To evaluate the predicting value of MUC1 expression in lymph node and distant metastasis of colorectal cancer (CRC).Pubmed/ MEDLINE and EMBASE were searched to identify eligible studies that evaluated the correlation between MUC1 and CRC. A meta-analysis was conducted to evaluate the impact of MUC1 expression on CRC metastasis.A total of 18 studies (n = 3271) met inclusion criteria and the mean Newcastle-Ottawa Scale (NOS) score was 6.3 with a range from 4 to 8. The pooled OR in the meta-analysis of 15 studies indicated that positive MUC1 expression correlated with more CRC node metastasis . The data synthesis of 6 studies suggested that MUC1 expression predicted more possibility of CRC distant metastasis . In addition, the combined OR of 7 studies showed that MUC1 expression indicated higher Duke’s stage . No publication bias was found in the mate-analysis by Begg’s test or Egger’s test with the exception of the meta-analysis of MUC1 with CRC node metastasis .Despite of some modest bias, the pooled evidence suggested that MUC1 expression was significantly correlated with CRC metastasis. Mucin 1 (MUC1) is a structural transmembrane protein with a heavily glycosylated extracellular domain, which is also known as episialin, CA5-3, DF3, PAS-O, PEM, H23Ag, EMA and MCA . The funColorectal cancer (CRC) is one of the most frequently diagnosed cancers and it is estimated to be the fourth most common cause of cancer death . Every yThis systemic review and meta-analysis was designed to clarify the role of MUC1 in CRC metastasis and evaluate the correlation of MUC1 expression with node metastasis, distant metastasis and Duke’s stage of CRC.According to a prespecified written protocol, the literature research was designed to identify available studies that evaluated the correlation between MUC1 expression and CRC metastasis. Specifically, CRC metastasis included node metastasis and distant metastasis. Additionally, Duke’s stage was chosen to estimate metastasis status because it was widely used in the diagnosis of CRC and could be easily divided into two categories according to metastasis or not. Duke’s stage C/D indicated node and/or distant metastasis while stage A/B indicated no metastasis. Therefore, several inclusion criteria were used to select eligible studies: (1)Patients with pathological diagnosis of colorectal cancer, regardless of pathological classification; (2) MUC1 expression in tumor tissue evaluated by immunohistochemistry (IHC) method with monoclonal antibody against MUC1; (3) Sufficient data to estimate the odd ratio (OR) and corresponding 95% Confidence Interval (CI) of the correlation of MUC1 expression with node metastasis, distant metastasis and/or Duke’s stage; (4) Case controlled studies. The excluded criteria included: (1) Patients diagnosed with recurrent CRC; (2) Full-text published in other languages rather than English or Chinese because of resource limitation.Studies were identified by searching the electronic databases of Pubmed/MEDLINE  and EMBASE . The keywords “MUC1”, “mucin 1”, “episialin”,“CA5-3”, “DF3”, “PAS-O”, “PEM”, “H23Ag”,“EMA”, “MCA”, “colorectal cancer”, “colorectal carcinoma”, “colon cancer” and “colon carcinoma” were used in various combinations. Published language was limited to English and Chinese because of time and resources limitations. The reference lists of identified studies were also searched manually as a complement the computer searches.Two independent reviewers screened the abstracts of primary identified studies and related references for eligibility. Full-text articles were read for further assessment if the eligibility was unclear by screening the abstracts. Discrepancies in the inclusion were resolved by discussion within the review team. For primary included studies, names of all authors and the medical centers involved were examined carefully to avoid repetitive data. Whenever studies pertained to overlapping patients, studies with larger sample size and more comprehensive data were retained.Two independent reviewers extracted the details of included studies with a standardized form. The following information was recorded: first author, year of publication, country of origin, sample size, number of male included, mean or median age, cancer characteristics, number of patients with node metastasis, number of patients with distant metastasis; number of patients with Duke’s stage C/D; antibody used; cut-off value of positive MUC1 expression and conclusions. The conclusions of each study were termed “positive” when positive MUC1 expression predicted more node metastasis, distant metastasis or Duke’s stage C/D, “negative” when positive MUC1 expression predicted less metastasis or Duke’s stage A/B, and “indeterminate” when no significant correlation between MUC1 expression and metastasis or Duke’s stage was found. Any discrepancy was resolved through discussion. The main outcome was the number of patients with node metastasis, distant metastasis or Duke’s stage C/D in MUC1 positive and negative group.According to the Cochrane Collaboration, the Newcastle-Ottawa Scale (NOS) was used to assess the quality of the included studies by judging on three board perspectives: the selection of study groups, the comparability of study groups and the measurement of exposure in study groups , 32.2)and I-squared (I2) value were computed to assess the heterogeneity of the included studies in each group. If significant heterogeneity was found i.e. P< = 0.10 and/or I2>50%, potential sources of heterogeneity between studies would be explored. If the heterogeneity could not be eliminated by subgroup analysis, the ORs of each group were combined respectively in a random effect meta-analysis using DerSimonian-Laird algorithm. If the heterogeneity was acceptable, Mantel-Haenszel algorithm was used in a fixed effect model. Meta-analyses were carried out with the Stata version 11.0 . The pooled OR>1 indicated more metastasis and Duke’s stage C/D in MUC1 positive group relative to MUC1 negative group, which would be considered significant if the 95%CI did not overlap 1, with p<0.05.The included studies were divided into three groups for analysis: those with data regarding node metastasis, those with data regarding distant metastasis and those with data regarding Duke’s stage. The correlation of MUC1 with node metastasis, distant metastasis and Duke’s stage in each study was estimated by OR and corresponding 95% CI. Heterogeneity chi-squared . In the sub-group analysis according to the monoclonal antibody against MUC1, a positive relationship between MUC1 expression and node metastasis was demonstrated by the combined OR of the 3 studies using KL-6 , and that of the 3 studies using Ma695  and the 2 studies using 139H2 . Meanwhile, the combined ORs of the studies using HMFC-2 and ICR2 were indeterminate with 95% CIs overlapping 1 and there was only one study using MUSE11, Ma552 and ZM-0391 respectively , as was shown in 2 = 0.47, P = 0.494, I2 = 0.0%, For the 6 studies estimating the correlation between MUC1 expression and CRC distant metastasis, the combination of ORs in fixed effect meta-analysis suggested that distant metastasis of CRC was more likely to occur when MUC1 expression was positive . In the sub-group analysis according to the antibody against MUC1, the combined OR of the 4 studies usingMa695  and the OR of the single studyusingKL-6  confirmed the correlation of MUC1 expression with Duke’s stage C/D. However, the ORs of the single study usingICR2 and HMFC-1 were indeterminate with 95% CIs overlapping 1 , distant metastasis p = 0.851) or Duke’s stage (p = 0.453), while Egger’s test revealed possible publication bias in the analysis of node metastasis (p = 0.000) but no bias in the analysis of distant metastasis (p = 0.811) or Duke’s stage (p = 0.729). In addition, both Begg’s test and Egger’s test showed no publication bias in the sensitivity analysis of node metastasis , distant metastasis  or Duke’s stage . The funnel plots of the Begg’s test and Egger’s test were shown in Figs 1 or DukePrevious studies found MUC1 was usually over expressed in CRC tissue and seemed to play a role in the tumor development and progression. This was the first meta-analysis to systematically evaluate the correlation between MUC1 expression and CRC metastasis. The results revealed that positive MUC1 expression in CRC tissue was strongly correlated with more metastasis of CRC, which was consistent with the conclusions of most included studies. Present evidence from the meta-analysis indicated that MUC1 might be a promising biomarker to predict the status of CRC metastasis at the time of diagnosis. The expression of MUC1 in CRC tissue can be determined by IHC method with monoclonal antibody against MUC1, which can be done simultaneously when pathologists conduct pathological diagnosis with tumor tissue obtained from surgery or biopsy. It is relatively easy to realize and popularize in clinical practice, which is of great significance to help surgeons determine treatment strategy. The establishment of the relationship between MUC1 expression and CRC metastasis may also help to clarify the metastasis risk of CRC patients at the time of diagnosis, especially those without symptoms or signs of metastasis. If MUC1 is highly expressed in a CRC patient without metastatic manifestation, it is worthy to do more detailed examination in search for any existing small metastasis. Patients with over expressed MUC1 may need more radical or aggressive treatment after diagnosis and more rigorous care after tumor resection due to a relatively high risk of metastasis. However, present study only proved the correlation between MUC1 expression and CRC metastasis. The positive rate of MUC1 expression in CRC patients with metastasis differed greatly in previous retrospective studies, and the sensitivity and specificity of MUC1 in predicting CRC metastasis have not been validated in prospective studies. Besides, MUC1 expression in CRC tissue cannot guide therapy before surgery or biopsy. Despite of these limitations, MUC1 deserves further investigation in CRC patients.Among the included studies in the meta-analysis, several monoclonal antibodies were used to detect the expression of MUC1. Although the expression level of MUC1 stained by different antibodies was not compared in any of the included studies, the positive rate of MUC1 expression was different among studies using different antibodies. In CRC patients with metastasis, the average positive rate was 44.6% (range from 20.0% to 62.7%) in the 6 studies using Ma695, 63.5% (range from 49.5% to 92.7%) in the 3 studies using KL-6 and 40.6% (range from 32.5% to 50.2%) in the 2 studies using HMFG-2. The difference may be attributed to many interference factors, but the various antibodies should be concerned first. Subgroup analysis was then performed according to the monoclonal antibody and the results or tendencies were basically consistent among different subgroups, which were also consistent with those of the overall meta-analysis. Together with sensitivity analysis, the results of subgroup analysis confirmed that positive MUC1 expression was correlated with more CRC metastasis in spite of the limited number of included studies.Node metastasis, distant metastasis and Duke’s stage were used in the meta-analysis to estimate the metastasis status of CRC because they are widely applied in clinical practice and closely correlated with TNM stage and prognosis of CRC patients. Given the difference in economy and medical technology, some small metastasis might not be detected at the time of diagnosis, which would cause interference to the results of retrospective case controlled studies. However, it in turn emphasizes that it is necessary to find a reliable approach to predict the metastasis status of CRC in advance. Prospective studies are needed to avoid this interference and evaluate the relationship between MUC1 expression and CRC metastasis, as well as the sensitivity and specificity of MUC1 in predicting CRC metastasis status.The potential role of MUC1 in malignancy has been widely investigated in recent years. Several lines of evidences have demonstrated that MUC1 expression is correlated with tumor proliferation, metabolism, invasion, metastasis, angiogenesis and resistance to apoptosis , 48–58. in vivo study showed that mice injected with MUC1 downregulated cells developed smaller tumors when compared to those injected with the control cells [Although a vast majority of studies indicated the proliferative and metastatic role of MUC1, several studies did indicate anti-proliferative and anti-metastatic effect of MUC1. Downregulation of MUC1 expression was found to increase proliferation and apoptosis in MKN45 gastric carcinoma cell line, but ol cells . In anotol cells . Furtherol cells . The conAlthough the systematic review and meta-analysis was conducted strictly in accordance with the Cochrane Collaboration, several limitations and potential bias should be taken into consideration when the results were interpreted. First of all, the most common pathological classification of CRC was adenocarcinoma with various differentiation degrees in the included studies, the correlation of MUC1 expression with other pathological types or certain differentiation degree was not evaluated sufficiently. As the biological properties of different types of CRC varied from each other, the results of this meta-analysis could not present the actual impact of MUC1 expression on certain subtype of CRC. However, a high qualified case controlled study with a large sample size explored the effect of MUC1 expression in CRC patients with different DNA mismatch-repair (MMR) status . The resSecondly, the NOS scores of the included studies ranged from 4 to 8 with a mean score of 6.3, suggesting the qualities were just acceptable but unsatisfactory. Based on the NOS scores, the main limitations were focused on the comparability between groups and non-response rate. The difference in confounding factors, such as age, sex, location and subtype of cancer, were not reported or compared between case group and control group, which might weaken the reliability of the results. Given that MUC1 expression was identified by researchers with experimental methods and was different from other exposure factors such as nicotine and alcohol that needed to ask patients to collect the information, the non-response rate might not necessary or applicable for the experimental studies here.Thirdly, random effect model was used in the meta-analysis of the 15 studies involving CRC node metastasis and the sensitivity analysis of the 3 studies involving Duke’s stage due to the potential heterogeneity between studies. However, the results were consistent with those of other analyses in fixed effect model, suggesting the heterogeneity may not make great differences in the analyses. Furthermore, publication bias was identified in the meta-analysis of node metastasis (p = 0.000) by Egger’s test while no bias was found by Begg’s test, which could be largely attributed to the limited number of included studies. The limitations in sample size, antibodies used and publication language should also be considered when interpreting the results in clinical practice.In conclusion, it is clear that MUC1 expression is significantly correlated with more CRC metastasis. Despite of some modest bias, the results of this meta-analysis has provided strong evidence that positive MUC1 expression indicates higher Duke’s stage and more possibility of node and distant metastasis in CRC patients. MUC1 can be used as a biomarker to identify the metastatic potential of CRC and also as a promising target for future immunotherapy to decrease the risk of metastasis and prolong the survival of CRC patients.S1 File(DOC)Click here for additional data file.S2 File(RAR)Click here for additional data file.


Post-transcriptional regulation of RNA facilitates the fine-tuning of gene expression. It occurs through multiple pathways that include the nuclear processing of mRNA and its precursors, mRNA silencing, regulation of mRNA decay, and regulation of translation. Poly(ADP-ribose) polymerases (PARPs), enzymes that modify target proteins with ADP-ribose, play important roles in many of the RNA regulatory pathways through multiple mechanisms. For example, RNA-binding PARPs can target specific transcripts for regulation, ADP-ribosylation of RNA-regulatory proteins can alter their localization, activity or RNA-binding, and non-covalent interactions of RNA-binding proteins with poly(ADP-ribose) can affect their function. In addition to regulating RNA during non-stress conditions, PARPs mediate RNA regulation during cellular stress conditions that are critical for the proper execution of a stress response. In this review, we summarize the current knowledge regarding PARP-dependent regulation of RNAs, and describe how by modulating RNA processing, translation and decay, PARPs impact multiple processes in the cell. Targets proteins are either mono(ADP-ribosyl)ated or modified with long polymers of poly(ADP-ribose) that play additional regulatory functions by binding and recruiting proteins through non-covalent interactions (Poly(ADP-ribose) polymerases (PARPs) use NADractions . In someractions , howeverractions . This isractions . In thisIn eukaryotes, mRNAs, initially transcribed as pre-mRNAs, are processed in the nucleus before the resulting mature mRNAs are exported to the cytoplasm . NuclearRNA binding proteins (RBPs) assist in each step of RNA metabolism and are the most important class of proteins that regulate RNAs. They are targeted for modification by PARPs, and in some cases their function is altered by non-covalent binding to poly(ADP-ribose). In addition, several PARPs are themselves RBPs and contain well-defined RNA binding domains.in vitro   Vyas et. In addiin vitro . RNA-binin vitro  , leading to structural stabilization of the domain . ProteinCCCH PARPs contain CCCH domains that are each similar in length and of generally high homology . PARP12 and PARP13 contain four CCCH domains, and PARP7 only one. The structure of the four tandem PARP13 CCCH domains has been solved . ExperimRNA recognition motifs (RRMs) consist of two alpha helices and four antiparallel beta strands. Several conserved aromatic residues protrude out of the beta strands and are essential for RNA recognition, although some exceptions exist . In geneThe RRM is one of the most abundant RNA binding domains, with a broad spectrum of target RNAs and sequences . In addiThe SAP domain contains several charged and hydrophobic residues that could be used to bind nucleic acids, but the precise mechanism of binding to RNA or DNA is unknown. This domain is primarily associated with DNA binding, although RNA binding has also been reported . Many SAVarious proteomic datasets are available that identify cellular targets of ADP-ribosylation. Given the manner in which the purifications were performed, some of these analyses cannot distinguish between ADP-ribosylated proteins and those that bind to ADP-ribose . TechniqSeveral categories of RNA regulatory proteins were enriched in each analysis suggesting that PARPs play different roles in RNA regulation. These include RNA metabolism , mRNA prPre-mRNAs synthesized during transcription must undergo splicing in order to remove non-coding introns prior to export to the cytoplasm . These pPARPs also play important roles in rRNA biology. Ribosomal RNAs (rRNAs) are key enzymatic and structural components of the ribosome. rRNA genes are transcribed as a 45S precursor that is processed into three species of mature rRNA  . rRNA syOne major component of nuclear pre-mRNA regulation by PARPs involves modulation of hnRNP – ribonucleoprotein complexes involved in multiple steps of RNA processing . These iSeveral hnRNPs  bind to poly(ADP-ribose) or PARP1  Figure . BindingUnder non-stress conditions, the majority of PARP1 and nuclear poly(ADP-ribose) is enriched in the nucleolus  Figure . PARP1 kPARP1 also regulates rRNA transcription by binding to TTF-1 interacting protein 5 (TIP5), a component of the nucleolar remodeling complex (NoRC) . It doesOnce transported to the cytoplasm, mature mRNAs undergo several fates that together determine the amount of transcripts available for translation. In addition to being directly translated into proteins, mRNAs can be degraded through RNA decay, or silenced via microRNA repression. Posttranscriptional regulation of mRNA functions as a critical layer of gene expression fine-tuning , and cytTranslation of mRNA to protein by the ribosome involves several steps. These include translation initiation, when a complex of proteins assembles onto the 5′ UTR of the mRNA to initiate ribosome assembly, and elongation, when the already assembled ribosome must be translocated to the next codon . Both ofCanonical mechanisms of translation initiation involves recognition of the 5′ cap of the mRNA, whereas internal ribosomal entry site (IRES) mediated translation initiation uses specific RBPs to target the ribosome to the mRNA independent of the 5′ cap . One of + as substrate, thereby inhibiting protein synthesis (Mono(ADPribosyl)ation of elongation factor 2 (EF2), a GTPase involved in ribosome translocation, results in the inhibition of protein synthesis , 1984b . Treatmeynthesis . The mecynthesis Several ribosomal proteins have been identified as PARP binding proteins, although the functional relevance of this binding is not understood. PARP1 has been shown to bind to the large ribosomal proteins L13, L14, L18a, L21, L22, L23a and L30,  as well A key component of the regulation of gene expression involves mRNA decay. The balance between transcription and the mRNA half-life or decay rate determines the steady-state concentrations of mRNAs available for translation in the cytoplasm. mRNA decay is a process regulated by the activity of specific proteins: 5′ to 3′ decay is mediated by the exonuclease XRN1, and 3′ to 5′ decay by the exosome complex (reviewed in ). RemovaPARP13 destabilizes specific mRNAs by targeting them for RNA decay  Guo et . PARP13 PARP14 also regulates mRNA stability . It destMicroRNA silencing is another mechanism used to regulate cytoplasmic levels of translatable mRNAs. Once loaded into the RNA Induced Silencing Complex (RISC), microRNAs bind to target mRNAs, leading to the translational repression and decay of these target mRNAs . ArgonauPARPs play important roles in the regulation of RNAs during cellular stresses such as DNA damage, oxidative stress, hypoxia or viral infection . In geneCytoplasmic stressors including hypoxia, oxidative stress, and heat shock, among others, lead to global translational repression. This repression is due to sequestration of mRNAs to cytoplasmic stress granules, structures that contain stalled translation initiation complexes, RNA and RBPs . RBPs thPARP5a, PARP12, PARP13, PARP14 and PARP15 as well as poly(ADP-ribose) and PARG are enriched in stress granules, and modulation of poly(ADP-ribose) levels by overexpressing or depleting PARPs or PARGs affects the assembly, maintenance and disassembly of these structures . The catViral infection induces the assembly of antiviral stress granules, structures that are highly similar to cytoplasmic stress granules and contain many of the same biochemical components , includiPARP13 was initially discovered as an antiviral factor in a cDNA expression screen to identify suppressors of murine leukemia virus (MLV) infection  Figure . It was PARP13-mediated regulation of microRNA activity also occurs during the antiviral response . Many anTranslation of viral proteins can also be inhibited by RNA binding PARPs including PARP7, PARP12, and PARP13. PARP12 binds to ribosomal proteins in polysome containing, actively translating fractions upon infection with the RNA virus Venezuelan equine encephalitis virus (VEEV) resulting in translation inhibition , 2014. TOne of the best studied PARP-mediated nuclear stress responses is heat shock. The heat shock response involves changes in transcription, but also altered processing of RNAs in the nucleus . In geneOne important PARP1 function during the nuclear stress response is poly(ADP-ribosyl)ation of poly-A-polymerase (PAP) . ModificThe increased PARP1 activity upon heat shock also regulates splicing by altering the RNA binding dynamics of hnRNPs  Figure . In Drosin vitro and RNA damage was not required to induce this increased poly(ADP-ribosyl)ation activity. These results suggest that PARP2 could function as a sensor for RNA accumulation in the nucleus  synthesis  Figure . The inc nucleus  and coul nucleus .RNA regulation is an exciting area of PARP biology that is just starting to hit its stride. Regulation of RNA processing by PARPs occurs at nearly all of the key regulatory steps, and its importance as a regulatory mechanism for gene expression during non-stress conditions, as well as during cell stress has become apparent. In general PARPs regulate RBP function either by directly modifying them with ADP-ribose, or by producing poly(ADP-ribose) that sequesters the RBPs through non-covalent binding interactions, decreasing their ability to bind RNAs. Nuclear and cytoplasmic regulation of RNAs by PARPs have many similarities, however one key difference is that cytoplasmic regulation involves RNA-binding PARPs that bind to and regulate RNAs in a sequence specific manner, whereas nuclear PARPs do not.The challenge ahead is to identify the specific mechanisms by which PARPs regulate RNA and RBP function. This will allow us to study equally important questions including potential coordination of PARPs in the regulation of key RNA regulatory steps and identification of common mechanisms of regulation, and to address the importance of RNA regulation by PARPs in cells, model organisms, and its potential importance in human disease. As a family of proteins PARPs have been shown to be druggable and PARP1 inhibitors are promising therapeutics for the treatment of cancer . TargetiRecent technological innovations in PARP biology, RNA analysis, and gene editing should lead to rapid progress in elucidating the function of the different RNA binding PARPs. These include the ability to identify specific amino acid residues on target proteins that are modified by ADP-ribose, allowing us to more closely examine the mechanistic consequences of ADP-ribosylation (reviewed recently in Vivelo and Leung 2014). They also include technical advances in the RNA field such as CLIP-seq  that wil


Although several methods have been suggested to identify RNA editing sites, there remains challenges to be addressed in distinguishing true RNA editing sites from its counterparts on genome and technical artifacts. In addition, there lacks a software framework to identify and visualize potential RNA editing sites. Here, we presented a software − ‘RED’ (RNA Editing sites Detector) − for the identification of RNA editing sites by integrating multiple rule-based and statistical filters. The potential RNA editing sites can be visualized at the genome and the site levels by graphical user interface (GUI). To improve performance, we used MySQL database management system (DBMS) for high-throughput data storage and query. We demonstrated the validity and utility of RED by identifying the presence and absence of C→U RNA-editing sites experimentally validated, in comparison with REDItools, a command line tool to perform high-throughput investigation of RNA editing. In an analysis of a sample data-set with 28 experimentally validated C→U RNA editing sites, RED had sensitivity and specificity of 0.64 and 0.5. In comparison, REDItools had a better sensitivity (0.75) but similar specificity (0.5). RED is an easy-to-use, platform-independent Java-based software, and can be applied to RNA-seq data without or with DNA sequencing data. The package is freely available under the GPLv3 license at  RNA editing is one of the post- or co-transcriptional processes with modification of RNA nucleotides from their genome-encoded sequence . In humaAZIN1, leading to a non-synonymous substitution (ser367gly) of AZIN1, is increased in hepatocellular carcinoma specimens. It was noted that the frequency of AZIN1 RNA editing increases during progression from cirrhosis and primary liver cancer to advanced hepatocellular carcinoma with recurrence and metastasis . Variation sites with LLR < m were excluded, where m is self-defined and m = 4 is suggested. The LLR ≥ 4 indicated that the probability of editing event happened is 104 times more than that of non-editing in reality.First, we integrated a likelihood ratio (ng sites . For a pLR) test  is a prong event . We needn(A) and n(G) for the given site was calculated based on the known RNA editing sites from the DARNED [In addition, we assessed the significance for a given RNA editing site by comparing its expected editing levels. The expected number of e DARNED  and RADAe DARNED . These ne DARNED .RED is designed to visualize and explore potential RNA editing sites with GUI at the genome and the site levels . The disToolbar panel: provides a convenient way to access commonly used functions, which can be accessed via the main menu or keyboard shortcuts.Directory panel: provides a quick overview of accessed objects, including annotation set, data set, site set and site lists. A site list includes the remaining variants after each step of filtering, which is shown in a hierarchical tree structure.Genome panel: provides an overview of human chromosomes . After RNA editing filters were applying, potential RNA editing sites would be shown in a karyogram overview.Chromosome panel: contains tracks of annotation and data. The annotation track shows gene features from NCBI RefSeq, and reference sequence. The data track presents all mapped RNA-seq reads from the BAM file with two different types: genomic interval and base status.Status panel: the left shows related information for a chromosome, a site or a feature when the mouse is over it, and the right is the usage of memory.RED was implemented with several key technologies, including: 1) fast data storage/retrieve and low memory usage with high-throughput next-generation sequencing data and variants data; 2) diverse filters to detect potential RNA editing sites with an improved performance; 3) information synchronization among the genome panel, annotation and data track; and 4) abundant presentation for data analysis and output.R (http://www.r-project.org) for statistical analysis. All functions related to MySQL and R were automatically executed in RED. RED can run on mainstream computational platforms, including Windows, Linux and Mac OS X.RED is mainly developed using Java programming language, together with MySQL for data management and To make the computation more efficient, everlasting and faster-saving, a widely used relational database management system (RDBMS) − MySQL − was employed to manipulate variants data , as well as files required for filtering. RED can speed up filtration by powerful database engine and make a quick query of the filtering result . The Java Database Connectivity (JDBC) was used in RED to connect a local or remote database.R, a language and environment for statistical computing, to perform statistical analysis, including FDR. A simple Java library  was used to call R commands and scripts from Java.RED also used functions in RED provides flexible exports for image and text output. Images generated from RED can be exported in the format of SVG or PNG. The distribution of RNA editing sites according to its editing type and chromosome were present in graph. Information regarding to all potential RNA editing sites can be exported to a tab-delimited text file, including chromosome, position, class ID, reference base, alternative base, quality, editing level, p-value and FDR. In addition, a RED project file, including potential RNA editing site lists, information of data set and annotation set, and software preference, can be saved at user’s preference.https://www.broadinstitute.org/gatk/guide/article?id=3891) with default settings. A Linux server of 256 GB memories and 4 CPU  Xeon(R) CPU E7- 8850 @ 2.00GHz) was used for the computation. A detail of the software and commands used in the analysis is present in theSharma et al.  identifide novo mode (MySQL (version 5.1.73), Java (version 1.8.0_25), and R (version 3.0.2)) detected 129 potential RNA-editing sites . REDItools in de novo mode (python version 2.6.6) identified 171 sites (P < 0.05). 121 sites (55%) were identified in two programs. Then, we tested the presence/absence of 30 potential RNA-editing sites that affecting amino acid [We counted the number if a given site was identified in any one of the three hypoxic samples. RED in de novo mode detected 18 out of 28 true RNA-editing sites  and one out of two false RNA-editing sites. For comparison, the de novo mode in REDItools detected 21 out of 28 true RNA-editing sites (P < 0.05) and one out of two false RNA-editing sites. The sensitivity and specificity of the two methods in identifying RNA editing sites are shown in indicating that REDItools had a better sensitivity but similar specificity. The Venn diagram (TMEM131) identified by RED is shown in We first tested 220 C→U (and G→A) sites listed in Supplementary Table S2 in Sharma et al. . Of 220  diagram  indicatewere approximately ten times higher than GATK ‘HaplotypeCaller’ process. It should be noted that the calling procedure in GATK has taken into account the information about intron-exon split regions.We noted that different numbers of RNA editing sites were identified between RED and REDItools, partly due to different number variants used in the programs. The sensitivity of variants called by GATK ‘HaplotypeCaller’ (used in RED) and ‘samtools mpileup’ (used in REDItools) differed, as shown in a recent study . AlthougThe comparison of computation costs for REDItools and RED in analyzing six RNA-seq samples  was showhttp://www.ncbi.nlm.nih.gov/bioproject/273421) [DNA-RNA mode with the parameters listed in We also identified potential RNA editing sites by comparing the RNA-seq and whole exome sequencing data in the liver normal tissue of a hepatocellular carcinoma patient (/273421)  by RED. R Environment (R 3.0.1 or later). REDItools requires only Python.RED is an integrated GUI for detecting and visualizing potential RNA-editing sites. RED provides multiple rules to filter out spurious RNA-editing sites and visualize the candidate RNA-editing sites. In addition, MySQL made the query of the filtering result efficient, and enabled the storage of RED results for each filtering step, which can be re-analyzed without running filters again. However, RED depends on three computational frameworks of Java Runtime Environment (jre or jdk with 1.6.0_43 or later), MySQL Database Management System (MySQL 5.1.73 or later) and https://github.com/REDetector/RED or SourceForge, https://sourceforge.net/projects/redetector.The RED package is freely available under the GPLv3 license at the Git repository web-based hosting service (github), Future additions to RED will include: 1) information of each site, including coverage, quality, position, and editing level, will be present in the chromosome panel; and 2) functional categories of RNA editing sites will be annotated, and information of chromosome cytoband will be added.In conclusion, RED is an effective software for the identification and visualization of RNA editing sites from next-generation sequencing data. It is highly flexible, including a variety of filters and stringent statistical assessment, and may provide very reliable sets of RNA editing candidate sites according to the user’s requirements.S1 Text(DOCX)Click here for additional data file.


Counteracting the worldwide trend of coral reef degeneration is a major challenge for the scientific community. A crucial management approach to minimizing stress effects on healthy reefs and helping the recovery of disturbed reefs is reef protection. However, the current rapid decline of the world’s reefs suggests that protection might be insufficient as a viable stand-alone management approach for some reefs. We thus suggest that the ecological restoration of coral reefs (CRR) should be considered as a valid component of coral reef management, in addition to protection, if the applied method is economically applicable and scalable. This theoretical study examines the potential applicability and outcomes of restocking grazers as a restoration tool for coral reef recovery—a tool that has not been applied so far in reef restoration projects. We studied the effect of restocking grazing fish as a restoration method using a mathematical model of degrading reefs, and analyzed the financial outcomes of the restocking intervention. The results suggest that applying this restoration method, in addition to protection, can facilitate reef recovery. Moreover, our analysis suggests that the restocking approach almost always becomes profitable within several years. Considering the relatively low cost of this restoration approach and the feasibility of mass production of herbivorous fish, we suggest that this approach should be considered and examined as an additional viable restoration tool for coral reefs. Coral reefs are considered to be among the most threatened and fastest deteriorating marine ecosystems . At presAn alternative approach, aimed at targeting this challenge, is coral reef restoration . Such degraded reefs are likely to remain in their unfavorable state if not inhabited by enough grazers. Since the natural recovery of grazing fish is very likely to take years  applied reef dynamic models  to compaWe examine the potential outcomes of fish restocking using modifications of mathematical models, consisting of differential equations, which have been used to examine reef resilience without intervention .C, M, T, P and R. The algal turf coverage refers to the proportion of seabed either covered by scant and small algae, such as might occur due to grazing of macroalge, or exposed hard substrate. We assume that the corals, macroalgae, and algal turfs are competing for seabed in a constant size location, and define the algal turf coverage to be 1-M-C. The variable P describes the abundance of grazing fish relative to the maximum capacity of grazing fish possible in the habitat, so that 0 < P < 1. Rugosity is defined as the ratio between the horizontal distance of two points in the reef and the length of a chain laid on the reef surface between those points, usually satisfying 1 < R < 3 , which is the product of a linear, increasing, function of rugosity and a Hill-Langmuir function of M + T (for details see g(P) is taken to be P. Thus, the grazing fish reduce the rate of macroalgae growth over corals, while simultaneously expanding algal turfs, which can provide seabed for coral growth . We used spillover rates estimated in δP to the initial value of P, namely P0. Since P is normalized to be between 0 (no grazers) and 1 , δP is given as a fraction of the maximal abundance of grazing fish possible in the modeled habitat.Terrain rugosity increases due to growth of corals, X as the size of the coral reef in km2; BC is the revenue, per km2 per year, resulting from coral coverage (excluding revenue from fishing); 2, and r′ is the economic discount rate. For t years, the difference in revenue for a coral reef with restocking versus a reef with no intervention can be estimated by : Cre(i) and Cno(i) are the coral coverages of reefs with and without restocking, at year i, respectively. Simply put, we calculate the difference in coral coverage between a reef with and without the restocking intervention. This difference is multiplied by the size of the reef and the financial benefit for each squared kilometer of the reef. The term is discounted with regard to inflation. Finally, the cost of restocking is subtracted. This is a conservative estimate, since in this model restocking increases coral coverage, and it is assumed that the benefit from coral reefs declines relative to the cost of the fish, due to discounting. The parameters and variables of the economic model describing the revenue are given in We model the economic impact of fish restocking using a cost-benefit analysis. We define f = 0 in all the following results. The state of a disturbed reef is represented by the initial conditions of the coral coverage (C) and macroalgae (M). The revenue of restocking  as wellved from ), are pr10 scale, with negative revenue replaced by zeros  is defined by the other two variables. The parameters used in P0 = 0.1, δP = 0.1, R0 = 1.6  was taken to be 15 km2, the estimated grazing fish number per δP = 0.1, we de facto add 300 fish per each km2 of reef area. The financial benefit from the coral reef (BC) was estimated from 2, which is a very conservative estimate  was set to 0.05, but our results remain robust when varying discount rates (The discount rate (nt rates .2), combined with the high revenue of coral reef area . Even if the reef does not restore to high coral coverage, the delay in its deterioration, enabled by restocking, will still be profitable for a large extent of the initial conditions. Similarly, even if the reef will eventually be restored without human intervention, restocking will shorten the period of time required to achieve this. This is shown in C), macroalgae coverage (M), and grazing fish (P) are plotted with and without restocking for two sets of initial conditions. C0 = 0.35, M0 = 0.05), in which the reef will be restored without intervention. Although both scenarios lead to an eventual high coral coverage, we can see that restocking will shorten the time to equilibrium to about 65% of this time in a system without restocking  in which the coral reef will deteriorate without intervention  to Reef II (downstream). Under this range of initial conditions we note five possible scenarios: (I) initial conditions under which in both reefs the system reaches a state with high coral coverage without restocking; (II) areas wherein one reef will reach high coral coverage without intervention, while the other reef will only succeed if restocking is applied; (III) areas wherein one reef will reach high coral coverage without intervention, while the other will reach the macroalgae state even if restocking is applied; (IV) areas in which both reefs will deteriorate to the macroalgae state without intervention, but restocking will salvage one of them; and (V) areas in which both rates will deteriorate and restocking will not help either reef. These areas are marked accordingly on CI, MI, PI and CII, MII, PII, are the coral coverage, macroalgae coverage and grazing fish, for the upstream and downstream reefs, respectively. We can see that restocking only the upstream reef can shorten the recovery time to about 60, for both the downstream and upstream reefs %, relative to the system without restocking  is a useful tool for the maintenance of coral cover , reef re suffice . In the The general notion that proactive human intervention will be critical for mankind’s survival, health, and prosperity, is becoming increasingly common among terrestrial ecology scientists and decision-makers . In contIn this work we used a mathematical model to examine the feasibility and potential efficiency of fish population restocking, aimed at accelerating coral reef recovery. The proposed ‘restocking’ tool, as applied to fishery enhancement management, is based on previous efforts to enhance wild fish populations by releasing cultured fish into aquatic environments . IdeallyB0   , which avirgatu) .2 for low-cost transplantations (2), and its potential benefit should at least incentivize the implementation of both tools concomitantly.Importantly, our results show that restocking is a financially beneficial method, due to the high economic value of coral reef services  and the ntations . TherefoIt is our expectation that future research will yield further ecological and economic estimates, which could help us to assess the efficiency of such interventions and of their combinations.It should be stressed that our proposed restoration approach is not presented as an alternative to protection. Moreover, we agree with the widely-accepted notion that protection  is the most important management tool by which to maintain reef health and to facilitate the fast recovery of reefs following wide-scale natural disturbances. We propose, nonetheless, that fish restocking, and possibly other ecological restoration tools in conjunction with conservations measures, be considered as an efficient and economically beneficial method for the rehabilitation coral reefs.10.7717/peerj.1732/supp-1Text S1Click here for additional data file.10.7717/peerj.1732/supp-2Text S2Click here for additional data file.10.7717/peerj.1732/supp-3File S3Click here for additional data file.


Ureteral obstruction with subsequent hydronephrosis is a common clinical complication. Downregulation of renal sodium transporters in obstructed kidneys could contribute to impaired urinary concentrating capability and salt waste following the release of a ureteral obstruction. The current study was undertaken to investigate the role of mitochondrial complex-1 inhibition in modulating sodium transporters in obstructive kidney disease. Following unilateral ureteral obstruction (UUO) for 7 days, a global reduction of sodium transporters, including NHE3, α-Na-K-ATPase, NCC, NKCC2, p-NKCC2, ENaCα, and ENaCγ, was observed, as determined via qRT-PCR and/or Western blotting. Interestingly, inhibition of mitochondrial complex-1 by rotenone markedly reversed the downregulation of NKCC2, p-NKCC2, and ENaCα. In contrast, other sodium transporters were not affected by rotenone. To study the potential mechanisms involved in mediating the effects of rotenone on sodium transporters, we examined a number of known sodium modulators, including PGE2, ET1, Ang II, natriuretic peptides , and nitric oxide synthases . Importantly, among these modulators, only BNP and iNOS were significantly reduced by rotenone treatment. Collectively, these findings demonstrated a substantial role of mitochondrial dysfunction in mediating the downregulation of NKCC2 and ENaCα in obstructive kidney disease, possibly via iNOS-derived nitric oxide and BNP. Obstructive kidney disease is a common clinical complication12356128Mitochondrial abnormality has been reported in obstructive nephropathy1011128In the present study, employing the mitochondrial complex-1 inhibitor rotenone, we investigated 1) whether inhibition of mitochondrial complex-I could affect the downregulation of sodium transporters in the obstructed kidney; and 2) whether some known diuretic factors were affected by rotenone treatment and potentially contributed to the effect of rotenone on regulating sodium transporters.To study the role of rotenone treatment in the regulation of sodium transporters in obstructive kidney disease, we examined the mRNA expression of sodium transporters including NHE3, α-Na-K-ATPase, NCC, NKCC2, and three ENaC subunits  via qRT-PCR. The data showed that NHE3, α-Na-K-ATPase, and NCC were markedly downregulated in obstructed kidneys, which was not affected by rotenone administration . In contThe immunohistochemistry results showed that the robust downregulation of NKCC2 protein expression in the obstructed kidney was entirely prohibited by rotenone treatment . SimilarWe further examined the protein levels of the collecting duct sodium channels ENaCα, ENaCβ, and ENaCγ. Similar to NKCC2, downregulation of ENaCα was reversed by rotenone treatment, as determined via immunohistochemistry and Western blotting . HoweverThrough Western blotting, we further examined the regulation of the NHE3 and α-Na-K-ATPase proteins. As shown in A review of the literature revealed that both PGE2 and Ang II have been reported to play roles in mediating the reduction of sodium transporters in obstructive kidney disease. Therefore, we examine the contents of PGE2 and Ang II in obstructed kidneys with or without rotenone treatment. Surprisingly, the induction of PGE2 and Ang II was not altered by rotenone administration . In agreRenal natriuretic peptides exhibit an established role in regulating sodium transporters in the kidney. Here, we detected the mRNA expression of three forms of natriuretic peptides . As expected, the three forms of natriuretic peptides were all markedly enhanced in the obstructed kidneys . RotenonTo further study the potential mechanisms involved in the effects of rotenone on regulating NKCC2 and ENaC, we examined nitric oxide synthases including iNOS, eNOS, and nNOS via qRT-PCR. As shown by the data, iNOS and eNOS, but not nNOS, were elevated in the obstructed kidneys . Followiet al. demonstrated that COX-2/prostaglandin E2 signaling might be a mechanism for inducing the dysregulation of renal sodium transportersObstructive kidney disease is a very common clinical complication. Kidney obstruction leads to a rapid downregulation of sodium transporters within 24 to 72 hours and time-dependent development of tubulointerstitial fibrosis81414Mitochondrial injury has been observed in obstructive kidneys11121317We further examined the kidney contents of PGE2 and Ang II and found that both were markedly elevated by kidney obstruction. However, neither was affected by rotenone. Meanwhile, the upregulation of angiotensinogen mRNA expression was not inhibited by rotenone. These data suggested that the effects rotenone on protecting NKCC2 and ENaCα downregulation are independent of PGE2 and Ang II. Due to the known role of ET1 in modulating renal sodium transporters1920Natriuretic peptides, including atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and C-type natriuretic peptide (CNP), are expressed in the kidneys and play important roles in regulating renal sodium transporters2223242527Another known regulator of renal sodium transporters is nitric oxide (NO)29303213In summary, employing the mitochondrial complex-1 inhibitor rotenone, we observed an interesting phenomenon, in which inhibition of mitochondrial complex-1 selectively reversed the obstruction-induced downregulation of NKCC2 and ENaCα in kidneys. The distinct responses of sodium transporters to rotenone treatment indicated that the downregulation of NKCC2 and ENaC might be associated with a different pathogenic mechanism than observed for other sodium transporters in obstructive nephropathy. The role of the mitochondria may be particularly important for the reduction of NKCC2 and ENaCα, but not the other transporter. These findings not only shed new light on our understanding of the mechanisms underlying the obstruction-related dysregulation of renal sodium transporters, but also suggest new targets for addressing renal concentrating defects in obstructive nephropathy.C57BL/6J mice were originally purchased from the Jackson laboratory. This mouse colony was propagated at Nanjing Medical University. In all experiments, 3- to 4-mo-old male mice were used. All of the mice were maintained under a 12:12-h light-dark cycle (lights on at 6:00 a.m. and lights off at 6:00 p.m.). Animal studies were performed under protocols in accordance with relevant guidelines and regulations and approved by the Nanjing Medical University Institutional Animal Care and Use Committee.Unilateral ureter obstruction was induced as previously described132O2 for 15 minutes at room temperature to block endogenous peroxidase activity. After boiling in antigen retrieval solution  for 15 minutes at high power in a microwave oven, the sections were incubated overnight at 4 °C with a rabbit anti-NKCC2 antibody  or a rabbit anti-ENaCα antibody . After washing with PBS, the secondary antibody was applied, and the signal was visualized using an ABC kit (Santa Cruz Biotechnology).The kidneys were fixed with 10% formalin and embedded in paraffin. Kidney sections (4-μm thickness) were incubated in 3% HThe whole kidney was lysed, and the protein concentration was determined using the Coomassie reagent. Protein (60 μg) from whole kidney lysates was denatured in boiling water for 10 min, separated via SDS-polyacrylamide gel electrophoresis, and transferred to nitrocellulose membranes. The blots were blocked overnight with 5% nonfat dry milk in Tris-buffered saline (TBS), followed by incubation for 1 h with rabbit anti-NHE3 , anti-α-Na-K-ATPase (Abcam), anti-NKCC2 , anti-ENaCα , anti-ENaCβ  or anti-ENaCγ  at a dilution of 1:1,000 After being washed with TBS, blots were incubated with a goat anti-horseradish peroxidase-conjugated secondary antibody  and visualized using ECL kits .http://frodo.wi.mit.edu/primer3/), and the sequences are shown in Table 1. qRT-PCR amplification was performed using SYBR Green Master Mix  and the Prism 7500 Real-Time PCR Detection System . The cycling conditions were 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min.Total RNA isolation and reverse transcription were performed as previously describedKidney tissue was homogenized in phosphate-buffered saline, followed by centrifugation for 5 min at 10,000 r.p.m. The supernatant was diluted 1:50 with enzyme immunoassay buffer. The concentration of PGE2 was determined using an enzyme immunoassay according to the manufacturer’s instructions . The kidney content of An II was measured with EIA kits .P < 0.05.All values are presented as the mean ± SE. Statistical analysis was performed using ANOVA followed by multiple comparison test. Differences were considered to be significant when How to cite this article: Zhang, Y. et al. Inhibition of Mitochondrial Complex-1 Prevents the Downregulation of NKCC2 and ENaCa in Obstructive Kidney Disease. Sci. Rep.5, 12480; doi: 10.1038/srep12480 (2015).


We also identify a residue on the ratchet helix that influences Mtr4 affinity for polyadenylated substrates. Previous work indicated that deletion of the arch domain has minimal effect on Mtr4 unwinding activity. We now show that combining the arch deletion with ratchet helix mutations abolishes helicase activity and produces a lethal in vivo phenotype. These studies demonstrate that the ratchet helix modulates helicase activity and suggest that the arch domain plays a previously unrecognized role in unwinding substrates.Mtr4 is a conserved Ski2-like RNA helicase and a subunit of the TRAMP complex that activates exosome-mediated 3′-5′ turnover in nuclear RNA surveillance and processing pathways. Prominent features of the Mtr4 structure include a four-domain ring-like helicase core and a large arch domain that spans the core. The ‘ratchet helix’ is positioned to interact with RNA substrates as they move through the helicase. However, the contribution of the ratchet helix in Mtr4 activity is poorly understood. Here we show that strict conservation along the ratchet helix is particularly extensive for Ski2-like RNA helicases compared to related helicases. Mutation of residues along the ratchet helix alters  To maintain correct gene expression in the cell, the integrity of RNA must be tightly regulated through RNA processing, turnover and surveillance pathways –3. Severin vitro observation is consistent with a UV cross-linking study in yeast, which determined that Trf4 substrates contain an average poly(A) tail length of 5 nucleotides, supporting the conclusion that poly(A) tail length is regulated in vivo (Kinetic studies indicate that Mtr4 has a higher affinity for RNA substrates containing a polyadenylated 3′ end relative to non-poly(A) sequences. Additionally, binding characteristics differ between the two types of RNA substrates when in the presence of nucleotides, suggesting that Mtr4 interacts with poly(A) sequences using a mechanism distinct from that employed to bind non(A) sequences ,28. Nota in vivo . These sin vivo  and non(A) substrates. The helix bundle domain (domain 4) of archaeal Hel308 PDB: 2P6R)  was usedB: 2P6R  WT) were made using a modified version of the QuikChange (Agilent) site-directed mutagenesis procedure (WT and mutant Mtr4 proteins was carried out as performed previously (R1030A-archless mutant exhibited poor solubility yielding much lower amounts of protein. Protein concentration was determined using a NanoDrop spectrophotometer (Thermo Fisher) with an extinction coefficient for Mtr4 of 89 450 M−1 cm−1  was obtained from the Jankowsky lab. All proteins were recombinantly expressed in an Escherichia coli BL21-codon+-(DE3)-RIL cell line (Stratagene). Cell lysis was performed by lysozyme treatment and sonication of frozen cell pellets. Cell lysis and clarification were performed separately for Mtr4WT and Trf4/Air2, at which point the soluble fractions were combined. Cobalt affinity, FLAG affinity and NAP-25 gel filtration was used to purify TRAMP complexes at 4°C. Final purification buffer consisted of 50 mM sodium phosphate (pH 7.0), 10% glycerol, 200 mM sodium chloride and 10 μM zinc chloride.Point mutations of Mtr4 wild-type (Mtr4rocedure . The expeviously . While mrotParam ). Expres  overhang), 5′-GCGUCUUUACGGUGCUUAAAAA-3′; R22R (non(A) overhang), 5′-GCGUCUUUACGGUGCUUGCCUG-3′. The 16 nucleotide top strand was radiolabeled using γ-32P ATP and T4 polynucleotide kinase and quenched by heating to 95°C before annealing. The RNA substrates were purified by native polyacrylamide gel electrophoresis, gel extraction and ethanol precipitation. Annealed substrates with the R22A bottom strand are referred to as a poly(A) substrate, while the annealed substrates with the R22R bottom strand are referred to as a non(A) substrate.All RNAs used in this study were purchased from Integrated DNA Technologies (IDT). The substrate sequences are as follows with duplex regions underlined: R16 , 5′-WT and saturating levels of ATP. Reactions were carried out at 30°C in a controlled water bath. The buffer used was 40 mM MOPS (pH 6.5), 100 mM NaCl, 0.5 mM magnesium chloride, 5% glycerol, 0.01% nonidet-P40 substitute (Amresco), 2 mM dithiothreitol and 1 U/μl of Ribolock (Thermo Fisher). Reactions were allowed to incubate for 5 min with ∼0.2 nM RNA  and the indicated concentration of Mtr4WT or Mtr4 mutant protein. Reactions were initiated by the addition of ATP and MgCl2 at saturating concentrations , and amplitudes (A) were performed using an integrated first-order rate law. Curve fits were made to data collected in triplicate, as employed previously (Fraction unwound = A(1−exp(-kunw*t))); where [E] is enzyme concentration, K1/2,E is functional affinity . At spekunw*t))),30,53. Tt curves , with thaffinity  and kunwWT and mutants were incubated with RNA substrate for 1–5 min to reach equilibrium before each measurement was taken. At all protein concentrations tested, the protein was in excess over RNA and the RNA was <10% of the Kd to allow for fitting of the data using a ligand binding, one-site saturation equation (F = [L]/(Kd + [L]), where [L] is the concentration of Mtr4). Anisotropy was measured on a Synergy H4 Hybrid Multi-Mode Microplate Reader (BioTek) with an excitation at 485 nm and an emission at 528 nm at 30°C. Sigmaplot (Systat Software) was used for curve analysis and Kd determination.Binding analysis of Mtr4 to RNA was carried out using fluorescence anisotropy. A fluorescein label was added to the 3′ end of the R16 RNA, and was annealed to the longer R22A substrate containing a 3′ poly(A) overhang. Binding reactions were buffered in 40 mM MOPS (pH 6.5), 100 mM sodium chloride, 0.5 mM magnesium chloride, 5% glycerol, 0.01% nonidet-P40 substitute and 2 mM dithiothreitol. Concentration of the fluorescently labeled duplexed RNA was held constant at 60 nM with increasing concentrations of protein (varied depending on the binding affinity of each mutant). Mtr42 (1.33 mM each) with time points taken at 0, 5, 10, 15 and 20 min. A 5X quenching solution (250 mM EDTA) was then mixed with each sample to reach a final concentration of 50 mM EDTA. The 2X malachite green solution  was added in a 9:1 excess to the sample and incubated for 17 min to complete the reaction before reading the absorbance at 650 nm. Initial rates ([Pi] μM min−1) were calculated by fitting a linear trend line to the absorbance values at the different time points using KaleidaGraph (Synergy Software). To determine the ATPase activity enhancement due to RNA with Mtr4WTand Mtr4 mutants, reactions without RNA were used to obtain background values.Mtr4 ATPase activity was measured using a malachite green assay, adapted from previously published protocols ,54. AbsoWTor Mtr4 ratchet helix mutants contained the same upstream promoter and downstream sequence as used previously , and pAplasmid; ) were usplasmid; . PlasmidSaccharomyces cerevisiae complemented with an Mtr4WT copy plasmid containing a URA3 selectable marker was transformed with Mtr4WT or Mtr4 ratchet helix mutant plasmids. Transformants were grown in Synthetic Complete-LEU (SC-LEU) liquid media overnight at 30°C to allow for random loss of the URA3 plasmid. Liquid cultures were serially diluted 5-fold and spotted onto control plates (SC-LEU) or 5-fluoro-orotic acid  and grown at 20, 30 or 37°C.An Mtr4 deletion strain of R/Rfree from the original Mtr4 structure (PDB: 3L9O), with final values of 0.248/0.299 . The revised coordinates have been deposited at the Protein Data Bank under accession code 4QU4, and are linked to the original submission (3L9O).During our comparison of the 3.4 Å apo Mtr4 structure (PDB: 3L9O) and the 2.9 Å RNA-bound structure (PDB: 2XGJ), it became apparent that improvements in sequence register and connectivity could be made in some regions of the lower resolution structure. Remodeled regions were primarily confined to the N-terminus, the fist (or KOW) region of the arch domain and a few loops . The electron density maps resulting from the improved model revealed additional electron density for several side chains, including several residues along the ratchet helix, which were then incorporated into the model. Model building was performed using Coot . PHENIX In the RNA-bound structure of Mtr4, two molecules are observed in the asymmetric unit . In bothWe next examined the conservation of the residues along the ratchet helix for the Ski2-like Mtr4, Ski2, Brr2 and Hel308 helicases, and the DEAH/RHA-box Prp22 and Prp43 helicases ,37,43,46Although no residue along the ratchet helix is universally conserved throughout Ski2 and DEAH/RHA-box helicases, conservation patterns are clearly evident. The most striking feature is that the fourth turn of the ratchet helix (counting from the N-terminus) is strictly conserved in a helicase-specific manner Figure . Mtr4 anS. cerevisiae Mtr4 to alanine . E1033 was also mutated to tryptophan (E1033W) to mimic the sequence observed in Hel308. Pre-steady state unwinding assays and calculations were performed using a helicase assay developed previously to characterize the unwinding activity of Mtr4 and other helicases .The interaction of R1030 and E1033 with RNA observed in the Mtr4 structures combined with the strong conservation at each of these positions in Ski2-like RNA helicases suggested that these residues might be important for Mtr4 activity. To assess the role of these residues in Mtr4 function, we mutated each position in elicases ,58. The kunw) for the Mtr4R1030A mutant at 800 nM protein than that observed for wild-type enzyme . In contrast, the Mtr4E1033A protein demonstrated a higher kunw at 800 nM than Mtr4WT . Mutation of E1033 to a tryptophan significantly impaired unwinding of a poly(A) substrate  at several enzyme concentrations were determined for the ratchet helix mutants to obtain the strand-separation rate constants at enzyme saturation (kunwmax)  between a poly(A) and a non(A) substrate for each individual mutant within the error reported. Additionally, all ratchet helix mutants displayed lower RNA enhanced ATPase activity than Mtr4WT, regardless of whether their unwinding rate constant was faster or slower , completely abolishes ATPase activity and unwinding activity (data not shown).Using a 3′ polyadenylated substrate (poly(A)), we observed a smaller unwinding constant  and non(A) substrates (R1030A mutant (Table R1030A is ∼2.5 fold slower than TRAMPWT.Since bstrates . We obseWT sequence preferences (WT and Mtr4E1033A enzymes showed an unwinding preference in kunwmax for the poly(A) substrate over the non(A) substrate at all enzyme concentrations tested  and non(A) substrates at each concentration  and a non(A) substrate  and non(A) substrate ) used recently to characterize Mtr4ferences . Mtr4WT d Figure  and C. Ie Figure . Thus, Rin vitro, we then asked how mutations at ratchet helix positions affect Mtr4 function in vivo. Mtr4 mutants, Mtr4R1030A, Mtr4E1033A, Mtr4E1033W and the double mutant Mtr4R1030A/E1033A were constructed, serially diluted and tested for viability at 20, 30 and 37°C in S. cerevisiae. Complementation with plasmid containing Mtr4WT was used as a positive control, whereas Mtr4archless and Mtr4D262A/E263A mutants were used to demonstrate a slow growth phenotype and an active site mutation, respectively. The ratchet helix mutations cause a slow growth phenotype at all temperatures tested when compared to Mtr4WT; however, this growth phenotype is less severe than that of Mtr4archless  on cell viability. Although the fist of the Mtr4 arch domain binds RNA in vitro  substrate compared to wild-type. In the case of Mtr4R1030A-archless, no binding was detected. Additionally, no binding was detected up to 20 μM by EMSA .Multiple domains contribute to RNA binding in Mtr4. The Mtr4 RNA-bound crystal structure  reveals s Figure . Mtr4WT WT (R1030A or the Mtr4E1033A ratchet helix point mutants with an arch deletion (Mtr4R1030A-archless or Mtr4E1033A-archless), unwinding activity is abolished (Table E1033A-archless (unwinding activity for TRAMPR1030A-archless was undetermined due to poor solubility of the complex). This loss of helicase activity is consistent with the in vivo data demonstrating severe growth phenotypes for both Mtr4R1030A-archless and Mtr4E1033A-archless  by ∼2-fold. For E1033, the effect on unwinding may be related to the size of the side chain. When the glutamate is mutated to a smaller alanine, kunwmax increases, whereas mutation to a larger tryptophan  has the inverse effect . The simplest model is that R1030 directly reads the RNA sequence, promoting preferential unwinding of substrates polyadenylated by Trf4 or Trf5. Given the preference for a 3′ tail containing five adenosines  tail. This result is consistent with the direct interactions observed between R1030 and an adenine base in the RNA-bound crystal structure of Mtr4 . Nevertheless, these data establish a relationship between the arch domain and the ratchet helix during unwinding events, and are consistent with the model that Mtr4 contains an extensive substrate binding interface that includes both RecA domains, the ratchet domain, and the arch, and that interactions with each of these domains contribute to unwinding (Figure The studies presented here highlight an unexpected relationship between the Mtr4 arch domain and unwinding activity. We previously reported  and confg Figure .Additional studies are needed to clarify the interactions between each domain and with RNA substrates. The dynamic relationship between domains and RNA-binding events is poorly understood. For example, conformational changes are observed between the apo- and RNA-bound forms of Mtr4, including the ratchet helix . Even laSupplementary Data are available at NAR Online.


The cytosolic antiviral innate immune sensor RIG-I distinguishes 5′ tri- or diphosphate containing viral double-stranded (ds) RNA from self-RNA by an incompletely understood mechanism that involves ATP hydrolysis by RIG-I's RNA translocase domain. Recently discovered mutations in ATPase motifs can lead to the multi-system disorder Singleton-Merten Syndrome (SMS) and increased interferon levels, suggesting misregulated signaling by RIG-I. Here we report that SMS mutations phenocopy a mutation that allows ATP binding but prevents hydrolysis. ATPase deficient RIG-I constitutively signals through endogenous RNA and co-purifies with self-RNA even from virus infected cells. Biochemical studies and cryo-electron microscopy identify a 60S ribosomal expansion segment as a dominant self-RNA that is stably bound by ATPase deficient RIG-I. ATP hydrolysis displaces wild-type RIG-I from this self-RNA but not from 5' triphosphate dsRNA. Our results indicate that ATP-hydrolysis prevents recognition of self-RNA and suggest that SMS mutations lead to unintentional signaling through prolonged RNA binding.DOI:http://dx.doi.org/10.7554/eLife.10859.001 Living cells produce long, strand-like molecules of RNA that carry the instructions needed to make proteins. Viruses also make use of RNA molecules to hijack an infected cell’s protein-production machinery and create new copies of the virus. RNA molecules from viruses have a number of features that distinguish them from a cell’s own RNAs, and human cells contain receptors called RLRs that can start an immune response whenever they detect viral RNAs. All of these receptors break down molecules of ATP, a process that releases useable energy. However, so far it is not understood how this activity helps the receptors to distinguish viral RNA from the cell’s own RNA molecules .Recently, some autoimmune diseases (including Singleton-Merten Syndrome) were linked to mutations in the parts of RLRs that allow the receptors to break down ATP. Now, Lässig et al. have studied the effects of specific mutations in an RLR called RIG-I in human cells. The experiments showed that mutations that disrupt RIG-I’s ability to bind to ATP also prevented the receptor from becoming activated. However, mutations linked to Singleton-Merten Syndrome don’t stop ATP from binding but instead slow its breakdown; this effectively locks the receptor in an ATP-bound state. Lässig et al. found that similar mutations in RIG-I caused human cells to trigger a constant immune response against the self-RNAs.Further experiments then suggested that the breakdown of ATP helps to remove RIG-I that has bound to double-stranded sections of self-RNAs. This activity frees the receptor, making it more able to detect double-stranded viral RNAs and preventing unintentional signaling. Lässig et al. also identified a specific double-stranded section of a human RNA that may be recognized by the mutated version of RIG-I in people with Singleton-Merten Syndrome.The next steps following on from this work are to extend the analysis to also include other RLRs and further explore the underlying mutations within the three-dimensional structures of the receptors and RNA molecules involved.DOI:http://dx.doi.org/10.7554/eLife.10859.002 The innate immune system provides a rapid initial reaction to invading pathogens and also stimulates the adaptive immune system . PatternRetinoic-acid inducible gene I (RIG-I), melanoma differentiation-associated gene 5 (MDA5) and laboratory of physiology and genetics 2 (LGP2) are three structurally related PRRs – denoted RIG-I like receptors (RLRs) – that recognize cytosolic foreign RNA. RIG-I senses RNA from a broad range of viruses including measles virus and Sendai virus (both paramyxoviridae), Influenza A virus, Japanese encephalitis virus and Hepatitis C virus, whereas MDA5 is activated for example by picornavirus RNA. LGP2 has augmenting and regulatory roles in MDA5 and RIG-I dependent signaling .RIG-I preferentially detects base-paired double-stranded RNA (dsRNA) ends containing either 5′ triphosphate (ppp) or 5′ diphosphate (pp) moieties  and not RLRs are members of the superfamily II (SF2) of ATPases, helicases or nucleic acid translocases. RIG-I and MDA5 consist of two N-terminal tandem caspase activation and recruitment domains (2CARD), a central ATPase/translocase domain and a C-terminal regulatory domain (RD). LGP2 lacks the 2CARD module but otherwise has a similar domain architecture. Binding of RNA induces a conformational change in RIG-I. If activated, the RD binds the ppp- or pp-dsRNA end, while the SF2 domain interacts with the adjacent RNA duplex and forms an active ATPase site . In thisThe SF2 ATPase domain plays a critical part in RIG-I activation, although the role of the ATPase activity is still debated. Mutation of the seven SF2 “helicase” motifs resulted in RLRs that are either inactive or signal constitutively . On the Several autoimmune diseases, including the Aicardi-Goutières and Singleton-Merten syndromes (SMS), were linked to single amino acid mutations in the SF2 domains of MDA5 and RIG-I . Two poiIn order to clarify the role of RIG-I’s ATPase in antiviral signaling and RLR associated human diseases, we engineered structure-derived and patient-identified mutations into RIG-I and tested the resulting proteins in different types of cell-based and in vitro analyses. Collectively, we find that SMS mutations phenocopy the structure-derived E373Q mutation in motif II, which is designed to trap RIG-I in an ATP-bound state. Freezing this state results in a dramatic autoimmune response because the enzyme binds self-RNA and signals. An unexpected, strongly enriched self-RNA is the ribosomal large subunit, which contains large, dsRNA expansion segments. Collectively, our results suggest that a biomedical and functional critical role of RIG-I’s ATPase is to prevent spontaneous and unintended activation by self-RNA. Thus, the SF2 translocase likely increases the sensitivity of the system by reducing background signaling. Furthermore, our studies suggest that in SMS, RIG-I is trapped in an ATP-bound state and signals through self-ligands.x served as guide for these mutations  prevent ATP binding and formation of a functional ATP-bound complex, ii) allow ATP binding and ATP-induced conformational changes but prevent ATP hydrolysis, or iii) disable interaction of the RNA with either the 1A or 2A domain of SF2 . The strtations  promoter activity assay carried out in HEK 293T RIG-I KO cells . OverexpTo rule out a “constitutive” active conformation of RIG-I E373Q due to an exposed 2CARD module (e.g. from an unfolded SF2) we performed small angle X-ray scattering with purified wtRIG-I and RIG-I E373Q demonstrating that both proteins have the same solution structure . In addiTo test whether E373Q signals in non-infected  because of interaction with self-RNA, we additionally introduced mutations in various RNA binding sites, in particular a ΔRD variant (RIG-I 1-798) and mutations in two RNA-interacting residues in domains 1A (T347A) and 2A (V699A) of SF2. The single mutation RIG-I T347A did not signal in either infected or non-infected cells, showing that the interaction of RNA with this specific amino acid is critical for signaling . InteresSince RD is also required for the displacement of the 2CARD module from SF2, we additionally analyzed a point mutation in RD. K888 mediates triphosphate binding in RD and mutations in this residue inactivate recognition of viral RNA . Of noteFinally, we addressed the effect of the Singleton-Merten mutations C268F and E373A. E373A is at the same position as our structure-derived E373Q mutant. Consistent with this, we observed that this substitution leads to a constitutive activation of the IFNβ promoter  Figure . InteresIn summary, our studies show that signaling of RIG-I requires both ATP and RNA binding. ATP hydrolysis, on the other hand, appears to be critical to dissolve the signaling state and to prevent activation of RIG-I by self-RNA.We hypothesized that E373Q traps RIG-I in an ATP bound high affinity conformation that is activated already by self-RNA. To test this idea, we immunoprecipitated RIG-I and its mutants from non-infected HEK 293T RIG-I KO cells or cells infected with measles or Sendai virus and analyzed the co-purified RNA molecules. Regardless of whether co-purified from infected or non-infected cells, the amount of RNA recovered from RIG-I E373Q was about 3 times higher than that from RIG-I . SimilarWhen analyzed on a Bioanalyzer RNA chip or on agarose gels, we found that the increased amount of RNA is to a large extent due to the presence of 28S rRNA, while 18S rRNA remains unaltered . ControlIn order to determine the immunostimulatory potential of the RNA co-purified from virus-infected cells, we back-transfected the RNA into HEK 293T ISRE-FF/RFP reporter cells , fluorescence anisotropy experiments and ATP hydrolysis assays in presence and absence of ATP or ADP·BeFpp-dsRNA , we alsopp-dsRNA . The hai3 than in its absence  or HEK 293T RIG-I KO cells . HEK 293Sequences encoding full-length human RIG-I or MDA5 with N- or C-terminal FLAG/HA-tag were cloned into pcDNA5 FRT/TO . Mutants were generated by site-directed mutagenesis with PfuUltra polymerase .6 HEK 293T or HEK 293T RIG-I KO cells were transfected with 10 µg pcDNA5 vector coding for different FLAG/HA tagged RLR proteins. Non-infected cells were harvested 24 h after transfection. Infections were carried out 6h after transfection with an MOI of 0.05 for measles virus or high MOI for Sendai virus and were allowed to proceed for 40 or 24 hr, respectively. Cells were harvested and incubated in Nonidet P-40 lysis buffer , pH 7.5) for 10 min on ice. Lysates were cleared by centrifugation and proteins were immunoprecipitated for 2.5 - –4 hr with anti-DDK magnetic beads  or anti-FLAG (M2) bound to magnetic protein G Dynabeads . Beads were washed five times with washing buffer  and incubated with proteinase K  for 30 min at 50 °C. RNA was isolated by phenol/ chloroform/ isoamyl alcohol extraction using Phase Lock Gel Heavy tubes . The quality of the isolated RNA was validated on an Agilent RNA 6000 Nano chip.6x105 HEK 293T RIG-I KO or 2.5×105 HEK 293T ISRE-FF/RFP reporter cells per well using Lipofectamine 2000  as transfection reagent according to the manufacturer's protocol. For downstream signaling assays HEK 293T RIG-I KO cells were co-transfected with 500 ng protein expression vector, 100 ng p125-luc, 10 ng pCMV-RL and 50 ng empty expression vector. For RIG-I E373Q/RIG-I Δ2CARD,E373Q competition assays HEK 293T RIG-I KO cells were co-transfected with 100 ng RIG-I E373Q expression vector, varying concentrations of the RIG-I Δ2CARD,E373Q expression vector, 100 ng p125-luc and 10 ng pCMV-RL. DNA concentrations were held constant by adding empty expression vector if necessary. For determination of the immunostimulatory potential of recovered RNA from co-immunoprecipitations, HEK 293T ISRE-FF/RFP cells were transfected with 250 ng RNA in Opti-MEM . For RNA stimulation of cells overexpressing RIG-I E373Q 2.5×105 HEK 293T RIG-I KO cells were transfected with 100 ng RIG-I E373Q expression vector, 100 ng p125-luc, 10 ng pCMV-RL and 1000 ng total RNA/ rRNA or ES hairpin RNA in Opti-MEM. All cells were harvested 24 h after transfection using 200 µL PLB  and subjected to immunoactivity experiments using the Dual-Glo luciferase assay system  as previously described (Immunoactivity experiments were carried out in 24-well plates seeded with 2.5×10escribed . The lucE. coli DH10MultiBac cells. Bacmids were extracted for transfection into SF9 insect cells and propagated virus was used for protein expression in High Five insect cells. Seventy-two hours after infection cells were harvested and flash frozen in liquid nitrogen. RIG-I Δ2CARD was expressed in E. coli BL21 Rosetta (DE3), using pET expression vectors as described earlier  the fluorescence signal was detected using a gradient from 5 °C to 100 °C with 0.5 K/30 s and one scan each 0.5 K in a real-time thermal cycler  using the FRET mode.Thermal stability of RIG-I or RIG-I E373Q in presence or absence of ATP was analyzed by fluorescence thermal shift assays. Proteins (20 µM) were incubated in 25 mM HEPES pH 7, 150 mM NaCl, 10 mM MgCl2, 5 mM β-Mercaptoethanol, 5% glycerol). RIG-I samples were measured at protein concentrations of 1.28, 2.65 and 8.35 mg/mL and RIG-I E373Q samples with concentrations of 0.87, 2.13 and 6.84 mg/mL. The respective scattering of the corresponding buffer was used for buffer subtraction. The samples did not show signs of radiation damage, which was assessed by automatic and manual comparison of consecutive exposure frames. The data was processed using PRIMUS from the ATSAS package .SAXS experiments were conducted at the PETRA3 P12 beamline of the European Molecular Biology Laboratory/ Deutsches Elektronen-Synchrotron, Hamburg, Germany. Samples were measured in absence or presence of 5 mM ATP in size exclusion buffer  for 15 min on ice, followed by disruption with nitrogen pressure  in a cell disruption vessel . The cell lysate was cleared  and the resulting supernatant was loaded onto a sucrose cushion (Buffer 1 supplemented with 35% sucrose). Subsequent spinning  was performed. After resuspension of the ribosomal pellet, a high-salt purification by centrifugation through a 500 mM sucrose cushion 2, 5 mM β-mercaptoethanol, 1 M sucrose, 1 µg/mL cycloheximide and 0.1% Nikkol) was conducted . The ribosomal pellet was resuspended in Ribosome Buffer 2, 1 mM DTT, 1/200 EDTA-free Complete protease inhibitor , 0.2 U/mL RNasin ), quickly centrifuged, frozen in liquid nitrogen and stored at -80 °C.HeLa S3 cells were harvested , washed with PBS  and incubated with 1.5x vol Buffer 1 5 HEK 293T were seeded per well of 24 well plates. After 24 h cells were harvested in PBS, collected by centrifugation and lysed in Nonidet P-40 lysis buffer for 10 min on ice. Supernatant was cleared by centrifugation and DNA was digested with TURBO DNase  for 3 min at 37 °C. Proteins were digested and RNA was extracted as described above. For ribosomal RNA isolation purified human ribosomes were proteinase K digested and RNA was extracted accordingly.For total RNA isolation 2.5 x 102, 2 mM DTT, 1 mM ATP, 0.1% DDM, 10% Glycerol) for 15 min at room temperature and then for 15 min at 4 °C. The mixture was loaded onto a sucrose cushion (binding buffer with 750 mM sucrose) and spun . Supernatant and pellet fractions were separated and TCA precipitated. The resulting samples were analyzed by SDS-PAGE and visualized using SYPRO Orange Staining .Human 80S ribosomes were incubated with or without 2.5x molar excess of RIG-I or RIG-I E373Q in binding buffer 2, 2 mM DTT, 1 mM ATP, 0.1% DDM, 5% glycerol) was applied to 2 nm pre-coated Quantifoil R3/3 holey carbon supported grids and vitrified using a Vitrobot Mark IV .5 OD/mL human 80S ribosomes were incubated with or without 2.5x molar excess of RIG-I E373Q. Each sample 0.5) of 17.7 Å and 21.9 Å, respectively. For comparison of the two final volumes, a statistical difference map between the two reconstructions was calculated.Data were collected on a 120 keV TECNAI SPIRIT cryo-electron microscope with a pixel size of 2.85 Å/pixel at a defocus range between 1.4 µm and 4.6 µm (with RIG-I E373Q ligand) or between 1.8 µm and 5.3 µm (without ligand) under low dose conditions. Particles were detected with SIGNATURE . InitialWe used the crystal structure of the human RIG-I protein (PDB code 3TMI)  and the 32P]ATP . 12 µM RIG-I or RIG-I E373Q were incubated in 50 mM HEPES, pH 7.5, 150 mM KCl, 5 mM MgCl2, 2.5 mM TCEP, 0.1 mg/mL BSA supplemented with 2.5 nM [α-32P]ATP for 10 min at room temperature in presence or absence of 100 nM RNA. 2.5 µL of reaction mixture was spotted on nitrocellulose membranes , air-dried and [α-32P]ATP was detected using a phosphor-imaging system .ATP binding was determined by DRaCALA using ATP . Proteins at different concentrations were pre-incubated with 100 nM RNA or purified ribosomes for 10 min at room temperature in EMSA buffer without ATP. The reaction was initiated by addition of 1.5 mM unlabeled and 10 nM [γ-32P]ATP and incubated for 20 min at 37 °C. Free phosphate was separated from ATP by thin layer chromatography in TLC running buffer  on polyethyleneimine cellulose TLC plates . [γ-32P]Pi and [γ-32P]ATP were detected using a phosphor-imaging system  and quantified using ImageJ.ATPase hydrolysis activity was determined using [γ- In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.eLife. Your submission has been favorably evaluated by John Kuriyan (Senior editor), a Reviewing editor, and three reviewers.Thank you for submitting your work entitled "RIG-I's ATPase prevents unintentional recognition of self-RNA" for peer review at The reviewers have discussed the reviews with one another and the Reviewing editor has drafted this decision to help you prepare a revised submission.Summary:RIG-I is a cytoplasmic viral sensor that induces antiviral IFN responses by recognizing non-self signatures in viral RNAs, such as 5' triphosphate and dsRNA structure. Recently, the correlation between autoimmunity and mutations in RIG-I family molecules has been reported. However, the mechanism of how mutant RIG-I leads to autoimmune symptoms is not well characterized. Here Lassig et al. investigated RIG-I mutations found in patients with Singleton-Merten Syndrome (SMS) and confirmed that the SMS mutant RIG-I (C268F and E373A) constitutively activate signaling. Moreover, they found that another constitutively active E373Q mutant is ATPase-deficient and exhibits prolonged binding with endogenous RNAs such as 28S rRNA, which does not activate wild-type RIG-I. These data suggest that abnormal immune response to endogenous RNA is a possible cause of SMS in patients with RIG-I C268F and E373A mutations. The authors also propose that ATP hydrolysis prevents unintentional recognition of self-RNA. The work is highly significant and the data are generally of high quality with some exceptions noted below.This manuscript contains important observations on RIG-I SMS mutants. However, to support their conclusion that constitutive activity of SMS mutants and E373Q mutant is due to hyper-responsiveness to endogenous RNAs, the following major concerns need to be addressed.Essential revisions:1) The role of ATP binding versus ATP hydrolysis:ATPase deficient RIG-I proteins cannot be activated by viral infection and act as dominant inhibitors. Consequently, in paragraph four, subheading “RIG-I ATP hydrolysis defective mutant E373Q shows increased interaction with ribosomal RNA”, the interpretation of In this regard, one important control lacking in the current manuscript is the characterization of the wt RIG-I in the presence of a non-hydrolysable analog of ATP such as ATP-gamma-S or AMPPNP. It would be meaningful to show, e.g., by EMSA, that wt RIG-I binds 28S rRNA in the presence of one of these analogs. This wound make it clear that ATP binding is required for binding RNA, yet ATP hydrolysis is needed to unbind cellular RNA.2) Activation by rRNA:It is known that binding to RNAs does not necessarily activate RIG-I. Thus, how can authors prove that 28S rRNA activates RIG-I E373Q for IFN signaling in the situation where no ATPase activity can be detectable in this mutant? For instance, can RIG-I E373Q stably expressing cells in RIG-I deficient cells respond to exogenous stimulation with rRNAs?In this regard, it will be helpful to add some more explanation about the nature of the 28S rRNA. How does it dominate the RNA binding site of E373Q mutant? Does the relative abundance/concentration amongst similarly structured RNA explain it? Is it the particular secondary structure of this RNA that makes it outcompete amongst other RNA molecules?3) The possibility that constitutive signaling exhibited by the SMS mutant and the E373Q mutant is due to a structural alteration that opens and exposes the CARD domain:When the RIG-I E373Q mutant responds to endogenous RNAs, how does it undergo the conformational change to elicit CARDs without ATPase activity? The authors should exclude the possibility that the mutant protein assumes an "active" conformation simply due to the amino acid substitutions without the need to bind to endogenous ligand, i.e., due to the constitutive exposure of CARD. It might be possible to resolve this important issue by using a construct of E373Q with the CARDs deleted; this construct should inhibit E373Q full-length mediated signaling in a dominant negative fashion because this mutant should bind to endogenous ligand but fail to trigger signaling due to the absence of CARDs.Major editorial details to address:1) The title of this manuscript is misleading since some known ATPase deficient mutants such as K270A lose their potential to activate IFN signaling in response to any ligand, and ATPase activity is required for wild-type RIG-I to transduce the conformational changes needed for signaling in response to viral dsRNA in a physiological condition. Thus, these well-established observations are contradictory to their conclusion as appears in the title of the manuscript.2) In 3) The quality of T347A,E373Q mutant protein was not provided. Assays performed in  Essential revisions:1) The role of ATP binding versus ATP hydrolysis:ATPase deficient RIG-I proteins cannot be activated by viral infection and act as dominant inhibitors. Consequently, in paragraph four, subheading “RIG-I ATP hydrolysis defective mutant E373Q shows increased interaction with ribosomal RNA”, the interpretation of 3 and added the data to the manuscript .As suggested, we performed EMSAs with RIG‐I and the ribosomal expansion segment derived hairpin RNA in presence of the non‐hydrolysable ATP analog ADP∙BeFnuscript . We alsocript (3 . In addi control . We find2) Activation by rRNA:It is known that binding to RNAs does not necessarily activate RIG-I. Thus, how can authors prove that 28S rRNA activates RIG-I E373Q for IFN signaling in the situation where no ATPase activity can be detectable in this mutant? For instance, can RIG-I E373Q stably expressing cells in RIG-I deficient cells respond to exogenous stimulation with rRNAs?In this regard, it will be helpful to add some more explanation about the nature of the 28S rRNA. How does it dominate the RNA binding site of E373Q mutant? Does the relative abundance/concentration amongst similarly structured RNA explain it? Is it the particular secondary structure of this RNA that makes it outcompete amongst other RNA molecules?We thank the referees for this suggestion. We performed co‐transfection experiments of RIG‐I E373Q with purified total RNA, ribosomal RNA and the expansion segment derived hairpin RNA and in all cases find an increase in signaling. These data are added to Even a weak activation of RIG‐I E373Q by 28S rRNA can lead to a considerable signal due to the huge amount of ribosomal RNA compared to other RNAs in the cytosol. Nevertheless we do not want to exclude that there are other double‐stranded RNAs that can activate RIG‐I E373 and stated this in the Discussion (paragraphs four and five).3) The possibility that constitutive signaling exhibited by the SMS mutant and the E373Q mutant is due to a structural alteration that opens and exposes the CARD domain:When the RIG-I E373Q mutant responds to endogenous RNAs, how does it undergo the conformational change to elicit CARDs without ATPase activity? The authors should exclude the possibility that the mutant protein assumes an "active" conformation simply due to the amino acid substitutions without the need to bind to endogenous ligand, i.e., due to the constitutive exposure of CARD. It might be possible to resolve this important issue by using a construct of E373Q with the CARDs deleted; this construct should inhibit E373Q full-length mediated signaling in a dominant negative fashion because this mutant should bind to endogenous ligand but fail to trigger signaling due to the absence of CARDs.To rule out a “constitutive” active conformation due to exposed CARDs we performed 3 experiments. We did small angle X‐ray scattering on RIG‐I and RIG‐I E373Q and demonstrate that both proteins have the same solution structure . We alsoMajor editorial details to address:1) The title of this manuscript is misleading since some known ATPase deficient mutants such as K270A lose their potential to activate IFN signaling in response to any ligand, and ATPase activity is required for wild-type RIG-I to transduce the conformational changes needed for signaling in response to viral dsRNA in a physiological condition. Thus, these well-established observations are contradictory to their conclusion as appears in the title of the manuscript.We thank the editor for this valuable advice and we changed the title to: “ATP hydrolysis by the viral RNA sensor RIG‐I prevents unintentional recognition of self‐RNA”.2) In We substantially revised our EMSAs, repeated them at least in triplicates and quantified the bands .We used different RNA types  in order to dissect differences in binding to the RNA end by RD vs. binding of the SF2 domain to the stem. We revised the text to clarify the use of different RNAs (first paragraph of “Specificity of RIG-I towards double-stranded RNA is increased in presence of ATP”).3) The quality of T347A,E373Q mutant protein was not provided. Assays performed in As suggested we purified RIG‐I T347A,E373Q and performed EMSAs with the ES hairpin RNA in presence and absence of ATP as well as ATPase assays (paragraphs three and four, subection “Specificity of RIG-I towards double-stranded RNA is increased in presence of ATP”;