Ureteral obstruction is associated with reduced expressions of renal sodium transporters, which contributes to impaired urinary concentrating capacity. In this study, we employed a synthetic mitochondrial superoxide dismutase 2 (SOD2) mimic MnTBAP to investigate the role of mitochondrial oxidative stress in modulating the sodium transporters in obstructive kidney disease. Following unilateral ureteral obstruction (UUO) for 7 days, a global reduction of sodium transporters including NHE3, NCC, NKCC2, and ENaCα was observed as determined by qRT-PCR, Western Blotting or immunohistochemistry. Among these sodium transporters, the downregulation of NHE3, NCC, and NKCC2 was partially reversed by MnTBAP treatment. In contrast, the reduction of ENaCα was not affected by MnTBAP. The β and γ subunits of ENaC were not significantly altered by ureteral obstruction or MnTBAP therapy. To further confirm the anti-oxidant effect of MnTBAP, we examined the levels of TBARs in the urine collected from the obstructed ureters of UUO mice and bladder of sham mice. As expected, the increment of urinary TBARs in UUO mice was entirely abolished by MnTBAP therapy, indicating an amelioration of oxidative stress. Meantime, we found that three types of SOD were all reduced in obstructed kidneys determined by qRT-PCR, which was unaffected by MnTBAP. Collectively, these results demonstrated an important role of mitochondrial oxidative stress in mediating the downregulation of sodium transporters in obstructive kidney disease. Unilateral ureteral obstruction (UUO) is a well-established experimental rodent model that mimics the complex pathophysiology of chronic obstructive nephropathy in an accelerated manner. Release of obstruction is often associated with diuresis or natriuresis, which is related to the downregulation of water channels and sodium transporters in obstructed kidneys . It has et al. demonstrated that NOX-derived ROS are partially responsible for the downregulation of renal tubular Na/K-ATPase expression in acute UUO rats [Oxidative stress contributes importantly to the pathogenesis of UUO –6. ROS pUUO rats . Our groUUO rats  and sodiUUO rats  in obstrTherefore, in the present study, we employed manganese (III) tetrakis (4-benzoic acid) porphyrin chloride (MnTBAP), a synthetic mitochondrial superoxide dismutase 2 (SOD2) mimic, to investigate whether mitochondrial ROS could affect the downregulation of sodium transporters in the obstructed kidneys.To detect the effect of MnTBAP therapy on the expression of sodium transporters in obstructed kidneys, we firstly examined the protein levels of NHE3 and NCC using Western blotting. As shown in Figure Next, we detected the effect of MnTBAP therapy on the protein expression of NKCC2. Consistent with previous reports , 12, 13,The epithelial sodium channel(ENaC) is expressed in the aldosterone sensitive distal nephron(ASDN) and consists of 3 subunits. In previous studies, we found distinct changes of three ENaC subunits in obstructed kidneys . Here, sTo clarify whether MnTBAP therapy affected the regulation of sodium transporters at a transcriptional level, we analyzed the mRNA expressions of individual transporters via qRT-PCR. In obstructed kidneys, NHE3, NCC, NKCC2, and ENaCα were all significantly downregulated Figure . HoweverAs a mitochondrial SOD mimics, MnTBAP showed a protective role against the downregulation of sodium transporters in obstructed kidneys, suggesting a possible defect of SOD-related antioxidant system. To validate the anti-oxidative effect of MnTBAP in this model, we measured the urinary levels of TBARS, a known marker of oxidative stress, using the urine collected from the obstructed ureters of UUO mice and bladder of sham mice. As shown in Figure Urinary tract obstruction is a serious disorder accompanied with impaired renal function such as a compromised ability to regulate the urinary excretion of water and sodium. It has been well documented that the abundance of sodium transporters was significantly reduced in response to ureteral obstruction, which at least partially contribute to the impaired urine concentrating capacity after the release of urinary tract obstruction , 13–15. et al. demonstrated that mitochondrial oxidative stress was associated with autophagy and apoptosis in tubular cells and represented an important mechanism of tubular decomposition and nephron loss during UUO [et al. reviewed that there existed an interaction among mitochondrial oxidative stress, apoptosis and inflammation during obstructive nephropathy [Oxidative stress was well described to contribute importantly to the pathogenesis of obstructive nephropathy , 18–22. ring UUO . Nishidaring UUO . Moreovehropathy . Our grohropathy . At the hropathy . Increashropathy , 33. OurAnother finding is that MnTBAP has impact only on the protein but not mRNA levels of NHE3, NCC, and NKCC2. This suggested that mitochondrial ROS may regulate these three transporters at posttranscriptional level. However, the exact mechanism mediating such an effect is still unknown. Then, we detected the expressions of SOD1-3 after kidney obstruction and MnTBAP administration. MnTBAP treatment normalized the oxidative stress marker TBARS in urine but did not affect the reduction of SOD1-3 during kidney obstruction, which might suggest that MnTBAP administration ameliorated oxidative stress during obstructive nephropathy without affecting the endogenous SOD.Taken together, our study showed that the mitochondrial SOD mimics MnTBAP partially but significantly attenuated obstruction-induced downregulation of NHE3, NCC, and NKCC2 at protein levels in kidneys. The reduced mRNA levels of these transporters were not influenced by MnTBAP therapy. Meanwhile, MnTBAP did not affect the expressions of ENaC subunits in UUO model. These findings suggested that mitochondrial oxidative stress might mediate the downregulation of sodium transporters except ENaC in obstructive nephropathy via a posttranscriptional mechanism. This study not only provided new insights into the understanding of the pathogenesis of obstructive nephropathy-related dysregulation of sodium transporters but also offered new therapeutic target for the management of obstructive nephropathy-related fluid imbalance.C57BL/6J mice were originally purchased from the Jackson laboratory. This mouse colony was propagated at the Nanjing Medical University. In all studies, 3- to 4-month-old male mice were used. All 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.). All experiments were approved by the Institutional Animal Care and Use Committee of Nanjing Medical University.N = 6 per group) were sacrificed 7 days after operation and the kidney tissues were harvested for further analysis.Unilateral ureteral obstruction (UUO) was induced as described previously , 31, 34.2O2 for 15 min. Antigen retrieval was carried out in boiling antigen retrieval solution  for 15minutes. Then the sections were incubated overnight at 4°C with rabbit anti-NKCC2 antibody  and at room temperature for 30 min with secondary antibody. Staining was visualized using an ABC kit (Santa Cruz Biotechnology).The kidneys were fixed in 10% formalin and embedded in paraffin. Kidney sections (4-μm thickness) were deparaffinized, hydrated in ethyl alcohol and washed in tap water. Endogenous peroxidase activity was blocked in 3% HProteins were extracted from the whole kidney and homogenized in protein lysis buffer. Equal amount of protein (60 μg) was subjected to SDS-polyacrylamide gel electrophoresis, and transferred to nitrocellulose membranes. The blots were blocked at room temperature (RT) for 1h with 5% nonfat dry milk in Tris-buffered saline (TBS) and probed with the following antibodies: rabbit anti-NHE3 , anti-NKCC2 , anti-NCC , anti-ENaCα  and anti-ENaCγ  at a dilution of 1: 1,000 at 4°C overnight. After incubated with secondary antibodies  at RT for 1h, membranes were visualized using Pierce™ ECL Western Blotting Substrate (Thermo Scientific). Quantification was performed using Image-Pro Plus 6.0 software.http://frodo.wi.mit.edu/primer3/) and the sequences are listed in Table Δ Δ CT method.Total RNA was isolated by using TRIzol (Invitrogen) and first-strand cDNA was synthesized from total RNA(4ug) using Superscript TM III first strand synthesis kit (Invitrogen). Quantitative PCR (qPCR) was performed using the SYBR Green Master Mix  and the ABI 7500 Real-Time PCR Detection System . Oligonucleotides were designed using Primer3 software (available at The measurement of urinary thiobarbituric acid-reactive substances (TBARS) was based on the formation of malondialdehyde by using a commercially available TBARS Assay Kit  according to the manufacturer’s instructions.p < 0.05 was considered significant.All results were presented as means ± SE. The statistical analysis was performed using ANOVA followed by Bonferroni’s test with SPSS 13 statistical software.


Human adenovirus 7 (HAdV-7) strains are a major cause of acute respiratory disease (ARD) among adults and children, associated with fatal pneumonia. An ARD outbreak caused by HAdV-7 that involved 739 college students was reported in this article. To better understand the underlying cause of this large-scale epidemic, virus strains were isolated from infected patients and sequence variations of the whole genome sequence were detected. Evolutionary trees and alignment results indicated that the major capsid protein genes hexon and fibre were strongly conserved among serotype 7 strains in China at that time. Instead, the HAdV-7 strains presented three thymine deletions in the virus associated RNA (VA RNA) II terminal region. We also found that the mutation might lead to increased mRNA expression of an adjacent gene, L1 52/55K, and thus promoted faster growth. These findings suggest that sequence variation of VA RNA II gene was a potential cause of such a severe HAdV-7 infection and this gene should be a new-emerging factor to be monitored for better understanding of HAdV-7 infection. Human adenoviruses (HAdVs) are pathogenic viruses that can usually cause acute respiratory disease (ARD), acute follicular conjunctivitis, haemorrhagic cystitis, gastroenteritis, myocarditis, meningoencephalitis, and even death . Outbreavs. epidemic, or mild vs. severe [The severity of HAdV-7 infection depends on many factors, such as virus virulence, viral transmission environment, or physical condition of the host, and dictates whether occurrence is sporadic . severe –10. Gene. severe , 12. A bTraditionally, capsid hexon and fibre genes have been used to detect genomic variation, as they also determine the HAdV serotypes. However, according to recent reports, the hexon and fibre genes of HAdV-7 were remarkably conserved across time and space . With thHere, we report a large-scale outbreak of severe acute respiratory disease associated with HAdV-7 that occurred in Wuhan, China, in 2015. To assess whether this outbreak was related to genetic variation of the VA RNA gene of the incriminated HAdV-7 strain, we analysed the whole genome sequence variation and viral growth of the Wuhan isolates. Knowledge of a link between VA RNA II genetic variation and HAdV-7 virulence may help us understand the function of this gene and take effective measures to control HAdV-7 prevalence.th of January and 21st of February, 2015. Health officials, medical staff, epidemiologists, and laboratory technicians were designated as an epidemic prevention team to investigate and control the outbreak. The names, onset times, clinical symptoms and informed consent for sample collection and subsequent research usage of the students were documented by trained medical staff and epidemiologists. Copies of the case records of the hospitalized students were provided by the corresponding doctors. All the protocols used in these investigations were approved by the Institutional Review Boards of the Centre of Disease Control and Prevention of China, and the Ethics Committee of Wuhan General Hospital.An outbreak of ARD was reported in a college in Wuhan city, Hubei Province, between the 20Bordetella pertussis, Legionella pneumophila, Chlamydophila pneumoniae, Streptococcus pneumoniae, Haemophilus influenzae, and Mycoplasma pneumoniae were detected with Seeplex®RV12 ACE and Seeplex® Pneumabacter detection kits . After confirmation of HAdV infection, real-time PCR primers for HAdV serotypes 7, 11, 14, and 55 were used for serotyping . Pure Link Viral RNA/DNA Mini Kits  were used to extract nucleic acids. Suspected pathogens, such as adenovirus, coronavirus, parainfluenza virus, influenza A virus, influenza B virus, respiratory syncytial virus A, rotyping .The samples of nasopharyngeal swabs were inoculated into A549 cells cultured in DMEM. The viruses were isolated by repeated freeze-thaw and preserved in -80°C. Primer sequences used for the amplification of hexon, fibre and VA RNA genes have been published elsewhere . The who5’-TGCAAAGAGCTCTAACGGGG-3’ and R: 5’-CTTCCAAGTACTCGCCCTCC-3’. Reference genes VA RNA II and E1 were detected using primers VA-F: 5’-GAGCCAGTGCTGCGTCAA-3’, VA-R: 5’- TTAGAAACGTCGCGGAAA-3’; and E1-F: 5’-CCACCTACGCTGCACGAT-3’, E1-R: 5’- ACCTCTGCCGCTTTCCAC-3’, respectively. KAPA SYBR FAST qPCR Kits  were used for amplification.Viral RNA was extracted using an Ultrapure RNA extraction kit . The primers used for the detection of L1 52/55K mRNA were F: 2 genome copies) were propagated in human A549 cells cultured in 12-well plates and were collected 6, 9, 12, 18, 21, 24, 36, 48, and 72 h after infection. The cultures were subjected to repeated freezing and thawing thrice and centrifuged at 12000×g for 10 min. Then, 200 μl of supernatants were used for DNA extraction using Pure Link Viral DNA Mini Kits and virus titres were determined by detecting the number of viral E1 gene copies by real-time PCR. The E1 gene fragment was cloned into the T vector  and diluted to 0.5 × 102–0.5 × 109 copies/μL as standard samples.Viruses  and were analysed using Student’s th of January and 21st of February, 2015  developed the illness and 169 of them were admitted to Wuhan General Hospital between the 20ry, 2015 . The aveAccording to case reports, patients presented typical respiratory infection symptoms, such as fever (92.3%), cough (83.4%), sore throat (62.1%), swelling of tonsils (47.9%), and a runny nose (26.0%). Among them, 92 patients (54.4%) were diagnosed with upper respiratory infection, 77 (45.6%) with pneumonia , and 121Of the 4113 students, 739 were identified as HAdV-7-positive by real-time PCR. The ratio among hospitalized patients was 76.9% (130 out of 169). Twenty viral strains were isolated from hospitalized positive patients and cultured in the laboratory; after which hexon, fibre, and VA RNA genes were amplified and sequenced. Alignments of these genes of the isolated 20 virus produced 100% similarity.Next, we used the BLASTn tool to find the strains with closest relation to the sequenced hexon, fibre, and VA RNA genes. Both hexon and fibre genes showed 100% identity with the HAdV-7 isolate CQ1198 (GenBank accession number JX625134), which belongs to the HAdV-7d type , revealeThen, we chose the Z9/WH strain (GenBank No. KX897164) of these Wuhan isolates as representative to make whole-genome sequencing, and the comparison result with strain CDC228 revealed that the Z9/WH strain had some other but insignificant mutations except for VA RNA gene .Phylogenetic trees based on hexon, fibre, and VA RNA gene sequences were constructed respectively. As all sequences were identical, one sample Wuhan strain (KU351170) was taken as representative. And the Z9/WH strain (GenBank No. KX897164) was chosen for the whole-genome sequence and DNA polymerase gene analysis  and other HAdV-7 strains found in China in recent years. The DNA polymerase gene and whole genome sequence of HAdV-7 found in China in recent years were located within the same branch but with a little difference. And the VA RNA I sequences from all serotype 7 strains clustered together and the sequences were highly conserved except for occasional mutations in the termination region , pneumonia (45.6%) and 8 ICU (intensive care unit) cases. To understand the reason behind this large scale outbreak, genomic variations were detected in our article.Hexon and fibre capsid proteins are the main antigenic determinants of HAdV serotypes. In a previous work on two other HAdV-7 outbreaks, we found that most genes had few or no mutations besides those in VA RNA genes, which displayed a 12-bp deletion . TherefoHow the lost T in the VA RNA II terminal region played influence on HAdV-7 is still unknown. Interestingly, the HAdV VA RNA genes are located right in front of the L1 52/55K gene , which pIn summary, we report here an uncommon outbreak of HAdV-7 among college students in Wuhan, China, in 2015. Sequence analysis revealed several missing T in the VA RNA II terminal region. This mutation, in turn, caused up-regulation of an adjacent gene, L1 52/55K. The latter promoted faster growth of the HAdV-7 strain, thus providing a causal link between the VA RNA II gene and a severe clinical outcome. Taking into account the phylogenetic results on the conserved nature of HAdV-7 hexon and fibre genes in China in recent years, VA RNA genes represent a new determinant factor for HAdV-7 virulence upon infection. However, further work, such as functional verification of the missing T bases by constructing a deletion mutant of strain CDC228 or reverse mutant of the WH strain, should be done to elucidate the function of the VA RNA II gene and its relationship to the increased growth capacity of the HAdV-7 strain.S1 AppendixFig A. Phylogenetic analysis of HAdV-7 hexon and fibre genes. Fig B. Phylogenetic analysis of DNA polymerase gene and VA RNA I gene of HAdV-7. Fig C. Sequence alignment of HAdV-7 VA genes. Fig D. Sequence alignment of VA II gene of HAdVs.(DOCX)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.


The RNA forms a branched network of stem-loops that almost all allocate near the capsid inner surface, while predominantly binding to coat protein dimers that are located in one-half of the capsid. This suggests that genomic RNA is highly involved in genome packaging and virion assembly.In single-stranded ribonucleic acid (RNA) viruses, virus capsid assembly and genome packaging are intertwined processes. Using cryo-electron microscopy and single particle analysis we determined the asymmetric virion structure of bacteriophage MS2, which includes 178 copies of the coat protein, a single copy of the A-protein and the RNA genome. This reveals that  MS2 is a single-stranded RNA bacteriophage that infects its host via adsorption to bacterial pili. Here the authors visualize the MS2 virion with asymmetric cryo-EM reconstruction, revealing that the genome of MS2 adopts a specific structure of asymmetrically distributed stem-loops connected to the capsid. Levivirus genus in the Leviviridae family of small, positive-sense, single-stranded ribonucleic acid (RNA) bacteriophages that infect their host via adsorption to bacterial pili. The MS2 virion consists of an RNA genome, encapsidated by a T=3 shell, containing coat protein (CP) and a single copy of the maturation or so-called A-protein (AP), which attaches to the viral RNA and binds the host receptor. The MS2 genome is one of the smallest known, comprising just 3,569 nucleotides, and was the first genome—of any life form—to be completely sequencedCP gene72)Bacteriophage MS2 ref.  is a spein vitro from CP alone at high enough concentrationin vivo. Encapsidation of its own genome depends on presence of the AP2  promote genome packaging13Although MS2 capsids spontaneously assemble AP2 ref. . Third, 2  extending outwards with a ∼30° angle from the surface from a two-fold symmetry axis position in the capsid, extending over a neighbouring hole at a three-fold axis.The outside of our asymmetric EM map of the MS2 virion  at 8.7 Å2, TR-CP2 X-ray model (PDB entry: 1ZDH) was fitted into the EM density map. In total, 44 SLs  ended at a CP2 RNA binding site, 2 SLs interacted with the AP  branched network of interconnected SLs . The RNAh the AP  and 7 we density , with so density . The obs2 interactions in the genome to the observed SLs in the EM map a higher resolution structure would be required. Nevertheless, the two regions known to connect to the APThe obtained resolution of the EM map is sufficient to visualize the double-stranded (ds) RNA SLs, including its helical nature; however, single RNA strands are not clearly resolved and therefore it was not possible to trace predicted secondary structures into the 3D map and to allocate predicted SLs in the MS2 genome into the density142 interactions promote capsid formation, the question is how the SL–CP2 interactions that we observed here influence virion assembly. To explore a potential role of the RNA genome structure in this process we investigated the distribution of PSs, interactions of SLs with CP2, over the capsid. From the 89 CP2, 44 (49%) have a connecting SL, 33 (37%) has crossing (ds) RNA density, while 9 (10%) does not have any adjacent density. Notably, these 44 PSs are distributed unevenly over the capsid, being localized predominantly on one side of the capsid  viruses. Even so structures of several other viruses have shown hints of (partly) ordered (ds)RNA28We reconstructed the MS2 virion, revealing the AP and the single conformation of the RNA genome E. coli strain XL1 blue was grown at 37 °C in LB medium until an OD600 of 0.5. Calcium chloride was added to a final concentration of 2 mM and the cells were infected with phage MS2 at a multiplicity of 10 and incubated for another three hours. Lysates with phage titres of approximately 1 × 10−12 were used for purification. Cellular debris was removed by centrifugation and phage concentrated by ultrafiltration using 15 ml 100 kDa cutoff Amicon concentrators. Phage was further purified by gel-filtration on sephacryl S500 column. Fractions were inspected for purity on coomassie-stained SDS-PAGE gel and negative staining EM.An overnight culture of Aliquots of purified MS2 were applied to glow-discharged holey carbon film supported by cupper grids (Quantifoil R2/2) after glow discharging with negative polarity for 1 min at 30 mA using a K950X carbon coater (Emitech). Grids were vitrified by plunging into a liquid propane/ethane mixture (2:1 v/v), which was cooled by liquid nitrogen. Samples were plunged using a Leica EM GP from room temperature and blotted for 1–2 s using filter paper. After vitrification, the grids were stored in liquid nitrogen until use.Data acquisition was performed on a Titan Krios transmission electron microscope (FEI) operated with Cs correction at 300 keV using EPU automated single particles acquisition software (FEI). Seven frames per images were recorded on a back-thinned Falcon II detector at a nominal magnification of 59,000 × with a sampling size of 1.14 Å per pixel.http://scipion.cnb.csic.es), which is an integrative image processing framework that currently mainly uses Xmipp (http://xmipp.cnb.csic.es/)http://www2.mrc-lmb.cam.ac.uk/relion/index.php/Main_Page)http://spider.wadsworth.org)http://blake.bcm.edu/emanwiki/EMAN2)http://www.cgl.ucsf.edu/chimera)Image processing was performed using Scipion platform .Supplementary Figures 1-4MS2 outside capsid view.  The asymmetric reconstruction of bacteriophage MS2 showing the capsid  and the AP (yellow).MS2 inside RNA view  Inside bacteriophage MS2 the AP (yellow) is connected to a structured genome (grey) that is composed of stem loop structures, in which the double stranded helices can be observed.MS2 RNA view.  Proposed AP binding site RNA densities of nucleotides 3510-3527 (magenta) and 388-414 (blue) with fit in density (red) the secondary structure of the SL.View of RNA-interacting capsid dimers.  Asymmetric distribution of 23 capsid dimers (red) to which the density of the RNA SLs (grey) fit the translational operator.Local resolution measurement. (to Supplementary figure 2) Resolution-coded colouring of the surface rendered RNA density.


Tombusviridae and other plus-strand RNA plant viruses.Plant viruses that contain positive-strand RNA genomes represent an important class of pathogen. The genomes of these viruses harbor RNA sequences and higher-order RNA structures that are essential for the regulation of viral processes during infections. In recent years, it has become increasingly evident that, in addition to locally positioned RNA structures, long-distance intragenomic interactions, involving nucleotide base pairing over large distances, also contribute significantly to the control of various viral events. Viral processes that are modulated by such interactions include genome replication, translation initiation, translational recoding, and subgenomic mRNA transcription. Here, we review the structure and function of different types of long-distance RNA–RNA interactions, herein termed LDRIs, present in members of the family  Plus-strand RNA viruses comprise the largest group of plant viruses, many of which are significant pathogens. During infections, the RNA genomes of these viruses serve multiple functions, including acting as messages for translation of viral proteins, templates for viral RNA replication and transcription, and genetic cargo for packaging. Collectively, these viruses use a wide variety of strategies to control these types of processes, and RNA elements within their genomes play integral roles in such regulation .Tombusviridae , represents, to date, the most extreme example of LDRI utilization  tail, which are typical terminal modifications required for efficient initiation of translation . These vFigure 1B). This PRTE–DRTE interaction spans ∼3.5 kb and is essential for efficient readthrough. Presumably, formation of this interaction directly or indirectly interferes with acquisition of translation release factors and/or facilitates recognition of near-cognate tRNAs that can pair with the p33 UAG stop codon. Interestingly, the 3′-proximal DRTE is integrated with a small RNA hairpin that is important for genomic minus-strand RNA synthesis . The UL–DL interaction is located between the PRTE–DRTE interaction; thus, it is hypothesized that the former assists in bringing the latter’s partner sequences into proximity, thereby promoting their pairing  . Disruption of this interaction inhibits VRC formation and genome replication, confirming the requirement for RII and RIV to be juxtaposed for function (ex (VRC) . The VRCex (VRC) . The firfunction . Hence, function . NotablyFigure 1D; Figure 1D; Proteins encoded 3′-proximally in the TBSV genome  are translationally silent within that genomic context. Consequently, their expression requires transcription of two sg mRNAs during infections . TombusvFigure 1A). In the predicted LDRI cascade, the 5′-UTR–3′-CITE LDRI would form first to allow for translation initiation and synthesis of p33 . Next, interacting PRTE–DRTE and UL–DL sequences would mediate readthrough production of the p92 polymerase . This would be followed by formation of the UL–DL interaction that directs VRC assembly needed for RNA genome replication . The AS–RS and auxiliary DE–CE interactions would then allow for transcription of the sg mRNAs , and their ensuing translation would be facilitated by their cognate 5′-UTR–3′-CITE LDRIs . Such structural choreography is envisioned to require a high level of genomic organization, and results from structural studies on the full-length TBSV RNA genome are consistent with this concept  tail structures and instead possess different types of 3′-CITEs     , Pea enaravirus) , and PelTombusviridae  and Red clover necrotic mosaic virus , tentatively identified translation-related 5′–3′-LDRIs were later proven to not be required for efficient translation of viral proteins  both require LDRIs between an RNA structure proximal to the readthrough site and their 3′-UTR for optimal readthrough of their RdRps ; however, their activities have yet to be confirmed empirically  and Dianthovirus   . In both, RCNMV) . Howeverus genus .cis-acting RNA elements positioned at different locations within the viral genome, and in some cases, like for TBSV, these RNA elements communicate through LDRIs  were demonstrated to utilize LDRIs to form their transcription attenuation structures  utilizes similar cap-independent translation initiation mechanism as members of the family Tombusviridae described in previous sections. Indeed, due to this and other likenesses, it has been suggested that BYDV and other luteoviruses should be reclassified as tombusvirids  (The usvirids . BYDV pousvirids  and baseeovirus) ; howeverBlackcurrant reversion virus  have a 3′-poly(A) tail, but lack a 5′-cap structure , Potato virus X (PVX), has an interesting genome replication and sg mRNA transcription mechanism that involves multiple LDRIs . These distance-spanning structures represent an additional layer of RNA-based control that provides novel regulatory mechanisms not possible with local RNA structures. Additionally, to ensure optimal function of all processes, LDRIs are highly integrated and coordinated with other RNA elements, as well as with the multiple functions of the viral genome. Accordingly, understanding this complex context in which LDRIs operate will require a holistic perspective when investigating viral genome structure and function.This survey of LDRIs in plus-strand RNA plant viruses illustrates both their prevalence in different virus classes and their diversity of function  it is now possible to gain insights into complete viral genome higher-order structures . Such glDe novo identification is possible using computational approaches, however, some LDRIs may be missed, and only a small subset is likely to be functional. Regardless, performing viral genome folding using RNA secondary structure predicting programs such as mFOLD can be informative regarding potential LDRIs (The discovery of new LDRIs also presents a challenge . Many ofal LDRIs . Additioal LDRIs . Any putA final interesting aspect, for which essentially nothing is known, is how LDRIs initially arise and how they evolve over time. With the popularity of high-throughput sequencing, which allows for facile discovery of new plant viruses, novel viral sequences are being published at an ever-growing rate . Among tBoth authors have made a substantial direct and intellectual contribution to the work and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Applications of these approaches to different cell lines and tissues have generated a rich resource for the study of RNA structure–function relationships at a systems biology level. In this review, we first introduce the designs of these methods and their applications to study different RNA structuromes. We emphasize their technological differences especially their unique advantages and caveats. We then summarize the structural insights in RNA functions and regulations obtained from the studies of RNA structuromes. And finally, we propose potential directions for future improvements and studies.RNA folds into intricate structures that are crucial for its functions and regulations. To date, a multitude of approaches for probing structures of the whole transcriptome,  In addition to transferring genetic information from DNA to protein, RNA can catalyze specific biochemical reactions, similar to the action of a protein enzyme. These RNA enzymes, One of the most significant findings in genomics in the last two decades is the discovery of pervasive transcription and the large number of non-coding RNAs (ncRNAs) in human transcriptome Fortunately, there has been a well-established general rule that sequence determines structure determines function, especially for proteins However, our current knowledge on RNA structures is very limited. Traditionally, structures of macromolecules are resolved with methods including X-ray crystallography, nuclear magnetic resonance (NMR) spectroscopy, and more recently cryo-electron microscopy (cryo-EM). Unfortunately, RNA molecules are usually much more flexible, dynamic, and thus structurally heterogeneous trans-acting factors like proteins, other RNAs, and small ligands, as well as other physiological conditions. As a consequence, they can only generate in silico secondary structural predictions of a given RNA alone. In addition, computational predictions do not work very well for big RNAs with complex structural elements like pseudoknots, kissing loops, or long-range interactions Thus, for many years, our understanding on RNA structures and their functional relevance relies primarily on computational predictions. These predictions typically use thermodynamic calculation to obtain secondary structure models with lowest free energy i.e., RNA structurome, in a single experiment.Fortunately, recently we have witnessed the fast development of a new type of approaches, resurging from RNA structure probing analysis with chemicals and enzymes developed as early as 1970s In this review, we will introduce the designs of these methods and their applications to study RNA structuromes in different species. We will emphasize their technological differences, especially their unique advantages and caveats. We will then summarize the structural insights into RNA functions and regulations obtained from the studies of RNA structuromes. And finally, we propose potential directions for future improvements and studies.in vitro to in vivo, and from one-dimension to two-dimension analyses  and double-stranded RNA (dsRNA). RNA structure probing approaches are designed to utilize these structural preferences. The technological development follows a path from low-throughput to genome-wide, from analyses .Endonucleases were found to have structural specificity decades ago Nuclease digestion is then followed by high-throughput sequencing to read cleavage sites . Parallein vitro RNA structuromes of multiple species have been successfully generated. In particular, deproteinized PARS is able to measure near in vivo RNA structures in vivo probing very challenging or impossible.Using the enzymatic cleavage methods, in vivo, in vitro, or after RNA denaturation Some chemicals can modify RNA nucleotides in specific structure contexts. Some of these modifications can block reverse transcription (RT) preceding the modified sites and thus allow for detection by reading RT stop sites. Nucleotide modifications mainly fall into two groups: base modification and backbone modification. Dimethyl sulfide (DMS) is a base modification reagent, which is frequently used to alkylate Watson–Crick (WC) face of unpaired adenine (A) and cytosine (C), while 1-cyclohexyl-3-(2-morpholinoethyl) carbodiimide metho-p-toluene sulfonate (CMCT) reacts with unpaired uracil and guanine (G) N-methylisotoic anhydride (NMIA), specifically modify the ribose of unstructured nucleotides without any bias toward one or more of the four nucleobases. Notably, complex structures besides canonical base pairing are also detectable by SHAPE reagents, allowing for probing more complicated interactions. SHAPE-seq was designed by adapting SHAPE methods in combination with high-throughput sequencing in vitro. But cell-permeable ones, e.g., 2-methyl-3-furoic acid imidazolide (FAI) and 2-methylnicotinic acid imidazolide (NAI), can be used for in vivo RNA structure probing in vivo click SHAPE (icSHAPE) uses an optimized SHAPE compound NAI-N3 with increased permeability and incorporates biotin-streptavidin isolation to enrich modified RNA fragments RNA base-pairing brings geometric constraints to ribose involved in secondary structure, and thus protects the backbone from chemical modification The two aforementioned types of methods only focus on detecting which regions of RNA are single-stranded and which are double-stranded. Information on another big part of RNA structure, the detailed intermolecular or intramolecular base-pairing pattern, however, is missing. Psoralens are well known mutagens that crosslink DNA or RNA duplexes by forming adducts with adjacent thymines (Ts) or uridines (Us) when activated by UV photon Apparently, proximity ligation cannot tell duplex regions from unpaired fragments. Various methods are thus used in order to enrich duplex regions to reduce background noise. PARIS involves 2D gel electrophoresis to separate duplexes from unpaired regions, which gets clean duplexes but lowers the yield at the same time RNA structure is crucial for gene function and regulation by influencing RNA transcription, processing, localization, translation, and degradation. The canonical roles of RNA structure in many different biological processes have been reviewed elsewhere The life cycle of a RNA molecule begins with its transcription. There is accumulating evidence showing that RNA folds into structures along with its transcription, and this co-transcriptional folding plays a critical role in defining RNA functions tryptophan (trp) operon showed that the formation of transient RNA structures is influenced by the interplay of co-transcriptional RNA folding and translation trp operon encodes a tryptophan-rich peptide. Its transcript can assume two alternative structural elements: the attenuator and the anti-terminator. While the attenuator structure prevents further transcription, the anti-terminator permits it. These two structure elements form co-transcriptionally and are regulated by the binding and activity of ribosomes to the leader.The relationship of RNA transcription and those transient intermediate structures remains elusive for most cases. A study in 1980s on the in vivo RNA folding pathways during transcription. None of the aforementioned high-throughput probing experiments has investigated the interplay between RNA structures and transcription. However, combining nascent RNA sequencing with structure probing, or using other methods to carefully isolate different stages of transcripts, we may be able to study their intricate relationships. In addition, recent progresses on in vivo methodology development are likely to improve our ability to interrogate the role of RNA structures in transcription, and vice versaWe currently do not have much insight into the cis-acting sequence elements. The basic ones are important for spliceosome binding and splicing reaction, including the 5′ splice site, the branch-point, and the 3′ splice site. In addition, several classes of auxiliary regulatory signals that play critical roles in splicing regulation have been defined as well. These include exon splicing enhancers (ESEs) and silencers (ESSs), as well as intron splicing enhancers (ISEs) and silencers (ISSs), categorized based on their location and their effects on splicing. Splicing factors that recognize and bind to the enhancer/silencer elements are defined as activator/repressor proteins, respectively. The structural effects of RNA on splicing involve these basic and auxiliary elements, as well as their interplay with the spliceosome complex and splicing factors.Most nascent RNAs are subjected to further processing, including capping, splicing, and polyadenylation before they are better prepared to meet their functional roles. Among those, alternative splicing (AS) is a widespread means that vastly increases transcript and protein diversity cis-acting elements i.e., forming  base-pairings that facilitate joining of a common exon to different alternative exons. An incredibly interesting example comes from the AS of the Drosophila gene encoding Down syndrome cell adhesion molecule (Dscam) Many examples have shown that RNA structure can regulate AS, by affecting spliceosome recognition of basic in vitro human structurome, RNA structure signals were screened across exon-exon junctions in already spliced mRNAs. It is observed that the splicing donor dinucleotides AG are more accessible compared to nearby nucleotides, whereas the acceptor nucleotide G/A tends to be more structured Arabidopsis structurome using ds/ssRNA-Seq confirmed this signature on splicing donor/acceptor sites with more details in flanking intronic regions Arabidopsis structurome, which is opposite in human structurome. In a later study of the in vivo Arabidopsis structurome, Ding et al. revisited RNA splicing using the Structure-seq. They found that in the region upstream of the splicing donor site, structures are less accessible for the unspliced events than for the spliced events in vivo Arabidopsis structurome for U12-type introns and constitutive introns RNA structure can regulate splicing by directly interfering with spliceosome binding sites. In the PARS study of the in vivo and in vitro RNA structures at Rbfox2-binding sites and found high level of differences, suggesting a strong structural effect of the splicing factor binding in vivo. The structural signatures and rearrangement were later shown to be effective in identifying true Rbfox2-binding sites 6A modification, RNA structures, and RBP binding sites 6A in the complementary strands of U-rich hairpins weakens the hairpin secondary structure and promotes hnRNPC binding. In addition, knockdown of two genes encoding m6A methyltransferases, METTL3 and METTL14, reduced hnRNPC binding and affected AS through disrupting hairpin structures RNA structures can also regulate splicing by directly affecting splicing factor binding. The icSHAPE study of the mouse structurome analyzed RNA structure signatures in auxiliary regulatory sites cis-acting elements present in the RNA, which encode the cellular “address” of the host transcripts. These cis-acting elements, primarily located within the 3′ UTR, are called “localization elements” or “zipcodes” The majority of RNAs are localized to distinct cellular domains with exquisite temporal and spatial control, providing an important mechanism for gene expression regulation bicoid mRNA from the nurse cells into the oocyte Accumulating evidences suggest that not only the sequences but also the structures of these zipcodes are critical for RNA localization. For example, a study showed that a stem-loop structural element, BLE1, is critical for the transport of the host Saccharomyces cerevisiae structurome has revealed that mRNA encoding proteins with specific sub-cellular locations or involved in some metabolic pathways are more structured in the coding region The experimental studies of RNA structures can potentially shed light on zipcode discovery. The analysis of the S. cerevisiae structurome, Kertesz et al. examined the structures of the transcripts that are predicted to encode a signal peptide. They found that the SSCRs and their proximity sequences have a lower PARS score, suggesting that specific secondary structures may assist RNA nuclear export RNA of some secretory proteins is exported from the nucleus by using a signal sequence coding region (SSCR) in the transcripts e.g., RNA thermometers or riboswitches, are able to affect mRNA translation i.e., a three-nucleotide periodicity in mRNA secondary structure in vitro and in vivo RNA structure probing experiments Many RNAs are made for translation. Long before the high-throughput probing experiments, it had been observed that RNA structure plays an important role in translation regulation. For example, the temperature-sensitive structures, The first whole-genome structural probing experiment was performed on HIV-1 genomic RNA using SHAPE technologies, focused on structure–translation relationships Caenorhabditis elegans, and human mRNAs agreed with the HIV-1 analysis, whereas opposite results were obtained for yeast and Arabidopsis. It is not entirely clear whether this is due to in vitro or in vivo structure features probed by different technologies or characteristics defined by species.However, following studies of PARS S. cerevisiae using PARS, Kertesz et al. observed that mRNA structure of the translation start site are negatively correlated with ribosome density throughout the transcript in vitro nature of the aforementioned study, this observation was later revisited and confirmed in the Arabidopsis structurome study in vivoNevertheless, the low structure contents around the start and stop codon, and also the three-nucleotide periodicity have been universally observed Arabidopsis structurome using ds/ssRNA-seq Moreover, an interesting positive correlation between the level of mRNA structure and its overall ribosome association has been revealed in another study of In vitro studies have suggested that the presence of RNA secondary structure promotes ribosome pausing in vivo may influence ribosome pausing, begging for more integrated quantitative studies. Interestingly, a mouse embryonic stem (ES) cell structurome study using icSHAPE technology in vitro when ribosome binding to mRNA is depleted, and in negative control sites with similar sequence context. This suggests that the RNA structure pattern of ribosome pausing sites is probably encoded in their sequences. Notably, the flanking 5′ region of the negative control sites showed the lack of typical three-nucleotide periodic signal, suggesting it may play some role in ribosome pausing regulation The translation rates are not uniform along the CDS region. in vivo could change translation efficiency, suggesting a potential mechanism for translation regulation. In summary, all these genome-wide studies show that mRNA structures exert a significant effect on its translation at multiple levels in various organisms.Recently, a structurome study of yeast and human using psoralen crosslinking reported again that dense structures around the start codon could inhibit RNA translation, whereas structures of long-range 5′-to-3′ interactions could promote translation RNA is degraded in a carefully-controlled way at the end of its life cycle. RNA structures are also found to be involved in RNA stability and degradation as well. For instance, in addition to regulating translation, the small structural elements of riboswitches can also influence RNA stability in vitro structurome of S. cerevisiae, the RNA folding energies were measured using PARTE, an expansion of the PARS technology by applying it at different temperatures i.e., less structured) decreased most rapidly in abundance following heat shock. Notably, inactivation of the exosome significantly decreased the degradation of mRNAs with unstable structures.It is then natural to speculate that RNA stability is positively correlated with RNA structure content in its 3′ end. In a study on E. coli. structurome in vitro using ds/ssRNA-seq. Del Campo et al. revealed that mRNA abundances are positively correlated with CDS secondary structures ArabidopsisThis finding is consistent with a study probing i.e., RNA structuromes, and studies of structure–function relationships have generated many new insights into RNA biogenesis, processing, localization, translation, and degradation. To date, studies have been focusing on general principles of the most basic biological processes. In the future, more in-depth investigations are needed for specific biological events in certain cell lines, tissues, environments, or conditions including human diseases. For example, what are the roles of structure in the infection of RNA virus? Is RNA structure an important regulator in early development when transcription is silent? Can we find any RNA structure biomarkers in human disease and could they be a diagnosis target for disease development? Comparative studies are especially required to uncover structures that may be causative factors or direct effectors. The future of these applications is to infer RNA functions based on data mining and classification of structure elements in different biological contexts, thus providing a knowledgebase for functional and mechanistic studies.Transcriptome-wide RNA structure maps, In addition to high quality data generation, well-designed bioinformatics analysis is the key to these applications. Technologies have been developed including a computational framework that processes sequencing data. The data processing follows normal RNA-seq analysis pipelines that include sequencing data quality control and trimming, reads alignment and abundance estimation. It also calculates a structure score to represent the preference for individual nucleotide or a sequence region to be single or double-strand. For enzymatic cleavage and nucleotide modification technologies, the structure score of a nucleotide is normally defined as the ratio of the number of RT stops or cleavage sites mapped to that position divided by the same number in a control experiment, in normal or log space Better structure probing technologies that combine the strengths of new chemicals and creative sequencing designs are also desired to provide more accurate and comprehensive structure information. For example, most probing methods, except for the SHAPE-MaP e.g., PARIS, SPLASH, and LIGR-seq Most probing methods that use enzymatic cleavage or base and sugar modifications only generate one-dimensional averaged structure information. Newly-developed methods that use UV or psoralen crosslinking can provide two-dimensional information, but their resolution and coverage are so far limited  complexes. To date, very limited number of RNA 3D experimental structures are available in the PDB database None.


Caenorhabditis elegans. While L1793P mutant myosin protein efficiently incorporated into the sarcomeric thick filaments, R1845W and H1901L mutants were prone to formation of myosin aggregates without assembly into striated sarcomeric thick filaments in cultured muscle cells. In C. elegans, mutant alleles of the myosin heavy chain gene unc-54 corresponding to R1845W, E1883K and H1901L, were as effective as the wild-type myosin gene in rescuing the null mutant worms, indicating that they retain functionality. Taken together, our results suggest that the basis for the pathogenic effect of the R1845W and H1901L mutations are primarily structural rather than functional. Further analyses are needed to identify the primary trigger for the histological changes seen in muscle biopsies of patients with L1793P and E1883K mutations.Myosin storage myopathy is a protein aggregate myopathy associated with the characteristic subsarcolemmal accumulation of myosin heavy chain in muscle fibers. Despite similar histological findings, the clinical severity and age of onset are highly variable, ranging from no weakness to severe impairment of ambulation, and usually childhood-onset to onset later in life. Mutations located in the distal end of the tail of slow/ß-cardiac myosin heavy chain are associated with myosin storage myopathy. Four missense mutations , two of which have been reported in several unrelated families, are located within or closed to the assembly competence domain. This location is critical for the proper assembly of sarcomeric myosin rod filaments. To assess the mechanisms leading to protein aggregation in myosin storage myopathy and to evaluate the impact of these mutations on myosin assembly and muscle function, we expressed mutated myosin proteins in cultured human muscle cells and in the nematode  The class II conventional muscle myosin exists as a hexameric protein composed of two myosin heavy chain (MyHC) subunits and two pairs of non-identical light chain subunits , 2. MyHCMYH7, is expressed in slow, type 1 muscle fibres as well as in the ventricles of the heart.There are several striated muscle MyHC isoforms encoded by different genes and expressed in a tissue and developmental specific manner –8. Slow/MYH7, predominantly located in the globular myosin head, are an important cause of hypertrophic and dilated cardiomyopathy. Mutations in the rod light meromyosin (LMM) region have been associated with skeletal myopathies with several distinct morphological and clinical phenotypes, depending on the mutated residue at the tail region ), ]), ]), respectively  showed a high degree of spontaneous motility, moving approximately 17 cm per hour. In contrast,  muscles  were parobserved , and whiobserved . The samectively .unc-54 null mutant partially rescued the motility defect. Importantly, the three mutant alleles, R1854W, E1892K and H1910L were as effective as the wild-type UNC-54 in rescuing the mutant , the transgenic worm of this variant was not generated . The gel-purified slow/ß-cardiac MyHC fragment with XhoI (5’) and EcoRI (3’) restriction sites was sub-cloned into the XhoI and EcoRI restriction sites of the pEGFP-N1 to generate WT-slow/ß-cardiac MyHCEGFP. All slow/ß-cardiac MyHC mutant constructs were generated in the pEGFP-N1 backbone . EGFP-tagged slow/ß-cardiac MyHC mutants  were generated through site-directed mutagenesis and cloning into E. coli using a Quick- ChangewSite-Directed Mutagenesis kit , as previously described ): unc-54 null mutant with the wild-type unc-54 gene on an extrachromosomal array; (8) unc-54; Ex (unc- 54(R1854W)): unc-54 null mutant with the mutated unc-54(R1854W) gene on an extrachromosomal array; (9) unc-54; Ex (unc- 54(E1892K)): unc-54 null mutant with the mutated unc-54(E1892K) gene on an extrachromosomal array; (10) unc-54; Ex (unc- 54(H1910L)): unc-54 null mutant with the mutated unc-54(H1910L) gene on an extrachromosomal array. Note that genotypes 8, 9 and 10 represent the closest genetic equivalent to animals homozygous for R1854W, E1892K and H1910L.Transgenic worms of the E1845W = E1854W, E1883K = E1892K and H1901L = H1910L mutations associated with either autosomal dominant or recessive MSM were generated. As the L1793 is not conserved in  mutants , 36; (3)C. elegans was performed using methods described previously [Transformation of eviously  28]. Tr. TrC. elE.coli lawn at room temperature. The animal positions were recorded every second minute to generate traces over 1 hour for each worm. At least five animals of each strain were analysed.Motility assay was performed as previously described . Brieflyhttp://www.ncbi.nlm.nih.gov/); the protein alignment was constructed by use of Pairwise Sequence Alignment, EMBOSS Needle (http://www.ebi.ac.uk/Tools/psa/emboss_needle/). BLAST analyses were performed using the NCBI BLAST Web site (http://www.ncbi.nlm.nih.gov/blast/bl2seq/wblast2.cgi). GenBank accession numbers for genomic unc-54 sequence was J01050 (GI: 156399) and for cDNA unc-54 sequence was NM_061195 (GI: 17509400).The sequence information was obtained from the National Centre for Biotechnology Information  The neck domain is shown in yellow and the C-terminal LMM segment of the rod domain is shown in orange. The heptad repeat motif forms the structural basis for coiled-coil dimer of the β-MyHC. (B) The positions of slow/β-cardiac MyHC mutations associated with MSM in the heptad repeat motif are indicated. (C) Differentiated human myotubes stained with MyHC show clear A-bands (white bracket).(TIF)Click here for additional data file.


Escherichia coli. We validated the network architecture at the molecular level by native polyacrylamide gel electrophoresis, as well as the network function at the bacterial population and single-cell levels with a fluorescent reporter. Our results suggest that it is possible to engineer complex cellular programs based on RNA from first principles. Because these networks are mainly based on physical interactions, our designs could be expanded to other organisms as portable regulatory resources or to implement biological computations.Synthetic gene circuits allow the behavior of living cells to be reprogrammed, and non-coding small RNAs (sRNAs) are increasingly being used as programmable regulators of gene expression. However, sRNAs  are generally used to regulate single target genes, while complex dynamic behaviors would require networks of sRNAs regulating each other. Here, we report a strategy for implementing such networks that exploits hybridization reactions carried out exclusively by multifaceted sRNAs that are both targets of and triggers for other sRNAs. These networks are ultimately coupled to the control of gene expression. We relied on a thermodynamic model of the different stable conformational states underlying this system at the nucleotide level. To test our model, we designed five different RNA hybridization networks with a linear architecture, and we implemented them in  Previous work on the design of synthetic regulatory RNAs in vivo has led to different ways of transferring information from small RNAs (sRNAs) to gene expression, such as those based on RNA regulators (in vivo extends such works by developing RNA mediators in such information transference (RNA regulation of the RNA regulator), something instrumental to increase the regulatory power of the system and previously achieved in vitro  RNA–RNA interactions are initiated by non-hybridized complementary regions called toeholds (iv) the assembly of multiple RNA strands is mostly hierarchical  toeholds , can be toeholds ,25; and archical ,24. We tarchical –29 to prin vitro –32.cis-repressed). Upon interaction with the trigger RNA, the domain to interact with the target RNA becomes active (ON state). In this article, we first present a general computational model for creating RNA hybridization networks. We then present the design of five minimal networks as a proof-of-concept of the approach. We finally show several experimental results that prove that these networks are functional at the molecular and cellular levels, which validate the predictability of the model.We focused on the particular case of a linear network implemented by RNAs with two interaction domains (i.e. an RNA hybridization chain reaction)  and thatWe exploited a thermodynamic model to design cascades of regulatory RNAs to finally control gene expression. The system is composed of three different RNA species: two sRNAs and one 5′ untranslated region (UTR). We constructed an objective function based on free energies and RNA structures , which wde novo sequence design. Rounds of random mutations were applied and selected with such energy-based objective function  and a kanamycin resistance marker  and cloned in a pSTC2-based plasmid that contained a pSC101m replication origin  was used for plasmid construction purposes as described in the manual (E. coli DH5α-Z1 cells (Clontech) or in E. coli K-12 MG1655-Z1 cells (both lacI+tetR+) for control over the promoters PLlacO1 and PLtetO1 . As ext2 (100 μM), MgSO4 (2 mM), and FeSO4 (100 μM). The kanamycin concentration was 50 μg/mL. Cultures were grown overnight at 37°C and at 225 rpm from single-colony isolates before being diluted for in vivo characterization. 1 mM IPTG (Thermo Scientific) was used for full activation of promoter PLlacO1 when needed, and 100 ng/ml aTc (Sigma-Aldrich) was used for full activation of promoter PLtetO1. For microfluidic cultures, cells were grown aerobically in fresh LB and in LB supplemented with 0.05% sulforhodamine B (Sigma-Aldrich), and IPTG + aTc (i.e. we used sulforhodamine B to monitor the presence of inducers in the chamber) , glycerol  as the only carbon source, CaClin vitro transcription, 3 μg of each pUC18-derived plasmid  in a 10% polyacrylamide gel, containing 8 M urea, TBE (1×). We cut the bands corresponding to the full-length RNAs for purification. The presence of RNA was confirmed by loading a small part of the purified preparations in another polyacrylamide gel.To perform the 2, 20 mM NaCl. The mix (20 μL) was denatured (1.5 min at 95°C) and slowly cooled (15 min at room temperature) .For the reaction of RNA–RNA interaction, we used ∼20 ng of RNA for each of the transcripts. The buffer of the reaction was 50 mM Tris–HCl pH 7.5, 10 mM MgClerature) . We thenImageJ was used to quantify the bands , which aCells were grown overnight in LB medium and were then refreshed by diluting 1:200 in M9 medium. They were grown for additional 2 h to then load 200 ml in each well of the plate (Custom Corning Costar). Appropriate inducers  were introduced when needed during refreshing. The plate was incubated in an Infinite F500 multi-well fluorometer (TECAN) at 37°C with shaking. It was assayed with an automatic repeating protocol of absorbance measurements (600 nm absorbance filter) and fluorescence measurements (465/35 nm excitation filter—530/25 nm emission filter for sfGFP) every 15 min. All samples were replicated on the plate from three different colonies.600 ≈ 0.5  and then dividing fluorescence by absorbance at OD00 ≈ 0.5 . CorrectThe design of our microfluidic device was performed in AUTOCAD (AUTODESK), and it was already applied to study a synthetic genetic oscillator . All ima600 of 0.2–0.4. Afterward, cells were diluted again in 1 ml PBS. All expression data were analyzed using a Becton-Dickinson FACScan flow cytometer with a 488 nm argon laser for excitation and a 530/30 nm emission filter (sfGFP). Gene expression of each sample was obtained by measuring the fluorescence intensity of thousands of cells. The data were analyzed using the Cytobank webserver by gating the events using scatter ranges, and then fluorescence histograms (without subtracting autofluorescence) plotted with MATLAB.Cells were grown overnight in LB medium and were then diluted 1:200 in fresh LB medium containing inducers  and incubated to reach an ODi and j) is shown in Figure #ijG) impinges on the progression of the reaction (ijG) has to be as negative as possible to ensure irreversibility in the intermolecular interaction (this represents the thermodynamic aspect of the reaction).We built a coarse-grained model, based on energies and structures, to describe the dynamic behavior a network consisting of an arbitrary number of different RNA molecules that can interact with each other Figure . Each noreaction . This isijG + Δ#ijG) of desired/undesired interactions  for each interaction, we can evaluate if a set of three arbitrary RNA molecules follows the energetic/structural requirements. In this work, we designed five different riboregulatory cascades: trigR31, trigR32, trigR1, trigR2, and trigR11 , in a loop , and in an inter-stem space . This stresses the high designability of RNA hybridization networks.in vivo, the RNA systems were implemented as separate transcriptional units (with their respective promoters and terminators) in plasmids  and anhydrotetracycline (aTc), respectively. We used a superfolder green fluorescent protein (sfGFP)   with a dIn addition, we investigated the effects of some of the attributes defining the molecular implementation of the systems. In terms of stationary behavior, it is expected that the stability of the output protein does not modify substantially the activation fold . We charL-based inducible promoters for the sRNAs, and a constitutive promoter for the mRNA). Computational simulations of cofolding using Vienna RNA . ,40. 3,40in vitro (for the sRNA species without transcription terminators), and then purified and quantified. We mixed the three individual species , and all combinations of two of these species. These mixtures, along with the individual RNAs as controls, were loaded on polyacrylamide gels and separated electrophoretically. The same amount of each RNA per lane was used. For systems trigR2  . We chos2 Figure  and trig2 Figure , native 2 Figure , lane 4,2 Figure , lane 5.2 Figure . The ele2 Figure .We also quantified the different species in the electrophoresis gels  by consir = 0.758, P = 0.004; Figure To validate the thermodynamic model, we balanced the computational and experimental results. r = –0.735, P = 0.002; Figure In addition, we quantified the activation fold for each induced state  relative to the uninduced state, according to our fluorescence data Figure . The expFinally, the higher basal expression in the case of trigR31 and trigR32  could be explained, at least in part, by the more negative free energies of hybridization between SR or SRR and SRRR. These systems also present larger toehold sequences. However, the contribution of the free energy of toehold hybridization to the reaction kinetics becomes saturated , which iE. coli)  . The cosFor illustrative purposes, we reshaped the network architecture of system trigR31 by incorporating a new RNA species  49). In. In49). trans and in a combinatorial manner  with small dynamic ranges . MoreoveE. coli (including eukaryotic hosts), as they are mainly based on physical interactions. This should be accomplished by only re-designing the interface with the output-protein expression machinery. Certainly, as our ability to design multifaceted RNAs increases, more complex bio-logical computing systems are expected to be developed.Finally, RNA hybridization networks might be very useful to perform bio-logical computations in living cells, due to their ability for storing and retrieving information e.g. . In addiSupplementary DataClick here for additional data file.


Here we establish an ultrasensitive quantitation assay for accurately determining N6-methyladenosine at one-nucleotide resolution in RNA. N6-Methyladenosine (m6A) is the most frequent post-transcriptional modification in RNA, and it plays a critical role in biological processes. The functions of m6A remain largely unexplored due to a lack of highly sensitive methods to quantitatively determine the m6A modification fraction at a precise location. Here, we first reveal that T3 DNA ligase has significant selectivity towards the m6A modification. On the basis of the new finding, we establish an ultrasensitive quantitation assay for accurately determining m6A at one-nucleotide resolution in RNA. With the proposed assay, as low as 4 fM RNA containing m6A can be determined and the selectivity is up to 54.1-fold to discriminate m6A against unmodified adenosine (A). The sensitivity has been improved about 106-fold so the proposed method can be successfully employed to accurately determine m6A in real biological samples, even in low abundance RNA. N6-Methyladenosine (m6A) is the most frequent form of post-transcriptional modification in RNA. Recent studies have shown that m6A occurs not only in eukaryotic and bacterial messenger RNA (mRNA)6A suggests that m6A modification is revisable and dynamically regulated, and further indicates that m6A modification is associated with gene regulation, including mRNA translation efficiency,6A modification plays a critical role in many biological processes and is linked to human health.6A remains largely uncovered, due to the lack of sensitive and robust methods to quantitatively determine the degree of m6A modification at a precise location.6A-specific methylated RNA immunoprecipitation with massively parallel sequencing (MeRIP-seq), S. R. Jafferey’s and D. Dominissini’s groups developed high-throughput sequencing methods to analyze m6A modification on a transcriptome-wide scale.6A and that the m6A sites preferentially appear near stop codons and in 3′ UTRs. However, the sequencing methods cannot quantitatively detect the m6A modification fraction at the precise location, which generally locate the m6A residues within approximately 200 nucleotides. As the MeRIP-seq methods offer the distribution of m6A at a transcriptome-wide scale, quantitative methods for the determination of site-specific m6A in RNA have become increasingly important, and are greatly significant for revealing the biological functions of m6A and the association between m6A and human disease and clinical diagnosis. Liu et al. first reported a RNase H cleavage-based method for quantifying m6A at a specific site, which required site-specific cleavage and radioactive labelling followed by ligation-assisted extraction and Thin Layer Chromatography (SCARLET).et al. have identified a DNA polymerase (Tth DNA Pol) with selectivity for the incorporation of thymidine opposite unmodified A over m6A.et al. have also revealed that Bst DNA polymerase can significantly hinder RNA-directed DNA synthesis.6A at specific sites. Although these methods also need electrophoresis separation and radioactive or fluorescent labels, the procedures have been simplified. Of particular note is that the sensitivity of all methods mentioned above is too low to quantify m6A in low abundance mRNA or lncRNA due to the lack of an amplification step, in which about 10 fmole RNA containing m6A can be detected in a large amount of total RNA samples. The reason may be that the products of probe extension and enzymatic cleavage are difficult to specifically amplify.In 2012, by combining met al. have described the T4 DNA ligase-based method to discriminate between A and m6A in RNA.6A in RNA samples, possibly due to the low selectivity of T4 DNA ligase (see below). In this work, we first reveal that T3 DNA ligase has strong selectivity to discriminate m6A from A in RNA. By ligating the DNA probes with T3 DNA ligase using a template of RNA and amplifying the ligated DNA products with PCR, as low as 4 fM (corresponding to 40 zmol in 10 μL solution) lncRNA containing m6A can be accurately determined and the selectivity for discriminating m6A in RNA can be greatly improved up to 54.1-fold.Generally, DNA ligase shows high specificity compared to DNA polymerase and the ligated DNA products can be specifically amplified. Dai 6A and RNA2488-A segments were synthesized by Integrated DNA Technologies (America). DEPC-treated water, RNAiso (lysis buffer), dNTPs and the DNA probes were obtained from TaKaRa Biotechnology Co. Ltd. . JumpStart™ Taq DNA Polymerase was purchased from Sigma-Aldrich . T3 DNA ligase, T7 DNA ligase, T4 DNA ligase and Taq DNA ligase as well as T4 RNA ligase 2 were purchased from New England Biolabs . Super Green I was purchased from Fanbo Biochemicals Co. Ltd. . Trichloromethane, isopropyl alcohol and ethanol were purchased from Sinopharm Chemical Reagent Co. Ltd. . All the solutions for reactions were prepared with DEPC-treated water.RNA2577-A, RNA2577-m2, 1 mM ATP, 1 mM DTT, and 7.5% polyethylene glycol (PEG 6000) at pH 7.6 and 25 °C) and an appropriate amount of the RNA target. The ligation reaction mixture B consisted of 0.9 U ligase and the ligation buffer. Mixture A was heated at 85 °C for 3 min and then incubated at 35 °C for 10 min; then the ligation reaction mixture B was added. The final volume of the ligation reaction mixture was 10 μL. Then the ligation reaction mixture was incubated at an appropriate temperature to carry out ligation. After ligation, the reaction mixture was put on ice immediately. The reaction conditions for T3 DNA ligase, T4 DNA ligase, T7 DNA ligase, T4 RNA ligase 2 and Taq DNA ligase were 35 °C for 15 min, 25 °C for 30 min, 35 °C for 15 min, 37 °C for 30 min, and 35 °C for 15 min, respectively.The ligation reaction mixture A consisted of 20 nM probe L, 20 nM probe R, the ligation buffer  gelatin). The PCR reaction was carried out with a StepOne Real-Time PCR System  using a hot start of 94 °C for 2 min, followed by 45 cycles of 94 °C for 20 s and 60 °C for 30 s. The real-time fluorescence intensity was monitored at 60 °C.A volume of 2 μL of the ligation product was transferred to a PCR reaction mixture with a final volume of 10 μL. The PCR reaction mixture contained a forward primer, a reverse primer (the concentration of each was 200 nM), 250 μM dNTPs, 0.4× Super Green I, 0.5 U JumpStart™ Taq DNA Polymerase and the PCR buffer  supplemented with 10% (v/v) Fetal Bovine Serum (HyClone), 100 U mLThe DMEM was removed from the Petri dish while the bottom of the Petri dish was filled with cells. After that the cells were washed three times with cold PBS buffer . Next, 6 mL RNAiso solution was added into the Petri dish, and then the cells were incubated at room temperature for 2 min. After incubation, the cell lysates were collected in a centrifuge tube which contained 1.2 mL trichloromethane. The centrifuge tube was shaken slightly at room temperature for 2 min, and then the cell lysates were centrifuged for 15 min at 12000 rpm at 4 °C, and the aqueous phase was collected in a new tube. Next, 3 mL isopropyl alcohol was added to the aqueous phase and the mixture was incubated at room temperature for 10 min. Then the mixture was centrifuged for 20 min at 12000 rpm at 4 °C. The precipitate was washed once with 75% ethanol and dissolved in DEPC-treated water. Finally, the concentration of total RNA was determined with a Nano Drop 2000 (Thermo Scientific).6A to be detected is located in MALAT1 lncRNA which has a polyA tail. Therefore, MALAT1 lncRNA can be obtained by the extraction of poly A+ RNA.The structure of most lncRNA is similar to that of mRNA containing a polyA tail.2, 10 mM Tris–HCl, 0.05 mM DDT, and 0.05% NP-40). Next, 500 μL STV–biotin binding buffer and 5 μL biotin–oligo (dT) (the concentration was 50 μM) were added into the tube. Then the tube was placed at 4 °C for 4 h with slight shaking, and thus the biotin–oligo (dT) bound onto the STV of the magnetic beads. The free biotin–oligo (dT) was washed out with washing buffer (10 mM Tris–HCl and 150 mM NaCl). Finally, 500 μL 2× binding buffer  was added into the tube.First, the magnetic beads in the tube were washed three times with an STV–biotin binding buffer .The magnetic beads and total RNA (the volume was 500 μL) were heated separately at 85 °C for 3 min, and after that the magnetic beads were transferred into the total RNA and the mixture was heated at 85 °C for another 3 min. Next, the mixture was shaken for 30 min at room temperature, and during that time the 3′ poly (A) region present in poly A+ RNA attached to the magnetic beads was eluted with DEPC-treated water. The concentration of poly A+ RNA was determined with a Nano Drop 2000 (Thermo Scientific) and the poly A+ RNA was stored at –80 °C.The magnetic beads were washed three times with washing buffer. Then poly ATm) of the hybrids of RNA targets and the DNA probes in the ligation reaction, 200 nM RNA target (RNA2577-A or RNA2577-m6A) was mixed with 200 nM probe L1 and 200 nM probe R1 D in a 10 μL mixture solution which contained T3 DNA ligase reaction buffer  and 0.4× Super Green I. The mixture was put into a StepOne Real-Time PCR System  to determine the fluorescence signal changes when increasing the temperature from 25 °C to 95 °C. Fig. S1(a) and (b)6A/(probe L1 – probe R1 D). After that, probe R1 D was replaced by probe R1 and we conducted the same experiment. The derivatives of the fluorescence intensity signal as a function of temperature, produced by RNA2577-A/(probe L1 – probe R1) and RNA2577-m6A/(probe L1 – probe R1) are depicted in Fig. S1(c) and (d),To determine the melting temperature  and probe R1 (right probe) were designed , 32.1 : 100 (T4 RNA ligase 2), 79.1 : 100 (T4 DNA ligase), 64.4 : 100 (T7 DNA ligase) and 77.7 : 100 (Taq DNA ligase), in which the T3 DNA ligase and T4 RNA ligase 2 show good selectivity for recognizing m6A from A in RNA. Using an RNA molecule as the template to ligate the DNA probes, it has been reported that modification with two ribonucleotides of one DNA probe at its 3′-terminus can greatly improve the specificity and efficiency of the ligase reaction.6A segment to that produced from the RNA2577-A segment reaches 6.87 : 100 (T3 DNA ligase) and 30.5 : 100 (T4 RNA ligase 2). These results indicate that T3 DNA ligase has the strongest selectivity to discriminate m6A from A in RNA and modification of probe R1 with two ribonucleotides can greatly improve the selectivity.The selectivity of the ligases for discriminating m6A modification is a single methylation of adenosine at the N6 position. The methyl group is small and chemically inert. More importantly, the methylated N6-position retains its ability to donate a hydrogen bond, so that m6A can still form Watson–Crick base pairs.6A from A in RNA.6A, and T3 DNA ligase shows the strongest selectivity, which is ascribed to its ability to recognize between m6A:T and A:T. We also demonstrate that T3 DNA ligase shows better selectivity to discriminate between m6A:U and A:U when probe R1 is modified with two ribonucleotides at its 3′-terminus. The melting temperatures (Tm) of the hybrids of the RNA segments and the probes were detected, and are 44.3 °C (RNA2577-A/probe L1 – probe R1D), 43.8 °C (RNA2577-m6A/probe L1 – probe R1D), 43.7 °C (RNA2577-A/probe L1 – probe R1), and 41.4 °C (RNA2577-m6A/probe L1 – probe R1)  scale" fill="currentColor" stroke="none"><path d="M0 1440 l0 -80 1360 0 1360 0 0 80 0 80 -1360 0 -1360 0 0 -80z M0 960 l0 -80 1360 0 1360 0 0 80 0 80 -1360 0 -1360 0 0 -80z"/></g></svg>U or A),6A are GGACU, AAACU, AGACU and GGACA. The core sequence of RNA 2577 is GGACU. To assess the effects of the core sequences on the specificity of the proposed ligase-dependent PCR assay, the core sequence of RNA 2577 (GGACU) is replaced with AAACU, AGACU, and GGACA, respectively. The corresponding RNAs containing A or m6A are respectively defined as RNA AAACU-A or RNA AAACU-m6A, RNA AGACU-A or RNA AGACU-m6A, and RNA GGACA-A or RNA GGACA-m6A. These RNA molecules were used to perform the specificity experiments. The experiments were performed with the same procedures using T3 DNA ligase as demonstrated above. The results are shown in 6A segment to that produced from the A segment reaches 7.20 : 100 (RNA AAACU), 7.70 : 100 (RNA AGACU), and 5.46 : 100 (RNA GGACA). These results indicate that core sequences have little effect on the specificity of the proposed ligase-dependent PCR assay.In 2012, Dan Dominissini 1.000000,.000000 s6A, and optimize the experimental conditions  are plotted against the logarithm (lg) of RNA2577-A concentration, as depicted in CT = –8.59 – 3.14 lg CRNA2577-A (M) with a corresponding correlation coefficient, R2, of 0.998. At the same time, as demonstrated in CT values produced by RNA2577-m6A at the same concentrations are much less than those produced by RNA2577-A. According to the calibration curve shown in CT values produced by 4 pM RNA2577-m6A correspond to the concentration of RNA2577-A at 73.9 fM. Therefore, the selectivity of the ligase-dependent PCR assay for detecting m6A in RNA is up to 54.1-fold. It is particularly worthwhile to note that the real-time fluorescence signal produced by 400 fM RNA2577-m6A is almost the same as the blank. That is to say, when the total concentration of the RNA target to be detected is less than 400 fM, RNA containing m6A produced a negligible signal, which will be of no contribution to the real-time fluorescence signal. The result means that the RNA-m6A concentration should be equal to that of the total RNA target concentration minus the RNA2577-A concentration when the total RNA concentration is less than 400 fM, which can be determined with the ligase-dependent PCR assay.With the proposed ligase-dependent PCR assay, we systematically investigate the influence of experimental parameters, including the ligation temperature, ligation time and amounts of T3 DNA ligase, on the selectivity and sensitivity for detecting m6A-containing RNA. The RNA2577-A segment and RNA2577-m6A segment were firstly mixed with a total concentration of 400 fM as the synthetic samples, in which the RNA 2577-m6A proportions were 0%, 10%, 30%, 50%, 80%, and 100%. Then, the RNA2577-A concentration was determined with the ligase-dependent PCR assay according to the calibration curve shown in 6A concentration was equal to 400 fM minus the RNA2577-A concentration. As shown in Table S2,6A concentrations are in good agreement with the added ones, indicating that the proposed assay can be used for quantitative evaluation of the extent of RNA containing m6A in RNA samples at a specific site when the total RNA concentration is less than 400 fM.Subsequently, we tested whether the ligase-dependent PCR assay can be employed for quantitative detection of m6A in real biological samples, the vital issue is how to obtain the total concentration of the RNA target. To address this issue, as demonstrated before, we selected an adenosine (A) site in the same RNA target molecule as the reference site, which is known to only contain A, such as the 2488th site in the MALAT1 lncRNA. The ligase-dependent assay can easily determine the total concentration of the RNA target molecule at the reference site, which can also control the total RNA target concentration at less than 400 fM for determination of real biological samples.For determination of RNA containing mCT value and lg of RNA2488-A concentration in the range from 4 fM to 4 nM. The correlation equation is CT = –6.39 – 3.16 lg CRNA2488-A (M) and the correlation coefficient, R2, is 0.990. Finally, we apply the ligase-dependent PCR assay to determine the m6A modification fraction at the 2577th site of the MALAT1 lncRNA in 85 ng poly A+ RNA extracted from HeLa cells. As shown in CRNA2488-A) in the sample with the ligase-dependent PCR assay at the 2488th site. As shown in CRNA2577-A) with the ligase-dependent PCR assay at the 2577th site. Therefore, the RNA2577-m6A concentration is calculated as 230.7 fM from the formula CRNA2488-A–CRNA2577-A, and the m6A modification fraction at the 2577th site is estimated to be 80.7%, which very much coincides with that obtained by the SCARLET assay (∼80%).6A modification fraction at the 2577th site of the MALAT1 lncRNA in poly A+ RNA extracted from HEK293T, and the m6A modification fraction at the 2577th site is estimated to be 51.3% (Fig. S56A in real biological samples.The designed RNA2488-A specific probe L2 and probe R2 are used to perform the ligation reaction by catalysis with T3 DNA ligase and then the ligated products are amplified with PCR. A synthetic RNA2488-A segment is employed to construct the calibration curve. As demonstrated in % Fig. S5, which a6A from A in RNA molecules. On this basis, a highly sensitive and selective ligase-dependent PCR assay has been established, which can accurately determine the m6A modification fraction at any specific site in RNA. Most importantly, owing to its ultrahigh sensitivity, the proposed assay can be used to quantify m6A in low abundance cellular RNA in real biological samples, which is currently not possible using existing techniques due to the lack of practical ability for nucleic acid amplification. In addition, the ligase-dependent PCR assay requires only common and available lab equipment and materials. Therefore, we believe that the proposed assay should be readily promising for applications in m6A related fundamental studies and clinical diagnosis.In summary, we have first revealed that T3 DNA ligase, a commercially available ligase, has strong selectivity for the discrimination of mThere are no conflicts to declare.Supplementary informationClick here for additional data file.