Objectives: The mainstream position on regret in psychological literature is that its necessary conditions are agency and responsibility, that is, to choose freely but badly. Without free choice, other emotions, such as disappointment, are deemed to be elicited when the outcome is worse than expected. In two experiments, we tested the opposite hypothesis that being forced by external circumstances to choose an option inconsistent with one’s own intentions is an important source of regret and a core component of its phenomenology, regardless of the positivity/negativity of the post-decision outcome. Along with regret, four post-decision emotions – anger toward oneself, disappointment, anger toward circumstances, and satisfaction – were investigated to examine their analogies and differences to regret with regard to antecedents, appraisals, and phenomenological aspects.Methods: Through the scenario methodology, we manipulated three variables: choice (free/forced), outcome (positive/negative), and time (short/long time after decision-making). Moreover, we investigated whether responsibility, decision justifiability, and some phenomenological aspects  mediated the effect exerted by choice, singularly or in interaction with outcome and time, on the five emotions. Each study was conducted with 336 participants, aged 18–60.Results: The results of both studies were similar and supported our hypothesis. In particular, regret elicited by forced choice was always high, regardless of the valence of outcome, whereas free choice elicited regret was high only with a negative outcome. Moreover, regret was unaffected by responsibility and decision justifiability, whereas it was affected by the three phenomenological dimensions.Conclusion: Our results suggest that (1) the prevailing theory of regret is too binding, since it posits as necessary some requirements which are not; (2) the antecedents and phenomenology of regret are broader than it is generally believed; (3) decision-making produces a complex emotional constellation, where the different emotions, singularly and/or in combination, constitute the affective responses to the different aspects of decision-making. The well-established psychological concept of regret based on decision theories strongly links the genesis of this emotion to the factual or counterfactual comparison between the actual outcome resulting from the option chosen in decision-making and the better outcome deriving from the foregone option . The corIt is worth noting that research on regret has come to a standstill in the last years, maybe because the wide consensus on the theory described above has led to the idea that the knowledge of this emotion does not need to be further developed. However, in this paper, we aimed to point out that the prevailing theory has not considered that an important source of regret is represented by forced choices, that is, by choices due to the constraint of external circumstances  that compel people to choose an option inconsistent with their intentions. A corollary of this assumption is that forced choices can produce regret even when their outcome is positive. Moreover, we aimed to show that the phenomenology of the regret elicited by forced choices is characterized by the awareness of having made an unsatisfactory but unavoidable choice. Finally, we aimed to reconsider analogies and differences between regret and other post-decision emotions.In the following sections, the state of the art of the literature on regret and other decision-related emotions is reviewed, with particular reference to the aspects that have been investigated in our studies.regret theory built by rational decision theorists in Economics , regret is considered unique for its relation with choice and responsibility e.g., .Compared to the position that ties regret to free choice and responsibility, the position that does not consider these requirements as essential, which we adopted in our research, is definitely not dominant. In addition to philosophers, such as status quo). On the whole, their findings show that responsibility can amplify regret but is not a necessary condition for its genesis and its experience, since regret can also be elicited by external decision-makers, such as a computer. Regret, as well as disappointment, increases especially when the outcome is worse than the status quo. Moreover, Connolly and colleagues have supported this position through some scenario-based experiments  where thAn attempt to conciliate the divergent positions about the role of agency and responsibility in the genesis and experience of regret has been the decision justification theory DJT, . AccordiHowever, an experimental study of Interestingly, some studies on real life or career regrets have questioned the relationship between the intensity of regret and the controllability of the eliciting events. For example, In summary, although some studies have challenged the idea that free choice and responsibility are a necessary condition for the onset of regret, to the best of our knowledge, there are no experimental studies that have systematically investigated the hypothesis that forced choice is an important source of regret and a core component of its phenomenology, regardless of the valence of the post-decision outcome. The studies presented here address this issue. As we will specify in the following section, we use the oxymoron “forced choices” to designate the choices for which no alternatives are available. In these types of choices, the decision-maker is not an external agent, as in the studies conducted by Connolly and colleagues , but theper se, independently of the outcome and the justifiability of decision-making, since the forced choice implies giving up the initial and preferred option. This effect should be particularly robust in self-relevant domains, due to the subjective importance of the preferred but not-chosen option. The “chosen” option, as a substitute for the former, would be inherently less attractive even if a positive outcome were derived from it. Moreover, in accordance with the The idea behind the present studies is that decision-making does not imply as a necessary condition that individuals decide freely. Indeed, people can be forced by external circumstances to choose an option that is inconsistent with one’s own intentions. In our opinion, this would elicit regret More specifically, the main hypothesis that was tested in our studies is the following: A forced choice should elicit the same high intensity of regret both in case of positive and negative outcome, whereas a free choice should produce more intense regret with a negative than a positive outcome.A corollary of this hypothesis concerns the characteristics of the phenomenology of regret: In the case of free choice, it should be marked by self-blame and the sense of personal responsibility; whereas when the choice was forced, it should be characterized by the awareness of having made an unwanted but inevitable choice. Consequently, responsibility should be not considered as a necessary requisite for the onset and the subjective experience of forced choice-related regret. Moreover, even the justifiability of the decision, which should decrease the intensity of regret , should Our hypothesis and their corollaries, which question the unicity of the genesis and experience of regret, raise the question of whether and on what criteria regret can be differentiated from other post-decision emotions.Many authors support the hypothesis that emotions arising from decision-making have specific antecedents, patterns of appraisal, and phenomenology . However, other authors  have poiWe adopt the perspective of appraisal theories , accordiWe assume that an important decision made in a self-relevant domain results in a broad emotional reaction, where the composing emotions capture different aspects linked to decision-making both in overlapping and in mutually independent ways, so that each of them should have a specific configuration.Along with regret, we investigated four emotions: disappointment, anger toward oneself, anger toward circumstances, and satisfaction. We focused our analysis on antecedents, and some appraisal and phenomenological aspects. In addition to disappointment, that is, the widely studied emotion that is closest to regret, the other emotions were chosen on the basis of the following criteria. Satisfaction, the only positive emotion, has been selected because it should increase with positive outcome. Moreover, since satisfaction refers to the affective reaction to one’s well-done actions , it doesBased on this analysis, we predicted some overlap between disappointment, regret, and anger toward circumstances, whereas the unique features attributed to regret should rather characterize anger toward oneself. In our view, the specificity of the nature of regret is that it can be elicited by opposite antecedents which, in turn, should generate contrasting subjective experiences.Since we set out to systematically test our hypothesis about the importance of forced choice on the most relevant phenomena highlighted by research on regret, we also included in our investigation two well-known phenomena: the effect of the temporal dimension and of the type of behavior (action or inaction) resulting from decision-making on the intensity of regret.We examined the first issue by considering the time elapsed from decision-making (a few days vs. a year) as a moderator of the effect of the type of choice. Instead, to investigate the second issue, we conducted two similar studies with two different samples: The only difference between them was that in the first study, the result of decision-making was an inaction, whereas in the second was an action.According to the temporal theory of regret , 1995, iIn the present studies, we adopted the scenario method, a widely used tool in the experimental studies on regret 2002. Thimagine-self instructions and imagine-target instructions  have used two types of instructions: ructions . In the imagine-self instructions produced more emotional involvement than imagine-target instructions  and what it might have been if he had made (or could have made) a different choice.For each study, we used five moderated mediation models  by means of one direct question for decision justifiability  and for responsibility , respectively. Instead, the phenomenological aspects were assessed by means of 14 questions, concerning feelings and factual and counterfactual thoughts about the type of choice made, its context, its degree of freedom, and its consequences. These questions were formulated for taking into account three dimensions: self-attribution, external attribution, and contentment. Note that we did not take into account all the aspects of the emotion phenomenology, but only those we judged relevant for investigating the subjective repercussions deriving from the type of choice. More specifically, we considered neither the intentions nor the action tendencies of the protagonist.We expected that, for both studies, the independent variable (choice) and the moderators (outcome and time) should differently affect both the five emotions and the mediators and that, in turn, the latter should affect the five emotions differently. Thus, choice should exert conditional (in interaction with moderators) direct and indirect (through mediators) effects on the five emotions. In the previous section, we delineated some of the principal effects we predicted and the reasons underlying these predictions.We conducted both studies in the career domain, which has been considered as one of the main domains where regret can be produced . The scerimpianto and rammarico, which are often used as synonyms, though their meaning varies slightly depending on the dictionaries . We decided to select the most frequently occurring term on the web, which is rimpianto. For disappointment, the corresponding Italian term is delusione.Finally, it is worth specifying that in Italian, there are two terms corresponding to English regret, namely, The study 1 investigated regret and other emotional reactions stemming from inaction.n = 42 for each condition). All participants were employed. Among the students, only student workers were selected. Most of the participants (63.1%) had a high school diploma, 28.9% had a college degree, and 8% had completed compulsory education.A 2 (choice: free vs. forced) × 2 (outcome: negative vs. positive) × 2 (time: short term vs. long term) between-subject design was carried out. Gender and age were considered as covariates. Three hundred thirty-six volunteer (unpaid) participants were recruited from different companies, sports centers, and universities in Campania. They were equally distributed by gender , were aged between 18 and 60 , and were randomly assigned to one of the 8 experimental conditions  and achieve a power of (1-β) of 0.95 with an error probability of 0.05, a minimum sample size of 327 participants was required. We decided to increase the number of participants to 336 in case we had to exclude someone for incomplete responses. Actually, no missing data were found. All participants executed the experimental task individually, at the place where they were recruited. They gave their informed consent before starting the experiment.To determine the sample size, an  G*Power . To deteIn conformity with the experimental conditions, eight scenarios were built with the same structure and three sources of variation: type of choice (free vs. forced), outcome (negative vs. positive), and time . In all scenarios, the protagonist is an executive of a company who has to decide whether to accept the offer to manage for 2 years a branch that the company is planning to open abroad. The job is very well paid, and the sector in which the branch will operate is very interesting; moreover, the protagonist is offered the possibility of a significant career advancement once (s)he returns. In all scenarios, decision-making results in inaction: The protagonist decides to stay in the same workplace, and the assignment is given to a colleague. In the free choice conditions, the protagonist does not have any constraints: (s)he is aware that the proposal to manage the branch abroad is a great opportunity for her career but prefers to remain in her workplace.In the forced choice conditions, a physical accident prevents the protagonist from choosing the desired option (going to manage the company branch abroad) and (s)he is therefore forced to choose to stay in the same workplace. In the positive outcome conditions, the protagonist gets a career advancement and a small pay raise. In the negative outcome conditions, the company decides to downsize some areas of production, including the one where the protagonist works, because the costs of opening the foreign branch have implied a restructuring of activities. Consequently, any possibility of career advancement disappears and the protagonist’s position in the company becomes much more marginal. In both types of outcomes, the colleague who has gone abroad is very happy with his/her work. The outcome is evaluated a few days (short term condition) or 1 year (long term condition) after the choice. For each experimental condition, in half of the scenarios the protagonist is a woman; in the other half, it is a man. However, the scenarios were assigned randomly to the participants, without doing any matching between the gender of the protagonist and the one of the participant.In all experimental conditions, participants received a two-page booklet, in the first of which one of the eight short stories was reported. After reading the scenario, they were asked to imagine as vividly as possible the thoughts and feelings of the protagonist, and then to evaluate, on a nine-point scale :how important she felt the object of the decision was for her life (“how important does the protagonist feel the decision whether to accept or refuse to manage a company branch abroad was for her life?);whether she perceived her decision as a free choice (“To what extent does the protagonist feel the choice of not accepting to manage a company branch abroad was a free choice?);whether she felt responsible for her own choice (“To what extent does the protagonist feel responsible for not accepting to manage a company branch abroad?”);whether she judged her decision as justifiable ;whether she judged her present working condition as positive (“To what extent does the protagonist judge her present working condition as positive?”).In this way, the effectiveness of the experimental manipulation was checked and two of the putative mediation variables between type of choice and regret, that is, responsibility and decision justifiability, were assessed. Henceforth, the five variables will be labelled as Intermediate Variables (InVs).Successively, participants evaluated on a nine-point scale  how intensely the protagonist was supposed to feel five emotions: anger toward circumstances, anger toward oneself, disappointment, regret, and satisfaction.Finally, they estimated on a nine-point scale  the probability with which the protagonist was supposed to agree with the 14 items of a questionnaire describing thoughts  and feelings related to the emotional experience. The questionnaire ;external attribution of the outcome due to forced choice ;affective evaluation of the current state .Four different random sequences including the five InVs, the five emotions, and the 14 items of the questionnaire were built, and the participants were pseudo-randomly assigned to the sequences.It is worthy to note that the we used two questions on responsibility. The first question asked how responsible the protagonist felt for her own choice when it was made and aimed to evaluate the perceived appraisal of the type of choice; the other question was in the phenomenology questionnaire and asked how responsible she felt for the situation in which she was in at the moment of the evaluation of the decision consequences.After completing the questionnaire, participants gave back the booklet. They were asked whether they had any questions and then were thanked for their participation.Before conducting the experiment, a pilot study was carried out with 20 unpaid volunteer employees in order to test the believability of the scenarios, the clarity of the instructions (with particular reference to manipulation check questions), and the perceived importance of the target of the decision-making. No changes were required after this study.All data analyses were carried out using SPSS 18.0 IBM software.In F = 0) and age of participants were included as covariates. The multivariate tests were significant for all the three IVs. The univariate tests revealed three main effects due to choice, outcome, and time, and two interaction effects: outcome x choice and time x choice. The main effects were examined through pairwise comparisons with Bonferroni correction; the interaction effects were examined through simple effects analysis with Bonferroni adjustment. Choice affected all the InVs, except importance: responsibility, perception of the freedom of the choice, and positive evaluation of present working condition increased with free choice, while decision justifiability increased with forced choice. Outcome affected only the positive evaluation of present working condition, which increased with positive outcome, and importance, which increased with negative outcome. Time affected the positive evaluation of present working condition, responsibility, and importance, all of which increased in long term. The outcome × choice interaction affected the positive evaluation of present working condition, which increased with positive outcome and free choice compared to positive outcome and forced choice. The time × choice interaction concerned importance, which increased with forced choice compared to free choice in short term, while in long term, there was no difference as a function of the type of choice. No effects were due to gender or age. In The first analysis, a 2 x 2 x 2 MANCOVA, was performed to check whether the manipulated experimental conditions affected the five InVs in the predicted directions. Gender  was reverse-coded. Varimax rotation was used after controlling, through Oblimin rotation, that there was no correlation higher than 0.30 among components. Three components with eigenvalue >1, explaining 65.11% of the total variance, were extracted. The three components and the items loading on each of them corresponded to those that had been hypothesized in the construction of the questionnaire. They were labelled To test the central hypothesis of the study, five moderated mediation analyses were performed. This analysis  examinesIn each analysis, we tested a model . Concerning the relationship between the IV and/or the moderators and the mediators , the results were similar to those of the MANCOVA and were reported in When mediators were included in the model, the results on the five emotions ; with forced choice, the probability of the emotion increased in the long term (positive interaction choice x time). Choice exerted indirect effects through self-attribution and external attribution and conditional indirect effect through contentment. More precisely, irrespective of the values of outcome and time, forced choice decreased the effect of self-attribution and increased the effect of external attribution. The former dimension decreased anger toward circumstances, whereas the latter increased it. Thus, the indirect effects became positive in both cases. Instead, the indirect effect through contentment was significant only for positive outcome, in both values of time: In these conditions, forced choice diminished contentment, which in turn diminished this emotion, making the conditional indirect effect positive.The probability of satisfaction decreased with negative outcome and increased with decision justifiability and contentment. Choice exerted only a conditional indirect effect through contentment. Indeed, with positive outcome, in both short and long term, forced choice diminished contentment, which in turn increased satisfaction. Thus, such an effect was negative.The probability of all emotions, except satisfaction, decreased in males compared to females. No significant effects were due to age.Study 2 was conducted to investigate regret and other emotional reactions that originate from decision-making resulting in action.It had the same structure as study 1, except for the scenarios.Once again participants were 336 unpaid volunteers, aged between 18 and 60 . They were recruited in the same way as study 1, and all were workers. Thus, also in this study, participants were equally distributed by gender. More than half of them (55.7%) had a high school diploma, 31.8% had a college degree, and 12.5% had completed compulsory education.The eight scenarios (choice x outcome x time) were built around the following plot: The protagonist chooses to leave a promising basketball career to work in a company. In the free choice conditions, s/he does not feel like facing the risks and uncertainties of a sports career and prefers to opt for a safer job. S/he therefore leaves basketball and finds a job with a solid company. In the forced choice conditions, a physical accident prevents the protagonist from continuing his sports career and he is therefore forced to seek a job that is compatible with his changed physical conditions. Finally, he finds employment with a solid company. In the positive outcome conditions, the protagonist realizes that the new job, although a bit monotonous, leaves him free time to cultivate his interests and is also well paid. In the negative outcome conditions, the protagonist realizes that her job does not highlight her skills, is monotonous, and does not provide for career advancement. In all conditions, the protagonist learns that his place on the basketball team has been taken by another player who has also turned out to be very good. The outcome is evaluated a few days (short-term conditions) or 1 year (long-term conditions) after the choice. For each experimental condition, half of times the protagonist is a woman; the other half it is a man.The procedure and the other materials were the same as study 1. Only the 5 manipulation check questions were slightly modified to adapt them to the different scenarios. For example, the question on the responsibility was the following: “To what extent does the protagonist feel responsible to take on another job instead of a sports career?” The question on the decision justifiability was the following “To what extent does the protagonist consider the reasons for taking another job instead of a sports career to be valid?”Also before this experiment, a pilot study was carried out with 20 volunteer employees in order to test the plausibility of the scenarios and the clarity of the instructions. No changes were needed after this study.The same statistical analyses of study 1 were performed also for study 2.In The results of the 2x2x2 MANCOVA conducted on the InVs , with age and gender as covariates, showed that multivariate tests were significant for age, for the three IVs, for choice x time and outcome x time interactions. The univariate tests revealed that choice affected all InVs; outcome affected decision justifiability and evaluation of present working condition; time affected the perception of the freedom of the choice; choice x time affected responsibility and perception of the freedom of the choice; outcome x time affected the evaluation of present working condition. Pairwise comparisons and simple effects analysis, both of them with Bonferroni adjustment, were used to investigate main and interaction effects. All InVs increased with free choice; decision justifiability and positive evaluation of present working condition increased with positive outcome; in short term, the perception of the freedom of the choice augmented. The interaction between choice and time revealed that with free choice, the responsibility did not vary as a function of time, whereas with forced choice, responsibility was higher in long than in short term. On the contrary, the perception of the freedom of the choice was independent of time in the forced choice condition, while it was higher in short than in long term with free choice. The outcome x time interaction showed that, with positive outcome, the evaluation of present working condition did not vary depending on time, whereas, with negative outcome, this evaluation was more positive in long than in short time. The evaluation of the protagonist’s present working condition was more positive as the age of the participants increased. No significant effects were due to gender. In The five 2x2x2 ANCOVAs conducted on the five emotions (with gender and age as covariates) to test the effects of the three IVs on their intensity showed that all emotions were affected by choice and by outcome; moreover, regret, disappointment, and anger toward oneself were also affected by choice x outcome interaction. More specifically, regret, disappointment, and anger toward circumstances increased with forced choice, whereas anger toward oneself and satisfaction increased with free choice; all emotions increased with negative outcome, except for satisfaction, which increased with positive outcome. The intensity of regret and disappointment increased with forced choice compared to free choice, when outcome was positive, while no significant difference depending on choice was found when the outcome was negative. Anger toward oneself always increased with free choice compared to forced choice but this effect was higher with negative than with positive outcome. Regret diminished when the participants’ age increased, whereas the opposite effect was found on satisfaction. No significant effects were due to gender. In The factor analysis performed to reduce the number of items presented in the questionnaire on the emotional experience yielded similar results to study 1.self-attribution, external attribution, and contentment. The values of Cronbach’s alpha for each dimension were 0.850, 0.704, and 0.831, respectively. The results are reported in Three components with eigenvalue >1, explaining 62.54% of the total variance, were extracted following the same procedure of study 1. The items loading on each component were the same of study 1. Once again, the components were labelled Also in study 2, five moderated mediation analyses were performed to investigate whether the effect of choice on the five emotions was moderated by outcome or time and was mediated by responsibility, decision justifiability, and the three dimensions extracted from the factor analysis, that is, self-attribution, external attribution, and contentment. The model 10 of the Process macro 3.1 for SPSS  was testAll results were interpreted with reference to the intercept .The results concerning the relationship between choice and/or the moderators and the mediators  were similar to those of the MANCOVA and were reported in After including the mediators in the model, the results on the five emotions were the following.The probability of regret increased with forced choice, negative outcome, self-attribution, and external attribution, while decreased with contentment, choice × outcome interaction, and the participants’ age. The conditional direct effect of choice on the probability of regret was moderated by outcome, being significant only for positive outcome (as the ANCOVA showed). Indeed, the negative interaction between choice and outcome indicated that with forced choice the effect of negative outcome decreased because in these conditions the probability of regret was high in both outcomes . The conditional indirect effects of choice through self-attribution and external attribution were not moderated by outcome and time, since they were significant for both types of outcome and both in short and long term. The signs of these effects were the same as in study 1. The conditional indirect effect of choice through contentment was moderated by outcome, being significant only with positive outcome, in both values of time. The sign of this effect was positive, as well as in study 1.The probability of anger toward oneself increased with negative outcome, responsibility, and self-attribution and decreased with decision justifiability, contentment, and with the choice x outcome interaction. The negative interaction replicated the results already showed by ANCOVA, that is, the probability of this emotion increased with negative outcome, when the choice was free, but this effect decreased when the choice was forced because the intensity of anger toward oneself was low in both outcomes. Choice exerted an indirect effect through self-attribution and a conditional indirect effect through contentment. More specifically, regardless of the values of outcome or time, forced choice decreased self-attribution, which in turn increased anger toward oneself: Thus, the indirect effect was negative and was not moderated by outcome or time. Instead, the indirect effect of choice was moderated by outcome, being significant only with positive outcome, both in short and in long term: Here, forced choice decreased contentment, which in turn decreased the probability of anger toward oneself, making the indirect effect positive.The probability of disappointment increased with negative outcome and external attribution, while decreased with contentment and with choice × outcome interaction. The latter indicated that with negative outcome, the probability of disappointment increased with free choice but that with forced choice this effect decreased because the intensity of disappointment was quite similar in both outcomes. Choice exerted a positive indirect effect through external attribution and a positive conditional indirect effect through contentment, as well as in study 1.The probability of anger toward circumstances increased with forced choice, negative outcome, external attribution, and decreased with responsibility and contentment. The effect of choice was moderated by outcome, being significant only with positive outcome in both values of time: With positive outcome, the probability of this emotion was higher in forced than in free choice, while with negative outcome, it was similar in both types of choice.Choice exerted an indirect effect through external attribution and a conditional indirect effect through contentment. More precisely, irrespective of the values of outcome and time, forced choice increased the effect of external attribution, which in turn increased the probability of this emotion: Thus, the indirect effect was positive. Instead, the indirect effect through contentment was significant only for positive outcome in both values of time: here forced choice diminished contentment, which in turn diminished the probability of anger toward circumstances, making the conditional indirect effect positive.The probability of satisfaction decreased with negative outcome and increased with contentment and the participants’ age. Choice exerted only a conditional indirect effect through contentment. Indeed, with positive outcome, in both short and long term, forced choice diminished contentment, which in turn increased satisfaction. Thus, such an effect was negative.The effects of age were found only on regret and satisfaction, whereas no significant effects were due to gender. These results are reported in The results of these studies largely support our hypothesis. We will first discuss the results that are common to both of them and then the differences between them. In particular, we will discuss the effect of the temporal dimension on the five emotions afterward, since it was somewhat different in the two studies.Both studies showed that a forced choice is a relevant source of regret. Regardless of whether the decision-making resulted in action or inaction, regret was more intense when elicited by a forced than a free choice. Indeed, while regret was always high when the outcome was negative, irrespective of the type of choice, forced choice-related regret did not diminish with a positive outcome, contrarily to regret elicited by a free choice. Actually, in both studies, this pattern was similar also for disappointment, a finding already found by some authors , suggestNote that in both studies, the manipulation of responsibility, and more generally of all intermediate variables, was successful: Responsibility increased with free compared to forced choice, suggesting that participants understood the meaning of the term correctly. As concerns decision justifiability, this variable increased with forced vs. free choice in the first study, whereas the opposite result emerged from the second study: Anyway, in both studies, the average scores were always high or medium-high, implying that participants felt that the reasons underlying the decision of the scenario’s protagonist were well-founded. Thus, the finding that two appraisal dimensions were irrelevant not only for disappointment but also for regret indicates that responsibility is not a necessary requisite for regret and that the DJT  does notIn both studies, the moderated mediation analyses showed that regret was the only emotion that increased with both self-attribution and external attribution, whereas decreased with contentment as well as all other negative emotions. In addition, it was the only emotion on which choice exerted both a direct (in interaction with outcome and/or time) and an indirect (through phenomenology) effect in both studies. In our opinion, this is the most relevant finding of these studies since it supports the hypothesis that regret derives from two types of choice and that the forced choice is at least as important as the free choice. In addition, it suggests that the emotional experience of regret is more complex than it is generally thought in literature.Disappointment increased with external attribution but was not affected by self-attribution. Consequently, although this emotion has the same relationship as regret to antecedents and the two appraisals, its phenomenology is primarily focused on awareness of forced choice. This result questions the dominant conception of disappointment because it reveals how central the relationship with the choice, not just with the outcome, is in its phenomenology.Anger toward circumstances was the other emotion with similar characteristics to regret. In both studies, it increased with forced choice, negative outcome, and external attribution. Nevertheless, in the first study, this emotion decreased with self-attribution, instead of increasing like regret; in the second study, it decreased when responsibility increased, whereas responsibility never affects regret.The negative emotion that most differed from regret was anger toward oneself. In both studies, it was the only emotion that increased with free choice in the presence of a negative outcome. With a positive outcome, in fact, the emotion decreased with both types of choice. Furthermore, it increased with responsibility, self-attribution, and decreased with decision justifiability, while it was not affected by external attribution. These results suggest that, at least for the participants in our studies, anger toward oneself is the emotion that encompasses the features that the mainstream of the economic and psychological literature attribute to regret. We will discuss this issue later.As to concerns satisfaction – the only positive emotion we took into account because of manipulation of the outcome – the results are congruent with the predictions derived by appraisal theories: In both studies, it increased with positive outcome, free choice, and contentment; in the first study, it also increased with decision justifiability. As expected, neither of the other two dimensions of phenomenology had any effect on it.Although the results of the two studies were very similar, some differences regarding the temporal dimension and the two covariates emerged: Now, we will briefly discuss them.In study 2, time did not affect the intensity of emotions, but did affect responsibility, self-attribution, and external attribution in interaction with choice. Summing up, when the choice was forced, responsibility and self-attribution tended to be lower in the short than in the long term, whereas with free choice, they were always very high. The probability of external attribution increased in the long term with free choice, whereas it was always high in both time measures when choice was forced.In study 1, the effect of time was more relevant, since it concerned responsibility, the same two phenomenological dimensions, and almost all emotions except disappointment. Responsibility, self-attribution, and external attribution increased in the long term, even if the effect on self-attribution was produced in interaction with choice. The effect of time on regret, anger toward oneself, and anger toward circumstances was moderated by the three-way interaction with choice and outcome. Thus, in neither study did we find sufficiently clear and robust results to make a congruent explanatory hypothesis. Although the temporal dimension was more significant when decision-making resulted in inaction rather than in action, our findings do not support the temporal theory of regret , 1995, aThe other differences between our two studies concern the effect of gender and age. In the first study, all emotions except satisfaction increased in females compared to males, whereas no significant effects were due to age. In study 2, gender did not affect results, but regret diminished with age, whereas satisfaction and contentment increased with it.To the best of our knowledge, only few studies found a gender difference between action and inaction regrets, in the opposite direction to our studies. In the context of romantic  or sexuaAs regards the effect of age found in study 2, which shows that as a function of age regret decreased whereas satisfaction and contentment increased, to our knowledge, no other studies have found this effect. The studies that focused on the relationship between regret and subjective well-being in the life cycle  or on coTo conclude this discussion, we would like to focus on two points.The first concerns the question of whether the difference between our results and the majority of those reported in the literature is merely a matter of terminology. As we wrote above, the studies dealing with terms from everyday life, as in our case, always have to deal with the problem that there is no one-to-one correspondence between the terms as they are used by lay people and as they are conceived in the specialist literature . This isrimpianto (or rammarico) and regret are not completely superimposable, nor are the English and Italian terms of the other emotions investigated, therefore in our studies, we have considered the appraisal and phenomenological aspects of emotions to overcome this possible issue. The success of the manipulation check suggests that participants found the proposed stories believable and that they interpreted them correctly. Thus, the emotional reaction attributed to the protagonists of the stories seems to be plausible, and this seems to confer ecological validity to the studies.Perhaps the terms The use of the scenario method is the final point on which we would like to focus.As we wrote in The rationale of the studies section, this method has the advantage of allowing an accurate manipulation of the experimental variables, especially if the object of the studies concerns decisions in a self-relevant domain and not trivial everyday decisions. The latter can easily be the object of behavioral experiments, because the manipulation of the variables related to them does not raise the ethical issues that would arise in manipulating self-relevant variables. However, the fact that emotional reactions are attributed to a third person and not felt in the first person could cast doubts on the correspondence between the participants’ real subjective experience and the one attributed to the scenario protagonist. We think there are several ways to respond to such an objection. The first is that perspective taking has been considered as a natural aptitude of living being, rooted in the brain. Notably, simulation theory  assumes Finally, even if one admits that there is not a total correspondence between the experience in first and in third person, the scope of our studies was to compare the emotional reactions elicited, or supposed to be elicited, by free vs. forced choice. Since these reactions were investigated through the same method, the comparison between them remains valid regardless of the possible difference between emotions experienced in the first or third person.Overall, we believe that our results can contribute to a deeper understanding of regret and other post-decision emotions along three directions: First, they suggest that the conception of regret derived from decision theorists is too binding, since it posits as necessary some requirements which are not. Second, they reveal that the antecedents and phenomenology of regret are broader than it is generally believed. Third, they reveal that decision-making  does not elicit a reduced number of sharply separated emotions; rather, it produces a complex emotional constellation, where the different emotions, singularly and/or in combination with the others, constitute the affective responses to the different aspects of the decision-making process. The emotions we took into account capture some of these aspects, although they are far from covering them all.In our opinion, the major limitation of our studies is that we did not consider the possible behavioral consequences of emotional experiences. In this way, we have omitted to investigate the motivational function of emotions  and, conIf we had asked the participants what the protagonist of the scenario was thinking of doing to deal with the situation she was experiencing, we would have had a more complete picture of the emotional experiences investigated, since the behavioral consequences would also have been included. Future research should investigate these aspects.https://doi.org/10.6084/m9.figshare.14444417.v1.The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found at: The studies involving human participants were reviewed and approved by Department of Psychology - University of Campania Luigi Vanvitelli. The participants provided their written informed consent to participate in this study.OM conceptualized the studies, analyzed the data, and wrote the manuscript. CG and BP conducted literature searches, collected the data, and prepared the figures and tables. LA and GN revised the manuscript. All authors contributed to the article and approved the submitted version.The department of Psychology of the University of Campania Luigi Vanvitelli paid the publication fee.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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


N6-methyladenosine (m6A) modification is a dynamically regulated RNA modification that impacts many cellular processes and pathways. This epitranscriptomic methylation relies on the participation of RNA methyltransferases (referred to as “writers”) and demethylases (referred to as “erasers”), respectively. We previously demonstrated that the Arabidopsis thaliana protein atALKBH9B showed m6A-demethylase activity and interacted with the coat protein (CP) of alfalfa mosaic virus (AMV), causing a profound impact on the viral infection cycle. To dissect the functional activity of atALKBH9B in AMV infection, we performed a protein-mapping analysis to identify the putative domains required for regulating this process. In this context, the mutational analysis of the protein revealed that the residues between 427 and 467 positions are critical for in vitro binding to the AMV RNA. The atALKBH9B amino acid sequence showed intrinsically disordered regions (IDRs) located at the N-terminal part delimiting the internal AlkB-like domain and at the C-terminal part. We identified an RNA binding domain containing an RGxxxRGG motif that overlaps with the C-terminal IDR. Moreover, bimolecular fluorescent experiments allowed us to determine that residues located between 387 and 427 are critical for the interaction with the AMV CP, which should be critical for modulating the viral infection process. Finally, we observed that atALKBH9B deletions of either N-terminal 20 residues or the C-terminal’s last 40 amino acids impede their accumulation in siRNA bodies. The involvement of the regions responsible for RNA and viral CP binding and those required for its localization in stress granules in the viral cycle is discussed. The corwriter”) . Other r6A marks . The thification .6A machinery have been discovered in Arabidopsis . The Arabidopsis genome encodes 14 homologs of Alk B family of “eraser” proteins   , of whic in vivo . Recentlstresses . Interesytoplasm , formingytoplasm .Arabidopsis genome has been described to encode more than 200 putative RNA-binding proteins (RBP)  . RBPs arns (RBP) . RBPs arns (RBP) . RNA recns (RBP) . Other Rns (RBP) , and thens (RBP) . Howeverns (RBP) . In manyns (RBP) . Among ons (RBP) . In thisns (RBP) . These pns (RBP) . Additions (RBP) . Similargranules . Moreovein vitro .Bromoviridae family and, like the rest of the members of this family, its genome consists of three single-stranded RNAs of plus polarity  belongs to the polarity . RNA1 anpolarity . We prevhe virus . Due to atALKBH9B interacts with the viral RNA, although the kinetic parameters of this interaction, as well as the identification of the RBD, were not analyzed  by incubating a constant amount (5 ng) of an RNA transcript, corresponding to the 3′ untranslated region of AMV-RNA3 (3′UTR-RNA3), with increasing concentrations of the glutathione S-transferase fusion protein (GST:atALKBH9Bwt)  . We chos lane 7, , suggest lane 7, . In any  lane 7,  and was plicates . The Kd  0.30 μM .atALKBH9Bwt with RNA-binding activity, we first designed GST:atALKBH9B deletion mutants lacking 160 amino acids at either the N- or C-terminal part , or 187 amino acids in the internal region of the protein (Δ187 Int)  (In order to determine the region of 187 Int) . Northweproteins . Thus, wT:Δ387Nt . NorthweT:Δ387Nt . To confGST:RBD) . As showatALKBH9Bwt, GST:Δ160Nt, GST:Δ258Nt; GST:Δ258Nt/Δ80Ct; GST:Δ258Nt/Δ40Ct and GST:Δ258Nt/Δ20Ct by incubating the viral RNA with different protein concentrations. As expected, EMSA showed a decrease in chemiluminescent signal intensity corresponding to the free viral RNA in mutants containing the RBD  in the presence of GST:RBD fusion protein. As shown in d value of the RBD fused to GST was 0.64 μM, indicating slightly less RNA-binding than the full-length protein (GST:atALKBH9Bwt). From the linear regression adjustment, a Hill coefficient c of 2.4 was obtained; this value above 1 (c = 1 indicates no cooperativity) would be taken as an indication of positive cooperativity.To validate the northwestern blot assays, EMSAs were performed with GST: the RBD , whereasatALKBH9B RBD with other RNA binding proteins (RBP) from the databank, but the alignment of sequences did not reveal any significant similarities. Nonetheless, a high percentage of IDRs has been reported in viral, prokaryotic, and eukaryotic RBPs   . Remarka427–467) .atALKBH9B efficiently cooperatively binds viral RNA through an RBD that has the characteristics of the IDRs present in other viral and eukaryotic RBPs.Taken together, these results indicate that atALKBH9B affected the infectivity of AMV but not of cucumber mosaic virus (CMV), correlating with the ability of atALKBH9B to interact (or not) with their coat proteins . For this purpose, we first designed three mutants corresponding to the N-terminal, central and C-terminal regions, fused to the N- and C-terminal parts of the YFP  after 48h. Leaves co-infiltrated with CYFP-9Bwt plus NYFP-CP, CYFP-9BΔ160Nt plus NYFP-CP, and CYFP:9BΔ187Int plus NYFP-CP rendered a strong YFP fluorescence signal in the cells, whereas no reconstituted YFP fluorescence was detected in leaves co-infiltrated with the pair CYFP-9BΔ160Ct plus NYFP:CP , forming biomolecular-condensates associated with stress granules  accumulated in cytoplasmic granules colocalizing with SGS3. In contrast, deletion of the N-terminal 20, C-terminal 40 or internal 186 residues  resulted in proteins showing a diffuse pattern throughout the cytoplasm . Moreover, EMSA analysis led us to determine that the Kd of the binding of the RBD alone   , Turnip –1.3 μM) , p7 MP o–1.3 μM) , AMV CP –1.3 μM) , or the –1.3 μM) . AdditioatALKBH9B sequence revealed no obvious structured RBD that could justify the RNA-binding properties described above. However, recently 6A-binding proteins, which undergo LLPS in the presence of polymethylated mRNAs. The resulting mRNA-YTHDF complexes form P-bodies and stress granules  and presents an RGxxxRGG motif between positions 469 and 465  to generate a construct with 9B merged to the C-terminal part of the GST. GST and GST:atALKBH9B fusion proteins were expressed in BL21 (DE3) E. coli cells and purified with glutathione Sepharose 4B beads  according to the manufacturer’s recommendations. All protein purification procedures were performed at 4°C.Full-length atALKBH9B purified proteins were electrophoresed in 12% SDS/PAGE and transferred to nitrocellulose membranes. Membranes were incubated overnight at 4°C in Renaturing Buffer . After this, membranes were incubated with 20 mL of the same buffer containing 50 ng/μL of the AMV sgRNA 4 labeled with digoxigenin for 3 h at 25°C. For the EMSA assay, 5 ng of 3′UTR of AMV RNA 3 transcripts were heated for 5 min at 85°C and cooled at room temperature for 15 min. Different amounts of purified GST:atALKBH9B fusion proteins were added and incubated for 30 min at 4°C in a 10-μl final volume of Union Buffer . Following incubations, the samples were separated through 1.2% agarose. RNAs were transferred to positively charged nylon membranes (Roche). RNAs were visualized on blots using DIG-labeled riboprobes corresponding to the 3’UTR of AMV RNA 3. Synthesis of the digoxigenin-labeled riboprobes, hybridization and digoxigenin-detection procedures were carried out as described in (Dilutions of GST or GST:ribed in .N–CYFP:atALKBH9B (wild-type and deletion mutants); GFP:atALKBH9B and mCherry:SGS3 fusion proteins were cloned using the Gateway System (Invitrogen) according to the manufacturer’s recommendations. Plasmid expressing the CPAMV merged to the N, or C-terminal part of the YFP (N–CYFP:CPAMV), was previously described in Agrobacterium tumefaciens C58 cells. Pairs of cultures carrying specific fusion proteins were mixed at an optical density of 0.5 each in infiltration solution  and co-infiltrated into N. benthamiana leaves. Laser-scanning confocal images were taken 48 h after agroinfiltration. Excitation and emission wavelengths were 488 and 508 nm for GFP, 514 and 527 nm for YFP, and 545 and 572 nm for mCherry. Expression of fusion proteins was corroborated by western blot analysis using anti-NtGFP or anti-CtGFP (Roche) antibodies were conducted following the recommendations of the manufacturer.The original contributions presented in the study are included in the article/VP and FA conceived the project and designed the experiments. LA-M conducted the experiments with assistance from JM-M and MM-P. LA-M, FA, and VP wrote the manuscript. All authors analyzed and discussed the results.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.


Pseudomonas aeruginosa the RNA chaperone Hfq and the catabolite repression control protein (Crc) govern translation of numerous transcripts during carbon catabolite repression. Here, Crc was shown to enhance Hfq-mediated translational repression of several mRNAs. We have developed a single-molecule fluorescence assay to quantitatively assess the cooperation of Hfq and Crc to form a repressive complex on a RNA, encompassing the translation initiation region and the proximal coding sequence of the P. aeruginosa amiE gene. The presence of Crc did not change the amiE RNA-Hfq interaction lifetimes, whereas it changed the equilibrium towards more stable repressive complexes. This observation is in accord with Cryo-EM analyses, which showed an increased compactness of the repressive Hfq/Crc/RNA assemblies. These biophysical studies revealed how Crc protein kinetically stabilizes Hfq/RNA complexes, and how the two proteins together fold a large segment of the mRNA into a more compact translationally repressive structure. In fact, the presence of Crc resulted in stronger translational repression in vitro and in a significantly reduced half-life of the target amiE mRNA in vivo. Although Hfq is well-known to act with small regulatory RNAs, this study shows how Hfq can collaborate with another protein to down-regulate translation of mRNAs that become targets for the degradative machinery.In  P. aeruginosa (Pae) features two extensive post-transcriptional networks that depend on Hfq . Whereas in Enterobacteriaceae and Firmicutes CCR is predominantly controlled at the level of transcription , in Pseual level . In Pae rred one . Several amidase ,22. As C amidase , respectibosomes .The response to different carbon sources is mediated through different levels of the regulatory RNA CrcZ ,26. As Cn or (ARN)n repeats, in which A is an adenine, R is a purine (A/G) and N is any nucleotide, which align to engage the six tripartite binding pockets on the distal face of Hfq , the Hfq/Crc/amiE6ARN assembly displayed an increased stability when compared with the Hfq/amiE6ARN complex alone  used in the single-molecule assays was produced in E. coli strain JW4130F′Δhfq. The cells were grown at 37°C in LB medium. At an OD600 of 0.7, the synthesis of Hfq* was induced by addition of IPTG and arabinose to a final concentration of 1 mM and 0.02%, respectively. The cells were harvested after 2 h, the cell pellet was resuspended in lysis buffer , and the cells were lysed by sonication. First, Hfq* was highly enriched by heating and ammonium sufate precipitation followed by three consecutive chromatography steps as described in detail by Beich-Frandsen et al.  as specified by the manufacturer. Then, given the difference in size between Hfq wt and HfqS65C-Flag, a western-blot analysis was performed with anti-Hfq antibodies to determine the ratio of Hfq wt and HfqS65C-Flag. Both, the column purified Hfq* and Hfq*, captured with the anti-Flag M2 magnetic beads, showed a 1:1 ratio of Hfq wt and HfqS65C-Flag  by Ni-affinity chromatography followed by removal of the Hisc et al. .in vitro transcription using T7 RNA polymerase, and then purified on 8 M urea 8% polyacrylamide gels. The DNA templates for transcription were obtained by extension of chemically synthesized overlapping oligodeoxyribonucleotides (Thermo Fisher Scientific) with Q5 DNA polymerase (NEB). The template for the amiE102 sub-sequence, which comprises nt –40 to +60 of amiE mRNA relative to the A (+1) of the AUG start codon, plus two additional Gs at the 5′end  and amiE_R (5′CCTGTGTCCTGTGTGTCCTGTCCAAAGTGTGTCGTCCTGTAGTTGACCACCGCCACTCCGACGGTGTCGTTGCTGCTGGAAATATCGCCG-3′) oligonucleotides, while the amiE_5′AANmut RNA (amiE_AANmut_F oligonucleotide (5′- TAATACGACTCACTATAGGTTTTTTCGTCCCGAAAAAATAGGTGATATCCATGC-GTCACGGCGATATTTCCAGCAGC-3′) in combination with the oligonucleotide amiE_R. The amiE_3′ARNmut RNA  and amiE_61-linker-R (5′-CCTGTGTCCTGTGTGTCCTGTCCA-AAGTGTGTCGTCCTGTAGTTGGAAATATCGCCGTGACGCATGGATATCACC-3′). The resulting RNAs contained the 3′end extension -//-5′-AGGACGACACACTTTGGACAGGACACACAGGACACAGG-3′, which is complementary to the SA5-Cy5 DNA oligonucleotide  microscope with an EMCCD camera (Andor). A 640 nm laser was used for Cy5 excitation. A selected field of view was recorded upon flow of Cy5-RNA for 3 minutes. The Cy5 emission signals were collected through a 60 × water immersion objective using custom software developed in the Ha Lab (smCamera).amiE102 RNA (250 nM) or variants thereof were added to the same concentration of the SA5-Cy5 DNA nucleotide and annealed by denaturation at 75°C for 5 min in 1× TNK buffer , refolded at 37°C for 15 min, and equilibrated at 20°C. DDS-coated quartz slides were pre-treated with biotinylated BSA (0.2 mg/ml), Tween-20 (0.2%) and Neutravidin (0.1 mg/ml) as previously described . The amplitude values are between 0 and 1.The dwell times were collected using custom Matlab code. To estimate the lifetimes and associated errors, un-binned distributions were fit using maximum likelihood methods  to equat3a was obtained by error propagation  of the AUG start codon was prepared by in vitro transcription using T7 RNA polymerase, and then purified on a 8M urea 8% polyacrylamide gel. The DNA template was amplified by PCR using the primer pair R185 (5′-TCTAGACGTAATACGACTCACTATAGGGCCTT-TTTTCGTCCCGAAAAAATAACAAC-3′) and Z172 (5′-GTAGTTGACCACCGCCACTC-3′), wherein the forward primer R185 contained the T7 promoter sequence. When compared with amiE102, the yield obtained with the amiE105 template was ∼2-fold higher. The RNA amiE105 is identical to amiE102 except for the 3′extension and that the artificial two G-nucleotides at the 5′-end of amiE102 RNA are replaced by 5′-GGGCC-3′.., which is identical to the authentic sub-sequence of amiE. For Hfq and Hfq/Crc assembly on amiE105, the RNA was annealed at 50°C for 3 minutes. Hfq and Hfq/Crc were mixed at 2:1 and 2:11 ratios prior to addition of amiE105 . After incubation on ice for 1 h, the mixture was diluted 7-fold before loading onto grids. Graphene oxide (GO) grids were prepared from regular Quantifoil r1.2/1.3 grids. A 2 mg/ml graphene oxide dispersion (Sigma Aldrich) was diluted 10-fold and spun at 300 × g for 30 s to remove larger aggregates. The dispersion was then diluted 10-fold before applying 1 μl to glow-discharged grids . After drying, 3 μl of the sample was applied to the GO grids and after 30 s of incubation, excess sample was blotted away and frozen in liquid ethane (Vitrobot markIV (FEI)). The grids were screened on a 200 kV Talos Arctica (FEI) , and datasets collected on a 300 kV Titan Krios (FEI) with a K3 (Gatan) direct electron detector . For the 2 Hfq/1 Crc/1 amiE105 dataset, a set of 1007921 particles was extracted by Warp  in the presence of 20 U RiboLock RNase Inhibitor (Thermo Fisher Scientific). 5 pmol of in vitro transcribed amiE-flag mRNA  and A131 (5′-TTTTTTCTATTATCAtcacttgtcgtcatcgtctttgtagtcCAGGATCGACAGCGG -3′). The forward primer contained a T7 promoter sequence and the reverse primer contained the Flag-tag encoding sequence (lower-case letters). The amplified PCR fragment was use as template in an in vitro transcription reaction using the AmpliScribe T7-Flash Transcription Kit (Epicentre Biotechnologies). Increasing amounts of purified Hfq protein as indicated in Figure TM Western ECL Substrate (BioRad). The quantification of the signals was performed with the Volume Tools of ImageLab software 5.2.1 (BioRad).amiE mRNA was determined in strains PAO1 . The samples were withdrawn at several times thereafter  as previously described  to the graphs determined in Excel. The amiE mRNA was not degraded with a first order kinetics in strain PAO1Δcrc. Therefore, the half-life was estimated by determining the slope (k*decay) from a linear trendline and the half-life was calculated by t1/2 = 50/k*decay.The stability of ins PAO1  and PAO1AO1  of the AUG start codon, two extra 5′-terminal G nucleotides, and a 3′extension for annealing to a Cy5 labeled DNA oligonucleotide. (hereafter amiE102); Figure amiE102 RNA comprises the proximal amiE6ARN motif as well as further AA(R)N motifs for the distal site of Hfq that are located downstream of the AUG  that contained both protomers in a 1:1 ratio  (∼60 s), indicating that photobleaching might lead us to slightly underestimate the lifetimes of the most stable complexes, but does not account for the short-lived complexes that we observed.The binding assays of w Figure . They retibodies . Maximume Figure . The unsamiE102. The presence of 100 or 500 nM Crc did not change the lifetimes of the stable and unstable Hfq*/amiE102 interactions, suggesting that Crc does not affect the mode of Hfq/RNA binding 3 motif and the distal hairpin comprising one putative ARN motif in the loop 3 motif. Nevertheless, most binding events between amiE_3′ARNmut and Hfq were transient and non-specific . This increased compactness of the Hfq/Crc/amiE105 assemblies was apparent from a decreased extendedness of the 2D class averages with 106 and 104 Å for the top and bottom class averages in Figure amiE105 assembly, which is significantly lower than the sizes measured from the 2D class averages of the Hfq/amiE105 assembly  so that the effector complex blocks translation more efficiently  in formation of stable Hfq/amiE102 complexes , which binds as a dimer to the operator on its own mRNA so that the N-terminal domain sterically prevents ribosome binding (E.coli ribosomal proteins, such as S4 and S15. The S4 autoregulatory feedback mechanism involves allosteric changes in the mRNA structure, affecting the distribution of translationally active and inactive mRNA (Autogenously acting translational repressors generally employ molecular mimicry and operate either by competing with initiating ribosomes or by an entrapment mechanism, whereby the formation of a productive translation initiation complex is impeded . A well- binding . The secive mRNA . Similarive mRNA . Regardlive mRNA  and of Tive mRNA  for theiive mRNA ,50. ThusamiE during CCR, or translational regulation by RsmA (Pae RsmA (P. fluorescens RsmE (amiE mRNA (Figures In contrast, effective translational repression by Hfq/Crc of undesirable mRNAs, such as  by RsmA  would be by RsmA  and ∼2-f by RsmA , respect by RsmA ,50. The  by RsmA  or dimerPae RsmA  and showens RsmE  appear t Figures .Although Hfq is well-known to act with sRNAs, this study shows how Hfq can collaborate with another protein to down-regulate mRNA expression. Thus, to effect robust translational control Hfq appears to need a partner—either a sRNA or another protein—to lock it onto the mRNA. Finally, assembly of Hfq/Crc on nascent mRNAs may provide a means for efficient competition with small RNA substrates of Hfq. In the complexes shown in Figure gkab510_Supplemental_FileClick here for additional data file.


Most protein-coding genes in eukaryotes possess at least two poly(A) sites, and alternative polyadenylation is considered a contributing factor to transcriptomic and proteomic diversity. Following transcription, a nascent RNA usually undergoes capping, splicing, cleavage, and polyadenylation, resulting in a mature messenger RNA (mRNA); however, increasing evidence suggests that transcription and RNA processing are coupled. Plants, which must produce rapid responses to environmental changes because of their limited mobility, exhibit such coupling. In this review, we summarize recent advances in our understanding of the coupling of transcription with RNA processing in plants, and we describe the possible spatial environment and important proteins involved. Moreover, we describe how liquid–liquid phase separation, mediated by the C-terminal domain of RNA polymerase II and RNA processing factors with intrinsically disordered regions, enables efficient co-transcriptional mRNA processing in plants. Arabidopsis genes have at least two poly(A) sites  polymerase 1 (Fip1), a core component of the CPSF complex) [A variety of growth defects have been reported in several polyadenylation factor mutants. For example, t plants . Meanwhicomplex) . Other dcomplex)  and altecomplex) . Furthercomplex) . Togethefip1-2 compared to the wild-type Arabidopsis [Pseudomonas syringae [Moreover, genome-wide induction of the 3′-untranslated region (UTR) extensions has been observed under conditions of dehydration , and incbidopsis , and AtCbidopsis  and in rsyringae . These fNascent RNAs undergo several processing steps, including 5′ capping, splicing, 3′ polyadenylation, and chemical modification, to become mature mRNAs. Emerging evidence has shown interconnections among these processes, which appear to occur in series but may happen co-transcriptionally and even be coupled or interact with each other ,38,39.To date, two models have been proposed to explain these coupling processes in yeast and mammalian cells. In the first model, called the recruitment model, transcription is central to the coupling of different RNA processing events, mainly through RNA polymerase II (RNAPII). RNAPII serves as a platform for the recruitment of various processing factors to a nascent RNA, including capping factors, splicing factors, and polyadenylation factors . For exaIn the second model, known as the kinetic model or kinetic competition model , the relCurrent evidence suggests that these two models function in an interdependent fashion; the transcription elongation rate could influence the recruitment of different processing factors, and, in turn, the transcription elongation rate could be influenced by the differential recruitment of factors that control the elongation rate ,46. StudArabidopsis thaliana (L.) Heynh. Here, we outline the current progress in understanding such coupling in plants, with a focus on polyadenylation.In addition to the coupling of transcription and RNA processing in mammals and yeasts, similar phenomena have been found in plants, particularly in studies of the model plant DOG1 expression is summarized in DOG1, which was first identified as a major quantitative trait locus for seed dormancy in natural variations of different Arabidopsis accessions [Arabidopsis thaliana (L.) Heynh and other plants [DOG1 has two alternative poly(A) sites: polyadenylation of DOG1 at the proximal site results in functional DOG1 (known as shDOG1), while polyadenylation at the less commonly used distal site results in the production of lgDOG1 [asDOG1 (or 1GOD) [DOG1, and its promoter is located in the region of intron 2 and exon 3 [DOG1 is highly and specifically expressed in seeds, while it is nearly undetectable in seedlings [asDOG1 is mainly expressed in seedlings and has been shown to suppress DOG1 expression in a cis manner during seed maturation [asDOG1 is expressed at a high level in seedlings and the expression of DOG1 is suppressed to promote germination; however, when a stressor is applied, asDOG1 is down-regulated and DOG1 accumulates to promote seed dormancy [DOG1 in Arabidopsis thaliana (L.) Heynh [DOG1 affects the expression of asDOG1 in Arabidopsis thaliana (L.) Heynh through APA, polyadenylation, and transcriptional co-regulation of seed dormancy   originatd exon 3 . DOG1 iseedlings ,52. Howeturation . Under ndormancy . Thus, A.) Heynh , and DOGdormancy A.Arabidopsis FLC antisense transcripts  site. This triggers the FLD-dependent demethylation of H3K4me2 in the FLC gene body, downstream of COOLAIR’s proximal poly(A) site, leading to the suppression of FLC expression and the promotion of flowering [FLC transcription are tightly coordinated, and both steps are affected by the status of the chromatin [FLC expression level increases, resulting in late flowering. Adaptive studies of Arabidopsis accessions have shown that single, natural noncoding polymorphisms can significantly change the splicing pattern of class II transcripts [COOLAIR [FLC expression. Taken together, these results suggest that a precise regulatory network exists during FLC transcription to couple polyadenylation and splicing.The most well-known model for the coupling of polyadenylation and splicing is the regulation of nscripts B. FLC, w in 2007 . Based olowering . Initiathromatin , which inscripts , resulti[COOLAIR  and the 6A functions as a stabilizing factor by suppressing local ribonucleolytic cleavage [6A can suppress the use of proximal poly(A) sites and that adenosines within the AAUAAA signal may be methylated [6A position [6A modification may either directly block the binding of CPSF30 and FLY to poly(A) signals or the binding of m6A by YTH domain-containing proteins, thereby inhibiting the recognition of poly(A) signals by CPSF30 and FLY [6A methyltransferase complex, which function in sporogenesis, can bind to mRNAs-encoding threonine proteases and NTPases at the early microspore stage, mediating their m6A modification and affecting their expression and/or splicing [6A modification is highly coordinated with APA site usage [6A modification in plants, evidence suggests that it plays a crucial regulatory role in the coupling of transcription with RNA processing.Recent data suggest that mcleavage . Other dthylated  , while RNAPII with a Ser5P CTD is required for co-transcriptional splicing; 5′ SS cleavage was achieved through an interaction between the spliceosome complex and Ser5P CTD during the elongation phase of transcription. In addition, RNAPII with a Ser2P CTD paused immediately downstream of the polyadenylation site [Arabidopsis thaliana (L.) Heynh, possesses a C-terminal interaction domain (CID), which is responsible for its interaction with the RNAPII CTD, while its C-terminal region mediates interactions with the polyadenylation factor Clp1-similar protein 3 (CLPS3) [A genome-wide analyses of ion site . Pcf11p- (CLPS3) ,67. RNAPAmong the proteins that are critical in coupling RNA transcription and processing is U1 snRNP, the most abundant snRNP in most eukaryotes . U1 snRNArabidopsis U1 (AtU1)A was found to be closely associated with AtU1-70K and AtU1C, confirming its essential role in pre-mRNA splicing in Arabidopsis thaliana (L.) Heynh [Most protein components of yeast U1 snRNP are conserved in plants ,78 Tabl, suggest.) Heynh .Previous studies also found that the interaction between CPSF and U2 snRNP contributes to the coupling of splicing and 3′-end formation in mammals and fungi ,84. U2 sU2AF65 homologues predicted in the Arabidopsis genome, but only two of them have a U2AF homology motif [Arabidopsis splicing factor 1. Intriguingly, plant SF1 homologues have an additional RNA recognition motif (RRM), which is not present in their yeast and mammalian counterparts [There are four gy motif   Heynh, Drosophila, and humans [In human and yeast cells, mRNA synthesis and processing are coupled to nuclear mRNA export, during which the TRanscription-EXport (TREX) complex plays an extraordinary role. The TREX complex is composed of the THO complex and a group of additional proteins that are conserved across species, including d humans . In yeasd humans . Intrigud humans . Moreoved humans . Meanwhid humans . Thus, tArabidopsis THO core complex consists of hyper recombination 1 (HPR1), TEX1 (THO3), THO2, THO5A/B, THO6, and THO7A/B [hpr1, tex1, and tho2 plants [Arabidopsis THO/TREX complex and splicing factors, consistent with observations in human and yeast cells [tex1 and hpr1, the majority of which were shared, suggesting their roles in polyadenylation [Arabidopsis ortholog of yeast Pcf11, possesses a CID as well, which can interact with the RNAPII CTD [Arabidopsis thaliana (L.) Heynh [trans-acting siRNAs [Notably, the  THO7A/B   Heynh . Howeverg siRNAs ,111.In addition to the proteins mentioned above, specific cytosol condensates may provide appropriate spatial environment for the coupling of transcription and RNA processing.Membraneless organelles formed through liquid–liquid phase separation (LLPS) may play a general role in the machinery that is responsible for co-transcriptional mRNA processing; they could provide spatial possibilities for diverse biochemical reactions to take place efficiently at the same time without disturbing each other or perturbing the intracellular environment. Over the past decade, phase separation has become an emerging field, with studies aimed at determining how cells organize diverse functions while maintaining cellular homeostasis .LLPS is a subtype of phase separation that is known to be sensitive to environmental changes; it can respond rapidly to intracellular fluctuations because of its reversible and dynamic nature . Phase sArabidopsis thaliana (L.) Heynh. The RNA-binding protein flowering control locus A (FCA) associates with a coiled-coil protein, FLX-like 2 (FLL2), to promote the formation of liquid-like bodies, which concentrate near 3′-end processing factors at specific poly(A) sites. Both FCA and FLL2 are IDPs with disordered domains [Arabidopsis proteome consists of approximately 30% IDPs [Various functions in plants rely on proteins with IDRs, including DELLA, CRY, BKI1, BAK1, and ELF3 ,122,123. domains . IDPs ar30% IDPs . These I30% IDPs . A conceArabidopsis thaliana (L.) Heynh through co-purification with RNAPII and transcription elongation factors [It is worth stressing that the membraneless compartments and important proteins mentioned above are not isolated from each other, but interrelated. For instance, Spt5 interacts physically with Prp40, a core protein in the U1 snRNP . Additio factors . The RNA factors . This ID factors .Arabidopsis thaliana (L.) Heynh  Heynh . Interesof mRNAs . Additioof mRNAs . Strikinof mRNAs . Apart fA model of the events involved in coupling transcription with RNA processing has emerged . In thisOnce a primary RNA transcript has been synthesized, it must be processed before becoming a fully functional mRNA. Processing includes 5′ capping, splicing within the transcript body, 3′ polyadenylation, and (sometimes) chemical modification. These complex events cannot be easily separated from each other in time and space; however, partitioned membraneless organelles formed by LLPS may greatly improve the accuracy and efficiency of this process.One interesting question is how plants achieve cleavage and polyadenylation at a precise position using conserved core factors when they lack the conserved PAS sequences found in animals and yeast. One possibility is that plant-specific proteins, such as plant-specific RBPs, are involved in the regulation of polyadenylation in plants. RBPs are of great importance in RNA–protein interactions, which are characterized by the recognition of a specific sequence or structural element in the target RNA by an RNA-binding domain (RBD) such as an RRM or K homology domain . In addi


The degradation of SARS-CoV-2 specific ribonucleic acid (RNA) was investigated by a numerical modeling approach based on nucleic acid amplification test (NAAT) results utilizing the SmartAmp technique. The precision of the measurement was verified by the relative standard deviation (RSD) of repeated measurements at each calibration point. The precision and detection limits were found to be 6% RSD (seven repeated measurements) and 94 copies/tube, respectively, at the lowest calibration point. RNA degradation curves obtained from NAAT data on four different temperatures were in good agreement with the first-order reaction model. By referring to rate constants derived from the results, the Arrhenius model was applied to predict RNA degradation behavior. If the initial RNA concentration was high enough, such as in samples taken from infected bodies, the NAAT results were expected to be positive during testing. On the other hand, if initial RNA concentrations were relatively low, such as RNA in residual viruses on environmental surfaces, special attention should be paid to avoid false-negative results. The results obtained in this study provide a practical guide for RNA sample management in the NAAT of non-human samples. The viral infection coronavirus disease 2019 (COVID-19) appeared in late 2019 and by 2020 had been classified a pandemic. The COVID-19 pandemic claimed millions of lives and infected hundreds of millions of people worldwide . UnfortuThe NAAT provides qualitative positive/negative results for detecting COVID-19 infection. False-negative results should be ruled out. However, ribonucleic acid (RNA) degradation is an unavoidable risk that is activated under wet conditions depending on ambient temperatures . A possiMany articles report that NAAT for the SARS-CoV-2 virus is the most effective preventative measure to combat the COVID-19 pandemic . There hThe golden standard for diagnosing COVID-19 is reverse transcription-polymerase chain reaction (RT-PCR) . In geneA SmartAmp SARS-CoV-2 kit commercialized by DANAFORM Inc. (Japan) in July 2021 for diagnosing COVID-19 was used. The effectiveness and validity of the SmartAmp assay for the diagnosis of COVID-19 was validated by Asai et al. . SmartAmA device package released by DNAFORM Inc. (Japan) as Life Case, including an RNA extractor, a real-time isothermal nucleic acid amplifier, a mini centrifuge (model Mini-6ks), and a circulator (model MiniT-C), were prepared. The real-time isothermal nucleic acid amplifier enables a test to be completed in 40 minutes with almost the same precision as a conventional PCR device. The Life Case package is intended for on-site testing, and all equipment and reagents required for the testing fit in three carry cases. In addition, a refrigerator (Medicool) for 4°C and a freezer (VT-78HC) for −80°C settings were purchased from SANYO Electric Co., Ltd. and Nihon Freezer Co., Ltd. (Japan), respectively.t.Swab samples are always treated with an inactivating agent for safety. Primarily, standard reagents were diluted on ice with an inactivation solution to 40 copies/ μl. Then, RNA samples were incubated using a circulator or refrigerator. RNA was purified using a smart extract kit for SmartAmp. t index determined from the SmartAmp assay profile.The concentration of SARS-CoV-2–specific RNA was obtained from fluorescent signals calibrations. Fluorescent signals from the amplified nucleic acid were processed with a fluorescent photon counting profile and the concentration of SARS-CoV-2 specific RNA was determined in each sample. The RNA concentration was obtained by calibrating the CTo confirm the measurable range and repeatability of RNA in this experiment, 250, 500, 2,000, 5,000, and 10,000 copies/μl were prepared by diluting a standard RNA solution  with an inactivation solution on ice. The calibration was performed using the same standard solution as described above in “RNA quantification” to ensure reliable quantification results. Each of the 500 μl of the above solution is concentrated and finally extracted with water at 50 μl. Since 10 μl is used for single SmartAmp test, the number of copies will be 500, 1,000, 4,000, 10,000, 20,000 copies/tube, respectively.v was assumed to proceed in a first-order reaction proportional to the reaction rate constant k with respect to the RNA concentration.In this study, the first-order reaction model was chosen as the initial speculation of the RNA degradation pathway . The RNAAs shown below, numerical analysis of the RNA degradation reaction was performed using the integral form of this equation.According to Eq , it can Integration of Eq  yieldsk from the experimental values, the nonlinear least-squares method was applied to Eq . The data show a sufficiently low detection limit (approximately 100 copies/tube), a high correlation coefficient (0.990), and a satisfactory RSD at 500 copies/tube (6.0%). The quantifiable range of this series of operations is in the range of 100 to 20,000 copies/tube. For the degradation efficiency experiment in the next section, 4,000 copies/tube was used in this study.As The rate constants shown in Half-life , it is common for a qualitative positive/negative determination to be provided. Therefore, false-negative results should be ruled out. To rule out a false-negative result, rigid control of temperature while testing is required. Yet, degradation of RNA in an aqueous solution is subject to ambient temperature , and ful105/swab . Anotherpatients . With reIf the initial RNA concentration is high enough, e.g., in samples taken from infected bodies, the results remain positive while testing. Therefore, when testing residual viruses on environmental surfaces at relatively low concentrations, the results need to take RNA degradation into account.The details of the RNA degradation mechanism remain unclear, however the activation energy of the major rate-determining reaction was estimated to approximately 50 kJ/mol from the Arrhenius model calculation. Arrhenius modeling indicates that higher temperature enhances the RNA degradation rate. Given such rapid RNA degradation under ambient temperature, our result provides a practical guide to RNA sample management in NAAT for residual viruses sampled from environmental surfaces under temperature-changing conditions.In this study, artificially synthesized RNA was used as an alternative to actual RNA in the SARS-COV-2 RNA test for safety concerns. For real samples, the situation would be a little more complicated due to other factors that affect RNA degradation. Unfortunately, the initial RNA copy count for environmental coronaviruses can vary depending on sampling conditions. The possible factors that may affect the sampling conditions include the presence of RNA-degrading enzymes , hydrolyt-test 99% confidence), respectively, at the lowest calibration point. The RNA degradation curves obtained from the SmartAmp measurements on four different temperatures  were in good agreement with the first-order reaction model. The Arrhenius model was used to predict RNA degradation behavior by referring to reaction rate constants derived from the results. If the initial RNA concentration was high enough, such as samples taken from infected bodies, the NAAT results were expected to remain positive when testing. On the other hand, RNA concentration in residual viruses on environmental surfaces would be relatively low, special attention should be paid to avoid false-negative results. This study provides a practical guide to RNA sample management in NAAT of non-human samples. The activation energy of the major rate-determining reaction was estimated to be approximately 50 kJ/ mol. Pursuit of RNA degradation mechanisms is ongoing and has been left to future research. The present article does not incorporate the negative effects of sampling conditions on NAAT results and can be considered the limitation of this paper. Further studies on sampling conditions remain as our future task.Degradation of SARS-CoV-2 specific RNA was investigated by a numerical modeling approach based on SmartAmp testing results. The precision of SmartAmp measurements was verified by the RSD of repeated measurements at each calibration point. The precision and detection limits were found to be 6% RSD (7 repeats) and 94 copies/tube (S1 File(DOCX)Click here for additional data file.


Decoding of genetic information into polypeptides occurs during translation, generally following the codon assignment rules of the organism’s genetic code. However, recoding signals in certain mRNAs can overwrite the normal rules of translation. An exquisite example of this occurs during translation of selenoprotein mRNAs, wherein UGA codons are reassigned to encode for the 21st proteogenic amino acid, selenocysteine. In this review, we will examine what is known about the mechanisms of UGA recoding and discuss the fate of ribosomes that fail to incorporate selenocysteine. The genetic code was well established by the mid-1960s and initially thought to be fixed and unalterable. This dogma was challenged in the 1970s and 1980s as a number of studies demonstrated that some mRNAs contain signals in addition to the 64 codons that could modify readout of the genetic code in various ways. The signals may involve intramolecular RNA structures such as hairpins or pseudoknots, combinations of specific nucleotides in tandem, recognition sites for RNA-binding proteins, and inter-molecular base pairing between mRNA and RNA within the ribosome ,3. Here,The potential for UGA recoding from termination to Sec incorporation occurs in a subset of organisms from all three domains of life. In all instances examined, an RNA secondary structure called the Sec insertion sequence (SECIS) marks the mRNA for recoding of UGA codons. The product of recoding is the co-translational insertion of Sec and the production of a selenoprotein. Most selenoproteins are oxidoreductases requiring Sec for their function ,5.Sec to the ribosome during UGA recoding. Consequently, it came as a surprise when it was shown that SECIS elements in eukaryotes are located in the 3′ UTR of selenoprotein mRNAs [In prokaryotes, the SECIS element is composed of a stem-loop located just downstream from the UGA codon A. The sein mRNAs , necessiOne consequence of eukaryotic SECIS action from a distance is that UGA recoding may occur at any in-frame UGA in the message ,11,12. NSec  demonstrated the importance of the stem, and of the sequences within the loop and the spacer separating it from the UGA-Sec codon to inhibit termination and stimulate Sec-incorporation [selenoprotein T (SELENOT) were also independently identified in a genome-wide search for deeply conserved RNA structures [SELENOS) mRNA have been shown to impact Sec-incorporation. The sequence motif referred to as the SELENOS positive UGA recoding element, or SPUR, has been shown to enhance Sec-incorporation efficiency [In cases of stop codon readthrough, where standard near-cognate tRNAs are used to decode the stop codon rather than Sec-tRNAmulatory ,28,29,30mulatory ,32,33. Pmulatory ,34 that poration ,36. The ructures . In addificiency ,39. The Sec specific elongation factor (EEFSEC) [Sec. This function is provided by EEFSEC.Just over 20 years ago, two eukaryotic trans-acting factors essential for Sec-incorporation were identified: the SECIS RNA-binding protein (SECISBP2), and the Sec-tRNA(EEFSEC) ,41,42. USec to the ribosome.EEFSEC differs from the standard eukaryotic elongation factor (EEF1A) in that it has the ability to interact with SECISBP2, has a higher affinity for GTP than GDP ,43, and Sec [Sec and would dissociate from EEFSEC upon delivery of Sec-tRNASec to the ribosome.SECISBP2 contains a canonical L7Ae RNA-binding domain ,46,47 thSec , suggestWhile SECISBP2 is clearly a central player in Sec-incorporation, studies of SECISBP2 deletions in mice  and cultSec. In the following section, we will discuss additional SECIS-binding proteins that have been proposed as part of the SECIS ribonucleoprotein complex, and how these may further modulate the efficiency of UGA-Sec recoding.Emerging from these early studies is a picture in which the SECIS elements act as a scaffold for RNA-binding proteins and this ribonucleoprotein complex recruits EEFSEC to accommodate UGA recoding by Sec-tRNALevels of complexity were added to the mechanism of Sec-incorporation with the discovery of several additional SECIS-binding proteins. The first of these, nucleolin (NCL), best known for its role in ribosome biogenesis, was shown to bind specifically to the SECIS element  around tIn contrast to NCL, the SECIS-binding ribosomal protein L30 contains an L7Ae domain and competes directly with SECISBP2-binding at the kink-turn motif of SECIS elements . The canSeveral years after the identification of L30 as a SECIS-binding protein, EIF4A3, a member of the DEAD-box family of RNA-dependent ATPases, was also shown to bind SECIS elements contained in selenoprotein mRNAs that are known to be sensitive to degradation and have reduced UGA recoding efficiency when selenium is limiting . MappingMost mRNAs contain a 5′ m7G cap that plays important roles in RNA processing and stability and is bound by the translation initiation factor EIF4E as a key step in the process of translation initiation . SeveralTGS1 is recruited to selenoprotein mRNAs by interactions between SECISBP2 and the survival of motor neuron protein complex (SMN). The SMN protein is a component of a ribonucleoprotein assembly chaperone pathway first described as being essential for assembly of small nuclear RNPs involved in splicing . In addiMost recently, the RNA-binding protein PTBP1 has been shown by RNA affinity chromatography to interact with 3′ UTR sequences of SELENOP in the U-rich region separating the two 3′ UTR SECIS elements . DeletioSec to the ribosome during UGA recoding, and how this process competes with the standard decoding process of translation termination. It has been clearly established through in vitro reporter assays and in vivo ribosome profiling experiments that UGA recoding is an inherently inefficient process , such that the majority of ribosomes that initiate translation on selenoprotein mRNAs fail to incorporate Sec and never reach the natural termination codon.Although many of the key components of the Sec-incorporation machinery have been identified, we have an incomplete picture of the dynamic nature of the Sec ribonucleoprotein complex, how it delivers EEFSEC and the Sec-tRNASeveral outcomes can be envisioned for ribosomes that fail to incorporate Sec: (1) ribosomes may decode the UGA-Sec codon as a stop codon and terminate translation C. RNAs tSaccharomyces cerevisiae).Every step of the information-flow pathway in a living cell is monitored and kept accurate by quality control mechanisms. Replication has DNA repair, transcription has proofreading Pol II, tRNA aminoacylation has noncognate hydrolysis, and ribosomes both execute and are subjected to multiple control mechanisms during translation. These quality control pathways are broad and arguably nonspecific, so naturally there are a multitude of exceptions that must either evade or modify specific components of quality control. As a case in point, the mechanism by which mRNAs containing a misplaced in-frame stop  codon are sensed and shunted to a degradation pathway was discovered in 1979 , and a vSec resulted in an overall 60–70% reduction of selenoprotein mRNA levels [Sec levels. In addition, there appeared a third case, SELENOT, where mRNA levels were reduced but the translational efficiency remained the same. Part of this complexity is undoubtedly due to the existence of an SECISBP2 orthologue, SECISBP2L, which is very likely responsible for supporting selenoprotein production when SECISBP2 is absent. Overall, it is clear that active Sec incorporation is essential for maintaining selenoprotein mRNA levels, albeit to varying extents, but the question remains as to how much higher they would be if there were no in-frame UGA codon. One study did tangentially address this question by stably transfecting cDNAs encoding the zebrafish and human versions of SELENOP into the human hepatocyte cell line HepG2. One version of the cDNA was wild-type, which contains multiple Sec codons, and the other had replaced those with Cys codons. Quantitative RT-PCR revealed that the steady state mRNA level for the Cys codon-containing construct was about twice that of the Sec codon counterpart [Selenoprotein mRNAs, with in-frame stop codons that are reprogrammed to allow Sec incorporation, stand as interesting case studies in the regulation of NMD. When selenium is replete, it is plausible to assume that efficient use of UGA as a sense codon would be sufficient to prevent NMD activation. However, it has long been reported that the efficiency of Sec incorporation is low, in the order of 10–25% ,32,35,71A levels . Howevernterpart . InterprSec, eRF1/eRF3, and PELO/HBS1. Although some attention has been paid to the mechanism of the interplay between termination and Sec incorporation [Sec to outcompete the access of eRF1 or PELO. The multitude of factors that would impact this competition include local RNA structure, codon sequence context, relative concentrations of the A-site binders, and the nascent peptide chain sequence that is known to regulate the processivity of ribosome transit during translation elongation [Sec complex, would represent an active competition. On the other hand, termination and NGD might be passively out-competed purely as a function of the relative concentrations of factors. Intriguingly, it has been reported that increasing the amount of eRF1 (but not eRF3) caused an increase rather than the expected decrease in Sec incorporation in transfected cells [As shown in poration ,32, veryongation . The majed cells . SimilarIn the context of considerable complexity regarding processes that monitor A-site occupancy during the translation elongation reaction, another quality control mechanism monitors the stalling of ribosomes. The ribosome quality control (RQC) system induces a general cellular stress pathway that is coordinated by activation of the heatshock protein Hsf1 when nascent chain peptides are stalled . The outIn this focused review, we have highlighted the fact that Sec incorporation must coexist with multiple quality control pathways. In theory, each of these pathways should effectively reduce Sec incorporation, but the Sec incorporation machinery co-evolved to deploy mechanisms that both subvert and exploit these pathways to optimize efficiency and signal stress conditions. The key for future exploration will be to decipher the mechanistic bases of the workarounds that organisms were required to evolve in order to effectively utilize Sec.