this interaction and the subsequent line broadening made formal determination of this conformation elusive by the traditional NMR structural technique of NOE analysis; however, to determine how backbone dynamics are altered, we performed  binding C. SpecifA and BFig. S5, ). There are no major differences between the spectra or the minimal perturbation maps. Two clusters of residues close to the C-clamp region of the core YTH domain show moderate changes. Binding of the 5′-end of the longer RNA in the C-clamp may involve additional contacts, altering the chemical environment of this particular region. Alternatively, an interaction of the RNA with the USR may contribute to these changes.The RNA used in NMR experiments (19 nucleotides) is longer than the RNA used in crystallographic experiments (7 nucleotides). To determine whether the conformation of the USR and N-clamp are influenced by RNA length, we compared spectra of the USR–YTH construct bound to 19- and 7-mer DSR-containing RNAs . We could model residues 312–488. In this structure, the longer RNA does not form any additional contacts with the YTH domain, and RNA binding is confined to the DSR motif. Furthermore, the conformation of the N-terminal USR is largely similar to those seen in the other structures.To determine whether nucleotides outside the UNAAAC motif are recognized by Mmi1, we determined a crystal structure of USR–YTH (residues 301–488) bound to an 11-mer RNA containing a DSR (B) We used truncation mutants to examine the effects of these regions on RNA binding in vitro. All truncation mutants could be purified after overexpression in Escherichia coli and were similar to the USR–YTH protein in CD and nano-DSF experiments .Together, binding assays, NMR, and X-ray crystallography suggested that residues 282–346 (which include the USR) contribute structurally to the interaction with RNA. In addition, the C-terminal residues of Mmi1 have a different chemical environment upon RNA binding B We usedA and Fig. 7B). When both the N and C termini are deleted , RNA binding is further abrogated . The RNA binding affinity of YTH-ΔNΔC, corresponding to the conserved YTH domain fold, is relatively weak (DK = 51 μm), underlining the key contribution of the low-complexity regions to RNA binding.A C-terminal truncation of the YTH construct  results in an ∼10-fold reduction in RNA-binding affinity compared with YTH (residues 327–488) . Although this low-complexity region contributes only two residues to RNA-binding (residues Asn-336 and Arg-338), our truncation experiments demonstrate that it is critical for the interaction with DSR RNA. Residues 282–346 most likely further stabilize a helical conformation in the bound RNA, and the resultant base-stacking may thermodynamically drive stable binding.Specific and high-affinity RNA binding are likely important for Mmi1 to efficiently repress expression of its target RNAs and prevent premature entry into meiosis. Here, we show that stable RNA binding by Mmi1 is dependent not only on its YTH domain but also on an upstream low-complexity region (residues 282–346). This N-terminal, low-complexity region does not adopt any formal secondary structure but forms a stably-folded platform encompassing the N-clamp and extending across the YTH domain to contact the C-terminal helix A. AlthouC), thus stabilizing its conformation . Interestingly the radius decreases to 2.6 nm in the presence of RNA suggesting a compaction of the low-complexity region. Finally, CD spectra recorded for all the constructs showed a similar line shape, although the magnitude of the signal at ∼225 nm increases as more of the low-complexity region is included . This can be characteristic of proteins with a molten-globule conformation  likely contributes to RNA binding indirectly. We were able to model 15 residues of the USR using crystal structures. The USR adopts a preferred conformation in solution: a network of interactions secures it onto the back of the core YTH domain . The remFig. 6ormation . Substanormation .The USR most likely enhances RNA binding by reducing the dynamic motion of the YTH RNA-binding groove. The USR interacts with residues of both the N- and C-clamps of Mmi1, thus stabilizing the conformation of residues within these regions that contact RNA. This is supported by comparative NMR analysis of USR–YTH and YTH, showing that the chemical environments differ for both N-terminal low-complexity residues and residues in the C-terminal helix. The interaction between Mmi1 and the 3′-end of the DSR RNA is stabilized by the hydrophobic interface between the low-complexity region and the N terminus of the core YTH fold .B), precluding any comparison of the dynamics of this region when bound to RNA. Given the close proximity of the USR to the C terminus on RNA binding, this may be due to the formation of a dynamic interface with RNA in this region. In agreement with this, the N-terminal (residues 322–338) and C-terminal (residues 483–488) regions were found to be disordered in a previous crystal structure . Regions rich in arginine and lysine can contribute to RNA binding directly. For example, serine/arginine-rich regions are intrinsically disordered but are important for functional RNA binding , 33 and Schizosaccharomyces Mmi1 orthologues , although a proximal intrinsically disordered region appears to be present in all YTH domain-containing protein clades examined . IDRs are not constrained by 3D structure, and therefore their sequence may evolve more rapidly than the sequence of structured domains  glycerol, 1 mm TCEP, protease inhibitor mixture (Roche Applied Science), and 0.4 mm phenylmethylsulfonyl fluoride), lysed by sonication, and cleared using ultracentrifugation. Proteins were purified from lysate using batch binding with nickel-nitrilotriacetic acid–agarose, washed with lysis buffer, and eluted in low pH buffer  glycerol, and 1 mm TCEP). The His tag was removed by overnight cleavage at 4 °C with 3C protease. Cleaved protein was diluted 2-fold in 20 mm BisTris, pH 6.0, and 0.5 mm TCEP buffer before loading on a 5-ml HiTrap S  cation-exchange column. Protein was eluted using a gradient of 0.15–1.0 m NaCl over 12 column volumes. Fractions containing Mmi1 were loaded onto a Superdex 75 26/60pg size-exclusion column equilibrated with 20 mm PIPES, pH 7.0, 150 mm NaCl, and 0.5 mm TCEP. Purified Mmi1 was concentrated using a 10,000 MWCO Amicon Ultra-15 concentrator (Millipore) to ∼5 mg/ml for the short constructs or >20 mg/ml for the longer constructs.Genes were cloned into a modified pET28 vector and expressed in BL21Star cells as N-terminally tagged 3C protease-cleavable hexahistidine fusion proteins. Proteins were expressed for ∼18 h overnight at 18 °C. Cells were resuspended in lysis buffer  for assays. Spectra were recorded over 260 to 190 nm with a data pitch of 0.5 nm and scanning speed of 50 nm/min. Spectra plotted in Fig. S1B are the average of 20 accumulated scans.Spectra were recorded on a Jasco J-815 CD spectrometer with a CDF-426S/15 temperature controller at 25 °C. Protein was diluted to 0.2 mg/ml in buffer (10 mMelt curves utilizing the intrinsic tryptophan and tyrosine fluorescence of the protein sample were recorded using a Prometheus NT.48 nano-DSF instrument (NanoTemper technologies). Protein was diluted to 0.2 mg/ml in binding buffer (as above) and recorded in nano-DSF grade standard capillaries at 50% fluorescence intensity between 15 and 95 °C with a temperature ramp rate of 2 °C/min.Experiments were performed using an Agilent 1200 series LC system with an on-line Dawn Helios ii system (Wyatt) with a QELS+ module (Wyatt) and an Optilab rEX differential refractive index detector (Wyatt). Detector 12 in the static light-scattering cell had been replaced with the QELS module. 100 μl of purified sample at 10 mg/ml was autoinjected onto a Superdex 75 increase 10/300gl column  and run at 0.5 ml/min. The molecular weights and hydrodynamic radii were calculated in ASTRA software (Wyatt).m PIPES, pH 7.0, 150 mm NaCl, 0.5 mm TCEP). 10-μl binding reactions were assembled with the following components: 2 μl of protein, 1 μl of 500 nm RNA probe, 1 μl of 10× buffer  glycerol), and 6 μl of diethyl pyrocarbonate H2O. After incubation for 15 min, samples were run on 10% TBE-PAGE. Gels were run at 100 V (∼15 mA starting current) for 90 min and visualized using a Typhoon FLA 7000.EMSAs were performed with fluorescently-labeled RNA oligonucleotides with increasing concentrations of Mmi1 YTH domain constructs. 2-Fold dilution series of proteins were prepared at 5× final concentration in protein dilution buffer . Protein was incubated at room temperature for 30 min with 0.1 nm 3′,6-carboxyfluorescein–labeled DSR-containing RNA (synthesized by IDT) in buffer containing 10 mm TAPS, pH 8.2 (+2 mm PIPES carried over from protein buffer), 40 or 140 mm NaCl (+10 mm NaCl carried over from protein buffer), 0.5 mm MgAc, 0.1 mm TCEP, and 0.01% Brij-35 in a total of 100 μl using 384-well low-flange black flat bottom nonbinding surface microplates (Corning). Fluorescence polarization was measured using a PHERAstar Plus (BMG Labtech). Dissociation constants were estimated using a quadratic function Prism 6.0 (GraphPad software). Error bars indicate the standard deviation of five biological replicates .Fluorescence polarization was performed using fluorescently-labeled RNA oligonucleotides. 2-Fold protein dilution series were prepared at 10× concentration in dilution buffer  Brij-35.) Automatic serial dilution to concentrations indicated in figures were made using the DRX instrument. Dynamic response of the chip was tested to determine the best measurement spots. Kinetic assays were designed in switchBUILD (Dynamic Biosensors) before being customized in switchCONTROL (Dynamic Biosensors). Binding experiments were performed at 20 °C at a flow rate of 30 μl/min with 5-min total association time measured using a single electrode. Dissociation kinetics were measured using binding buffer flowed over the chip surface for 10, 15, or 420 min (depending on the affinity). Data curves were fitted with single-phase exponential decay curves in Prism 6.0 (GraphPad Software), with associated standard errors. On-rates were calculated from three different concentrations. Dissociation constants were calculated using the observed on- and off-rates.Kinetic measurements were performed using a DRX series instrument (Dynamic Biosensors) with a MPC-48-2-Y1 chip (Dynamic Biosensors). Protein stock at the highest concentration to flow onto the chip was made in binding buffer  before data collection. To improve back-exchange of deuterated protein, samples were incubated overnight at 20 °C in buffer containing 4 m urea, 20 mm PIPES, pH 6.8, 150 mm NaCl, 0.5 mm TCEP before undergoing two rounds of dialysis to remove the urea. For protein–RNA complexes, samples were mixed at a 1:1.2 protein/RNA molar ratio before dialysis.Isotopically-labeled proteins were overexpressed in a modified K-MOPS minimal medium  containi1H{13C,15N} triple-resonance cryoprobes with 10% D2O added to each sample as a lock solvent. 1H-15N band-selective excitation short-transients transverse relaxation-optimized spectroscopy (BEST-TROSY) experiments were collected with an in-house optimized pulse sequence CA with 1024*64*128 complex points in the 1H, 15N, and 13C dimensions; and trHNCAB and trHN(CO)CACB with 1024*64*110 complex points in the 1H, 15N, and 13C dimensions. Backbone assignment was additionally aided by collection of nitrogen correlating trHN[CAN]NH and trHN[COCA]NNH spectra with 1024*80*80 complex points. Each backbone dataset was collected with nonuniform sampling set at 30–50% and processed using compressed sensing (1H)2 + (0.2(Δδ15N)2). 15N{1H}-hetNOE measurements were carried out using standard Bruker pulse programs, with interleaved on- or off-resonance saturation. The hetNOE values were calculated from peak intensities by taking the ratio Ion/Ioff.Assignment of backbone amide peaks was completed using the following standard TROSY triple resonance spectra: trHNCO and trHN(CA)CO experiments collected with 1024*64*128 complex points in the  sensing . All spe sensing . Weightem BisTris, pH 5.5, and 0.2 m MgCl2. Crystals were cryo-cooled in mother liquor supplemented with 20% (w/v) glycerol before data collection at 100K at DLS beamline I03. Data were processed using the XIA2 pipeline (2YU6). Initial automated model building was carried out using phenix.autobuild  concentrated to 5 mg/ml were grown using sitting drop vapor diffusion with a reservoir solution containing 20% (w/v) PEG 3350, 0.1 pipeline  and AIMLpipeline . The strpipeline  using anutobuild  and Buccutobuild . This wautobuild  and phenutobuild .m MgSO4, and 0.1 m Tris, pH 8.0, for the 7-nt RNA or 15% (w/v) PEG 8K and 0.1 m CHES, pH 9.5, for the 11-nt RNA. Diffraction data were recorded at ESRF beamline ID30-A or DLS beamline I02 for the 7-nt RNA and 11-nt RNA crystals respectively. Data were processed using GrenADES implementing XDS  or an 11-nt DSR RNA (CUUUAAACCUA) at a 1:1.2 protein/RNA ratio. Screens were set up at ∼15 mg/ml, and crystals were grown in sitting-drop vapor-diffusion plates in 1:1 drops with reservoir containing 20% EtOH, 10 mting XDS  or XIA2 ting XDS  implementing XDS , for indting XDS  for scalting XDS , and modting XDS  and phenting XDS . RNA wasting XDS , 61 afteVisualizations of structures were made using PyMOL. For structural alignments, all deposited Mmi1 structures were aligned against chain A of the YTH domain bound to 11-nt RNA using UCSF Chimera . This ch6FPP, 6FPQ, and 6FPX.Backbone assignments of the USR–YTH protein in the absence and presence of the 19-mer RNA have been deposited with BMRB accession codes 27398 and 27399, respectively. Crystal structures were deposited in the PDB with accession codes J. A. S., S. M. F., and L. A. P. conceptualization; J. A. S., J. L. W., C. H. H., M. Y., S. H. M., and S. M. F. formal analysis; J. A. S., J. L. W., and S. M. F. investigation; J. A. S., J. L. W., and L. A. P. visualization; J. A. S., J. L. W., C. H. H., M. Y., S. H. M., and S. M. F. methodology; J. A. S. and L. A. P. writing-original draft; J. A. S. and L. A. P. project administration; J. A. S., J. L. W., C. H. H., S. H. M., S. M. F., and L. A. P. writing-review and editing; S. M. F. and L. A. P. supervision; L. A. P. funding acquisition.


The fungal cell wall and membrane are the most common targets of antifungal agents, but the potential of membrane lipid organization in regulating drug-target interactions has yet to be investigated. Energy-dependent lipid transporters have been recently associated with virulence and drug resistance in many pathogenic fungi. To illustrate this view, we discuss (i) the structural and biological aspects of ATP-driven lipid transporters, comprising P-type ATPases and ATP-binding cassette transporters, (ii) the role of these transporters in fungal physiology and virulence, and (iii) the potential of lipid transporters as targets for the development of novel antifungals. These recent observations indicate that the lipid-trafficking machinery in fungi is a promising target for studies on physiology, pathogenesis and drug development.  These drug classes are ineffective in a number of cases, which is linked to toxicity, low bioavailability in target tissues and antifungal resistance . VKAS is supported by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior. MLR is supported by grants from CNPq  and Fiocruz (grants VPPCB-007-FIO-18-2-57 and VPPIS-001-FIO-18-66). LN is supported by grants from CNPq (grant 311179/2017-7 and 408711/2016-7). The authors also acknowledge support from the Instituto Nacional de Ciência e Tecnologia de Inovação em Doenças de Populações Negligenciadas(INCT-IDPN). MLR is currently on leave from the position of Associate Professor at the Microbiology Institute of the Federal University of Rio de Janeiro, Brazil.JR acknowledges support from the Brazilian agency The authors declare no conflict of interest.


RNA-seq is the most commonly used sequencing application. Not only does it measure gene expression but it is also an excellent media to detect important structural variants such as single nucleotide variants (SNVs), insertion/deletion (Indels) or fusion transcripts. However, detection of these variants is challenging and complex from RNA-seq. Here we describe a sensitive and accurate analytical pipeline which detects various mutations at once for translational precision medicine.The pipeline incorporates most sensitive aligners for Indels in RNA-Seq, the best practice for data preprocessing and variant calling, and STAR-fusion is for chimeric transcripts. Variants/mutations are annotated, and key genes can be extracted for further investigation and clinical actions. Three datasets were used to evaluate the performance of the pipeline for SNVs, indels and fusion transcripts.For the well-defined variants from NA12878 by GIAB project, about 95% and 80% of sensitivities were obtained for SNVs and indels, respectively, in matching RNA-seq. Comparison with other variant specific tools showed good performance of the pipeline. For the lung cancer dataset with 41 known and oncogenic mutations, 39 were detected by the pipeline with STAR aligner and all by the GSNAP aligner. An actionable EML4 and ALK fusion was also detected in one of the tumors, which also demonstrated outlier ALK expression. For 9 fusions spiked-into RNA-seq libraries with different concentrations, the pipeline was able to detect all in unfiltered results although some at very low concentrations may be missed when filtering was applied.The new RNA-seq workflow is an accurate and comprehensive mutation profiler from RNA-seq. Key or actionable mutations are reliably detected from RNA-seq, which makes it a practical alternative source for personalized medicine.The online version of this article (10.1186/s12920-018-0391-5) contains supplementary material, which is available to authorized users. Somatic mutations are a hallmark of a tumor and inherited mutations cause certain genetic disorders. Characterization of these mutations and exploration of their clinical relevance constitute a critical part of personalized medicine. Mutations present in multiple forms and common ones include single nucleotide variants (SNVs), short insertions/deletions (indels), or fusion transcripts. SNVs or indels are primarily detected from DNA sequencing such as whole-genome, exome-sequencing, targeted sequence or amplicon. However, RNA-seq is the most popular sequencing application as it contains much richer genomic information. Not only does it measure gene expression but it also can detect important structural variants such as SNVs, indels or fusion transcripts, some of which are known actionable mutations for tumor treatment. A good example of this is EGFR single base mutation L858R) in exon 21 and in-frame deletions (ranging from 12 to 18 bases) in exon 19, both can be targeted by EGFR tyrosine kinase inhibitors, such as gefitinib and erlotinib with clear clinical benefits to patients , use of 8R in exoMany RNA-seq workflows have been developed –9, but tA.Sequence read alignment: This pipeline includes two aligners i.e. GSNAP [e. GSNAP  and STARe. GSNAP , with STe. GSNAP . GSNAP ie. GSNAP . Read gre. GSNAP .B.Aligned read preprocessing for SNV/Indel detection: RNA-Seq variant calling is much complex than DNA-seq and the gapped alignment also causes incompatibility with existing variant callers. This module prepares the aligned bam file for next variant calling step. SplitNCigar, Indel Realignment and Base Recalibration are done by GATK tool kit [tool kit . In the C.Variant/somatic mutation Calling: Our previous work showed GATK [wed GATK  haplotypwed GATK  was bettwed GATK . They arD.Variant annotation: Functional annotation is a key step for identified variants to understand the potential clinical impacts. Annovar [ Annovar  is a ligE.Fusion Transcripts: Fusion transcripts are characteristic of tumors and highly relevant to targeted therapy [ therapy , 19. It F.Gene Expression: The gene level quantification is done using featureCounts software [software . The genG.Summary Report and output structure: The output file structure is illustrated in Fig. PanMutsRx is implemented modularly using Python 3 and shell scripts for various operations such as input/output file operations, log file management, submitting jobs to cluster , tool execution and integration. The main operation of the workflow is summarized in Fig. The workflow has flexibility to execute individual modules separately and appropriate log files are generated for troubleshooting. Additional options are provided to run the workflow in the open grid engine parallel cluster environment, but depending on the other grid engine types changes may need to be made. Parameters used for all steps are provided in Additional file To evaluate the performance of PanMutsRx in SNV/Indel and fusion detection, we used 3 datasets.https://www.encodeproject.org/; ENCFF377UIC with 147 million pair-end reads at 100 bp read length) and analyzed through our pipeline for SNVs and Indels and compared with the benchmark DNA variants. As variants from RNA-seq are only possible from coding regions and only expressed genes can be assessed, the comparison was limited to the genomic positions with at least 10X coverage in the RNA-seq where variants are reported in the reference dataset from GIAB. The sensitivity was calculated as the percent of correct calls in RNA-seq at these positions in comparison with variants in DNAs (SNVs or indels separately). For specificity, we extracted all positions in RNA-seq with at least 10X coverage but there are no variants in DNA as defined in GIAB benchmark set (true negatives or TN). Any variants in these positions reported from RNA-seq were considered as false positives (FP) and the specificity was obtained by the formula: TN/(TN + FP).Genome in a Bottle (GIAB) consortium released a benchmark SNP and indel dataset for sample NA12878 by integrating multiple DNA sequence data sets including whole genome sequencing . For theWe also run sample ENCFF377UIC by other public tools and compared the relative performances for the SNV and Indel detection. Opossum is a RNA-seq preprocessing tool before variant calling by either Platypus or GATK haplotype caller and demonstrates a good performance in SNV detection . In addiLung cancer is one of tumors harboring a high number of mutations  and someThis publicly available dataset is created for the community to evaluate fusion detection algorithm where 9 well known oncogenic fusion transcripts were spiked into RNA-seq libraries at wide range of molarities . We downIt took about 20 h for PanMutsRx to complete all the processing and analyses for the sample with 150 million reads , with STAR alignment by Opossum preprocessing and GATK haplotype caller (STAR_OPOSSUM_GATK), STAR alignment by Opossum preprocessing and PLATYPUS variant calling (STAR_OPOSSUM_PLATYPUS), and MAP-RSeq with RVBoost (MAPRSeq_RVBoost). MAP-RSeq use Tophat alignment and GATK unified genotyper. As Tophat is gapless alignment and RVBoost only works with SNVs, our comparison with this was limited to SNVs only. PanMutsRx obtained very similar sensitivities for both SNVs and Indels with more complexed Opossum pre-processing along with GATK haplotype caller . Opossum along with Platypus demonstrated the lowest sensitivities for SNVs and Indels, 86% and 75% respectively Fig. . AlthougThe lung adenocarcinoma dataset SRA ERP001058 contains 41 tumors with known targetable or oncogenic mutations in 6 genes: EGFR, KRAS, NRAS, MET, BRAF and CTNNB1. The most notable are EGFR micro deletion at exon 19 (7 tumors) and single nucleotide substitution L858R at exon 21 (13 tumors) as they are targetable clinically. Among the 41 mutations, 39 were detected by the PanMutsRx pipeline with STAR aligner. Careful examination of the two tumors whose mutations were missed showed that both had very low mutation frequency (one with 1 and another with 3 mutated reads). As demonstrated in our previous evaluation, GSNAP is marginally more sensitive in indel detection compared to STAR, and this was corroborated using GSNAP for alignment, which indeed was able to detect both mutations as hypothesized  or RNA-seq, the important and actionable mutations are often conserved in RNA-seq. This suggests we can extract useful and relevant information to reduce the complexity of multi-genomic information from RNA-seq. We provide a post-processing script to extract SNVs, Indels, fusion transcripts, or expression for a list of genes users provide.Available RNA-seq workflows mostly focus a particular function for example, gene expression, SNV or fusion transcript detection, which has its advantages of easy management. However, conducting analysis for each separately needs redundant work with significant effort for the RNA-seq data. PanMutsRx aimed to perform all clinical relevant tasks at once by selecting high performing tools for each application. RNA-seq alignment by different aligners makes much less difference for SNVs than for Indels and our selection of STAR and GSNAP as part of PanMutsRx was based on our comprehensive comparison among several tools . Our curThe missed calls in RNA-seq can be several reasons. We found majority of them were caused by insufficient alternative allele and although could be called but filtered out. These positions can be recovered by reducing filtering stringency but the trade-off would be increased false positives. Although Indel detection performs reasonably well, there is room for further improvement.We have developed a sensitive and comprehensive RNA-seq analytical pipeline which can capture multiple mutations simultaneously  and can be potentially used in clinical practice and precision medicine.Additional file 1:Table S1. Parameter Settings used for alignment, data pre-processing and variant calling. Table S2. PanMutsRx Run time in each step of processing. (DOCX 18 kb)


Paralichthys olivaceus) infected with viral hemorrhagic septicemia virus (VHSV)” The data presented here are related to the research article entitled “Temperature-dependent immune response of olive flounder ( Specifications TableValue of the data●The information of unigenes expressed at colder and warmer temperature helps us to know how the response of host transcriptome varies with respect to surrounding environment.●The comparison of modulated genes during VHSV infection at 13 and 20 °C helps for management measures in olive flounder aquaculture.●The identification of affected signaling pathways in the head kidney of VHSV infected olive flounder sheds new light on the investigation of disease pathogenesis and for novel treatment targets.1Temperature-dependent immune response of olive flounder  infected with viral hemorrhagic septicemia virus (VHSV)” The data presented here are related to the research article entitled “22.1Olive flounder of average weight 39.7 g were purchased from a commercial fish farm (Geoje Island) without any history of VHSV. Animals were maintained at 11–13 °C, and acclimated for one week.2.24 TCID50 per fish or control media in 0.1 ml and acclimatized at 13 and 20 °C, separately. Total RNA was isolated from the head kidney of three individual VHSV-infected olive flounder cultured on 13 and 20 °C. At 4 h post infection, three individuals were randomly collected from each group and head kidneys were excised for gene expression analysis. Kidney tissue samples were stored at −80 °C until RNA isolation. Total RNA was isolated using a standard Trizol extraction protocol  according to the manufacturer׳s instructions. The concentration and integrity of the RNA were assessed with a Thermo Scientific NanoDrop 8000 Spectrophotometer and Agilent 2100 Bioanalyzer, respectively. RNA with an OD260/280≥1.8 and an RNA integrity number ≥7.0 was used in subsequent experiments. Equal amounts of high quality RNA from each sample were then used separately for cDNA synthesis and sequencing. The cDNA library was prepared with ~1.0 μg of total RNA following manufacturer׳s recommendations of TrueSeq RNA library Preparation Kit . The library was then amplified, and the final library yielded ~ 500 ng of cDNA with an average fragment size of ~ 350 bp. The resulting cDNA libraries were then paired-end sequenced (2 × 100 bp) with HiSeq. 2500 platform . All sequencing reads were deposited in the NCBI Sequence Read Archive (SRA) under the accession number SRP102673.The fish of each groups were intraperitoneally injected (Isolate: FDC-VHS2014-5) with a VHSV dose of 1×102.3The raw reads of fastq format were undergone pre-processing and high quality sequences were subject to de novo assembly using Trinity software


LHT1 paralogs might have the ACC-transporter activity. Here, we took a gain-of-function approach by transgenic complementation of lht1 mutant with a selected set of amino acid transporters. When we introduced transgene into the lht1 mutant, the transgenic expression of LHT2, but not of LHT3 or amino acid permease 5 (AAP5), restored the ACC resistance phenotype of the lht1 mutant. The result provides genetic evidence that some, if not all, amino acid transporters in Arabidopsis can function as ACC transporters. In support, when expressed in Xenopus laevis oocytes, both LHT1 and LHT2 exhibited ACC-transporting activity, inducing inward current upon addition of ACC. Interestingly, the transgenic expression of LHT2, but not of LHT3 or AAP5, could also suppress the early senescence phenotypes of the lht1 mutant. Taking together, we propose that plants have evolved a multitude of ACC transporters based on amino acid transporters, which would contribute to the differential distribution of ACC under various spatiotemporal contexts.1-Aminocyclopropane-1-carboxylic acid (ACC), a biosynthetic precursor of ethylene, has long been proposed to act as a mobile messenger in higher plants. However, little is known about the transport system of ACC. Recently, our genetic characterization of an ACC-resistant mutant with normal ethylene sensitivity revealed that lysine histidine transporter 1 (LHT1) functions as a transporter of ACC. As amino acid transporters might have broad substrate specificity, we hypothesized that other amino acid transporters including  Transporters of plant hormones play a pivotal role in cell-cell communication, underpinning tissue-specific differentiation and environmental adaptation of higher plants, as exemplified by various defects of hormone-transporter mutants throughout the life cycle . For theetr1-1 mutant -constitutive triple response1-ethylene insensitive 2-EIN3 binding F-Box 1 (EBF1)/EBF2-ethylene insensitive 3 (EIN3)/EIN3-like  by ACC oxidase. ACC is synthesized by ACC synthase (ACS) using S-adenosyl methionine, supplied reaction . Synthesreaction , while ureaction . As suchreaction . Besidesreaction .lysine histidine transporter 1 (LHT1) in Arabidopsis exhibited dose-dependent resistance to exogenous ACC, but not to gaseous ethylene. In the agreement, the mesophyll protoplast of the lht1 mutants was impaired in the uptake of 14C-ACC. The findings that the lht1 mutant showed a normal ethylene response in the presence of higher concentrations of ACC and it retains, although partial, uptake activity of 14C-ACC implicated the presence of additional transporters. Since LHT1 belongs to a large gene family encoding amino acid transporters, which comprised more than 100 members in Arabidopsis , lht2-1 (SAIL_222_C12), and lht1-101LHT1ox-1 transgenic plants have been previously described , using primers listed in d-TOPO (Invitrogen). The resulting entry clone was recombined into a gateway-compatible plant expression vector, pH2GW7  or of d-amino acids (Sigma-Aldrich). The seedlings were then further grown under continuous light or dark after irradiation for 12 h. For amino acid competition assay with ACC, seedlings were grown on MS media containing 1 μM of ACC with mock or 100 μM of amino acid for 3.5 days in the dark after irradiation of white light for 12 h. The length of hypocotyls and roots was measured using ImageJ software (http://rsbweb.nih.gov/ij). Seedling images were scanned with an Epson scanner .To test the response of seedlings to ACC or ™ M-MuLV Reverse Transcriptase . The 10-fold diluted cDNA was subject to qRT-PCR analysis using specific primers listed in ™ Real-Time PCR System  using an EvaGreen® qPCR Supermix . The reactions were performed in technical triplicate for each gene. The comparative ΔCt method was used to evaluate the relative quantities of each amplified product in the samples, as described (PP2A (At1g13320), a reference gene  analysis was performed with total RNA. After extraction, DNase I–treated total RNA was reverse transcribed by using RevertAidescribed . Relativnce gene .LHT1 or LHT2 were subcloned into the pGEM-HE vector to be expressed in oocytes using Nde1 restriction enzyme site. After sequence verification, the cRNAs were transcribed from the linearized cDNAs using transcription kit  with T7 RNA polymerases. The RNA was dissolved in nucleotide-free water and diluted at a final concentration of 1 μg/μL and then aliquoted and stored at −80°C until use. The nanoinjection of cRNAs (40 ng) into the vegetal or animal pole of each oocyte was carried out using a microinjector . The nanoinjector pipettes were pulled out by the glass capillary tubes that were used for the electrodes and polished to a ∼20-μm outer diameter.The cDNAs encoding Xenopus laevis oocytes and preparation of single cells were described in the previous study . The matured oocytes were selected and incubated in ND96 containing: 96 mM NaCl, 1 mM MgCl2, 2 mM KCl, 1.8 mM CaCl2, and 20 mM HEPES at pH 5.6 with 1% penicillin and streptomycin (Sigma). Two electrode voltage clamp experiments were carried out after 48 h for each of the RNA-injected oocytes  in two amino acid transporter family, e.g., LHT and AAP family. When the transgene was introduced into the lht1 mutant, the resulting 17 of 19 T2 lines with 35S::LHT2 showed normal triple response to ACC as wild type did. In contrast, none of more than 20 T2 lines with 35S::LHT3 or 35S:AAP5 exhibited defective triple response to ACC as the lht1-101 mutant did. After confirmation of the elevated transcript level of each amino acid transporter , senescence related gene 1 (SRG1), senescence associated gene 12 (SAG12), phytoalexin deficient 4 (PAD4), isochorismate synthase 1 (ICS1), NDR1/HIN1-like 25 (NHL25) in the lht1-101 mutant  did not induce any current in the control oocytes , the Rural Development Administration , and by the Ministry of Science, ICT & Future Planning (2015R1C1A2A01052950).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


One approach for estimating a function of a lncRNA is the identification of its interaction target because functions of lncRNAs are expressed through interaction with other biomolecules in quite a few cases. In this paper, we developed “LncRRIsearch,” which is a web server for comprehensive prediction of human and mouse lncRNA-lncRNA and lncRNA-mRNA interaction. The prediction was conducted using RIblast, which is a fast and accurate RNA-RNA interaction prediction tool. Users can investigate interaction target RNAs of a particular lncRNA through a web interface. In addition, we integrated tissue-specific expression and subcellular localization data for the lncRNAs with the web server. These data enable users to examine tissue-specific or subcellular localized lncRNA interactions. LncRRIsearch is publicly accessible at  Long non-coding RNAs (lncRNAs) were initially considered to be transcriptional noise or experimental artifacts, but recent research has revealed that lncRNAs play important roles in various biological processes, such as cell differentiation  and SERPINE1 (ENST00000223095) interaction, and human 1/2-sbs RNA and ANKRD57 (ENST00000356454) interaction    are included in the dataset. On the other hand, for the K562 cell-line, five types of RCI data  are additionally included in the dataset. These subcellular localized RNA-RNA interactions are also provided in LncRRIsearch  and the energy threshold , and then inputs name or ID of genes or transcripts . LncRRIsLncRRIsearch helps users to investigate lncRNA-RNA interactions exhibiting tissue-specific expression patterns . Users cAfter selecting the tissue-specific expression pattern, the corresponding query and target RNAs predicted by RIblast are listed. In this step, once a query RNA is selected, the list of possible target RNAs is automatically updated for the selected query. By selecting the tissue-specific query and target RNAs, detailed information about interactions between the query and target RNAs is provided . In addiUsers can investigate subcellular-localized human lncRNA-RNA interactions . Users fWe developed LncRRIsearch, which is a web server for comprehensive prediction of human and mouse lncRNA-mRNA and lncRNA-lncRNA interactions including tissue-specific expression and subcellular localization data. There are two advantages of LncRRIsearch over other lncRNA-RNA interaction databases or web services; the comprehensiveness of interaction prediction and the ability to investigate tissue-specific or subcellular localized interaction patterns.We envision three future improvements of LncRRIsearch. The first is the development of real-time RNA-RNA interaction prediction software. Although LncRRIsearch provides comprehensive human and mouse lncRNA-RNA interaction based on GENCODE version 25 and M14, novel lncRNAs will be discovered in the future. Real-time prediction would be useful for the discoverers of new lncRNAs to investigate their interactions. The acceleration of RNA-RNA interaction prediction is still an important research topic. One possible direction is the simplification of the energy model. RIblast uses a complete nearest-neighbor energy model in the search step, but some researchers have reported that the use of an approximated energy model produces a marked increase in the calculation speed in exchange for only a slight decrease in the prediction accuracy (Tafer et al., The second improvement is the integration of the results of RNA-RNA interaction detection experiments. Recently, several high-throughput sequencing methods for the exhaustive identification of RNA-RNA interaction sites have been developed, including PARIS (Lu et al., The third improvement is an increase in the number of target species. This improvement would enable us to not only investigate the lncRNA interactions of newly added species but also compare lncRNA interactomes between species. Nguyen et al. recently showed that the conservation of experimentally confirmed lncRNA-RNA interaction regions is high, although lncRNA generally lacks sequence conservation (Nguyen et al., http://rtools.cbrc.jp/LncRRIsearch/.All datasets analyzed for this study are included in the manuscript and the TF, JI, and MH conceived the study and wrote the manuscript. TF, JI, and YO processed the data. YO constructed the database. TF and JI equally contributed to this work. MH supervised this study. All authors read and approved the final manuscript.YO was employed by company IMSBIO CO., LTD. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.