Although self-rated health (SRH), a subjective measure of overall health status, associates with metabolic abnormalities, studies on the relationship between SRH and non-alcoholic fatty liver disease (NAFLD), a hepatic manifestation of metabolic syndrome, are limited. In this study, we evaluated whether or not SRH predicts the risk of incident NAFLD. This cohort study was performed in a sample of 148,313 Korean adults free of ultrasound-diagnosed NAFLD at baseline with annual or biennial follow-up for a median of 3.7 years. SRH and NAFLD were measured at baseline and follow-up visits. NAFLD was determined based on the ultrasound-diagnosed fatty liver without excessive alcohol consumption or any other cause. Hazard ratios with 95% confidence intervals were estimated via a parametric proportional hazards model. During 522,696.1 person-years of follow-up, 23,855 individuals with new-onset NAFLD were identified . After adjustments for possible confounders including total calorie intake, sleep duration, and depressive symptoms, the multivariate-adjusted hazard ratios  for incident NAFLD comparing good, fair, and poor or very poor SRH to very good SRH were 1.06 (0.97–1.14), 1.18 (1.09–1.27), and 1.24 (1.13–1.37), respectively. This association of SRH with incident NAFLD remained significant after accounting for changes in SRH and confounders during follow-up and was similar across clinically relevant subgroups. In a large-scale cohort study of apparently healthy Korean adults, poor SRH was independently and positively associated with incident NAFLD risk, indicating a predictive role of SRH as a health measure in NAFLD. In addition to liver-related adverse outcomes3, NAFLD is also associated with metabolic abnormalities, renal disorders and cerebrovascular or cardiovascular disease (CVD)6 and is considered a hepatic manifestation of metabolic syndrome (MetS) as well as a multisystem disease9. Given the increasing burden of NAFLD and its liver and extrahepatic complications, it is crucial to develop preventive measures for targeting individuals at risk for NAFLD.Nonalcoholic fatty liver disease (NAFLD) is the most common liver disease globally and encompasses the bland steatosis to nonalcoholic steatohepatitis (NASH) that has potential to develop into progressive liver disease including cirrhosis, hepatic failure, or liver cancer10. Indeed, SRH has been reported to be independently predictive of various adverse health outcomes such as CVD, cerebrovascular disease, respiratory disease, functional deterioration, depressive symptoms, and all-cause mortality11. SRH is likely to capture the net effects of various measured and unmeasured risk factors12. Previous studies demonstrated relationships of poor SRH with unhealthy lifestyle15 and metabolic derangements such as obesity, diabetes, MetS, and metabolically unhealthy status, all of which are closely related to insulin resistance and are also risk factors of NAFLD18. Additionally, poor SRH has been associated with elevated inflammatory markers and insulin resistance, important key factors for NAFLD20. Until now, there are few studies to evaluate whether or not SRH predicts NAFLD.Self-rated health (SRH) is a reliable indicator to assess personal perceptions of subjective overall health status by a single question and is widely used in various health research fieldsThus, we investigated the longitudinal relationship between SRH and new-onset NAFLD in a large cohort of apparently healthy Korean adults who underwent a health check-up examination with repeated measurements of SRH, NAFLD and other confounders including lifestyle habits, comorbidities, and metabolic parameters.21. Most participants were employees of companies and local governmental organizations and their spouses. In Korea, annual or biennial health screening exams of the employees are required by the Industrial Safety and Health Law. The rest of the examinees voluntarily underwent self-paid health checkups.The present study is part of the Kangbuk Samsung Health Study, a cohort study of Korean men and women aged 18 years or older who annually or biannually underwent comprehensive health examinations at the Kangbuk Samsung Hospital Health Screening Centers in Seoul or Suwon, Republic of Korea2; positive serologic markers for hepatitis B or C virus ; use of steatogenic medications within the past year such as valproate, amiodarone, methotrexate, tamoxifen, or corticosteroids 2; history of liver cirrhosis or ultrasound-diagnosed liver cirrhosis, or any type of liver disease . Some individuals met more than one exclusion criterion, yielding a final analytic sample of 148,313 . Participants were excluded for the following reasons at baseline: missing information on SRH or abdominal ultrasonography ; history of cancer ; type 2 diabetes ; fatty liver on ultrasound ; significant alcohol consumption  The study was approved by the Institutional Review Board of Kangbuk Samsung Hospital, which waived the requirement for informed consent as we used only de-identified data routinely collected as part of a health screening program.22. Health behaviors and socioeconomic status were categorized as follows: smoking status , alcohol consumption (≤10 g/day and >10 g/day), physical activity ), sleep duration , monthly household income (<6 million Korean won (4907 USD) or ≥6 million Korean won), job status (full-time job or part-time job) and education attainment (less than college graduate or college graduate or more). HEPA was defined when either of two criteria were satisfied: (i) vigorous-intensity activity on ≥3 days per week accumulating ≥1,500 metabolic equivalent task (MET) min/week; or (ii) seven days of any combination of walking, moderate-intensity, or vigorous-intensity activities achieving at least 3,000 MET min/week23. Usual dietary intake was assessed via a 106-item, semi-quantitative, self-administered food frequency questionnaire for Korean24. Depressive symptoms were assessed using the Korean version25 of the Center for Epidemiologic Studies Depression (CES-D) Scale26 and were categorized as CES-D score <16, 16–24, and ≥25.Abdominal ultrasonography, blood tests and physical parameters were routinely measured as part of the basic health check-up program at baseline and follow-up visits. Data on demographic factors, socioeconomic status, health behaviors, medical history and medication use were collected by standardized, structured, self-administered questionnaires28. There have been a number of studies investigating the content validity of SRH across different ethnicities and populations29.SRH was assessed using a self-administered questionnaire in Korean at baseline and follow-up visits and was defined by responses to a single question such as “In general, how would you rate your health?”, with the possible choices of “very good” (1), “good” (2), “fair” (3), “poor” (4) or “very poor” (5)2 according to criteria for obesity in Asians30. Blood pressures (BP) were measured using an automated oscillometric device  by trained nurses while examinees were sitting with the arm supported at heart level. Hypertension was defined as BP ≥140/90 mmHg or current use of BP-lowering agents. Type 2 diabetes mellitus was defined as fasting serum glucose ≥126 mg/dL, hemoglobin A1c (HbA1c) ≥6.5%, or current use of insulin or medications for diabetes.Anthropometric parameters were measured by trained nurses. Obesity was defined as body mass index (BMI) ≥25 kg/mFasting blood measurements included glucose, HbA1c, alanine aminotransferase (ALT), insulin, high sensitivity C-reactive protein (hsCRP), and lipid profiles including total cholesterol, low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), and triglycerides. The homeostatic model assessment – insulin resistance (HOMA-IR) was calculated as follows: fasting blood insulin (uU/ml) × fasting blood glucose (mg/dL)/405.31 Ultrasonographic diagnosis of fatty liver was based on standard criteria, including diffuse increase of fine echoes in the liver parenchyma compared with the kidney or spleen parenchyma, deep beam attenuation, and bright vessel walls.32 The inter- and intra-observer reliability for diagnosis of fatty liver was very high 33. Since we had excluded participants with significant alcohol consumption and other causes of hepatic steatosis at baseline (see further details in the exclusion criteria), incident cases of fatty liver were considered NAFLD.Fatty liver was assessed based on abdominal ultrasound by experienced radiologists who were not aware of the study purpose with participants in the supine position with the right arm raised above the head.stpm command in Stata)34.SRH was categorized into four groups: (1) very good, (2) good, (3) fair, or 4) poor or very poor. Only a small proportion of participants responded very poor health, which was combined into the poor category. Characteristics of the study participants were presented by SRH. The development of NAFLD was the primary outcome. The incidence rate was expressed as the number of new-onset NAFLD cases per 1000 person-years. Since we knew that NAFLD had occurred at some point between the two visits but did not know the precise timing of NAFLD development, we used a parametric proportional hazards model to account for this type of interval censoring (The adjusted hazard ratio (aHR) and 95% confidence interval (CI) for incident NAFLD were estimated using a parametric proportional hazards model. Models were initially adjusted for age and sex and then further adjusted for year of screening examination, center, education attainment, monthly household income, employment status, smoking status, alcohol consumption, physical activity, sleep duration, CES-D, total calorie intake, BMI, history of CVD, and history of hypertension (multivariable-adjusted model). To incorporate the change of SRH and confounders during follow-up, we performed time-dependent analyses while introducing SRH and other covariates as a time-varying covariate in the models. We assessed the proportional hazards assumption by examining graphs of estimated log (−log) survival; no violation of the assumption was found.2). Likelihood ratio tests comparing models with and without multiplicative interaction terms were used to evaluate interactions by subgroup characteristics. Statistical analyses were performed using STATA version 15.0 . All p-values were two-tailed, and p-values < 0.05 were considered statistically significant.In addition, we performed subgroup analyses by age (<50 vs. ≥50 years), sex (men vs. women), current smoking (yes vs. no), alcohol consumption (<10 vs. ≥10 g/day), HEPA (no vs. yes), and BMI (<25 vs. ≥25 kg/mThe average (SD) age of participants was 38.3 (7.7) years Table . Age, maP for trend <0.001). After further adjustments for possible confounders including socioeconomic status , health behaviors, sleep duration, and depressive symptoms, the multivariable-adjusted hazard ratios (95% CIs) for NAFLD in good, fair, and poor or very poor SRH categories were 1.06 (0.97–1.14), 1.18 (1.09–1.27), and 1.24 (1.13–1.37), respectively, compared with the very good SRH category (multivariable-adjusted model). Further adjustment for either HOMA-IR, hsCRP, or lipid profiles including LDL-C, HDL-C, and triglycerides slightly attenuated the association between SRH and incident NAFLD but did not eliminate it (data not shown). When updated status in SRH and confounders over follow-up were treated as time-varying covariates, SRH independently associated with incident NAFLD in time-dependent models.During a median follow-up period of 3.7 years , 23,855 new cases of NAFLD were identified, with a corresponding incidence rate of 45.6 per 1,000 person-years. Poorer SRH was associated with a higher risk of developing NAFLD (Table Association between SRH and incident NAFLD was similar across various subgroups without significant interactions by age (<50 vs. ≥50 years), sex (women vs. men), current smoking (no vs. yes), HEPA (no vs. yes), or obesity (no vs. yes) Table .Table 3HIn this cohort study of 148,313 Korean adults free of ultrasound-diagnosed NAFLD at baseline, lower SRH was associated with an increased risk of developing NAFLD in a dose-dependent manner. This association was independent of potential confounders, including employment status, education attainment, household income, depressive symptoms, comorbidities, and lifestyle factors and was consistently observed when updated status of SRH and confounders at follow-up visits were incorporated as time-varying covariates. Therefore, the findings of the present study support an independent and predictive role of SRH as a health measure in the development of NAFLD. SRH may help identify people who have a high risk of future NAFLD.35. In a cross-sectional study of a nationally representative population in the United States, assessment of SRH was included as a subscale of health-related quality of life through the question, “Rate your health status as poor, fair, good, very good or excellent”35. In that study, NAFLD was associated with poorer SRH35. However, SRH was compared among three groups  without adjusting for lifestyle factors, depression, or laboratory testing, which are considered important confounders37. Therefore, it is unclear whether or not that observed association between SRH and NAFLD is independent due to lack of adjustment of potential confounders and it is also limited by their temporal ambiguity between SRH and NAFLD. Another study examined the relationship between NAFLD and self-reported general health perception in a sub-sample (213 participants) of the Israel national health and nutrition examination survey38. That study showed an inverse association between NAFLD and the prevalence of “very good” self-reported health perception, but this association was no longer observed after adjustment for BMI. Furthermore, in that study, the presence of NAFLD compared to the normal liver at the baseline survey did not predict perceptions of health deterioration after seven years of follow-up38. However, contrary to our study, that study focused on the impact of NAFLD on health perception while treating NAFLD as a predictive variable and SRH as a primary outcome. Before the present study, no cohort studies had examined the prospective association between SRH and new-onset of NAFLD. This is the first cohort study to show a positive and independent association between poorer SRH and incident NAFLD after adjusting for possible subjective and objective confounding factors such as demographic factors, lifestyle behaviors, depressive symptoms, comorbidities, insulin resistance, and systemic inflammation among 148,313 young and middle-aged adults without ultrasound-diagnosed NAFLD at baseline using a prospective cohort study design and repeated measurements including SRH, fatty liver status, and other covariates.Some studies exploring the relationship between SRH and NAFLD have shown conflicting results41. Furthermore, SRH has been reported to be associated with behavioral factors such as physical activity, smoking, dietary habit, alcohol drinking, and excessive adiposity, all of which are closely correlated with NAFLD15. SRH may comprehensively reflect lifestyle-related health conditions42. However, the relationship of poorer SRH with NAFLD risk, in our study, was observed even after adjustments for various lifestyle factors as well as depressive symptoms. Poor SRH has been reported to be associated with elevated inflammatory markers, another important key factor for NAFLD20. Thus, poor SRH might reflect the inflammatory milieu in favor of development of NAFLD and be related to symptoms related to systemic inflammation such as fatigue, malaise, sleepiness and reduced energy availability, which might prevent individuals from engaging in physical activity or adopting healthy lifestyles and affect perceived physical functioning, one of the subscales of SRH43. However, in our study, further adjustment for lifestyle factors, hsCRP, and HOMA-IR did not fully attenuate the association between SRH and incident NAFLD. Even though SRH is easily assessed through a single-item question about “general health,” this question can be used to assess different dimensions of individual health status according to subjects’ own definition of health, which may include overall subjective and objective health at that point, possibly capturing an overall picture of individual’s health beyond the conventionally measured health conditions. Indeed, the pathogenesis of NAFLD is not clear and seems to be multifactorial with multiple proposed mechanisms44. Our study findings support SRH as a predictor of incident NAFLD independent of known NAFLD risk factors to help identify individuals at high risk for NAFLD, with the goal of implementing strategies to prevent incident NAFLD. However, further studies are required to determine what aspects of health experience can affect self-rated health, to determine whether sub-scales of SRH provide a sensitive marker of NAFLD risk, and to clarify whether the use of SRH as a part of actual consultation in clinical settings can improve patient health outcomes including NAFLD.The mechanisms underlying the association between SRH and the development of NAFLD are not fully elucidated yet. NAFLD is attributed to unhealthy health behaviors that are closely associated with excessive adiposity, unfavorable lipid profiles and insulin resistance45, it cannot reliably detect fatty infiltration of the liver below the threshold of 20 to 33%47. Second, health behaviors were assessed based on self-administered questionnaires; thus, some degree of residual confounding might have been introduced by social desirability bias or measurement errors. Third, anxiety has been linked to poorer SRH as well as subjective well-being in other population-based studies50. However, information on anxiety symptoms, in our study, was not available until 2017, limiting our ability to consider it in the analysis. Thus, we cannot exclude the possibility of residual confounding related to measurement errors and unmeasured confounders that might explain the observed association between SRH and incident NAFLD. Finally, our study population is much younger than those in previous studies on SRH: only 6.3% of the study population was 50 years old or older. Even though the interaction by age on the association between SRH and incident NAFLD was not statistically significant in our study, the association between SRH and incident NAFLD tended to be stronger in those 50 years or older (vs. younger group). Previous studies have shown that the role or meaning of SRH in predicting health outcomes might differ by age group52. Therefore, study findings derived from young and middle-aged Koreans who attended health-screening examinations might not be generalizable to other populations with different characteristics such as age, race/ethnicity, or socioeconomic status. On the other hand, the relatively young age of our cohort participants may be a strength because our results have implications in young adults with poor SRH for future risk of NAFLD in relatively short-term follow-up and long-standing NAFLD since young adulthood, which might adversely health outcomes such as liver- and non-liver complications.We note several limitations of the present study that are important to consider during the interpretation of the findings. First, we used ultrasonography to diagnose NAFLD without confirmation by liver biopsy, a gold standard for diagnosis of NAFLD. However, liver biopsy was neither feasible nor ethical in our large, low-risk individuals due to its invasive nature. Although abdominal ultrasound is widely used in population-based epidemiological studies with an acceptable diagnostic accuracy for diagnosing hepatic steatosisIn conclusion, individuals with poorer SRH had a higher risk of new-onset NAFLD, indicating that SRH is useful as a predictive health measure for various adverse health outcomes including NAFLD. Health care providers should consider the predictive role of SRH in incident NAFLD among preventive strategies for NAFLD. Further prospective studies are warranted to better understand the mechanism by how SRH predicts incident NAFLD.Supplementary Table 1.


ATP‐binding cassette (ABC) transporters are fascinating molecular machines that are capable of transporting a large variety of chemically diverse compounds. The energy required for translocation is derived from binding and hydrolysis of ATP. All ABC transporters share a basic architecture and are composed of two transmembrane domains and two nucleotide binding domains (NBDs). The latter harbor all conserved sequence motifs that hallmark the ABC transporter superfamily. The NBDs form the nucleotide binding sites (NBSs) in their interface. Transporters with two active NBSs are called canonical transporters, while ABC exporters from eukaryotic organisms, including humans, frequently have a degenerate NBS1 containing noncanonical residues that strongly impair ATP hydrolysis. Here, we summarize current knowledge on degenerate ABC transporters. By integrating structural information with biophysical and biochemical evidence of asymmetric function, we develop a model for the transport cycle of degenerate ABC transporters. We will elaborate on the unclear functional advantages of a degenerate NBS.  NBD, nucleotide binding domainNBS, nucleotide binding siteTMD, transmembrane domainResearch into ATP‐binding cassette (ABC) transporters has started with the initial discovery of cancer cells showing resistance to colchicine treatment, and it was early on discovered that the protein responsible for the resistance must be an ATP‐driven transporter termed permeation glycoprotein (P‐glycoprotein), later systematically named ABCB1 . SequencATP hydrolysis is carried out by composite ATP hydrolysis sites or nucleotide binding sites (NBSs) that are symmetrically located in the interface between both NBDs and formed by residues from both NBDs. This interface harbors the conserved sequence motifs of this superfamily, which are all involved in binding and/or hydrolysis of the bound nucleotide triphosphate. On a molecular level, one NBS is formed by the Walker A and B motif, the A‐, Q‐ and H‐loop of one NBD and the signature sequence, and the X‐loop of the second NBD. ATP acts as a molecular glue that brings both NBDs together and holds them in close association, while the D‐loops of both NBDs stabilize the dimer by multiple interactions in the center of the NBD dimer interface. The glutamate residue in the Walker B motif was shown to be essential for ATP hydrolysis, and consequently, its mutation to glutamine leads to functionally impaired transporter variants . WithoutThe NBDs of ABC transporters consist of two subdomains: a larger Rec‐like domain and a smaller helical domain Fig. . InitialThe human genome encodes 48 ABC genes: Four of them are proteins involved in ribosome function and regulation (ABCE1‐3 and ABCF1), while 44 encode transmembrane proteins. These assemble into 42 membrane proteins, because four of these genes encode half‐size transporters that associate to form heterodimeric transporters (ABCB2/B3 and ABCG5/G8). Thirty‐four of the membrane proteins are transporters, while one is an ATP‐gated chloride ion channel (ABCC7) and two (ABCC8 and ABCC9) are sulphonylurea receptors that regulate the potassium ion channels Kir6.1 and Kir6.2.Sequence comparisons of ABC transporters show that the TMDs have heavily diverged during evolution, which is most likely associated with the broad spectrum of transported substrates that ranges in size from a single ion to an astonishing 1.5 MDa . In contMutations of the signature sequence in NBD2 are present in all degenerate NBSs of the human genome. The second most frequently observed change, which results in a degenerate NBS, is the exchange of the C‐terminal glutamate residue of the Walker B motif to an aspartate. This is a very conservative mutation, because it replaces the negatively charged glutamate with the negatively charged aspartate, which only differs by reduction of the length of the amino acid side chain by one methylene moiety. The NBSs carrying such a mutation seem still able to hydrolyze ATP , 42, butATP hydrolysis is asymmetric in ABC transporters with a degenerate site, leading to a high energy state , 45 in tThe currently available structures of ABC exporters Table  with a tIn contrast to type I exporters, the heterodimeric type II exporter ABCG5/G8 shows symmetric NBD separation similar to ABCG2, despite the degeneration of NBS1. Structures of transporter from the ABCA (ABCA1) and ABCG (ABCG2 and ABCG5/G8) subfamilies show additional extensive interaction surfaces of their NBDs at the C‐terminal ends, which clamp the NBDs together, similar to ABC importers. A mechanical consequence is a relocation of the main axis of NBD rotation that runs parallel to the membrane in ABCA and ABCG transporters, but perpendicular in ABCB and ABCC and most likely ABCD transporters Fig. . Additio2+. In contrast, the consensus NBS2 requires the presence of MgATP to reach the closed state. Interestingly, an inward‐facing structure showed only one molecule of AMP‐PNP bound to the degenerate NBS1, while being very similar to the apo conformation. An exception is thereby the D‐loop of TM288, which reaches over and interacts with AMP‐PNP bound to NBS1, markedly deviating from the typical D‐loop conformation. Typically, the D‐loop shows no direct interaction with the nucleotide in the occluded state. It will remain to be seen if this feature of the D‐loop is TM287/288 specific or generally relevant for sensing the presence of ATP in the inward‐facing transporter and promoting NBD closure. This interpretation is supported by alanine mutations of the interacting aspartate of the D‐loop of TM288, which showed reduced ATP affinity and lower ATP hydrolysis activity , va, va3) mo in NBS2 , and ADP in NBS2 . ADP is  in NBS2 , which i in NBS2 . Importa in NBS2 . NBS1 anKM and Vmax, suggesting that the role of the Q‐loop in ABCC1 is not directly linked to ATP hydrolysis or transporter asymmetry [Introduction of the canonical glutamate in place of the aspartate of the degenerate Walker B motif in NBS1 enhanced its hydrolytic capacity and the affinity for ADP, but markedly decreases transport activity, while a leucine, glutamine or asparagine mutant had little effect on function , 104. Musymmetry . Mutatiosymmetry .These biochemical data clearly show that under physiological conditions, ATP remains bound to NBS1 and maintains association of the NBDs, while the allosterically coupled NBS2 is hydrolyzing ATP in consecutive transport cycles. The affinity of most nucleotides is higher for NBS1. While very speculative and awaiting verification by independent experimental approaches, it is tempting to deduce from the vanadate data a backward reaction in which ABCC1 can be trapped by MgADP and vanadate without ATP hydrolysis. This would imply that NBS2 should not separate much during the transport cycle, but contemporaneously it does not mean that ABCC1 could function as an ATP synthetase.While activation by phosphorylation is a prerequisite for channel function, the focus of this review is on the role of the NBDs on ATP binding‐dependent channel opening. It has been shown that NBS2 hydrolyzes ATP, which leads to channel closure. Similar to ABCB2, isolated NBD1 forms homodimers in an ATP‐dependent manner . ABC proThermotoga maritimus is unknown. Under conditions of transporter overexpression, a resistance toward daunomycin was detected as well as transport of 2,7‐bis(carboxyethyl)‐5(6)‐carboxyfluorescein, while classic multidrug resistance transporter substrates such as Hoechst 33342 stimulated the ATPase activity [The transported substrate of TM287/288 from the thermophilic bacterium activity . Chemicaactivity , suggestactivity .Thermus thermophilus has one degenerate NBS with TmrA carrying the canonical Walker B. Mutation of the canonical Walker B glutamate of TmrA to glutamine inactivates the transporter, but the transporter retains residual ATPase and transport activity [TmrAB of the thermophilic Gram‐negative eubacterium activity , with a activity .S. cerevisiae is the prototype of the pleiotropic drug resistance (PDR) subfamily of transporters [in vitro system and structural information is required. A step toward establishing an in vitro system for Pdr5 has only recently been accomplished [Pdr5 from sporters  that aresporters . The samsporters . When alsporters , ATPase sporters , a resulsporters . Howevermplished , but we These biochemical data show clear differences in the function of NBS1 and NBS2, while the affinity for MgATP to the degenerate NBS1 is comparable to the canonical NBS2. The data for ABCC1 and ABCC7 suggest that MgATP remains bound to NBS1 over many ATP hydrolysis cycles in NBS2. Also, homodimerisation of isolated NBSs of ABCB2 in the presence of MgATP highlights the role of the degenerate NBS1 for MgATP‐dependent dimerization. Interestingly, insertion of canonical residues in the degenerate motifs of NBS1 leads to a loss of function, which contrasts with the assumption that NBS1 and NBS2 share their function. The degenerate NBS1 seems to have developed a new function that is associated with stable binding of ATP, whereby MgATP serves as a stationary hub connecting NBD1 with NBD2. It remains to be seen if NBS1 acquired the function of an ATP sensor, which regulates ATP hydrolysis in NBS2 according to cytosolic ATP levels.−1), which served as interaction hub for the closely engaged NBD dimer by interacting with both NBDs. Also, MgADP stabilized the closely engaged NBD dimer, though to a lesser extent than MgATP. In contrast, the ATP hydrolysis products MgADP and Pi represented a high energy state, which primed the NBD dimer for separating NBD1 and NBD2. Simulations of the NBDs of MJ0796 showed stable NBD dimers in the presence of two ATPs, but when one of the ATP was replaced by ADP, a rotation of the helical domain relative to the core domain led to the opening of the respective NBS [Simulations using isolated NBDs investigated the stabilization by nucleotides of the NBD dimer of ABCB1 , 45 and tive NBS , 121.Simulations of full‐length ABC transporters from the ABCB subfamily reported that the closed NBD dimer is only stable if ATP is present in NBS1 and NBS2 . It was observed for ABCB1 that the NBD dimer became asymmetric in the presence of MgATP . A similExtensive simulations of ATP‐bound TM287/288 observed a spontaneous conversion from the inward‐facing state to the outward‐facing state in a subset of simulations. Starting from the asymmetric inward‐facing state, he NBDs reached a configuration with symmetrically bound ATPs to the NBD dimer interface . A similSimulations of inward‐facing ABCC7 showed that the structures determined by cryo–EM were not stable when inserted in a membrane environment , 133 leaSequence analysis, structure determination, and simulation data have shown that the TMDs of ABCC7 are asymmetric with respect to the exposed charged residues proposed to be involved in chloride conductance. A chloride conductance path was mapped from the intracellular side of the membrane to the extracellular side , 135. WhThe similarity of NBD sequences across all ABC proteins and the appearance of a large number of NBDs which were all very similar to each other stimulated the suggestion of a generalized transport cycle mechanism applicable to all ABC transporters , 139, whIn contrast to the above models that propose a full separation of the NBDs during the transport cycle, the sequential model  requiresMost of these models were proposed before full transporter structures became available in multiple conformations and were largely derived from ABC transporters with two canonical NBSs. These models are generally not fully compatible with all the data derived from transporters harboring a degenerate NBS1, suggesting a variation of the transport cycle with a noncanonical NBS. Some transport models were also specifically proposed for transporters harboring a degenerate site. A simplistic transport cycle model was proposed for ABCC1 using information from several cryo‐EM structures  and predIn the transport model proposed for heterodimeric TM287/288 , ATP remi is released, which triggers asymmetric opening of the NBDs to conclude the transport cycle. The rate limiting step is the exchange of the nucleotide triphosphate in the inward‐facing conformation [The transport model proposed for heterodimeric TmrAB deviates from the constant contact model as proposed for TM287/288. To derive the model, eight structures of TmrAB reconstituted into nanodiscs were used to trace a continuous path through the transport cycle . The modormation  that leaThe alternating access model  has beenWe can infer from the available structures of ABC transporters that not all ABC exporters use the same mechanics for cargo translocation, which is linked to differences in transporter folds. The available structures suggest that in the ABCB and ABCC subfamilies, the main axis of rotation runs approximately parallel to the main axis of the transporter and therefore normal to the membrane, while the conformations observed for ABCG2  and ABCGATP‐binding cassette transporters are characterized by a twofold symmetry axis which implies that every structural element is present twice. NBS1 and NBS2 are identical in homodimeric transporters and remain very similar in heterodimeric or full transporters. Of note, 21 of the 29 human nonhomodimeric ABC transporters have a degenerate NBS. It seems therefore likely that the degenerate NBS has an evolutionary advantage.We can infer from sequence alignments that the degenerate NBS has acquired a new function, because it remained under evolutionary pressure, and its sequence has not further diverged as would be expected for a site that has lost any function. The consistent motions and concurrent direction of rotation of the NBDs relative to each other Fig.  suggest Motions in the NBDs are tightly coupled to motions in the TMDs to allow for substrate transport. In transporters with a degenerate NBS, the asymmetry of the NBDs most likely translates into asymmetric motions of the TMDs Fig. . If TMDsOne of the key differences between primary active and secondary active transporters is the directionality of transport. In the latter, directionality is controlled by the concentration gradient of the cotransported ion and an inverted transport direction can be physiological relevant. In contrast, ATP‐driven, primary active transporters show unidirectional transport. Two types of mutations were reported, which converted unidirectional ABC transporters to a bidirectional ATP‐gated facilitator: (a) mutations in the putative substrate‐binding/translocation path of the TMD of ABCB1 resulted in a transporter variant that shows concentration gradient dependent substrate translocation , and (b)While the results for the different ABCB1 variants could potentially be explained by increased access from the extracellular site or a lack of gate closure, thereby compromising directionality by enhanced accessibility to the substrate‐binding cavity from the extracellular site, the effect of the D‐loop mutants is more likely linked to ATP‐dependent NBD dimerization. This interpretation is consistent with the notion that nucleotides control transporter conformation such as the exporter is inward‐facing in the apo state, while outward‐facing, when two ATP are bound. Sequence comparison Fig.  shows anIt remains debated if all ABC exporters follow the same transport cycle. As outlined above, it is unlikely that type I ABC exporters (ABCB/C/D subfamilies) share the mechanism of transport with type II ABC exporters (ABCA/G subfamilies). When focusing on type I ABC exporters and specifically on the ABCB and ABCC subfamilies, these transporters share a number of features: The difference in sequence between the ABCB and ABCC subfamilies is small and, accordingly, structure determinations have shown that they share the same fold. While all transporters from the ABCC subfamily have a degenerate NBS1, also some belonging to the ABCB subfamily harbor a degenerate NBS1. The conformations of the closed NBD dimer are basically the same, if a nonhydrolyzable ATP analog is bound or if the glutamate of the Walker B motif is mutated. In contrast to the structural similarity, biochemical data have clearly shown that wild‐type transporters harboring a degenerate NBS (such as ABCC1 ) bind twWith respect to the human transporters, these data could be interpreted in the following way: The motions of the NBDs of transporters with a degenerate NBS operate similar to a clamshell, while transporters with two canonical NBSs follow a seesaw motion of NBD rotation, but without a clear sequential order Fig. . A transATP in NBS1 is most likely serving as a hinge point for the transport cycle in transporters with a degenerate site, where it remains stably bound for 20–30 cycles. Comparing transporters with one or two canonical ATPases, a single loop through the transport cycle could be very similar, except that hydrolysis can occur stochastically in either of the two NBSs, if NBS1 is also ATP hydrolysis competent. The nonhydrolyzed ATP in the second NBS could serve as a hinge to guide the rotational motions. The fact that two nucleotides could not simultaneously be trapped in both NBSs of ABC transporters with two canonical NBSs suggests that ATP in the second NBS can probably not or less efficiently serve as a motion‐stabilizing hinge. Instead, both NBSs primarily serve the role of providing energy for ATP binding and hydrolysis, thus suggesting that full NBD separation might occur more frequently, if ADP/ATP exchange is not fast enough. It seems plausible that the D‐loop in the center of the NBD‐NBD interface could contribute to the hinge and/or serve as spacer, thereby shifting the hinge point from ATP in NBS1 of degenerate NBSs toward the center of the NBD dimer in transporters with two canonical NBSs.In conclusion, the available structural and dynamic data suggest a common conformational path through the transport cycle, though ABC transporters with a degenerate NBS have a stronger tendency for rotation relative to each other. Biochemical data are not clear enough to affirm or reject the hypothesis of a partially shared mechanism. From the currently available data, we can infer that ATP in the degenerate NBS1 has acquired the role of stabilizing the closed NBD dimer and to serve as a hinge for conformational changes, although it remains unclear if ATP plays the same role in transporters with two canonical NBSs.TS, RG, and LS drafted the manuscript. RG generated the figures. TS and LS edited the manuscript. The final version was approved by all authors.


Apis mellifera). To test the potential of DWV to be utilized as a vector, the 717 nt sequence coding for the enhanced green fluorescent protein (eGFP), flanked by the peptides targeted by viral protease, was inserted into an infectious cDNA clone of DWV in-frame between the leader protein and the virus structural protein VP2 genes. The in vitro RNA transcripts from egfp-tagged DWV cDNA clones were infectious when injected into honey bee pupae. Stable DWV particles containing genomic RNA of the recovered DWV with egfp inserts were produced, as evidenced by cesium chloride density gradient centrifugation. These particles were infectious to honey bee pupae when injected intra-abdominally. Fluorescent microscopy showed GFP expression in the infected cells and Western blot analysis demonstrated accumulation of free eGFP rather than its fusions with DWV leader protein (LP) and/or viral protein (VP) 2. Analysis of the progeny egfp-tagged DWV showed gradual accumulation of genome deletions for egfp, providing estimates for the rate of loss of a non-essential gene an insect RNA virus genome during natural infection.We developed a honey bee RNA-virus vector based on the genome of a picorna-like Deformed wing virus (DWV), the main viral pathogen of the honey bee ( Vectors based on the genomes of RNA viruses of animals ,2 or plaInsect-specific RNA virus vectors have been designed using genomes of the arboviruses, members of Flavivirus, Alphavirus and Nodavirus groups ,7,8. Curegfp gene insertion and showed the expression of the egfp reporter in the cells of honey bee pupae infected with the egfp-tagged DWV RNA vectors by using fluorescent microscopy and Western blot. Analysis of the genetic stability of the DWV vector provided novel insights into the ability of RNA viruses to retain non-essential sequences, contributing to a better understanding of their evolution.In this study, we develop a virus expression system for the widespread virus DWV type A  using a egfp insertions at the leader protein-viral protein 2 (LP-VP2) border by overlap extension polymerase chain reaction (PCR) and subcloning, using pDWV-304 [egfp amplification, respectively; the primers are listed in The full-length infectious cDNA clone of DWV type A, pDWV-304 (GenBank accession number MG831200)  was usedpDWV-304  and pAcPpDWV-304  as the tegfp-tagged DWV cDNA plasmid constructs were linearized using the PmeI restriction site located at the 3′ end of DWV cDNA, downstream of the poly A sequence, to produce the templates for a full-length 10.9-kb transcript. The in vitro RNA transcripts from pDWV-L-GFP, pDWV-S-GFP and the parental pDWV-304 were produced using the HiScribe T7 High Yield RNA Synthesis Kit (New England Biolabs) according to the manufacturer’s instructions. After 3 h of incubation at +37 °C, the DNA templates were digested using TURBO DNase (Life Technologies). The in vitro RNA transcripts were purified using the RNeasy mini kit (Qiagen), 13.2 × 1012 copies in 10 µL of phosphate buffered saline (PBS), or 10 µL of PBS were injected intra-abdominally into the hemolymph of honey bee pupae at the pink eye stage that were not exposed to Varroa mite feeding, using syringes with a 0.3-mm needle G31 (BD Micro-Fine) as described previously [g for 5 min and filtration through a 0.22 µm nylon filter (Millipore). The DWV concentration in the extracts was quantified by RT-qPCR, and the extracts were stored at −80 °C before they were used.The eviously . The extVarroa mite infestation rate from the apiaries located in the USDA Bee Research Laboratory (BRL) in Beltsville, Maryland (sourced in September 2019), and in the USDA Honey Bee Breeding, Genetics and Physiology Research Laboratory in Baton Rouge, Louisiana USA (sourced in November 2019). Pupae at the pink-eyed stage were pulled out of cells using soft tweezers 3 h before their use in the injection experiments, which were carried out as described previously [Honey bee pupae were obtained from strong colonies with low (less than 1%) eviously . Newly eVarroa mites were quantified by reverse transcription-quantitative PCR (RT-qPCR), which included cDNA synthesis using random hexanucleotides. RT-qPCR and cloning of the RT-PCR fragments were carried out essentially as previously described [Total RNA extracted from individual honey bee pupae using Trizol reagent (Ambion) or CsCl density gradient fractions were further purified using an RNeasy kit (QIAGEN) according to the manufacturer’s instructions. Quantification of the DWV and GFP RNA and actin mRNA loads in individual honey bee pupae and associated escribed , using pNicotiana benthamiana, agroinfiltrated with the construct expressing 27 kDa monomeric GFP [Western blot analysis was carried out using a WesternBreeze kit (Invitrogen), following the manufacturer’s instructions. The aliquots of homogenized honey bee pupae protein (50 μg) or 20 µL aliquots of CsCl gradient were heated at 95 °C for 10 min in a sodium dodecyl sulfate (SDS) sample buffer  and separated on SDS 4%–12% Bis-Tri -polyacrylamide gel electrophoresis gels (Invitrogen). Polyclonal rabbit anti-GFP antibodies  or rabbit polyclonal antisera raised to DWV virus particles purified using CsCl gradient centrifugation  were useeric GFP , was useeric GFP  overnighegfp-tagged DWV constructs, proteolytic processing of the viral polypeptide between eGFP and VP2 was expected to take place, ensuring the generation of wild-type structural viral proteins required for the encapsidation of DWV RNA.A full-length infectious cDNA clone of a US strain of wild-type DWV type A, pDWV-304 , was useegfp target by RT-qPCR  per pupa, and DWV-S-GFP, ranging from 10.33 to 10.66 log10 GE per pupa  and the DWV target  were not significantly different in the pupae injected with the recombinant egfp-tagged DWV transcripts  between the genes for LP and VP2 did not prevent replication of the modified viruses, i.e., by disrupting potential RNA elements essential for virus RNA replication [Cell cultures are commonly used for studying the clone-derived RNA viruses of vertebrates and invertebrates, but this option is not readily applied to cloned DWV. Honey bee cell cultures suitable for work with DWV are not readily available. A recently reported honeybee cell line is infected already with DWV  and no o RT-qPCR  showed tper pupa . Levels nscripts  and contlication .10 GE/pupa; 11.14 ± 0.338 log10 GE/pupa, mean ± SD, by 72 hpi; P = 0.0398, df = 7, ANOVA; P = 0.0505, df = 8, ANOVA; 10 GE/pupa; 5.79 ± 0.801 log10 GE/pupa; mean ± SD) were significantly lower (tens of thousands of times) compared to those in any of the transcript-injected groups . As expected, the egfp RNA was not detected in the buffer control (PBS)- and the DWV-304-injected bees  tissues expressing monomeric GFP of 27 kDa , potentially increasing the length of the modified genomic RNA beyond the RNA size limit for encapsidation. In addition, the insertion of a foreign sequence might potentially disrupt yet unknown RNA signals required for the formation of stable virus particles [egfp-tagged DWV and to compare them with wild-type DWV particles. To do this, we carried out cesium chloride (CsCl) gradient centrifugation of the filtered extracts from honey bee pupae 72 hpi with the transcripts from the cDNA clones DWV-S-GFP and DWV-304 (unmodified DWV). Following ultracentrifugation, the fractions with densities ranging from 1.22 g/cm3 to 1.58 g/cm3 were collected and the copy numbers of DWV and GFP RNA targets were quantified for each fraction by RT-qPCR  [egfp gene and the sequences coding for duplicated flanking peptides) was encapsidated into particles with a stability and buoyant density similar to those of unmodified DWV.We found that in the case of DWV-S-GFP, the 1.34 g/cm targets A. In thefraction B, which tion (3) . In addiegfp inserts, both DWV-L-GFP and DWV-S-GFP, was further confirmed by infectivity of the filtered tissue extracts prepared from the transcript-infected honeybee pupae sourced at 72 hpi. Extracts selected for injection had nearly equal copy numbers of DWV and GFP RNA by RT-qPCR , and the levels of DWV-S-GFP ranged from 9.42 to 10.12 log10 GE/pupa , P = 0.275, dF = 12; Encapsidation of DWV genomes with analysis A. Honey egfp inserts in the pupae injected with DWV-L-GFP and DWV-S-GFP-derived extracts by Western blot analysis with the anti-GFP antibodies. Specifically, at 72 hpi monomeric eGFP bands of the same size were observed in both the extract-injected pupae and in the pupae injected with corresponding transcripts  than those of GFP indicating accumulation of DWV variants without egfp insert. Both egfp-tagged DWV variants showed similar levels of GFP and DWV at 72 hpi , and similar difference between DWV and GFP copy numbers in individual pupae . In the case of DWV-S-GFP injection, pupae were also sampled at 6, 24 and 48 hpi, showing non-significant differences between DWV and GFP copy numbers within each time group at 6 and 24 hpi, while at 48 dpi the copy number of DWV RNA significantly exceeded that of the GFP RNA , it became obvious that the proportion of the mutants with deletions of egfp was increasing with time. The DWV/GFP copy number ratio per injected honey bee was increasing with time after infection  when the protease . The expprotease , and by protease A. The rey 72 hpi , which iegfp insertion could affect the encapsidation of the viral RNA. It was previously demonstrated that an RNA size limit for encapsidation exists for single stranded RNA viruses with icosahedral symmetry of virus particles structurally resembling those of Iflaviruses, such as Turnip crinkle virus [egfp inserts took place in in the pupae injected with the DWV-S-GFP and DWV-L-GFP in vitro RNA transcripts. We found that DWV genome with egfp insertion was packaged into stable virus particles and had the same physical properties as those containing unmodified DWV RNA  was designed in this study, including other cloned DWV-like viruses [Varroa mites [Varroa mite vector [egfp during their replication would allow to determine if replication of any variants of DWV-like viruses occur in their Varroa mite vector and pinpoint the Varroa mite cells where replication could take place.One of the aims of this study was to develop a tagged DWV vector, which could be used for tracking virus replication in the cells and tissues of the DWV host(s). Indeed, we observed bright GFP fluorescence in the cells of the pupae infected with the extracts containing recovered DWV-L-GFP or DWV-S-GFP. Different cell-types showed GFP fluorescence, which was consistent with the previously reported presence of DWV throughout the honey bee body in various cell types . Importa viruses ,30,31, woa mites  and the e vector ,34,35,36egfp tagged DWV constructs. While deletion of foreign genes from RNA virus vector genomes was reported previously [egfp deletion mutant genomes generated at 72 hpi in the course of DWV-S-GFP infection in the extract-injected bees still had 200–300 nt-long parts of the eGFP-coding sequence (We also analyzed the genetic stability of our eviously ,38, thiseviously . Interessequence A. It is egfp took place, this process was gradual and did not happen immediately. We showed that the egfp-tagged viruses replicated in the injected pupae, increased their loads thousands of times (It should be emphasized that although the accumulation of the progeny with the deletion of non-essential of times C, suggesof times C. It is egfp reporter gene using a novel arrangement, which has not been applied to constructing picorna-like vectors previously [In this study, the DWV vectors were constructed using a full-length infectious cDNA clone of DWV  by insereviously , and inceviously , and it


Self-rated health (SRH), a subjective perception of an individual’s overall health, is widely used in public health assessment. The transition from adolescence to young adulthood is a critical period involving substantial changes in lifestyle and dietary habits. Therefore, it is important to understand SRH among young Japanese females. The present study aimed to investigate the relationships between SRH among female healthcare students and their lifestyle factors, such as living status , smoking habit, alcohol consumption, frequency of breakfast consumption (FBC), physical activity, insomnia, and physique recognition.A cross-sectional survey was conducted on 1101 female healthcare students in Japan. The body mass index was calculated from the body height and weight using data from periodic health examinations. Self-reported sociodemographic, lifestyle or dietary habits, physical activity, and SRH were used through a self-administered questionnaire. Participants were classified as having either good SRH  or impaired SRH (fair or poor). Multivariate logistic regression analysis was performed to investigate the independent relationships between SRH and lifestyle factors.A total of 11.4% participants demonstrated impaired SRH. Multivariate logistic regression analysis showed that the University of California Los Angeles activity score, Athens Insomnia Scale, and physique recognition were associated with SRH.It was suggested that public health activities that consider physical activity, sleep, and physique recognition may help maintain and improve SRH in female university students in Japan. Self-rated health (SRH), a subjective perception of an individual’s overall health, is a widely used measure in public health assessment. Typically, SRH is examined using a single question, e.g., asking respondents to rate their overall health on a scale from excellent to poor . AccordiSRH of the general population in Japan is officially assessed every 3 years via the Comprehensive Survey of Living Conditions (CSLC), a nationwide cross-sectional survey of a representative sample of the country’s general population. The CSLC findings from 2016 found that 86.7% of males and 84.4% of females reported excellent, very good, or good SRH , and mean BMI was 20.9 .Table The present study examined the relationships between SRH and lifestyle habits of Japanese female healthcare students. The results indicated that the UCLA activity score, AIS score, and physique recognition were significantly associated with impaired SRH. Overall, this cross-sectional observational study of lifestyle habits among Japanese female healthcare students showed that lower physical activity and insomnia were significantly positively associated with impaired SRH. Further, the desire to be underweight showed a significant negative relationship with impaired SRH.Insomnia has been identified as the most common sleep disorder . VariousThe UCLA activity score is used to evaluate the physical activity of patients with joint replacement . SRH wasMost study participants were either normal weight or underweight, with very few being overweight. However, majority of them wished to have a thinner physique. It is noteworthy that the group who desired to be underweight still considered itself to be healthy. Baba et al. reported that the desire to be thinner was positively correlated with a sense of gain or loss about one’s body, being praised, and sex role acceptance; however, it was negatively correlated with self-esteem and stress . These pThis study had some limitations. First, it was conducted in a medical university and was thus not population based. These participants may have followed healthier lifestyles and been more health-conscious than the general population of the same age. Therefore, caution should be exercised when generalizing these results to the general population, even among those in the same age group. Although the study’s convenience sampling may have contributed to selection bias, the students showed high levels of incorrect physique recognition, suggesting that the results are likely to be valid. Second, in the present study, we asked participants if they would like to gain weight from the BMI actually obtained by physical examination and further classified each student’s ideal body physique by BMI and their self-report. However, this survey method is a new approach that incorporates the survey methods of Noh et al.  with refThird, this study was conducted using a self-administered questionnaire; therefore, there may be a recall bias, and our perception of subjective health may be different. It was unclear whether subjects had low SRH because they had symptoms and illness despite being satisfied with their body shape or had higher SRH because they had no symptoms or illness despite being dissatisfied with their body shape in this study. In the future, it is a more detailed examination based on the concept of health is warranted. Fourth, in the present survey, participants were asked to complete a questionnaire about their desired body physique; this might lead to a social desirability bias, which projects a favorable image of themselves to avoid receiving a negative evaluation. Finally, because this was a cross-sectional study, causal relationships could not be determined. In the future, investigation is warranted to ascertain whether physical activity, sleeping, and physique recognition are the results or causes of SRH.We conducted a cross-sectional study to investigate the relationships between SRH and lifestyle among female healthcare students at a Japanese medical university. The UCLA activity score, AIS score, and physique recognition were all significantly associated with impaired SRH. Therefore, it was suggested that public health activities that consider physical activity, sleep, and physique recognition may help maintain and improve SRH of female university students in Japan.


RNA silencing is a posttranscriptional gene silencing mechanism directed by endogenous small non-coding RNAs called microRNAs (miRNAs). By contrast, the type-I interferon (IFN) response is an innate immune response induced by exogenous RNAs, such as viral RNAs. Endogenous and exogenous RNAs have typical structural features and are recognized accurately by specific RNA-binding proteins in each pathway. In mammalian cells, both RNA silencing and the IFN response are induced by double-stranded RNAs (dsRNAs) in the cytoplasm, but have long been considered two independent pathways. However, recent reports have shed light on crosstalk between the two pathways, which are mutually regulated by protein–protein interactions triggered by viral infection. This review provides brief overviews of RNA silencing and the IFN response and an outline of the molecular mechanism of their crosstalk and its biological implications. Crosstalk between RNA silencing and the IFN response may reveal a novel antiviral defense system that is regulated by miRNAs in mammalian cells.  RNA silencing is a posttranscriptional gene silencing mechanism that is conserved among diverse organisms and is induced by microRNA (miRNA), an approximately 22-nucleotide (nt) long endogenous non-coding double-stranded RNA (dsRNA) . The humIn mammalian cells, endogenous or exogenous RNAs with typical structural characteristics are accurately recognized by specific types of RNA-binding proteins acting in one of the two pathways ,23,24,25The human genome encodes 1917 miRNA precursors (pre-miRNAs), from which 2656 mature miRNAs are generated according to miRBase, a searchable database of published miRNA sequences and annotations, release 22 . miRNAs In the RNA silencing pathway in the cytoplasm, multiple essential or important modulator proteins are involved. Trans-activation response (TAR) RNA-binding protein (TRBP) is a dsRNA-binding protein, which interacts with Dicer to enhance processing of pre-miRNA into miRNA duplexes ,58. TRBPExogenous dsRNAs, such as viral RNAs, are sensed by host pattern recognition receptors (PRRs), inducing IFN production in mammalian cells. Toll-like receptor 3 (TLR3)  and retiAfter production, IFN is secreted from the cell and recognized by IFN-α/β receptor (IFNAR) 1 or IFNAR2 on the cell surface in a paracrine or autocrine manner . Then, IE)-value, which is a parameter describing the number of hits that can be expected by chance in database searches used as a benchmark of sequence similarity, revealed that the amino acid sequences of the ATPase/helicase domains of Dicer and RLRs are similar. In particular, those of Dicer and LGP2 showed strong similarity , Piwi/Argonaute/Zwille (PAZ), RNase IIIa/b, and an RNA-binding domain (RBD). Both RIG-I and MDA5 consist of two N-terminal CARDs, an ATPase/helicase domain, and a C-terminal domain (CTD), while LGP2 lacks CARDs A. The Prmilarity A. FurtheBoth RNA silencing and the IFN response are induced by dsRNA, although the molecules involved can accurately distinguish between endogenous and exogenous dsRNA. Pre-miRNA is RNA approximately 70-nt in length and typically forms a stem-loop secondary structure with 3′ overhang and 5′-monophosphate, with a stem region that usually includes mismatch(es) and/or bulge(s). Dicer, a pre-miRNA processing enzyme, recognizes the 5′-monophosphate and 3′ overhang of pre-miRNA as a substrate of its PAZ domain ,7,8. AGOFurthermore, several mechanisms prevent indiscriminate recognition by the molecules in each pathway. ADAR1 has been reported to edit adenosine to inosine in > 200-nt long dsRNA formed within the 3′ UTR of endogenous mRNAs, destabilizing the dsRNA and preventing indiscriminate recognition by MDA5 as exogenous RNA . MethylaAs noted above, both Dicer and RLRs are DExD/H box-containing RNA helicase family proteins, and the ATPase/helicase domains of Dicer and LGP2 are similar. Dicer interacts with TRBP through its ATPase/helicase domain  and our PKR is an IFN-inducible, serine-threonine kinase that can be phosphorylated by binding to dsRNA through N-terminal dsRBD1 and 2 or autophosphorylated through dimerization  Figure B. PhosphIn addition, TRBP and PACT reportedly interact with LGP2 to regulate IFN production during infection with Theiler’s murine encephalomyelitis virus (TMEV) or encephalomyocarditis virus (EMCV), but not during SeV infection ,29,30; tBoth RNA silencing and the IFN response are induced by dsRNAs in the cytoplasm, but they have been considered two independent pathways in mammalian cells. RNA silencing is a posttranscriptional gene silencing mechanism conserved in diverse organisms. By contrast, the IFN response is an antiviral response of the innate immune system that is conserved among higher vertebrates. The mutual regulation of RNA silencing, directed by endogenous miRNAs, and the IFN response, directed by exogenous RNAs such as viral RNAs, might be a conserved mechanism specific to mammalian cells. Moreover, several proteins with functions in RNA silencing or the IFN response interact with multiple proteins: (i) TRBP interacts with Dicer, PACT, PKR, and LGP2, (ii) LGP2 interacts with RIG-I, MDA5, and TRBP, and (iii) PKR interacts with TRBP, PACT, and ADAR1. RNAs appear to be clearly distinguished as endogenous or exogenous for incorporation into each pathway, irrespective of the complicated interactions among proteins. Endogenous RNAs such as miRNA, mRNA, rRNA, and tRNA do not usually activate the IFN response in human cells, and their misrecognition by virus sensor proteins is prevented by the proteins ADAR and MTr1.Crosstalk between RNA silencing and the IFN response through protein–protein interactions may have several advantages for mammalian cells: (i) introduction of exogenous viral dsRNA into the cell triggers the crosstalk response through viral sensor proteins, (ii) expression levels of the responsible molecules differ among tissues or cell types, allowing tissue- or cell-specific functions, (iii) such a complicated response may support flexible responses depending on the species of virus, and (iv) the balance of cell survival and death among virus-infected cells can be regulated based on the infecting virus or the expression levels of each molecule. The proteins described above form heterodimers or homodimers in vitro, but it remains unclear whether they form a complex in any combination in vivo. RLRs, PKR, and ADAR1 p150 are IFN-inducible genes, indicating that their expression levels are upregulated during viral infection. By contrast, Dicer, AGO, TRBP, PACT, and ADAR1 p110 are not induced by IFN, but their expression levels are known to differ among types of tissues or cells . DiffereIn plants and invertebrates, antiviral RNA interference (RNAi) directed by viral siRNA functions as an antiviral defense system by cleaving viral RNAs. However, whether antiviral RNAi occurs in mammalian cells remains controversial. Previous reports have shown that IFN represses antiviral RNAi in mammalian cells ,107,108,Recent works from our group and others worldwide have revealed that RNA silencing directed by endogenous miRNAs and the IFN response directed by exogenous dsRNAs such as viral RNAs are a mutually regulated antiviral defense system in mammalian cells. Although the protein–protein interactions involved in RNA silencing and the IFN response are complicated, the elucidation of such interactions may shed light on the newly discovered crosstalk as a common mechanism conserved among mammalian cells. Further studies are needed to reveal the antiviral effects of this crosstalk during viral infection of mammalian cells.


Nevertheless, some organisms lack ProQ and/or Hfq homologs, suggesting the existence of other RBPs involved in sRNA function. Along this line of thought, the global regulator CsrA was recently shown to facilitate the access of an sRNA to its target mRNA and may represent an additional factor involved in sRNA function. Ribonucleases (RNases) can be considered a class of RNA-binding proteins with nucleolytic activity that are responsible for RNA maturation and/or degradation. Presently RNase E, RNase III, and PNPase appear to be the main players not only in sRNA turnover but also in sRNA processing. Here we review the current knowledge on the most important bacterial RNA-binding proteins affecting sRNA activity and sRNA-mediated networks.Small non-coding RNAs (sRNAs) are critical post-transcriptional regulators of gene expression. Distinct RNA-binding proteins (RBPs) influence the processing, stability and activity of bacterial small RNAs. The vast majority of bacterial sRNAs interact with mRNA targets, affecting mRNA stability and/or its translation rate. The assistance of RNA-binding proteins facilitates and brings accuracy to sRNA-mRNA basepairing and the RNA chaperones Hfq and ProQ are now recognized as the most prominent RNA matchmakers in bacteria. These RBPs exhibit distinct high affinity RNA-binding surfaces, promoting RNA strand interaction between a  The majority of small non-coding RNAs (sRNAs) interact with a complementary mRNA through an antisense mechanism, leading to the formation of a duplex sRNA-mRNA region. Consequently, expression from the target mRNA is affected and frequently repressed . sRNA-meRNA chaperones constitute a specific group of RBPs that transiently bind and induce structural changes in RNA substrates by melting RNA secondary structures . Such stRibonucleases (RNases) are another group of specific RBPs that interact with sRNAs. These enzymes are responsible for the catalytic cleavage of all classes of RNA . The staEscherichia coli, and ∼30% rely on Hfq to carry on their functions (trans-encoded sRNAs (which are expressed from a different genomic region than their mRNA targets), stabilizing the imperfect basepairing between sRNA/mRNA pairs. At least in Gram-negative bacteria, Hfq primary role is to promote the annealing of sRNA-mRNA duplexes, acting as a molecular “matchmaker,” but its role in Gram-positive bacteria is more controversial  with paralogs  found in different Pseudomonas species  possibly by altering the secondary structure of the rpoS mRNA in E. coli  and the StpA protein. CSPs are a group of small proteins that display the RNA-binding cold shock domain (CSD)  and can  fashion . CspA ac fashion . In Stapal sRNAs . Accordial sRNAs . For exa E. coli  and affelmonella . E. colitivities , 1996. Itivities . Importapression .E. coli RNase E have been identified in the majority of Proteobacteria classes  synthase. RapZ was recently found to be the receptor for GlcN6P . g domain . RNase Ig domain . The tarepairing . In B. spA toxin . In StreNase III .E. coli, PNPase is the main enzyme involved in the degradation of sRNAs that are not bound to Hfq, as shown for the regulation of different Hfq-dependent sRNAs, such as MicA, GlmY, RyhB, and SgrS levels  is a highly conserved 3′–5′ exoribonuclease that processively degrades RNA . PNPase S levels , 2013. TS levels . AdditioISPR RNA .Sinorhizobium meliloti it was shown that YbeY could cleave sRNA-mRNA pairs , involved in protein quality control and ribosome recycling (bsrG mRNA that together constitute a temperature-dependent type I toxin/antitoxin system in Bacillus subtilis (Although to a lesser extent, additional RNases are involved in the regulation of sRNAs. The degradative enzymes YbeY and RNase R are illustrative examples. YbeY is a highly conserved endoribonuclease commonly associated with rRNA processing . HoweverNA pairs . Inactive stress . Additios Qrr1-4 , which as Qrr1-4 . RNase Rs Qrr1-4 . There aecycling . RNase Rsubtilis .RNA chaperones can modify sRNA structure, facilitate the basepairing of sRNAs to their target mRNAs and together with RNases control sRNA stability . HoweverJA outlined the manuscript. AQ, AS, and RS prepared the figures. CA and JA supervised the work. All authors wrote and participated in preparation of the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The 31 nucleotide (nt) long highly conserved stem-loop 5a (SL5a) is located within the 5′-untranslated region (5′-UTR) important for viral replication. SL5a features a U-rich asymmetric bulge and is capped with a 5′-UUUCGU-3′ hexaloop, which is also found in stem-loop 5b (SL5b). We herein report the extensive 1H, 13C and 15N resonance assignment of SL5a as basis for in-depth structural studies by solution NMR spectroscopy.The SARS-CoV-2 (SCoV-2) virus is the causative agent of the ongoing COVID-19 pandemic. It contains a positive sense single-stranded RNA genome and belongs to the genus of Betacoronaviruses. The 5′- and 3′-genomic ends of the 30 kb SCoV-2 genome are potential antiviral drug targets. Major parts of these sequences are highly conserved among Betacoronaviruses and contain The online version contains supplementary material available at 10.1007/s12104-021-10007-w. SCoV-2 is a member of the Betacoronavirus family and contains a large single-stranded (+) RNA genome with a length of approx. 30,000 nucleotides (nts) . Sub-elements are joined to the SL5 basal stem by a four-helix junction. These sub-elements are termed SLs 5a, 5b and 5c. SL5a consists of 31 nucleotides and represents the largest of the three stem-loops. Intriguingly, the apical loop sequences of SL5a and SL5b are identical (5′-UUUCGU-3′) and belong to the 5′-UUYCGU-3′ motif, which is also found in Alphacoronaviruses. This high level of sequence conservation suggests functional importance, e.g. in viral packaging Masters . Thus, wEcoRI and NcoI sites of an HDV ribozyme encoding plasmid  according to the manufacturer’s instructions and linearized with HindIII prior to in-vitro transcription using the T7 RNA polymerase P266L mutant, which was prepared as described in . Preparative transcription reactions (6 h at 37 °C and 70 rpm) were terminated by addition of 150 mM EDTA. SL5a RNA was purified as follows: RNAs were precipitated with one sample volume of ice-cold 2-propanol. RNA fragments were separated on 15% denaturing polyacrylamide (PAA) gels and visualized by UV shadowing at 254 nm. SL5a RNA was excised from the gel and eluted using the following protocol: The gel fragments were granulated in two gel volumes 0.3 M NaOAc solution, incubated for 30 min at − 80 °C, followed by 15 min at 65 °C. The RNA was further eluted from gel fragments overnight by passive diffusion into 0.3 M NaOAc, precipitated with EtOH and desalted via PD10 columns . Residual PAA was removed by reversed-phase HPLC using a Kromasil RP 18 column and a gradient of 0–40% 0.1 M acetonitrile/triethylammonium acetate. After freeze-drying of RNA-containing fractions and cation exchange by LiClO4 precipitation (2% in acetone), the RNA was folded in water by heating to 80 °C followed by rapid cooling on ice. Buffer exchange to NMR buffer  was performed using Vivaspin centrifugal concentrators (2 kDa molecular weight cut-off). Purity of SL5a was verified by denaturing PAA gel electrophoresis and homogenous folding was monitored by native PAA gel electrophoresis, loading the same RNA concentration as used in NMR experiments.RNA synthesis for NMR experiments: For DNA template production, the sequence of SL5a together with the T7 promoter was generated by hybridization of complementary oligonucleotides and introduced into the 15N- and a 680 µM uniformly 13C,15N-labeled sample, were prepared and used for the assignment presented herein.Using this protocol, two NMR samples of SL5a, an 810 µM uniformly 15N-labeled RNA were carried out at the Karolinska Institute (KI) using a Bruker AVANCEIII 600 MHz NMR spectrometer equipped with a 5 mm, z-axis gradient 1H -QCI cryogenic probe. All NMR experiments with the 13C,15N-labeled RNA were conducted at the Center for Biomolecular Magnetic Resonance (BMRZ) at the Goethe University (GU) Frankfurt using Bruker AVIIIHD NMR spectrometers from 600 to 800 MHz, which are equipped with the following cryogenic probes: 5 mm, z-axis gradient 1H -TCI cryogenic probe (600 MHz), 5 mm, z-axis gradient 1H -QCI cryogenic probe (700 MHz) and 13C-optimized 5 mm, z-axis gradient 13C, 15N [1H]-TXO cryogenic probe (800 MHz).NMR experiments using the www.logs.repository.com). 1H chemical shifts were referenced externally to DSS, and 13C and 15N chemical shifts were indirectly referenced from the 1H chemical shift as described earlier  and 3.6.2 (KI). The chemical shift assignment was conducted using Sparky , 1H,13C-HSQC spectra of the ribose region of SL5a and a 14 nt RNA with a 5′-cUUCGg-3′ tetraloop  yielded a similar peak pattern , N7: 100% and N9: 100%) and pyrimidines (N1: 95% and N3: 80% (cytidines)) were successfully assigned to a large extent. Within the ribose moieties, 91% of the H1′ and 91% of the C1′ atoms were assigned. Within the remaining ribose carbon atoms C2′–C5′, 77% were assigned. In summary, we assigned 97% of the 1H  and 92% of the 13C , C1′) atoms, which are considered most important for an in-depth structural characterization. We updated the BMRB deposition with code 50346.The nearly complete resonance assignment of SL5a builds on the imino resonance assignment published earlier Below is the link to the electronic supplementary material.


Furthermore, transcriptome-wide mapping has found that the aberrant deposition of mRNA modification, which can disrupt the gene regulatory network and lead to uncontrollable cancer cell growth, is widespread across different cancers. In this review, we try to summarize the recent advances of these three mRNA modifications in maintaining the stemness of cancer stem cells and discuss the underlying molecular mechanisms, which will shed light on the development of novel therapeutic approaches for eradicating cancer stem cells.Despite their small numbers, cancer stem cells play a central role in driving cancer cell growth, chemotherapeutic resistance, and distal metastasis. Previous studies mainly focused on how DNA or histone modification determines cell fate in cancer. However, it is still largely unknown how RNA modifications orchestrate cancer cell fate decisions. More than 170 distinct RNA modifications have been identified in the RNA world, while only a few RNA base modifications have been found in mRNA. Growing evidence indicates that three mRNA modifications, inosine, 5-methylcytosine, and N With the rapid development of high-throughput sequencing technologies, more than 170 types of post-transcriptional RNA modifications have been detected so far  and have6-methyladenosine (m6A), N1-methyladenosine(m1A), Inosine (I), Pseudouridine (Ψ), 5-methylcytosine (m5C), 5-hydroxymethylcytidine (hm5C), N6,2′-O-dimethyladenosine (m6Am), 7-methylguanosine (m7G), and N4-acetylcytidine (ac4C). Despite the low frequency in the human genome, they affect almost every step of mRNA biogenesis and degradation. For example, mRNA modifications extensively modulate a vast pool of biochemical events surrounding mRNA metabolisms, such as mRNA splicing [Emerging RNA immunoprecipitation-sequencing methods have provided a detailed understanding of the genome-wide landscape of RNA modifications in human cells –12. Howesplicing , 14, RNAsplicing , 16, stasplicing –21, mRNAsplicing –24, and splicing , 26.Growing evidence indicates that mRNA modifications display dramatic and dynamic variations during lineage commitment and cell reprogramming –29, suggThe concept of cancer stem cells (CSCs) was proposed in the 1970s . AnalogoA recent breakthrough of the high-throughput sequencing platform has illustrated the detailed epigenetic landscape in CSCs . The epi5C), and N6-methyladenosine (m6A). In this review, we will provide an update of how these three mRNA modifications orchestrate regulatory gene networks within CSCs  editing is one of the most prevalent RNA modifications. This process involves hydrolytic deamination of adenosine, catalyzed by the ADAR family members  . The newPrevious studies demonstrated that A-to-I modifications could modulate the stemness of hematopoietic malignancies. For example, ectopic expression of ADAR1 potentiated malignant myeloid progenitor expansion through promoting alternative splicing of GSK3β, which enhanced the production of a misspliced form of GSK3β . In vivoE912 mutant, potentiates self-renewal gene expression and suppresses the biogenesis of stem cell inhibitory microRNA let-7 [In addition to mRNA, growing evidence demonstrates that ADAR1 also hinders the biogenesis of tumor-suppressive miRNAs, thereby driving leukemia stem cell self-renewal. Wild-type ADAR1, but not the editing-defective ADAR1NA let-7 . SubsequRecent studies have provided substantial new insights into how A-to-I modifications regulate RNA splicing. mRNA maturation involves serial processing steps which structurally alter the newly synthesized RNA transcripts, such as 5′ end capping, RNA splicing, RNA editing, and 3′ end polyadenylation. Among these molecular processes, RNA splicing is a well-documented molecular event that is tightly regulated by A-to-I modification , 66. TheIn addition, emerging evidence suggests that A-to-I editing plays a role in regulating RNA stability. In human B cells, DNA and RNA sequencing data showed that the expression levels of thousands of genes were modulated by ADAR proteins . FurtherIn summary, ADAR1 plays a pivotal role in maintaining the stemness of hematopoietic malignancies. Through enhancing self-renewal gene expression and impairing the biogenesis of tumor-suppressive miRNAs, ADAR1 is indispensable for normal hematopoietic stem cell maintenance and leukemia stem cell self-renewal. This suggests that ADAR1 may play an important role in a wide spectrum of hematopoietic disorders which have acquired aberrant stem cell self-renewal features.5C) was first identified in tRNA and rRNA [5C profile has shown that m5C modifications are preferentially located in the vicinity of the translational start codon of mRNA [5C modification is predominantly catalyzed by the RNA methyltransferase, NSUN2, and the m5C sites are recognized by the m5C reader protein, ALYREF  does NSUN2 exert opposite roles in different cancer types, and (2) does m5C methylation either promote or inhibit protein translation based on different microenvironments? It is not rare that a single gene can have dual roles as either a tumor suppressor or an oncogene. For instance, the stem cell marker gene, KLF4, has contrasting roles in various cancer types as reported in previous studies [The above contradictory findings raised two fundamental questions for NSUN2 and m studies , 88. Bec5C modification promotes or suppresses mRNA translation is still under debate. It was reported that NSUN2-mediated m5C modification can either promote CDK1 and IL-17A translation or attenuate p27KIP1 translation [Besides, whether the mnslation –91. In tnslation .5C has a sophisticated role in governing mRNA translation and further investigation is necessary to further clarify its mechanism of action.Taken together, these contrasting findings indicate that m5C modification alters mRNA expression. Recent findings suggest that m5C might enhance mRNA stability. It was found that YBX1 preferentially recognized mRNA with m5C modifications and subsequently stabilized target mRNAs. In zebrafish early embryos, m5C-modified maternal mRNAs displayed enhanced stability when compared to non-m5C-modified mRNAs [5C-modified mRNAs through cooperation with mRNA stabilizer Pabpc1a. This highlights an essential role of m5C modification in RNA metabolism and zebrafish embryo development. In human bladder cancer cells, it was reported that many oncogenic mRNAs were hypermethylated by NSUN2. As an m5C reader, YBX1 recognized m5C sites within HDGF mRNA transcripts and then recruited mRNA stabilizer HuR, leading to enhanced mRNA stability [Currently, it remains largely unknown how med mRNAs . Subsequtability . High ex5C affects not only mRNA stability but also mRNA splicing. It was reported that the distribution of m5C sites partially overlapped with the binding sites of some RNA binding proteins. By analyzing the PAR-CLIP data from public databases, scientists uncovered that m5C sites were enriched in the binding regions of the mRNA splicing factors SRSF3 and SRSF4 [5C modification in modulating mRNA alternative splicing. Interestingly, a recent publication revealed a previously unknown role of m5C modification in HIV infection through modulation of RNA splicing and translation [5C methyltransferase NSUN2. High-throughput sequencing data subsequently confirmed an m5C site located in the vicinity of the A2 splice site within the Vif gene. Knockout of NSUN2 reduced the use of the D1/A2 splice junction and altered the RNA splicing of HIV RNA transcript. Moreover, loss of NSUN2 reduced m5C occurrence on HIV RNA transcripts and hampered HIV RNA translation, suggesting an important role of m5C modification in the life cycle of HIV. Collectively, these data suggest that m5C modification may be involved in mRNA splicing, although there are many questions that must be addressed by scientists in the coming future. For instance, it is still unclear which reader proteins recognize m5C sites and thereby mediate mRNA splicing. Since only a small number of mRNA splicing factor binding sites overlap with the m5C region, is it a specific phenomenon that occurs in some particular RNA transcripts? Further studies are needed to elucidate the functional role of m5C in alternative splicing.Besides, some recent papers indicate that mnd SRSF4 , indicatnslation . It was 5C sites in eukaryotic cells. Moreover, the identification of m5C writers and readers has aided our understanding of the functional roles of m5C modifications in the regulation of RNA stability, alternative splicing, and RNA translation. Although scientists have illustrated the genome-wide m5C distribution at single-nucleotide resolution, the role of m5C in mammalian cells remains unclear. For instance, the m5C eraser is still unknown and how m5C mediates other RNA processing steps is to be further explored. Therefore, it is necessary to identify novel m5C-interacting proteins or enzymes, which will further elucidate the functional roles of m5C in various biological events and human diseases.High-throughput sequencing has provided a detailed mapping of m6-methyladenosine (m6A), occurring at the N6 position of adenosine, is the most pervasive and abundant post-transcriptional modification in eukaryotic cells. By using the antibody-enrichment sequencing method, m6A sites were found in all areas of mRNA transcripts but displayed significant enrichment near the stop codon and 3’UTR region [6A modification [6A sites are present in a consensus motif DRACH  [6A RNA modification is evolutionarily conserved across different species [NR region . It was fication . Subsequ C or U) . Further species .6A in mRNA is mediated by methyltransferase complexes, such as METTL3/14, VIRMA, RBM15/15B, WTAP, HAKAI, and ZC3H13, called ‘writers’. The removal of m6A from mRNA transcript is catalyzed by ‘eraser’ demethylases, FTO and ALKBH5  have abundant m6A modifications on their RNA transcripts, which eventually impairs mRNA stability and induces RNA degradation [6A methyltransferase complex. Complete depletion of m6A in METTL3-null mice led to early embryonic lethality owing to prolonged RNA half-life of core pluripotency genes, resulting in a delay in initiation of differentiation programs [6A in RNA transcripts is essential for the maintenance of self-renewal capacity during embryo development.As the most prevalent post-transcriptional modification, mgramming . Transcrradation , 104. MEprograms . Therefo6A profiles frequently occur in a variety of cancer types [6A methylation have been reported in different types of cancer, such as liver cancer and acute myeloid leukemia (AML) [6A methylation levels of BCL2 and c-MYC transcripts and thus enhanced their translation [6A modification within the mRNA transcript of SP1, an oncogene in AML which modulates c-MYC expression [6A demethylase FTO played an oncogenic role in AML through reducing the m6A levels of ABS2 and RARA, which led to decreased mRNA levels of these two targets and eventually contributed to leukemogenesis [Emerging evidence indicates that aberrant mer types . Unexpecia (AML) . In AML,nslation . METTL3 pression . On the ogenesis .6A demethylation actively helps to maintain the self-renewal capacity of cancer cells  have been identified by serial biochemical approaches. Subsequent studies have highlighted the biological and pathological importance of these proteins. However, the underlying molecular mechanisms of m6A modifications need to be further explored. In conclusion, the central questions remain about how m6A is added on or removed from target mRNAs, and how m6A modulates RNA metabolism.As the most prevalent RNA modification form in eukaryotic mRNAs, the mPrevious studies highlighted mRNA modifications as key modulators in determining cell fate transition during embryonic development . Recentl6A in synthetic RNA molecules [Recent breakthroughs in epitranscriptome sequencing technologies have enabled scientists to decode mRNA modifications in mammalian cells, which strengthen our current understanding of the distribution and function of various mRNA modifications. However, although more than 170 RNA modifications have been identified , only a olecules , 119. Th5C methyltransferase NSUN2 has been characterized, we still do not know the parallel demethylases which are responsible for the removal of m5C [In addition to novel sequencing strategies, the corresponding RNA modifying enzymes remain largely unknown. For instance, although the ml of m5C . MoreoveIn conclusion, the aberrant deposition of RNA modifications is tightly linked to the stemness of CSCs. The underlying molecular mechanisms show that RNA modifications orchestrate almost every step of mRNA metabolism, ranging from mRNA biogenesis to mRNA decay, which can eventually converge to determine the cancer stem cell’s fate and tumor progression.


Animal epidemic diseases caused by RNA viruses are the primary threat to the livestock industry, and understanding the mechanisms of RNA virus clearance from target cells is critical to establish an effective method to reduce economic losses. As an SF-1, ATP-dependent RNA helicase in the UPF1p family, MOV10 participates in the RNA degradation of multiple viruses mediated via miRNA pathways and therefore contributes to a decrease in the replication of RNA viruses. This review primarily focuses on the bioactivity of MOV10, the mechanism of RNA virus removal, and the potential roles of MOV10 in RNA virus clearance. In addition, clues are provided to reduce animal diseases caused by RNA viruses. Animal epidemic diseases caused by RNA viruses are the primary threat to the livestock industry , and undAnimal epidemic diseases are the primary threat to the livestock industry. Of the veterinary pathogens that cause these diseases, viruses are responsible for approximately half of the most important animal diseases, according to the OIE's  classification of terrestrial and aquatic notifiable animal diseases . Animal Compared with DNA viruses, RNA viruses have a higher mutation rate and cause more serious economic damage in the livestock industry . Lower mRNA interference (RNAi) is an important mechanism in mediating the clearance of RNA viruses . Viral Rin vitro and a decrease in virulence in mice . Sp. Sp46). Though the indirect effects of miRNAs on RNA viral infection were widely confirmed and recognized, the direct effects of miRNAs were still controversial. This controversy was mainly concentrated on several factors. Firstly, miRNAs were reducible generated from the cells after the viruses infection, but these miRNAs were hardly presented its' directly target effects on viral genome because of the low level of miRNAs produced by the target cells. The other factor is the potential escaping mechanism of viral and the cells complex immune system. The mismatch of miRNAs and viral genome mutation also reduced its inhibition effects.MOV10 is an important SF-1, ATP-dependent RNA helicase in the UPF1p family –55, origThe antiviral activity of MOV10 has been evaluated and detected in many studies. The infectivity of different viruses is regulated by the MOV10 helicase, for example, human hepatitis delta virus , human iIn a recent study of MOV10 that focused on animal disease, MOV10 directly inhibited replication of the PRRSV by retaining the viral nucleocapsid protein in the cytoplasm of Marc-145 cells . MOV10 aAs mentioned above, MOV10 antiviral activity is expressed through the activation of different genes and pathways, of which miRNA pathway-mediated viral inhibition is important in restricting viral invasion. In the entire synthesis process of miRNAs, MOV10 not only functions in pri-miRNA production in the cell nucleus  but alsoThe generation of pri-miRNAs and pre-miRNAs is the first step in the production of miRNAs, which is regulated by Drosha and DGCR8 enzymes and is also controlled by the MOV10 gene . GeneralThe pre-miRNAs generated from the canonical and non-canonical pathways are exported from the nucleus via Exportin 5 and then cleavage by Dicer occurs within the RISC loading complex (RLC) . Then, tMOV10 is also important in the regulation of mRNA degradation and stability. A MOV10 protein, typically in the cytoplasm, combines with an Argonaute protein to form a complex that was recognized as an important component stored in a mRNA processing body (the P body) . In a prThe virulence and infectivity of RNA viruses are influenced by MOV10-mediated changes in miRNA/mRNA. In the third stage of HIV infection, HIV infectivity is weakened because of miRNA expression in target cells . MOV10 aThe antiviral activity of MOV10 during RNA virus infection is discussed in this review. Overall, the antiviral capacity of MOV10 is primarily reflected in two important mechanisms. First, MOV10 weakens viral virulence by binding the related protein, thereby activating IFN signal and cell autophagy pathways. Second, MOV10 functions as an RNA helicase during RNA virus infection. MOV10 regulates miRNA and mRNA generation, maturation, and degradation by miRNA pathways, which influence virus replication and packaging. The two mechanisms are typically coordinated in different virus infection stages. In addition, the number of studies is increasing that confirm MOV10 not only has important roles in infection by human viruses, such as HIV and human hepatitis delta virus, but also shows antiviral activity against animal RNA viruses. The antiviral ability of MOV10 and the related pathways during animal RNA virus infection are worthy of future attention. It is also can be used as an important tools for reduction of animal RNA viruses infection in future.FS, XL, and YJ conceived the study and wrote the manuscript. All authors contributed to the article and approved the submitted version.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.


The main modules of RNAWRE not only allow browsing and downloading the RNA modification effectors but also support the BLAST search of the potential RNA modification effectors in other species. We hope that RNAWRE will be helpful for the researches on RNA modifications.RNA modifications are involved in various kinds of cellular biological processes. Accumulated evidences have demonstrated that the functions of RNA modifications are determined by the effectors that can catalyze, recognize and remove RNA modifications. They are called ‘writers’, ‘readers’ and ‘erasers’. The identification of RNA modification effectors will be helpful for understanding the regulatory mechanisms and biological functions of RNA modifications. In this work, we developed a database called RNAWRE that specially deposits RNA modification effectors. The current version of RNAWRE stored 2045 manually curated writers, readers and erasers for the six major kinds of RNA modifications, namely Cap, mDatabase URL: http://rnawre.bio2db.com Since the first kind of RNA modification was discovered over 60 years ago , approxiThe advent of next-generation sequencing technology facilitated the transcriptome-wide detection of several major kinds of RNA modifications , 10. In Besides dynamically installing, removing and interpreting RNA modifications, these effectors are also found to be linked with neurological diseases, metabolic diseases and mitochondrial-related defects . HoweverAlthough a huge amount of researches has been focused on RNA modifications , to the 1A, m6A, m5C, ψ and Poly A. The construction of RNAWRE is illustrated in Stimulated by a recent work , in the 1A, m6A, m5C, ψ and Poly A from GenBank. To identify the potential effectors for the other species, we also collected the protein sequences of animals from Ensembl  were used for the statistic page. All proteins were stored in a MySQL database. An interactive web interface was constructed to enable users to conveniently access the RNAWRE and obtain the information needed either for basic research applications or biological analysis through any modern browser on their devices. C#, HTML and JavaScript were implemented to transmit user query information and rapidly extracted data from MySQL databases management to generate report pages.The RNAWRE website was implemented by performing a variety of common software packages in the WINDOWS system, including Internet Information Services, MySQL database management, ASP.NET, HTML, CSS and JavaScript . The datThe RNAWRE consists of nine main modules, i.e. Home, Browse, Search, Blast, Statistic, Download, Links, Help and Contact.1A, m6A, m5C, ψ and Poly A. The detail number of effectors related to each kind of modifications are 1057, 352, 283, 185, 95 and 73, respectively. The detail information was enumerated in At present, the RNAWRE database contains 2045 manually curated writers, readers and erasers for the six major kinds of RNA modifications, Cap, mBos taurus’, the RNA modification protein is displayed in the list on the right. By clicking on the ‘more’ button in the list, the detailed information of these proteins, such as UniProtID link to the UniProt database, review status, subcellular location if any, GO cellular component if any, Ensembl ID, GeneID if any link to the NCBI Gene database, length and sequence will be displayed in a new page ; Natural Science Foundation for Distinguished Young Scholar of Hebei Province (C2017209244).