These results indicate that individual differences of depressive symptoms affect choice-induced preference change. We discuss the mechanisms underlying the relation between depression and choice-induced preference change.Most experimental studies of depressive symptom effects on decision-making have examined situations in which a single correct answer exists based on external circumstances . In addition to such decision-making, for decision-making of other types, no correct answer exists based on external circumstances . For internally guided decision-making, a phenomenon is known by which preference for the chosen item increases and preference for the rejected item is decreased after choosing between two equally preferred items which is designated as choice-induced preference change. Recent reports suggest that this phenomenon is explainable by reinforcement learning theory just as it is with externally guided decision-making. Although many earlier studies have revealed the effects of depression in externally guided decision-making, the relation between depressive symptoms and choice-induced preference change remains unclear. This study investigated the relation between depressive symptoms and choice-induced preference change using the blind choice paradigm. Results show that depressive symptoms are correlated with change in preference of rejected items (Spearman’s  Depression is among the world’s greatest public health problems. The World Health Organization  reportedMany studies of depression effects on decision-making have used tasks in which a particularly more or less predictable answer is available, called externally guided decision-making ,13. In sIn addition to externally guided decision-making, decision-making also exists for which no correct answer is available for a subject based on external circumstances ,13,20–26ed the same decision when they are compelled to make a decision based on a perceptually ambiguous stimulus with no performance feedback. Importantly, they showed that the underlying choice repetition is explained well by the RL-based model in which the likelihood of choosing an option is updated by the choice itself. Although Akaishi et al. [Recently, an RL-like mechanism is thought to explain how choice-induced preference change occurs –44. Akaii et al.  used a pi et al. ,45–47, wi et al. ,48,49. BThis preliminary study investigated the relation between depression and choice-induced preference change. We predict that individuals with strongly depressive symptoms tend to show less choice-induced preference change. As a first step, for this study, we examined depressed participants in a non-clinical sample. Many reports of the literature have described that non-clinically depressed people show similar symptoms in major depressive disorders. Indeed, studies of externally guided decision-making have revealed that depressed participants in a non-clinical sample exhibit difficulty with reinforcement of items according to their own choices, to the same degree as major depressive disorder patients ,6,9,17. We used the blind choice paradigm  instead n = 69 participants . The result of choice-induced preference change from our EEG experiment was presented in our previous paper [In this experiment, 51 Japanese undergraduate and graduate students participated. Furthermore, when we examined the relation between depression and choice-induced preference change, we added data about the change in ratings and CES-D scores  of 33 Japanese undergraduate and graduate students from our earlier EEG experiment using the same paradigm as that of the present study . The totus paper . TherefoAll participants had normal or corrected-to-normal vision. No participant was taking any medication. None had a history of neuropsychiatric disorders. The study was approved by the ethics committee of the Graduate School of Education, Hiroshima University. All participants gave written informed consent before the start of the experiment.I was bothered by things that usually do not bother me.”), with responses made using a four-point scale from “rarely or none of the time” to “most or all the time.” Higher scores indicate a higher level of depressive symptoms.Each participant’s tendency to depression was assessed using the Japanese version of the Center for Epidemiological Studies Depression Scale . The score range in our study participants was 2–41. We found no gender difference in CES-D scores (t(67) = 2.10, p = .35). The CES-D also provides cutoff scores of 16 or greater, which aid in identifying individuals at risk for clinical depression. Of our study participants, 24 participants had scores higher than the cutoff scores.The average of CES-D scores is 15.1 . The experiment was conducted on a PC using software  with Psychtoolbox –60.From images presented by Konkle, Brady, Alvarez, and Oliva , 140 colBefore the experimental task, participants completed questionnaires related to depressive symptoms. The CES-D score was used to evaluate levels of self-reported depressive symptoms.After completing the questionnaire, participants performed three experimental tasks  on number pad buttons of a keyboard. The image was presented for 3 s on the screen. No time limit was applied for the ratings. Participants performed the ratings for the 140 images. The order was randomized across participants.In the blind choice task, participants were told that this experiment was conducted to investigate the implicit preference judgment process. Participants were instructed that two images that were visible in the pre-choice rating task would be presented for approximately 7 ms in each trial. In fact, however, the two dummy images were presented subliminally. After the two dummy images on the right and the left sides of the screen were presented, the two masking stimuli were presented for 1 s. The masking stimuli were followed by the words “choose” on the screen, which asks participants to judge which images they prefer by pressing the keyboard keys. The dummy images were presented. Therefore, participants’ judgments could not be guided by their own preferences. However, participants were told that “it is well known that people can judge the preference intuitively for subliminally presented stimuli.” After participants made a choice, the feedback  presented in the pre-choice rating task was presented for 3 s along with the red rectangle which surrounds one of these images indicating a blindly chosen image. Two conditions were used during the blind choice task: a critical condition and a non-critical condition. In the critical condition , the two images with the same pre-choice rating score were presented in the feedback. In this condition, the chosen image was determined randomly. It was presented with a red rectangle on the same side as the participant’s pressed key. In the non-critical condition , two images with different pre-choice rating scores were presented in the feedback. In the non-critical condition, the highly rated image was always marked as the chosen image, and was presented on the same side as the participant’s pressed key. This condition was used to induce participants to believe that they were able to judge their preference intuitively, even for subliminally presented stimuli.After the blind choice task, participants were again asked to evaluate subjective preferences for the 140 images. The post-choice rating task procedure was the same as that of the pre-choice rating task.We computed the mean-corrected ratings to analyze the choice-induced preference change by following procedures described by Sharot et al. . For eacn = 69  = -0.50, p = .62). Additionally, we conducted one-sample t tests to ascertain whether each change in ratings of chosen and rejected images was significantly different from zero. Results show no significant change either for chosen images or rejected images (chosen images: t(43) = 0.07, p = .95; rejected images: t(43) = 1.10, p = .28). Actually, those results were not surprising because the effect size of choice-induced preference change is small [is small .p-values using the FDR method to prevent an increase in the risk rate by multiple comparison. Results show that CES-D scores were positivity correlated with the change in ratings of rejected images . In addition, to explore sex-related differences of correlation between depressive symptoms and choice-induced preference change, we conducted partial correlation analysis when controlling for sex. Results show that significant correlation between CES-D scores and change in ratings of rejected items remains when controlling for sex .Although no significant choice-induced preference change was found for the entire group of participants as a whole, significant correlation was found between choice-induced preference change and depressive symptoms. To assess the relation between choice-induced preference change and depression, we conducted Spearman’s rank correlation analysis. We corrected These results demonstrate that depressive symptoms are related to choice-induced preference change. Specifically, individuals with strongly depressive symptoms tend to show no reduced preference for rejected items.This study was conducted to investigate the relation between depression and choice-induced preference change. In this study, we observed no choice-induced change using the blind choice paradigm. However, as we had expected, results show that the degree of choice-induced preference change for a rejected item varies according to individual differences of depressive symptoms. Depressive symptoms in non-clinically diagnosed participants tend to show less decreased preference of rejected items. This correlation was persistent after controlling for sex. These results, which indicate that individual differences strongly affect the occurrence of choice-induced preference change, are the first ever reported demonstrating that depressive symptoms are associated with choice-induced preference change. Based on results of their meta-analysis, Izuma and Murayama  reportedResults show that depressive symptoms are related to the preference change of rejected items, but no significant relation with depressive symptoms was found for the chosen items. These results suggest that depressive symptoms are related selectively to updating of the preference of rejected items, but they are not related to updating of the preference of chosen items. Although respective underlying processes of the preference change in chosen and rejected items remain unclear at this stage, our results underscore the need for individual explanations for the learning processes of chosen and rejected items. Choice-induced preference change is explained traditionally by cognitive dissonance theory  and selfCan the RL theory explain our results? Although different factors related to depression  have been known to affect the increasing and the decreasing item values, the present results cannot be explained fully based on notions about RL theory examined in studies of externally guided decision-making. In studies of the relation between depression and externally guided decision-making, depressed individuals are known to show impaired ability to increase the item value through RL in decision-making tasks with reward-maximization goals ,10, but The difference between internally guided decision-making and externally guided decision-making is that the correct answer is determined by one’s own criteria or external circumstances ,13. In iIn contrast, the decrease of self-relatedness for the rejected item is thought to function as devaluation of the item. Consistent with this assumption, earlier studies using the free-choice paradigm demonstrated that striatal activity for rejected items is lower . In the In this manner, an RL-like mechanism might engage in internally guided decision-making, but the value-updating process in internally guided decision-making cannot be explained simply using the RL theory, as externally guided decision-making. The characteristics of the task and reinforcer in internally guided decision-making differ from those in externally guided decision-making. Although it is unclear whether self-relatedness for chosen and rejected items mediates RL as we have discussed above, it is possible that investigating the mechanism behind the relation between the depression and choice-induced preference change is one means of clarifying how the choice-induced preference change emerged.What is the implication for depression? The results of the present study showing that depressed individuals show less updating of the value of rejected items are expected to provide clues to elucidate the mechanism behind the indecision of depressed individuals. Results of prior studies suggest that procrastination is associated with depression, and that depressed individuals tend to delay making decisions related to important matters –72. BaseDespite the importance of our data for revealing the relation between depression and choice-induced preference change, several limitations of this study must be considered. First, our study was conducted with the participation of nonclinical undergraduate and graduate students. Although our use of the CES-D points to examination of depressed participants in a non-clinical sample, it is possible that the mechanisms of preference updating in nonclinical depressed individuals and in major depressive disorder patients differ. Future research must be undertaken to elucidate the association with choice-induced preference change in major depressive disorder patients.Secondly, although we examined the relation between choice-induced preference change and RL theory, the reinforcement process of chosen and rejected items remains unclear. Our data imply that reinforcement processes in internally guided decision-making differ from those of externally guided decision-making, and that this difference derives from characteristics of the task and reinforcer. Future studies must be conducted to explore how the RL theory framework is applicable to internally guided decision-making.only affect preference updating of rejected items. They do not affect the preference updating of chosen items. Although the underlying processes of preference updating in chosen and rejected items remains unclear, it is possible that depression affects choice-induced preference change just as it affects externally guided decision-making. However, because of the differences in characteristics of these two types of decision-making, updating value processes in internally guided decision-making might not be explained simply and solely by RL theory, unlike externally guided decision-making. To elucidate the mechanism underlying the relation between depression and choice-induced preference change, future studies must be conducted to investigate the reinforcement process, with consideration of the characteristics of internally guided decision-making.This study investigated the relation between depression and internally guided decision-making. Our results demonstrated for the first time ever reported that depressive symptoms S1 File(XLSX)Click here for additional data file.


C9orf72 gene. The non-exclusive pathogenic mechanisms by which C9orf72 repeat expansions contribute to these neurological disorders include loss of C9orf72 function and gain-of-function determined by toxic RNA molecules and dipeptides repeats protein toxicity. The expanded repeats are transcribed bidirectionally and forms RNA foci in the central nervous system, and sequester key RNA-binding proteins (RBPs) leading to impairment in RNA processing events. Many studies report widespread transcriptome changes in ALS carrying a C9orf72 repeat expansion. Here we review the contribution of RNA foci interaction with RBPs as well as transcriptome changes involved in the pathogenesis of C9orf72- associated FTD/ALS. These informations are essential to elucidate the pathology and therapeutic intervention of ALS and/or FTD.The most frequent genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) is intronic hexanucleotide (G4C2) repeat expansions (HRE) in the  C9orf72) gene, referred as C9-FTD/ALS  is a devastating adult-onset neurodegenerative disorder which involves upper and lower motor neuronal loss that finally leads to paralysis and death. Frontotemporal dementia (FTD) is a complex dementia disturbing language, cognitive and behavioral skills. Both are fatal within 3–5 years of symptoms onset , and is indistinctly related to the differentially expressed in normal and neoplastic cells (DENN) family of GDP–GTP change factors activating Rab GTPases 23 RNA incubated with nuclear extracts from human embryonic kidney (HEK) 293 cells. Subsequently, they identified 20 top candidate RBPs including hnRNPs, splicing factors and mRNA-binding proteins. A subset of these proteins was further investigated by immunohistochemical analysis on patient hippocampus. This analysis showed hnRNPA3 as a component of the neuronal cytoplasmic and nuclear inclusions. These results suggest that binding of the hnRNPA3 to G4C2 repeats initiate aberrant export of C9orf72 pre-mRNA to the cytosol for RAN translation or its subsequent degradation. However, the co-localization between hnRNPA3 and nuclear RNA foci in patient cerebellum . They found that only hnRNPH, serine-arginine-rich splicing factor 1 (SRSF1), and serine-arginine-rich splicing factor 2 (SRSF2) co-localized with intranuclear G4C2-positive RNA foci. Further, they showed that only hnRNPH binds the G4C2 repeats in co-immunoprecipitation assay and co-localizes with 70% of RNA foci detected in patient cerebellum. The authors went further to show that hnRNPH sequestration in RNA foci decreases the alternative splicing of TARBP2, a known RNA target of hnRNPH, implying the loss of hnRNPH function. It has also been demonstrated that hnRNP H co-localizes with RNA foci in NSC34 and HeLa cells  as well as in the motor cortex of C9-FTD/ALS patients. Knock-down of ADARB2 drastically reduces the formation of RNA foci, suggesting the functional role of ADARB2 in C9orf72-mediated RNA toxicity.Donnelly et al.  employedHaeusler et al.  also ideCooper-Knock et al.  carried Despite, several studies identify and discuss repeat- binding RBPs, yet there is disparity among each study, and the results were not always replicated in other animal models or patient tissues. This discrepancy could be due to the variations in methodologies used. The different cellular models, mouse lysates or patient tissues used for these studies might express a different group of proteins than that observed actually in human CNS and therefore could result in strong false positive hits. Moreover, the lengths of the repeats studied are not similar to what was observed in patients. All of these factors could contribute in biasness of the results. Moreover, the role of different RBPs to the pathologies of the disease is also not very clear. It is still unclear whether the disruption of RBPs function is because of their binding or co-localization with RNA foci. Detailed follow-up studies are needed to show that these events affect RBPs endogenous functions and contribute to pathology.C9orf72 repeat expansion exerts direct effect on the transcriptome via the formation of RNA foci , Ingenuity Pathways Analysis (IPA:www.ingenuity.com), and KeyMolnet (www.km-data.jp/keymolnet) to study molecular networks engaged in C9-FTD/ALS by utilizing three different C9orf72 omics datasets. These data sets were: (i) proteome of C9orf72 HRE RBP, which provides the most important biochemical information of C9-FTD/ALS (Cooper-Knock et al., in vivo source (Highley et al., To identify biologically relevant pathways in C9-FTD/ALS pathology, Satoh et al.  used difC9orf72 HREs involvement in the ribosome function, spliceosome, and post-transcriptional modification of RNA. Essentially, the proteome is enriched of RBPs having RNA-recognition motifs and prion like domains. Similarly, a network analysis of differentially expressed genes in iPSNs of patients with C9-ALS shows that the majority of genes identified were under expressed, namely the genes encoding for extracellular matrix proteins and matrix metalloproteinases. Moreover, the authors did not observed any significant differences in splicing patterns of C9-ALS patients and controls. In addition, the authors also reported that the post-transcriptional RNA processing, cytoskeletal dynamics and intracellular molecular transport have been affected in C9-ALS patients.The results of this study reveal Overall, the potential pathomechanisms associated with C9-FTD/ALS may represent possible therapeutic interventions. Considering that the RNA foci and/or DPRs are important components of the disease, selectively inhibition of their transcription and translation might reduce the disease burden. Significant advances have been achieved in this direction including utilization of antisense oligonucleotides Figure  and RNA C9orf72 repeat expansion contributes to neurodegeneration through the formation of nuclear RNA foci that sequester, and cause functional loss of key RBPs results in the defects in RNA processing and gene expression. Several RBPs co-localize with RNA foci in C9-FTD/ALS brain tissues and/or neurons differentiated from iPSCs. Furthermore, sequestration of RBPs may change the cellular transcriptome which could offer a better therapeutic intervention. Importantly, disease models such as cultured cells or motor neurons does o not cover the different brain regions affected in C9-FTD/ALS, thus may not reflect disease processes accurately. Future studies seeking the cause of pathomechanisms in disease-relevant models and patient tissue will be crucial to understand disease pathogenesis and prevention. At present, it seems that we have just begun to understand the disease pathogenesis. As our understanding of pathomechanisms underlying C9-FTD/ALS pathogenesis grows, so also will the possibility of developing effective therapeutic strategies.There are intense discussions in the C9orf72 field on the pathomechanisms and as reviewed here, there is possibility that a combination of multiple mechanisms may be involved in causing the disease. For example, the reduced level of functional C9orf72 could increase neuronal susceptibility to RNA foci or DPRs. Overall, disturbance of normal RNA processing is one of the key pathological events of C9-FTD/ALS. RNA metabolism and RNA-binding proteins are recurrent themes in neurodegeneration and is crucial for neuronal survival. The VK designed the topic; VK, GH, and MH wrote the paper.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.


Compared with orthoretroviruses, our understanding of the molecular and cellular replication mechanism of foamy viruses (FVs), a subfamily of retroviruses, is less advanced. The FV replication cycle differs in several key aspects from orthoretroviruses, which leaves established retroviral models debatable for FVs. Here, we review the general aspect of the FV protein-nucleic acid interactions during virus morphogenesis. We provide a summary of the current knowledge of the FV genome structure and essential sequence motifs required for RNA encapsidation as well as Gag and Pol binding in combination with details about the Gag and Pol biosynthesis. This leads us to address open questions in FV RNA engagement, binding and packaging. Based on recent findings, we propose to shift the point of view from individual glycine-arginine-rich motifs having functions in RNA interactions towards envisioning the FV Gag C-terminus as a general RNA binding protein module. We encourage further investigating a potential new retroviral RNA packaging mechanism, which seems more complex in terms of the components that need to be gathered to form an infectious particle. Additional molecular insights into retroviral protein-nucleic acid interactions help us to develop safer, more specific and more efficient vectors in an era of booming genome engineering and gene therapy approaches. Spumaretrovirinae subfamily of retroviruses . ,2. Spumaaviridae .In infected cells, FVs assemble new virions by a type B/D strategy, which separates capsid assembly and budding processes in a spatio-temporal manner a 4]. In. In4]. Ipol open reading frame (ORF), since FV Pol is not expressed in an orthoretroviral-like Gag-Pol fusion protein but as a separate Pol precursor protein from a spliced mRNA (Although assembly of FVs is similar to prototypic B/D type orthoretroviruses like Mazon-Pfizer monkey virus (MPMV) or mouse mammary tumor virus (MMTV), the FV replication cycle bears unique features not observed in any other retrovirus during these steps . For exaced mRNA  . Fr. Fr19]. proteins . Expresscis-acting sequence (CAS) elements that are essential for its successful transmission to susceptible hosts and subsequent cellular replication of the virus  ,22gag ORS-I RNAs a 23]. S. Sgag ORlication ,25. Intee below) ,29,30,31The CAS-III element located at the 3′ end of the FV genome harbors the LTR U3- and R-regions, which like the respective 5′ LTR sequences are involved in proviral integration and minus-strand switch during reverse transcription, respectively . Furthercis-acting RNA sequence element, termed CAS-II ,4948,49]FV Gag transcription and translation are regulated at different levels. gag 5′ UTR sequences is the MSD, and was briefly mentioned above when discussing CAS-I  site or a complete intron sequence, but not by adding such sequences downstream of an authentic PFV gag ORF with 5′ UTR sequences lacking a SD site ..52].Gag), encoded in full-length vgRNA containing the authentic gag 5′ UTR, appears to use a ribosomal shunting mechanism , Gag precursor gets cleaved by the viral PR only once during particle morphogenesis, resulting in a large 68 kDa cleavage product (p68Gag) and a small p3Gag peptide  and a nuclear export signal (NES) are located at the N-terminus of PFV Gag ,61. The The temporary nuclear localization of Gag has long been recognized as a hallmark in FV infected cell cultures ,63. A puDuring mitosis, authentic PFV Gag attaches to host cell chromatin via a chromatin binding signal (CBS) and remains in the nucleus after cell division ,67. The In regard to Gag interacting with nucleic acids, the C-terminal region of the protein stood in the limelight early on. Whereas in the orthoretroviral Gag protein, single or double cysteine-histidine zinc finger motifs (Cys-X2-Cys-X4-His-X4-Cys) localized in the C-terminal NC domain were characterized to be essential for vgRNA recognition and selective packaging, FV Gag proteins lack these motifs . Insteadin vitro binding assays with recombinant C-terminal PFV Gag protein domains and nucleic acids assigned a function in vgRNA binding to GR-I . S. SPol ise domain . Ultimategration .Pol precursor is thought to occur predominantly during or shortly after encapsidation into newly assembled capsids [Pol dimerization through interfaces within the C-terminal IN domain. The second hypothesis proposes dimerization of pr127Pol and/or p85PR-RT on RNA secondary structures encoded in the CAS-II element of the viral genome as the main trigger for PR activation [Autocatalytic processing of the pr127 capsids . PFV PR  capsids , and twotivation ,86.cis-acting viral sequences summarized above, it appears that Pol interacts with specific PES elements contained within CAS-I and CAS-II of the FV genome [Pol precursor but not the mature p85PR-RT and p40IN is efficiently encapsidated into assembling PFV capsid structures [PR-RT subunit RNA binding activities have been described [All experimental evidence accumulated until now strongly supports a Pol encapsidation mechanism that is linked to viral genome packaging, although Gag-Pol protein-protein interactions may be involved as well, but seem to not be alone sufficient ,39,43,74V genome ,39,42,44ructures . This maescribed ,88. Alteescribed ,86.Our knowledge about cellular cofactors involved in FV protein-vgRNA interactions and vgRNA encapsidation is extremely limited. As mentioned above, a complex comprising the karyopherin CRM1, the adaptor proteins ANP32A and B, and the RNA binding protein HuR was shown to be responsible for nuclear export of Gag  Figure . ProbablThe only additional co-factor described to date of being involved in vgRNA encapsidation, is the DEAD-box RNA helicase DDX6  Figure . EndogenIn the past two decades the molecular details of selective vgRNA encapsidation in orthoretroviruses, pioneering in MLV and HIV-1, became fully appreciated. However, this appreciation only emerged after the eminent role of secondary RNA structures in the retroviral genome was recognized, and laborious molecular and cellular analysis of RNA and Gag interactions were conducted ,91,92,93cis-acting genome sequences compared to orthoretroviruses. With special emphasis on Gag and Pol interacting with nucleic acids during particle morphogenesis, we conclude that the way we picture FV Gag to engage, bind and package RNA has changed over time. Abandoning the view of individual GR boxes having functions in RNA interactions, and focusing more on a general RNA-binding Gag C-terminus, now paves the route in addressing further mechanistic and molecular details of vgRNA encapsidation.In FV research, orthoretroviral relatives almost always serve as primary reference models for investigating the molecular details of their replication cycle. Whereas the knowledge obtained from many studies on orthoretroviruses helped to understand the FV biology, the differences in their replication strategies are now more and more challenging to expand the current picture on FV replication. Here we provided a condensed account on the similarities, and more importantly of the differences, of the FV Gag and Pol biosynthesis as well as the essential To fully comprehend the picture of vgRNA packaging, we also need to address the other side of the process: understanding which vgRNA motifs are essential to specifically bind Gag. As mentioned above, several reports highlight sequences in CAS-I and II within the FV genome important for vgRNA encapsidation . HoweverAs outlined above, the expression of FV Pol from an individual transcript is not shared by orthoretroviruses, but observed for hepadnaviruses, which share some replication characteristics with FVs ,3. This Elucidating the details of FV RNA packaging is not a mere scientific footnote. It might help to improve FV vector design, which in the recent past showed promising properties for application in gene therapy, and bears potential to safeguard genome engineering techniques by its beneficial integration profile.


Electrostatic interactions complemented by specific rRNA-protein interaction drive the ribosome-protein aggregation process. Under similar conditions the rRNA, especially the large subunit rRNA and in vitro transcribed RNA corresponding to domain V of 23S rRNA (bDV RNA) stimulates lysozyme aggregation leading to RNA-protein aggregate formation. Protein aggregation during the refolding of non-disulfide containing protein BCAII at high concentrations also induces ribosome aggregation. BCAII aggregation was also stimulated in presence of the large subunit rRNA. Our observations imply that the specific sequestration of the translation machine by aggregating proteins might contribute to their cytotoxicity.An understanding of the mechanisms underlying protein aggregation and cytotoxicity of the protein aggregates is crucial in the prevention of several diseases in humans. Ribosome, the cellular protein synthesis machine is capable of acting as a protein folding modulator. The peptidyltransferase center residing in the domain V of large ribosomal subunit 23S rRNA is the centre for the protein folding ability of the ribosome and is also the cellular target of several antiprion compounds. Our  Protein aggregation has been recognized as a hallmark of numerous disease conditions in humans such as Alzheimer, Parkinson, Type II Diabetes and prion diseasesE. coli and B. subtilis, archaebacteria; eukaryotes like yeast, rat liver, wheat germ and bovine mitochondria) and with proteins belonging to wide range of structural classes (ref. 7in vitro transcribed RNA corresponding to the domain (bDV RNA) also exhibits this activity. Studies on the mechanisms of bDV RNA protein folding activity showed that it is a two-step process involving its two sub-domains RNA1 and RNA2. The initial binding of the unfolded proteins takes place with the RNA1 sub-domain and the RNA2 region of this domain is responsible for releasing the bound protein which subsequently folds into its native structurein vivo and in vitro leading to nucleation of folding of the nascent polypeptide chain synthesized by the ribosome111314151615in vitro transcribed bDV RNA could bind to and interfere in the aggregation of partially folded BCAII (bovine carbonic anhydrase II) and reduced denatured hen egg white lysozyme (lysozyme)Ribosome, the cellular protein synthesis machine is also the platform for folding of newly synthesized polypeptide chains3Lysozyme is a small globular protein with 4 disulfide linkages. Since its pI is 11.35, it is predominantly positively charged at physiological pH. This protein has been extensively used to understand the mechanism of protein folding, misfolding and amyloid fibril formationAs stated earlier, when lysozyme is exposed to high concentration of the reducing agent DTT, disulfide bond breakage and scrambling leads to the formation of loosely packed amorphous aggregatesLysozyme (10 μM), was incubated in 20 mM DTT at physiological pH  in the absence or in the presence of the 70S ribosome (Methods section) and the change in turbidity was followed for a period of 60 min. As shown in The constituents of the aggregate then needed to be further analysed. The aggregate formed upon incubation of lysozyme (10 μM) and ribosome (0.1 μM) for 45 min under reducing conditions was separated by centrifugation (Methods section). The aggregate pellet and the supernatant were analysed by denaturing polyacrylamide gel electrophoresis. Unexpectedly, as shown in Our next set of experiments was performed to study the effect of lysozyme on the eukaryotic yeast 80S ribosome under reducing conditions. Incubation of lysozyme (10 μM) with substoichiometric concentration (0.1 μM) of yeast 80S ribosome under reducing conditions also showed enhanced increase in turbidity and presence of ribosomal components in the aggregates by SDS-PAGE analysis . This stNext, to determine the extent of ribosome aggregation with respect to the total amount of ribosome present in the experiment, 0.1 μM of the 70S or 80S ribosome was incubated with lysozyme (10 μM) in presence of DTT . The rRNTo follow the time course of the ribosome aggregation, lysozyme (10 μM) was incubated with 70S ribosome (0.1 μM) in presence of DTT in separate reactions. The reaction mix was centrifuged at different time intervals and the rRNA in the aggregate analysed by agarose gel electrophoresis. The intensity of the RNA band in the aggregate would be a measure of the extent of ribosome aggregation induced at the specified time points . A semi-It is well established that an increase in protein concentration leads to increased protein aggregation. Hence, further studies were conducted to observe whether the facilitation of protein-protein interaction at higher protein concentration could lead to reduced lysozyme-ribosome interaction and hence reduced ribosome aggregation. As shown in in vitro translation assay leads to significant reduction in translation of the GFP reporter gene and this observation has been attributed to a dysfunctional consequence of tau–ribosome association in Alzheimer’s patientsTo test whether a preformed protein aggregate is also capable of triggering the ribosome aggregation process, 65 μM lysozyme was incubated in 20 mM DTT for 30 min. Lysozyme undergoes almost complete aggregation under these conditions. The lysozyme aggregates obtained by centrifugation was incubated with 0.65 μM ribosome for 45 minutes. SDS PAGE analysis shows that the preformed lysozyme aggregates also had the ability to stimulate ribosome aggregation . The looin vitroLysozyme is predominantly positively charged at the pH used in our study and the ribosome surface has a highly negative electrostatic potential arising primarily from the phosphodiester backbone of the RNA5Our next objective was to investigate the effect of ribosome associated polyanions like mRNAs and tRNAs on lysozyme induced ribosome aggregation. The model mRNA used in this experiment contains a Shine Dalgarno sequence that would position an AUG codon at the P-site of the ribosome. In our earlier studies, this mRNA was used to target the binding of deacylated Met-tRNA to the P-site under appropriate conditionsSince the rRNA plays a major role in determining the anionic surface of the ribosome5in vitro synthesized bDV RNA on the aggregation of lysozyme under reducing conditions. Light scattering studies showed that when lysozyme (2 μM) was incubated in 20 mM DTT, stoichiometric concentrations of the bDV RNA could stimulate protein aggregation  of lysozyme (2 μM) in presence of bDV RNA (2 μM) was analysed by SDS-PAGE and agarose gel electrophoresis. As shown in To check whether any entity other than the rRNA could also stimulate lysozyme aggregation the following experiment was performed. Lysozyme aggregation at 10 μM concentration was allowed to proceed in 20 mM DTT in the presence of increasing concentration of native BCAII. The aggregation mix was centrifuged after 45 minutes (Methods) and the supernatants and the pellets were analysed separately on an SDS PAGE. As shown in Earlier studies have demonstrated that the RNA1 sub-domain of bDV RNA is involved in the initial interaction with unfolded polypeptide chain and the RNA2 sub-domain enables the subsequent release of the proteinin vitro transcribed bDV RNA, mDV RNA, RNA1, RNA2 and heparin for 45 min. Similar experiment was also performed in presence of a 165 nucleotides long mRNA corresponding to the rmf (Ribosome Modulation Factor) gene . The comparable aggregation stimulating effect of mDV RNA, bDV RNA and the RNA1 sub-domain alone indicated that this effect depended only on binding of the RNA to the protein and not on its subsequent release. Also as shown in in vitro transcribed bDV RNA or its derivatives themselves did not scatter light in presence of 20 mM DTT for the duration of the experiments (The bar diagram in eriments .in vitro transcribed bDV RNAThe observed effect of bDV RNA on lysozyme aggregation (stated above) however appeared contradictory to our earlier observation in which lysozyme aggregation during its refolding from the reduced denatured state was suppressed in presence of the ribosome or the To investigate whether the formation of protein-ribosome aggregates observed with lysozyme is unique to disulfide containing proteins carrying a net positive charge or is a general phenomenon, the following experiments were performed with the BCAII protein. Bovine carbonic anhydrase II (BCAII) is a small globular protein having no disulfide linkages. Since its pI is 6.93, it carries a negative charge (−2.3) at physiological pH. BCAII has been used as a model in protein folding studies and is also capable of amyloid formation31When BCAII is denatured with 6 M GuHCl and diluted 100-fold such that the final protein concentration is 2 μM, the protein undergoes aggregation as revealed by both the increase in turbidity and light scattering at 450 nm . As withProtein misfolding and aggregation have an adverse effect on all living organisms and is also associated with several neurodegenerative diseasesin vitro transcribed RNA corresponding to domain V of 23S rRNA (bDV RNA) stimulate lysozyme aggregation. The aggregation of the non-disulfide containing protein BCAII also induces ribosome aggregation indicating that the induction of protein aggregation is a general phenomenon and not specific for disulphide containing proteins. The large subunit rRNA could also stimulate BCAII aggregation. In this context reference can be made of earlier studies demonstrating that the total RNA extracted from yeast or bovine liver can promote the assembly of the Tau protein into the Alzheimer’s disease associated paired helical filaments (PHFs). The cellular RNA isolated from neuroblastoma cells (N2a RNA) is also capable of inducing accumulation of Prp-RNA aggregates both in vitro and in vivo42As stated earlier lysozyme forms amorphous aggregates under reducing conditions used in our study39in vivo stress conditions in which there is an increase in population of both non-translating ribosomes  and protein aggregates44in vivo6As stated earlier, the ability of the bacterial ribosome to significantly improve the refolding yield of proteins is well established. It is to be noted that in these studies the ribosome and the substrate proteins were present in stoichiometric concentrations under conditions conducive to their folding. In our studies the effect of proteins undergoing aggregation on non-translating empty ribosomes has been analysed. This situation mimics the in vivo accumulation of protein aggregates that are predominantly composed of ribosomal components has been reported earlier. Mutant yeast cells lacking gene for Tsa1 protein (a ribosome associated molecular chaperone having peroxidase activity) were found to accumulate aggregated proteins and this was exacerbated when the cells were subjected to reductive stress in presence of 20 mM DTT475050in vitro studies suggest that the misfolded aggregation-prone proteins, whose cellular levels are likely to increase under all the aforementioned conditions, might target the non-translating ribosomes or its subunits4748495052in vitro assay due to the addition of tau protein oligomers also points towards the targeting of the translating machine during aggregate mediated cellular toxicityThe et al.In this context, a recent review by Yang E.coli tRNA (f-Met deacylated tRNA) were purchased from M.P Biomedicals. Heparin and the yeast Saccharomyces cerevisiae 80S ribosome were kind gifts from the laboratories of Dr. S. N. Kabir and Dr. Jayati Sengupta (IICB). Nitrocellulose filter was purchased from Millipore and reagents for molecular biology like T7 RNA Polymerase and RNase free DNase I were purchased from Fermentas. All the other chemicals used were local products of analytical grade. Data analysis for turbidity measurements and light scattering experiments was performed using OriginPro8 software and statistical analysis for bar diagrams performed using Sigma Plot 13. The densitometry analysis was done using Quantity One (Bio-Rad).Bovine carbonic anhydrase II (BCAII), hen egg white lysozyme, GuHCl, DTT, Tris-base and total 2, 100 mM NaCl192, 30 mM KCl, 70 mM NH4ClThe buffers used in this study are the following: Buffer A: 50 mM Tris-HCl (pH 7.5), 10 mM MgClE. coli large subunit RNA (bDV RNA) and bovine mitochondrial large subunit RNA (mDV RNA), cloned in plasmid pTZ57R/T were kind gifts from the laboratory of Professor Chanchal Dasgupta . The mDV RNA has a truncated RNA2 sub-domain and therefore shows a delay in releasing the bound proteinE. coli domainV was cloned into the pTZ57R/T vector downstream to the T7 Polymerase promoter. The RNA corresponding to mDV RNA, bDV RNA, RNA1 and RNA2 were synthesized by run-off transcription and prepared as described earlier101928in vitro transcription of the E.coli Ribosome modulation factor gene cloned in pET 28a vector available in the laboratory) on lysozyme and BCAII aggregation.The DNA corresponding to domainV of the E. coli MRE 600 cells and the ribosomal subunits were prepared as described in ref. Ribosomes were purified from E. coli tRNA to the free or empty ribosome was performed in Buffer-P as described earlierThe binding of total Partially unfolded lysozyme aggregates under reducing condition and is a widely used model for protein aggregation as stated earlier. In our studies the aggregation behaviour of lysozyme and bovine carbonic anhydrase II was monitored in presence of the ribosome and bDV RNA by turbidity measurements at 450 nm in Hitachi spectrophotometer (U-1900) and by scattering measurements in a Hitachi spectrofluoremeter (F-2700) with excitation and emission set at 450 nm respectively in Buffer A. Since the ribosome shows light scattering at 450 nm, all the time course studies in presence of the ribosome were done by measuring the change in turbidity at the mentioned wavelength.Hen egg white lysozyme (2 μM or 10 μM or 65 μM) was incubated in 20 mM DTT  in the absence or presence of 70S ribosome (1 μM or 0.1 μM) and rRNA (0.02 μM–2 μM). For studies with BCAII the protein (200 μM) was denatured with 6 M guanidine hydrochloride and 3.5 mM EDTA for 3 hours and then diluted 100 fold in the absence or in the presence of ribosome (0.02 μM except mentioned in the figure legend) and rRNA (0.02 μM–2 μM) in Buffer A to study the aggregation process. The denatured and reduced lysozyme was prepared by incubating lysozyme (200 μM) with 6 M guanidine hydrochloride and 100 mM DTT for 3 hours. The aggregation process of denatured and reduced lysozyme was studied by 100 fold dilution of the denaturants in the absence or in the presence of ribosome (0.2 μM). Protein aggregation was also monitored in the presence of rRNA extracted from the ribosomal subunits using lithium chloride. The rRNAs were present different stoichiometric ratios with respect to the protein as indicated in the Figures.in vitro transcribed bDV RNA  or stoichiometric concentrations of RNA1, RNA2 and mDV RNA and the aggregation was followed using light scattering measurements as stated above. Similar experiments were also performed to study the effect of equimolar concentrations of heparin (2 μM) on lysozyme aggregation. The increase in light scattering was followed for a period of 45 min and 20 min for reduced lysozyme or denatured BCAII respectively and the increase in scattering intensity in presence of heparin and the different RNA variants were plotted as bar diagrams for comparison. The light scattering experiments performed with the rRNA and the bDV RNA variants were repeated five times and the data represents the average of all these experiments.To study the effect of bDV RNA on 2 μM of lysozyme (under reducing conditions) and or 2 μM denatured BCAII (under refolding conditions) were incubated with The proteins (lysozyme and BCAII) were incubated with the 70S ribosome, 80S ribosome, bDV RNA for 45 minutes or as mentioned in the figure legend (lysozyme) or for 20 minutes (BCAII). The insoluble aggregates formed were separated by centrifugation at 21,380 g for 20 minutes at 4 °C. The pellet was resuspended in the Buffer A. The supernatant was concentrated using Amicon Ultra-10K filter. The resuspended pellet and supernatant were loaded on 12% SDS –PAGE for analysis of the proteins constituting the aggregates. The RNA incorporated in the aggregate was analysed by 1% agarose gel electrophoresis of the pellet resuspended in Buffer A containing 1 M urea. For all agarose gel experiments, the concentration of 70S or 80S ribosome used was 0.1 μM.For the control experiment where the effect of native BCAII was tested on lysozyme aggregation, 10 μM lysozyme in presence of DTT was incubated with or without 2 μM, 1 μM, 0.5 μM BCAII in Buffer A containing 20 mM DTT for 45 minutes at room temperature and centrifuged at 21,380 g for 20 minutes. The pellet fractions were resuspended in 40 μl of Buffer A and the supernatant fractions were concentrated as described above before loading on a 12% SDS - PAGE.32P-labelled bDV RNA was obtained by in vitro transcription (stated above) in presence of a 32P-UTP followed by DNase I digestion and precipitation using ethanol. Lysozyme was incubated with equimolar 32P-bDV RNA in the presence of 20 mM DTT for various time intervals and filtered through pre-soaked nitrocellulose filter paper (Millipore) with pore size of 0.22 μm. The filters were dried and 32P counts were taken in a liquid scintillation counter (Perkin Elmer). The RNA bound to the protein was retained on the filter while the free RNA passed through it. The percentage of radioactivity retained on the filter paper, calculated considering the radioactive count incorporated in the total RNA to be 100%, was plotted against time.Filter binding studies were performed as described in refs Lysozyme (10 μM) was incubated with 70S ribosome (0.1 μM) for different time intervals  in presence of 20 mM DTT and the samples were diluted 10-fold in Buffer A before preparation of EM grids were prepared immediately as stated in ref. How to cite this article: Pathak, B. K. et al. Sequestration of Ribosome during Protein Aggregate Formation: Contribution of ribosomal RNA. Sci. Rep.7, 42017; doi: 10.1038/srep42017 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


C-to-U RNA editing in mitochondria and chloroplasts and the nuclear-encoded, RNA-binding PPR proteins acting as editing factors present a wide field of co-evolution between the different genetic systems in a plant cell. Recent studies on chloroplast editing factors RARE1 and CRR28 addressing one or two chloroplast editing sites, respectively, found them strictly conserved among 65 flowering plants as long as one of their RNA editing targets remained present.Arabidopsis thaliana. We found CLB19 consistently conserved if one of the two targets was retained and three independent losses of CLB19 after elimination of both targets. The Ericales show independent regains of the ancestrally lost clpPeU559HY editing, further explaining why multiple-target editing factors are lost much more rarely than single target factors like RARE1. The retention of CLB19 despite loss of both editing targets in some Ericaceae, Apocynaceae and in Camptotheca (Nyssaceae) likely represents evolutionary transitions. However, the retention of CLB19 after a phylogenetic deep loss in the Poaceae rather suggests a yet unrecognized further editing target, for which we suggest editing event ndhAeU473SL.Extending the earlier sampling to 117 angiosperms with high-quality genome or transcriptome data, we find more evidence confirming previous conclusions but now also identify cases for expected evolutionary transition states such as retention of RARE1 despite loss of its editing target or the degeneration of CRR28 truncating its carboxyterminal DYW domain. The extended angiosperm set was now used to explore CLB19, an “E+”-type PPR editing factor targeting two chloroplast editing sites, rpoAeU200SF and clpPeU559HY, in Extending the scope of studies on plant organelle RNA editing to further taxa and additional nuclear cofactors reveals expected evolutionary transitions, strikingly different evolutionary dynamics for multiple-target editing factors like CLB19 and CRR28 and suggests additional functions for editing factor CLB19 among the Poaceae.The online version of this article (10.1186/s12862-018-1203-4) contains supplementary material, which is available to authorized users. The simultaneous existence of three separate genomes in the chloroplast, mitochondrion and nucleus in every plant cell requires co-adaptation and co-evolution for successful and co-ordinated gene expression . GeneticArabidopsis thaliana, more than 70 PLS-type editing factors addressing individual or multiple sites in chloroplasts or mitochondria have been identified.A particularly wide field of investigation for nucleus-organelle co-ordination and co-evolution has emerged with the identification of specific RNA editing factors addressing the numerous sites of C-to-U RNA editing in plant chloroplasts and mitochondria. Essentially, the individual sites of RNA editing in the two organelle transcriptomes are targeted by a special class of RNA-binding pentatricopeptide repeat (PPR) proteins , 4. ThesPhyscomitrella patens carry the full suite of carboxyterminal domains E1-E2-DYW [The ultimate carboxyterminal DYW domain in most editing factors has received particular attention owing to its evident similarity with cytidine deaminases –11 and i1-E2-DYW –16, manyArabidopsis thaliana, Nicotiana tabacum or Oryza sativa with some 30–50 editing sites, editing is much more abundant in the chloroplast transcriptomes of early-branching angiosperms such as Amborella trichopoda with more than 130 chloroplast edits [Arabidopsis (20 of 32) were also identified in Amborella, possibly indicating their very ancient origin among angiosperms. Moreover, those shared chloroplast editing sites included some, for which a specific editing factor had already been identified in Arabidopsis, including CRR28 and RARE1, which opened the possibility to identify editing factor orthologues and trace their evolutionary history. Defining a 65 taxon-set of angiosperms with high-quality genome (and/or transcriptome) data available, we indeed consistently identified CRR28 and RARE1 orthologues in all angiosperms where a requirement for editing remained at the respective chloroplast targets. Intriguingly, RARE1 concomitantly disappeared with loss of its editing target accDeU794SL through C-to-T conversion at DNA level at least 14 times independently [Cicer arietinum) once both original editing targets were simultaneously converted into thymidines in the cpDNA making C-to-U editing at these two sites obsolete. These observations suggested that the nuclear-encoded RNA editing factors in plants may disappear surprisingly quickly once the necessity for editing at their organelle target sites is lost and that a single-target editing factor like RARE1 gets lost more frequently than a multiple-target RNA editing factor like CRR28.The growing list of flowering plants for which high quality genome (and/or transcriptome) data have become available should allow to trace the co-evolution of editing factors and their cognate RNA editing targets. Of interest in that regard are ancient RNA editing factors emerging early in the evolutionary history of flowering plants that allow to track their evolutionary fate and those of their cognate editing sites for ca. 140 million years of flowering plant diversification. We recently found that in contrast to only moderate chloroplast RNA editing in typically investigated angiosperm models like st edits . Interesendently . In contTo further evaluate those conclusions, we investigated additional high-quality angiosperm genome and transcriptome data that have become available, thus significantly extending the original 65 taxa sampling into a 117 angiosperm species data set. The widely extended taxon sampling largely corroborates the above conclusions. We identified four new cases for simultaneous loss of editing site accDeU794SL and its corresponding editing factor RARE1. In contrast, we found that CRR28 was consistently retained in seven new cases of losing either the one or the other of its two editing targets ndhBeU467PL or ndhDeU878SL.Arabidopsis thaliana: clpPeU559HY and rpoAeU200SF [Given the intriguing results on CRR28 we now additionally investigated CLB19 as another chloroplast editing factor previously shown to address two chloroplast editing sites simultaneously in AeU200SF . We idenwww.ncbi.nlm.nih.gov). Ultimately, we thus expanded our sampling to now comprise 117 angiosperms included in the cladograms discussed in the following , cardoon (Cynara cardunculus) and lettuce (Lactuca sativa). Other important addendums are the elevated number of Caryophyllales species, especially of the Silene genus which is known to be highly variable in mitochondrial RNA editing [Arachis hypogea and Gossypium hirsutum were replaced with closely related species A. duranensis, A. ipaensis, G. arboreum and G. raimondii, respectively, which offered yet better sequence qualities.We re-applied our previous criteria originally resulting in a 65-angiosperm data set  to identng Figs. . The new editing , 20 and Eucalyptus, Fragaria, Cicer, Nicotiana tabacum and in the palms  we now observed five additional independent losses of this editing site in Azadirachta, in Cannabis and Humulus, in Dianthus and Silene, in Bienertia and in Ipomoea . In all these additional cases of losing either the one or the other editing target site, CRR28 orthologues are retained, evidently because the respective other editing site needs to be addressed. The extended CRR28 protein phylogeny is in full agreement with the species phylogeny indicating common orthologue ancestry  remains the only case of a double loss of both RNA editing sites and the only case where no CRR28 homologue could be detected , however, are truncated behind the so-called “PG-box” at the beginning of the DYW domain , in Lactuca, in the Ranunculales (Aquilegia), in the Myrtales (Metrosideros) and in the Fagaceae (Castanea and Quercus). Note that instead of a C-to-T conversion in the chloroplast gene copy, the loss of editing may alternatively occur (e.g. in Poaceae or in Trifolium subterraneum) as a result of endosymbiotic gene transfer of accD to the nucleus [Morus, the extended sampling identified a total of ca. 20 independent losses of the accDeU794SL editing site  and chestnut (Castanea), the two Fagaceae species  in the extended taxon sampling, although a C-to-T conversion in their cpDNAs makes RNA editing obsolete at the previous accDeU794SL editing site  for RNA target recognition, see below) arguing for a comparatively recent evolutionary loss of the editing target by C-to-T conversion or accD transfer to the nucleus. This is a likely scenario for the dynamic cpDNA evolution in the genus Trifolium [Castanea and Quercus ancestor would be dated to approximately 4 mio. Years ago ([www.timetree.org).In the clear majority of the more than 50 angiosperms in our sampling which lack the accDeU794SL editing site, this is linked to the simultaneous absence of editing factor RARE1 in the genomic data. However, we now find that RARE1 is retained in the genomes of oak  and Chloranthus spicatus , representing other early-emerging flowering plant lineages. This clearly revealed that the clpP and rpoA mRNAs are typical examples for chloroplast genes more affected by RNA editing in the early-branching lineages with three or even four (in Illicium) additional sites of editing in clpP and rpoA , in Camptotheca acuminata , in Vaccinium macrocarpon , in the Apocynaceae  and in Tarenaya hassleriana . In three of the above cases – Vaccinium macrocarpon, Amaranthus hypochondriacus and Tarenaya hassleriana – the simultaneous loss of both editing sites has obviously resulted in the loss of CLB19 orthologues from the nuclear genomes  where the ancestrally lost editing site clpPeU559HY has been regained  and the retention of the editing factor for a certain period of evolution after loss of both target sites  before its disintegration and loss (in Andromeda and Vaccinium).The loss of the rpoAeU200SF editing site following the earlier loss of the clpPeU559HY editing target that was initially identified in pon Fig.  appears pon Fig. . An indened Fig. . Hence, www.timetree.org, [Oryza sativa . Accordiva Fig. . An A-toiva Fig. . In conttes Fig. .Fig. 5AlCoffea and Catharanthus is accompanied by C-to-A conversion seven nucleotides upstream of the former editing sites opposite of PPR S-4. Conversely, C-to-U transition six nucleotides upstream of the rpoAeU200SF edit (corresponding to PPR P2–3) would improve target recognition, but this change is shared between Coffea retaining the editing site and Catharanthus, which has lost both CLB19 editing targets.The situation is similar in the case of the Gentianales Fig.  althoughThe widely extended inspection of flowering plant genome and transcriptome data reported here has, on the one hand, fully corroborated the previous insights on editing factors CRR28 and RARE1 . The sinCicer arietinum hence remains the only evident example for loss of CRR28 upon serial loss of both its editing sites.In contrast to RARE1, the dual-target editing factor CRR28 is retained also in all the now identified additional cases of losing either the one or the other of its editing site targets  in the sister species A. tricolor suggesting a rapid loss of CLB19 within the genus Amaranthus. Likewise, evolution of CLB19 and its targets is particularly interesting among the Ericales  but also the retention of CLB19 after loss of both targets , which is to be expected as an evolutionary intermediate state , at least one taxon featuring only edit clpPeU559HY (Erica), several genera retaining CLB19 without both targets  and finally those having lost CLB19 after loss of the two targets (Andromeda and Vaccinium).Very much like in the CRR28 case, we identified several flowering plants retaining CLB19 if only the one or the other of its editing target site was lost Fig. . Similarles Fig. . Not onlate Fig. . Moreoveate Fig. . This rerpoA target was found to be preferred over the clpP target in the EMSA studies by Kindgren et al. [rpoA target completely [Whereas the above scenarios fit evolutionary expectations, the long-term retention of CLB19 despite an early loss of both known editing targets among the Poales likely calls for additional explanations. Although RNA-binding to its likely targets could initially not be demonstrated for CLB19 , it has n et al. , somewhan et al. , 40, 41.mpletely , as predmpletely .Coffea  implemented in a new version of our PREPACT service (www.prepact.de) [Amborella trichopoda [We here suggest an explanation for the retention of the CLB19 orthologue in Poales where both editing targets identified in pact.de) , we findichopoda , but losCamptotheca lineage  [http://blast.ncbi.nlm.nih.gov/Blast.cgi). Qualities of genome and/or transcriptome data were evaluated for the presence of three conserved nuclear protein genes: PPR proteins VAC1/ECB2 and PDM1/SEL1 and the arginine decarboxylase ADC previously used for phylogenetic studies [Arabidopsis proteins as most similar homologues. Alignments initially obtained with the MUSCLE tool integrated in MEGA were manually edited. Alignments are available from authors upon request._196831) , CRR28 (_196831)  and CLB1_196831)  were use_196831)  against  studies . The qua studies . The MEG studies  was usedhttp://iqtree.cibiv.univie.ac.at/) [Final alignments were used for calculation of ML (Maximum Likelihood) phylogenetic trees using the IQ-tree webserver (.ac.at/) . The JTTaccD, clpP, ndhB, ndhD, and rpoA) were ideally collected corresponding to the nuclear genome taxon sampling or taxonomically as closely as possible  or data from available closely related sister taxa were employed as e.g. Agave americana or Dianthus longicalyx.Wherever available, data from complete chloroplast genome assemblies were used. Nucleotide coding sequences for chloroplast target genes  and Go-Taq polymerase (Promega) or Q5 polymerase (New England Biolabs) approaches. PCR products were isolated from agarose gels using the NucleoSpin Extract II Kit (Macherey & Nagel) and sequenced directly or after ligation into the pGEM-T Easy vector (Promega). Commercial Sanger sequencing was done by Macrogen Europe . A compilation of cDNA results obtained in the course of this work, in previous studies [Plant material for rotocols , 50. RNA studies , 37, 51  studies , 52 is gAdditional file 1:Phylogeny of the CRR28 orthologs in angiosperms. Shown is a Maximum Likelihood tree see . Additional file 5:Table of essential positions for RNA recognition in the PPRs of CLB19 and the corresponding RNA targets. (XLSX 40 kb)


The selection of a future breeding dog is a complicated task, in which disease characteristics and different traits have to be combined and weighed against one another. Truncation selection, that is the exclusion of affected animals, may be very inefficient when selecting on a large number of traits, and may result in a reduction of the genetic diversity in a population or breed. Selection could be facilitated by the use of a selection index that combines multiple traits or breeding values into one score. This however requires a consideration of their relative value according to their economic weight, which is difficult to express in monetary units for health traits. The use of a choice experiment to derive non-market values might be a solution to this problem. This is a pilot study to assess the potential use of choice experiments to ascertain the public preference and relative importance attached to health- and conformation traits in the selection of a Cavalier King Charles spaniel. The focus was on two prevalent disorders, mitral valve disease and syringomyelia, and on several important conformation traits such as muzzle length and eye shape. Based on available prior information, a Bayesian D-optimal design approach was used to develop a choice experiment and the resulting choice sets.Every participant (breeder or owner) in the choice experiment was presented with a total of 17 choice sets, in which at most four traits could vary to reduce the cognitive burden. A total of 114 respondents participated in the choice experiment and results showed that respondents (breeders/owners) current attitudes were directed towards health , followed by eye shape and level of inbreeding.This approach identifies the value breeders and owners attach to certain traits in the breeding objective. The resulting relative weights, represented as the logworths obtained from the choice experiment, could be an alternative to economic weights. They could be implemented as a weight when breeding values are available, but more study on this topic will be necessary. A challenge in this approach is to scale up the experiment with additional traits. Moreover, for other traits, the genetic parameters and correlations should be known first, in order to include them in the health selection index as well. When selecting a future breeding dog, different disease characteristics and other traits have to be balanced against one another, which makes it a complicated task. In the case of selecting for a large number of traits, the exclusion of all affected animals might be very inefficient, since this may reduce the genetic diversity in a population or breed. A solution could be the use of a selection index, in which all traits of interest are combined into one single value according to their relative weight. For this however, traits need to be evaluated according to their economic weight, which is difficult for health traits. This study examined breeders and owners attitudes and the importance they attached to three health- and three conformation traits in the selection of a Cavalier King Charles spaniel, and this using a choice experiment. The focus lay on two very prevalent disorders, mitral valve disease and syringomyelia, and on several important conformation traits such as muzzle length and eye shape. Results indicated that breeders and owners valued health traits highest followed by eye shape and level of inbreeding.Many modern dog breeds suffer from multiple heritable diseases with varying prevalence and severity. The choice of potential breeding animals involves the consideration of a multitude of traits and disease characteristics. The combination of all desired characteristics seriously complicates the selection of potential breeding animals. Selection is further complicated by the need to maintain the genetic diversity of a breed as the exclusion of animals with any undesired trait will lead to a genetic bottleneck in the breeding population .The Cavalier King Charles spaniel (CKCS) breeders have been strongly criticized since the BBC documentary “pedigree dogs exposed” BBC, 2008). CKCS are prone to several genetic diseases, such as mitral valve disease (MVD) and syringomyelia (SM). MVD is a heart disease in which the mitral valve closes insufficiently leading to mitral regurgitation 008. CKCS. A higheThe use of a selection index would aid the search for the most optimal breeding animal as it combines health and conformation traits into one score by considering the relative values of each trait according to their economic weight . HoweverThis paper assesses the potential usefulness of a choice experiment to ascertain the relative importance breeders and owners attach to different traits within the Cavalier King Charles spaniel (CKCS) This study originated as a response to a recently invoked law that will oblige Flemish CKCS breeders to screen their dogs prior to breeding. We believe this pilot study is the first to investigate the potential of choice experiments in the domain of dog breeding.Conducting a choice experiment consists of different steps. First, the attributes (traits) and attribute levels need to be defined. Secondly, a suitable experimental design needs to be constructed: alternatives need to be selected for presentation to the respondents and they need to be combined in choice sets in order to generate as much information as possible concerning the respondents’ preferences. Next, the questionnaire has to be developed. Finally, after collecting the responses, they need to be analysed statistically using a suitable choice model .The potential traits of interest to CKCS breeders, including general characteristics of the breed standard as well as disease traits, were listed in consultation with stakeholders and experts in CKCS breeding, including the Belgian Cavalier Kennel club and the president of the Cavaliers for Life foundation. As the number of attributes in a choice experiment is limited to reduce the cognitive burden of the respondents , only foFor the design of the choice experiment, the module “Design of experiments” of the JMP software was used . Based on the multinomial logit model for choice data, a Bayesian D-efficient partial profile design was constructed, optimised for 17 choice sets. This D-efficient design takes into account the available prior knowledge about the preferences of the respondents. Thereby, the D-efficient design avoids uninformative choice sets in which one alternative completely dominates the other . A pilotThe choice experiment was presented as a questionnaire. We provided respondents with a web link, allowing them to carry out the experiment at their convenience. Questionnaires were sent via email by the Belgian Cavalier club “Pejatoyspa” to a sample of breeders and owners, and it was advertised on the website of the Cavaliers for Life foundation. To help respondents, we presented an explanation of the experiment to familiarize them with a choice experiment and the 8 selected attributes and their levels. We asked the respondents to choose between two different dogs they would use as a breeding animal, displaying a number of attributes. Additionally, general information was collected about the respondents’ age, sex, country of origin, occupation , the number of dogs owned, the number of CKCS owned, and whether their CKCS is affected by one of the diseases. To avoid fraudulent respondents, a captcha verification was used before starting the questionnaire.10  [The data analysis was performed using the “choice modelling” platform in JMP . The relative importance of each trait was quantified by estimating the multinomial logit model in the eight attributes using a maximum likelihood approach .This reod test) . To testBetween March 2016 and September 2016, internet questionnaires from different CKCS owners and breeders were collected. The open distribution of the questionnaire did not allow a classical calculation of the response rate. However, a total of 207 respondents visited the website online, of which 114 fully completed the survey and 87 filled it in partially. Respondents had to make 17 choices each, which resulted in a dataset of 1938 choices. Only the fully completed questionnaires were considered for further analysis.One part of the survey was intended to collect generation information concerning the respondents. Out of the 114 respondents who completed the questionnaire, 103 (90.37%) were women and 10 were men (8.77%). One respondent did not fill in this question. Almost all respondents were dog owners themselves (111 respondents or 97.37%), 46 (40.35%) were also dog breeders, and 11 (9.65%) were show jury members. 86.85% (99) of the respondents had a CKCS at home, and 63% of these already had been confronted by a genetic disorder . Respondents originated from different countries, with 28 (25%) coming from Belgium, 20 (18%) from Denmark, 18 (16%) from USA, 22 (19%) from UK, 5 (4%) from the Netherlands and 19 (17%) from other countries. Two respondents (2%) did not fill out this question.At first, the effect of being an breeder was investigated by including interaction term of breeder × attribute in the model. None of these interaction terms reached significance  and mitral valve disease (MVD) are the most important attributes in the choice of a dog. SM is even twice as important as MVD (logworth of 130 compared to 54 respectively). Three other traits, eye shape, level of inbreeding and eye disease, have an equal importance , followed by muzzle length, and coat colour which had almost no importance (logworth of 5 only).The marginal utility values for each trait show that, for the included disease traits, the values for “tested dogs free from the disease” are positive, whereas those for “tested and disease present” or “not tested” are negative. This indicates that participants attribute a higher utility value to a tested dog compared to both the non-tested animals and the tested animals that were affected by the disease. The marginal utilities for non-tested animals are less negative than for animals known to have a disease, indicating that the respondents prefer non-tested animals over animals with the disease. When comparing the values for the “tested and disease present” status among the included diseases, the marginal utility value for SM is highest, followed by MVD and eye disease, indicating that respondents value SM as the worst disease. Concerning the level of inbreeding, the marginal utilities become negative when exceeding an inbreeding level of 6% or more.This study invited breeders and owners to express their attitudes towards the importance of traits in the selection of a CKCS by means of a choice experiment. This approach has its advantages. It presents the respondents with different alternatives and choosing the preferred alternative is a more realistic task compared to for example conjoint analysis, in which respondents are asked to rank the different profiles . A critiThe final study was completed by 114 respondents, of which 90% were female. This bias is frequently seen in other questionnaires applied to dog owners –21 and cThe choice experiment showed that no difference in preference between breeders or owners, they both attached most importance to the health traits such as SM and MVD. This was partly an expected result, as the CKCS breed is known to suffer from these diseases , 6. BothThe importance that is attached to the health traits in this study is not surprising, given the importance that is attached to it in the breeding recommendations prior to breeding by the British Kennel Club (an assessment by the British Veterinary Association / Kennel Club breeding scheme). However, these preventive screening measures for SM and MVD are not compulsary. In Belgium, preventive measures are only recently mandatory and this only for half of the country (recommended by the Flemish government since 2016). Furthermore, the importance attached to health traits can also be reflected by the high prices of the treatment of these traits. For example SM can be treated medically or by surgical management, which is costly . Our stuThe choice experiment which was applied in this study was a first investigation in the rational thoughts of breeders and owners towards some non-economic traits such as desired conformation or beauty traits in the selection of a CKCS. A next step could be to include other possible traits of interest. Our study approach resulted in logworths, that give an indication of the relative importance of each trait and can be a first step towards creating a comprehensive total selection index, similar to a multi-trait index in cattle, which can be used to ameliorate the breed . HoweverThere is an additional challenge when an overall index would become available. Ideally selection should be balanced with the rate of inbreeding, applying for example optimal genetic contribution. A step towards balanced breeding might be to restrict the use of sires at an international level. Regulations should be made to promote using seldom-used lines or animals, and to limit the use of popular sires. The use of popular sires has been shown to lead to a dissemination of genetic disorders . Also moAs a conclusion, this study assessed the priorities and relative importance of traits in the selection of a CKCS as breeding or companion animal using a choice experiment. The marginal utility of each trait can be used as a weighing factor to define a total selection index, including health- and non-economic traits.


At the epicenter of RNA epigenetics, m6A influences essentially all stages of RNA metabolism. As a result, m6A modulates cell differentiation and pluripotency, cell cycle and tumorigenesis, and several types of stress responses, etc. A recent report by Shi and colleagues uncovers a novel pathway in which m6A RNA, its associated enzymes, and DNA polymerase κ constitute an early-response system that confers cellular resistance to ultraviolet irradiation, separate from the canonical nucleotide excision repair (NER) pathway that normally repairs UV-induced DNA damage.N6-methyladenosine (m Among the trio of linear heteropolymers that enable organic life as we know it, RNAs are recognized as the most versatile and self-sufficient. In order to serve its primary directive in the faithful preservation and propagation of genetic information to subsequent generations, DNA assumes a protective, inert duplex structure where sequence information is carefully concealed in is hydrophobic, stacked central axes. Further, the genetic information is maintained in duplicates, in the form of two paired strands, each replete with the 4-bit informational strings. Despite such extraordinary measures to preserve the genetic information, cellular DNA is subjected to a constant onslaught of chemical and environmental insults that create lesions that interfere with its replication, transcription, modification, etc. Depending on the source  and type of DNA damage , cells employ a battery of DNA repair pathways to detect and correct the damages. These include base excision repair (BER), nucleotide excision repair . To remedy such limitations, RNA has evolved more than 100 types of chemical elaborations on its nucleobase and ribose moieties. For instance, an average 12% of nucleotides on tRNA (~8 per tRNA molecule) are post-transcriptionally modified. RNA posttranscriptional modifications confer numerous advantages and nuances in modulating RNA structure and function, some of which are further dynamically coupled with the metabolic state of the cell.6A), N1-methyladenosine (m1A), pseudouridine (ψ), 5-methylcytosine (m5C), and 2′-O-methylation (2′-OMe), etc. [6A, or N6-methyladenosine modification, is the most abundant internal modification in eukaryotic mRNA (~3–5 sites per mRNA) [6A plays wide-ranging roles in essentially all known steps of RNA metabolism, affecting RNA splicing and processing, nuclear export and localization, translation and stability, etc. These effects are manifest in phenotypic effects on cell differentiation, pluripotency, tumorigenesis, circadian rhythm, etc. [6A is involved in several types of stress responses, including heat shock, nitrogen starvation, oxidative stress, hypoxia, etc. [6A modification can lead to immediate remodeling of RNA secondary and tertiary structures, as m6A weakens duplex formation and stabilizes stacking within single-stranded regions. Such thermodynamic effects not only modulate RNA structure, but also regulate its access by RNA-binding proteins such as HNRNPC, constituting “m6A switches” [6A modification is well suited for fast-acting responses to cellular and environmental stimuli.Using genome-wide RNA sequencing methods that can detect and enrich specific modified RNA, it was recently discovered that in addition to tRNAs, rRNAs, snRNAs, etc., that are canonically modified, mRNAs are also elaborated with a number of distinct chemical modifications including N6-methyladenosine (mer mRNA) . m6A plaia, etc. . The depwitches” . Taken t6A acting as early responders of stress response, Shi, He, and colleagues have recently reported a remarkable discovery that m6A in RNA, its associated enzymes Poly-ADP ribose polymerase (PARP), N6-adenosine-methyltransferase heterodimer (METTL3/14), Fat mass and obesity-associated protein (FTO), and DNA polymerase κ (Pol κ), form a novel, dynamic pathway that launches a rapid response (within 2 min) to UV-induced DNA damage [Along the emerging theme of mA damage .6A, Xiang et al., detected rapid, reversible accumulation of m6A RNA at the sites of irradiation [6A mark at these DNA damage sites , an inability to recruit downstream effector Pol κ to the damage site, significantly delayed re-initiation of nascent transcription after DNA damage, and ultimately drastically reduced cell survival . Remarka6A plays and makes a surprising connection between m6A RNA biology and DNA repair. Like any other transformative discovery, this work raises more questions that it answers. For example, are specialized transcripts or all nearby RNAs responsible for aiding DNA damage repair? The authors address this question by capturing the GGACU motif used for METTL3 recruitment and demonstrate the enrichment of UV-induced m6A in 5′ untranslated regions (UTR) in a metagene analysis. This finding echoes an earlier analysis of the heat shock response, where increased m6A in 5′ UTR promotes cap-independent translation [6A-marked transcripts play a more direct role besides recruiting METTL3? Besides known protein-binding primary sequence motifs, could the RNA secondary, tertiary, or quaternary structures  contribute to factor recruitment or other functions in this context? Which protein reads the UV-induced m6A deposition and is this protein or the RNA or both that ultimately recruit Pol κ? What is special about Pol κ that contributes to its unique recruitment and activity at the damage site, not possessed by other DNA polymerases such as Pol δ, ε, η, ι, etc.?This timely work expands the already impressive repertoire of cellular functions mnslation . Are theNevertheless, what is clear is that this landmark paper leaves us with a fertile area for future research and makes one wonder what other pathways would be next to cross paths with RNA epigenetics and noncoding RNA biology.


The CRISPR/Cas system, in which the Cas9 endonuclease and a guide RNA complementary to the target are sufficient for RNA-guided cleavage of the target DNA, is a powerful new approach recently developed for targeted gene disruption in various animal models. However, there is little verification of microinjection methods for generating knockout mice using this approach. Here, we report the verification of microinjection methods of the CRISPR/Cas system. We compared three methods for injection: (1) injection of DNA into the pronucleus, (2) injection of RNA into the pronucleus, and (3) injection of RNA into the cytoplasm. We found that injection of RNA into the cytoplasm was the most efficient method in terms of the numbers of viable blastocyst stage embryos and full-term pups generated. This method also showed the best overall knockout efficiency. Mouse is the most widely used mammalian model organism. However, conventional gene targeting methods using homologous recombination in mouse embryonic stem (ES) cells are generally used for genetic research. The recent development of site-specific endonucleases for selective genome cleavage has been an important advancement in genome engineering. These enzymes include zinc-finger nucleases (ZFN)4in vitro transcribed RNA would be a better alternative. However, Cas9 RNA and the gRNA work in different locations, the cytoplasm and pronucleus, respectively. Theoretically, it would be best to inject each RNA into the cytoplasm or pronucleus, but, in practical terms, such injections are very difficult. Here, we report a comparison of three different injection methods: (1) injection of DNA into the pronucleus, (2) injection of RNA into the pronucleus, and (3) injection of RNA into the cytoplasm. We found that the injection of RNA into the cytoplasm was the most efficient method and yielded the greatest numbers of normal blastocyst stage embryos and full-term pups. This method also showed the best overall knockout efficiency.However, this study left a number of issues unresolved. For example, which microinjection method is the most successful for generating knockout mice. Two components are introduced into oocytes to make knockout mice by the CRISPR system. One is the Cas9 nuclease RNA and the other is a gRNA complementary to the target DNA. The most direct method would be to inject the vectors for Cas9 and gRNA into the pronucleus; however, this method has the risk of integration of the vectors into the chromosomes, even when using circular plasmidsTet1 gene, which encodes a member of the tet methylcytosine dioxygenase family (Tet1 gene whose 16 bp sequence (N14GG) did not cross-react with any other sites in the mouse genome was selected and used to construct the gRNA expression vector. To examine the knockout efficiency of the gene, ES cells were co-transfected with the Cas9 expression vector and the gRNA vector targeting Tet1. The cells were collected 48 h after transfection and DNA was extracted. The targeted locus contains a Sac I restriction site; therefore, successful targeting is indicated by a disruption of the Sac I site. The digested product was subjected to electrophoresis and the knockout efficiency of the Tet1 gene was determined (Tet1 loci was successful targeted (55%), as indicated by disruption of the Sac I site, while cells transfected with the Cas9 expression vector and a control gRNA vector showed no disruption of the Sac I site. Thus, this gRNA was applicable for making knockout mice.We designed a gRNA for exon 4 of the e family . Tet protermined . The TetTet1 gene. Similar results were also obtained for this target (We compared three methods for injection: (1) injection of DNA into the pronucleus, (2) injection of RNA into the pronucleus, and (3) injection of RNA into the cytoplasm. RNAs for injection were transcribed in vitro and DNAs were circular plasmids. The nucleic acids were injected into the cytoplasm or pronucleus of 1-cell stage C57BL/6 embryos. After injection, the zygotes were immediately transferred into pseudopregnant female mice. The developmental efficiency to the blastocyst stage is expressed as the percent of the total injected embryos developing to the blastocyst stage. At the blastocyst stage, the developmental efficiency for methods (1), (2), and (3) was 24%, 33%, and 65%, respectively , Table 1s target .Tet1 was amplified by PCR. The PCR products were digested with Sac I and subjected to gel electrophoresis for genotyping. There were no apparent phenotypic differences among the knockout mice generated by the three methods (Tet1 knockout mice and found that the homozygote showed significantly lower birth weight than wild-type mice as reported previouslyTet1 newborn mice showed that percentages of gene disrupted mice per newborn pups were similar among three injection conditions (1) injection of DNA into the pronucleus (80%), (2) injection of RNA into the pronucleus (100%), and (3) injection of RNA into cytoplasm (100%)  , Table 2 methods , Table 2Tet1 exon 4 from five pups per injection method. PCR products were TA cloned and sequenced. The Tet1 sequences of the two alleles of each pup are shown in To determine the mutations generated by CRISPR in individual founders, we analyzed the sequence of the CRISPR/Cas is a rapid and efficient method for many species in which genome engineering has been difficult11121314151617The injection of RNA into the cytoplasm showed the best overall knockout efficiency normalized by the number of transferred zygotes. This result indicates that cytoplasmic injected guide RNAs can interact with the chromosomes and function as a guide to cleave chromosomal DNA. There are two possible mechanisms for this process. The guide RNAs may enter the pronucleus, or, alternatively, cleavage may occur after pronuclear envelope breakdown. If the latter is true, it could explain why the cytoplasmic RNA injection showed the best overall knockout efficiency. The total amount of RNA injected is larger in zygotes subjected to cytoplasmic injection because the volume of cytoplasm is larger than the nucleus. After pronuclear envelope breakdown, the pronucleus and cytoplasm are no longer separate and the amount of RNA injected determines the concentration of injected RNA. However, the percentage of pups produced by cytoplasmic injection  and the The percentages of homozygous knockouts per KO mice were significantly lower in the mice that received an injection of DNA into the pronucleus, compared to the other two groups . InjecteInterestingly, most of the biallelic mutant mice have the same deletions . All of 19http://www.addgene.org/41824/) was linearized using Afl II. The 100 bp DNA fragment was incorporated into the vector using Gibson assembly (NEB).Two oligonucleotides  were annin vitro transcription (IVT) using mMESSAGE mMACHINE T7 ULTRA kit (Life Technologies). A four nucleotide spacer (GCGT) and the T7 promoter were added to the gRNA template plasmid by PCR amplification using the Tet1 primers (Tet1 gRNA PCR product was gel purified and used as the template for IVT using a MEGAshortscript T7 kit (Life Technologies). Both the Cas9 mRNA and the gRNAs were purified using a MEGAclear kit (Life Technologies) and eluted into RNase-free water. The qualities of the RNAs were checked by gel electrophoresis.A three nucleotide spacer (GCG) and the T7 promoter were added to the Cas9 coding region by PCR amplification using the Cas9 primers  with pCA primers . The ampC57BL/6J mice were purchased from CLEA Japan. All animal experiments were approved by the Animal Care and Experimentation Committee of Gunma University and were carried out in accordance with the approved guidelines.in vitro transcribed Cas9 mRNA (50 ng/μl) and gRNA (20 ng/μl)2 in air. For the production of mutant mice, 1-cell stage embryos were transferred into the ampulla of the oviduct (9–18 embryos per oviduct) of pseudopregnant Jcl:ICR (CLEA Japan) females. To determine the developmental rate in vitro, embryos were cultured until the blastocyst stage (4.5 days).All animal procedures were approved by Animal Care and Experimentation Committee at Gunma University and the National Cerebral and Cardiovascular center, and carried out in accordance with approved guidelines. Female C57BL/6J mice (CLEA Japan) were superovulated by the injection of 7.5 units of pregnant mare's serum gonadotropin  followed by 7.5 units of human chorionic gonadotrophin  48 h later. Females were mated overnight with C57BL/6JJcl male mice. The following day, fertilized embryos were collected from the oviducts. For DNA injection, the Cas9 expression vector (2.5 ng/μl) and gRNA expression vector (2.5 ng/μl)To detect small genome modifications, PCR was performed using primers flanking the targeted region . The PCRBody weights of pups were analyzed with one-factor ANOVA, followed by Tukey-Kramer test. Percentages of blastocysts, pups and mutants were analyzed by chi-square test. A probability value of P < 0.05 was considered significant. All statistical analyses were performed using software (statcel2).Supplementary Figures


On August 10, 2016, 2 years after the most recent wild poliovirus (WPV) case was reported in Nigeria (in July 2014) . Efforts by the Nigeria military have resulted in improved accessibility in Borno during the last year, although assessments in November 2016 indicate that 39% of settlements are still inaccessible because of insurgency-related insecurity  in a healthy contact, aged 6 years, of one of the polio patients from Monguno LGA. cVDPVs are genetic variants of the oral vaccine virus that emerge and can cause paralysis indistinguishable from WPV disease in unimmunized or underimmunized populations  (containing types 1 and 3 OPV) were conducted during August 15–18, 2016, in Jere and Gwoza LGAs and in three additional LGAs with substantial IDP populations. Five rounds of regionally synchronized SIAs also were implemented, targeting children aged <5 years in five Lake Chad basin countries  at risk for poliovirus transmission because of large population movements. SIA quality was evaluated using lot quality assurance sampling (LQAS) methodology. Overall, approximately 30 million children in 18 Northern Nigeria states were vaccinated with bOPV. In addition, one dose of inactivated polio vaccine (IPV) was administered to children in Borno State to boost immunity . A separAFP surveillance. In 2016, a total of 614 AFP cases were reported in Borno State, a 73% increase from 354 reported cases in 2015. Improvements in AFP reporting in 2016 can be attributed to increased surveillance activity in the wake of the March 2016 cVDPV isolation, as well as improving access to populations in previously inaccessible areas of the state.Environmental surveillance. Following the reported WPV circulation in Borno, the number of environmental sampling sites in Borno was increased from four to six. In March 2016, the frequency of sample collection at existing sites in Maiduguri  was increased from monthly to weekly following the reported cVDPV2 isolation  infection was reported in Nigeria in July 2014, the World Health Organization removed Nigeria from the list of endemic countries because of the high likelihood that endemic WPV circulation had been interrupted in Nigeria. However, Borno State in northeastern Nigeria has experienced years of armed insurgency, which has hampered implementation of effective polio eradication activities.During August and September 2016, four WPV cases and one circulating vaccine-derived poliovirus (cVDPV) isolate were reported in accessible areas of Borno State. Analysis of the WPV isolates showed limited genetic relationship, indicating prolonged undetected transmission. In response, regionally synchronized supplementary immunization activities were conducted in five Lake Chad basin countries, and >30 million children in 18 northern states in Nigeria were vaccinated. Additional measures to strengthen polio surveillance quality were implemented in accessible areas of Borno State. Ongoing conflict-related insecurity continues to restrict polio workers’ access to populations in insurgent-held areas.Although the areas that are insurgent-held have diminished over the last year, about 40% of communities in Borno State remain inaccessible. Response to the detection of WPV and cVDPV was highly successful in accessible areas. Increasing polio vaccination coverage and improving surveillance quality among cohorts of unimmunized and underimmunized populations is a critical public health need in Borno and the Lake Chad region.