Normal cellular metabolism and exposure to ionizing and ultraviolet radiations and exogenous agents produce reactive oxygen species (ROS). Due to their reactivity, they can interact with many critical biomolecules and induce cell damage. The reaction of ROS with free nucleobases, nucleosides, nucleotides, or oligonucleotides can generate numerous distinct modifications in nucleic acids. Oxidative damage to DNA has been widely investigated and is strongly implicated in the development of many chronic-degenerative diseases. In contrast, RNA damage is a poorly examined field in biomedical research. In this review, I discuss the importance of RNA as a target of oxidative damage and the role of oxidative damage to RNA in the pathogenesis of some chronic-degenerative diseases, such as neurological disorders, atherosclerosis, and cancer. Furthermore, I review recent evidence suggesting that RNA may be the target for toxic agents and indicating RNA degradation as a powerful tool to treat any pathology in which there is an aberrant expression of mRNA and/or its gene products. Some enzyme activities generate O2•− and ROS are released from phagocytes. ROS can also be produced by ionizing and ultraviolet radiations and exogenous agents following a redox cycle induced by cell metabolism. ROS play a physiological role as they are involved in destroying cells infected with viruses or bacteria [Under most conditions, normal cellular metabolism is the source of endogenous reactive oxygen species (ROS). Multiple steps in the electron transport chains involve the formation of the Obacteria  and in cbacteria . Oxidatibacteria , 5. In cIn this review, I discuss the involvement of RNA in the oxidative damage and the critical role that oxidative damage to RNA plays in the pathogenesis of several chronic-degenerative diseases. Furthermore, I review recent studies reporting that RNA may be the target for different toxic agents and identifying RNA inactivation as an effective strategy to treat any pathology where an aberrant expression of mRNA and/or its gene products is observed.Under most conditions, generation of ROS is due to metabolic reactions and is a part of the aerobic energy building process. The main source of ROS in human cells is mitochondria, where the rate of ROS production is proportional to the rate of mitochondrial respiration .At low levels, ROS play a pivotal role in cell division and survival, cell signaling, inflammation and immune functions, autophagy, and stress response. However, a redox imbalance caused by exposure to oxidant agents alters a biological system's ability to detoxify ROS or to repair any damage caused by them .• rising from O2•− and H2O2 by the Fenton and Haber-Weiss reactions. The reaction of ROS with free nucleobases, nucleosides, nucleotides, or oligonucleotides can generate numerous distinct modifications in nucleic acids. Many different types of oxidatively altered bases have been recorded in DNA [Damage to RNA can be produced by HOd in DNA , 7. Guand in DNA . The mosd in DNA .• and H2O2 can damage RNA [• followed by oxidation or by reaction of guanine with singlet oxygen followed by reduction [Although both HOmage RNA , 8-hydroeduction . This oxeduction . Alterateduction .Oxidative damage can alter RNA structure and function and interfere with the interaction between RNA and other cellular molecules. As an example, oxidative damage to RNA template produces the block of reverse transcription . MoreoveNoncoding RNAs, such as rRNAs and tRNAs, and small nuclear RNAs, have a normal half-life much greater than 3 h. Two different studies reported the presence of 8-hydroxyguanosine in RNA, with a half-life of ~12.5 h , 17. HigCellular RNA could theoretically be more prone to oxidative damage than DNA for different reasons. For instance, mammalian cells have RNA levels 4.4 times higher than that of DNA by weight. RNA is less associated with proteins. Moreover, its bases are not protected by hydrogen bonding and are thus more easily accessible to ROS. RNA has an extensive cytoplasmic distribution in close proximity of mitochondria , where t versus 8-oxo-7,8-dihydro-2′-deoxyguanosine content in DNA has been performed following treatment with H2O2. In this experimental system, the level of RNA damage per nucleoside resulted in 14–25 times greater than that in DNA [Consistent with the above reported observations, several studies have shown that the levels of oxidative damage in RNA can be 10–20 times higher than that of DNA of the same source in different models, including rat liver , human lt in DNA . in vitro studies demonstrated that rRNA had higher iron binding than tRNA or mRNA. Accordingly, iron-rich rRNA had a 13-fold greater formation of 8-oxo-7,8-dihydroguanosine in oxidation experiments as compared to the iron-poor tRNA [Another factor that may account for the high susceptibility of RNA to oxidative damage is the iron-binding properties of certain classes of RNAs. Iron catalyzes Haber-Weiss and Fenton reactions that produce ROS . Purifieoor tRNA .The higher levels of oxidization observed for RNA could also be due to different rates of removal of RNA and DNA damage. Indeed, in contrast to the redundant repair mechanisms for DNA, little is still known about how and to what extent oxidatively damaged RNA is removed .Some observations support the hypothesis that oxidized RNA is not tolerated by cells and can be cleared from the functional RNA pool by RNA surveillance mechanisms. In mammals, the levels of RNA oxidative damage have been found to be reduced in 24–72 h after removal of the oxidative stress insult , 24.Different studies reported mechanisms for the turnover of oxidized free nucleotides and the turnover of damaged RNAs. The polynucleotide phosphorylase, a 3′-5′ exoribonuclease, binds an oligoribonucleotide containing 8-oxo-7,8-dihydroguanosine with a much higher affinity than it binds to an undamaged RNA . Of noteIn humans, MTH1, a nucleotide pool sanitization enzyme, hydrolyzes 8-oxoGTP. The resulting 8-oxoGMP cannot reenter the nucleoside triphosphate pool because guanylate kinase lacks activity on 8-oxoGMP . A seconSelective degradation of oxidized RNA requires the ability to distinguish between oxidized RNA and normal RNA. RNases lack such selective degradation. Different studies have reported the existence of RNA-binding proteins that specifically recognize oxidatively damaged RNA molecules and effectively target the damaged RNA to degradation by RNases , 29, 30.Mechanisms for the degradation of mRNA have been reported, where ribosome plays a role in recognizing oxidatively damaged mRNA during translation . The humStable RNAs represent the majority of cellular RNA and thus should contain the majority of damaged lesions in RNA pool. For rRNA, some studies report degradation following oxidative damage. As an example, Martinet et al. have shown that a reduction of 18S and 28S rRNA levels parallels high 8-hydroxyguanosine RNA levels in human atherosclerotic plaques . SimilarFor tRNA, studies on yeast and human cells have found that oxidative stress induces the accumulation of cleaved tRNA halves . HoweverOxidative RNA damage has been recently found to be involved in the pathogenesis of several human diseases, especially chronic degeneration . OxidatiAn increased level of oxidative DNA and RNA damage and alteration of ribosome function was reported in postmortem brains of patients with Alzheimer's disease and Parkinson's disease , 18, 40.Notably, the presence of 8-hydroxyguanosine was also observed in cerebrospinal fluid from patients with Alzheimer's disease and Parkinson's disease  and in sOn the whole, the observations above reported could suggest the use of 8-hydroxyguanosine as a biomarker to catch some neurodegenerative disorders, such as Alzheimer's disease, in their early phase.Similar RNA oxidative damage was observed in brain samples of patients with dementia with Lewy bodies , Creutzf substantia nigra in Parkinson's disease, and in the motor cortex and spinal cord in amyotrophic lateral sclerosis; no alteration in 8-hydroxyguanosine levels was detected in the cerebellum in Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis compared with the controls [Of particular interest, different studies detected a regional distribution of RNA oxidative damage that mirrors the selective neuronal vulnerability that characterizes each neurological disease. Indeed, an increase in the levels of 8-hydroxyguanosine was observed in the hippocampus and cerebral neocortex in Alzheimer's disease, in thecontrols , 48–51.  postmortem tissues from patients with Alzheimer's disease and amyotrophic lateral sclerosis evidenced that mRNA oxidation does not represent a random phenomenon but is highly selective. The hypothesis that the selective mRNA oxidation was due to the abundance of mRNA species was excluded. Indeed, although β-actin and MAP-2 mRNAs are abundant mRNA species, only very small amounts of their oxidized products were detected [The analysis ofdetected .Oxidative RNA damage can cause an impairment of translation due to both mRNA damage and decreased ribosome function. The observed correlation between oxidation of some mRNAs in affected neurons of patients affected by Alzheimer's disease and low protein expression for those genes  lends fuRibosome stalling is another consequence of mRNA oxidation , as suggEpilepsy is a chronic condition whose incidence increases with age . RecentlSc and lead to the propagation of the disease-associated conformational changes [C protein expression in vitro [The involvement of RNA mutations induced by ROS has been suggested as a possible initial event in the etiology of sporadic prion disease. The hypothesis is based on the evidence that, in Prp-expressing cells, mutations occur in the Prp mRNA more than in the corresponding two copies of the Prp gene. Moreover, the lack of RNA repairs systems similar to those found for DNA mismatch correction results in a higher rate of RNA mutations. Thus, RNA mutations induced by ROS could trigger miscoding that can transiently generate Prp changes . In thisin vitro  and woulin vitro  and thusIn addition to neurological diseases, oxidative RNA damage has been described in advanced human atherosclerotic plaques , 59. In Although, preliminary, some experimental observations suggest a potential involvement of oxidative RNA damage in cancer development . 2-Nitroα and IL-6 production from nonirradiated keratinocytes and peripheral blood mononuclear cells. The release of cytokines from nonirradiated cells was due to UVB-induced modifications in the double-stranded domains of some noncoding RNAs of irradiated keratinocytes and was dependent on Toll-like receptor 3 (TLR3) and Toll-like receptor adaptor molecule 1 (TRIF) [α plays in apoptosis, inflammation, and immune surveillance [Excessive exposure to solar UVB radiations is a key risk factor for skin cancer in humans, operating through DNA damage , damage repair, and photoimmunosuppression . A very 1 (TRIF) . Taking eillance , the TLReillance . in vivo significance of the mechanism of RNA repair. In some cases, the functional consequence of RNA damage has been explored and includes reduced protein synthesis as a result of ribosomal cross-linking and/or mRNA oxidation. As RNA damage has been found to be associated with different chronic-degenerative diseases, a better understanding of how RNA damage impacts cell function is needed. Recently, different classes of small noncoding RNAs such as miRNAs, siRNAs, and piRNAs have been discovered. They are not translated into proteins but act through complementary base pairing with target RNAs [A growing collection of findings suggests that high levels of RNA oxidative damage may significantly affect its function and cause detrimental effects to cells and organisms. The existence of mechanisms to control the levels of RNA oxidation detected from bacteria to humans indicates that RNA damage is a significant issue. However, the molecular details about RNA oxidative damage and its consequences remain to be explored as well as theget RNAs . Along t2O2, an oxidizing agent that also raises the levels of PtdInsP3 and activates downstream signaling; doxorubicin, which acts as both an alkylating and an oxidizing agent; spermine, one of the few NO-donors releasing authentic NO and ROS; and S-nitroso-N-acetylpenicillamine, a donor of NO [ vice versa.From a toxicological point of view, the emerging role that oxidative RNA damage can play in several chronic-degenerative diseases should be carefully considered and encourage the inclusion of the analysis of RNA damage in current testing strategies for better defining the toxicity profile of xenobiotics. Interestingly, some recent studies showed that cellular RNA is a sensitive target for different xenobiotics with different mechanisms of action, such as Hor of NO –72. Giveor of NO , lacked or of NO , 75, werFrom a pharmacological point of view, it is noteworthy that cancer cells are characterized by multiple DNA lesions resulting in transcription of large amounts of nonsense mRNA that is translated to proteins. Indeed, protein synthesis in cancer cells is stretched to its limit in contrast to normal cells . The desRNA oxidation strategy can also help destroying viral RNAs by blocking the expression of virus-encoded essential proteins. An RNase P complexed with an external guide sequence was used with the aim of targeting the mRNA encoding the protease of human cytomegalovirus and thus blocking its expression and growth. The study reported a decrease of 95% in the protease expression and a decrease of 4 000 times in viral growth in cells infected with cytomegalovirus expressing the RNase P complexed with the external guide .C, to the aberrant prion protein, PrPSc [Moreover, it has recently been suggesting that host-encoded RNA molecules may play a role in the pathogenesis of the prion disease for the conversion of the normal prion glycoprotein, PrPn, PrPSc . This meIn conclusion, damage to RNA still represents a maturing approach and its true cellular potential remains to be deeply explored in both toxicological context and pharmacological context.


On the contrary, in RNA the anti conformation is predominant in Watson-Crick base pairing. In this paper two 8-substituted guanosine derivatives, 8-methoxyguanosine and 8-benzyloxyguanosine, were synthesized and incorporated into oligoribonucleotides to investigate their influence on the thermodynamic stability of RNA duplexes. The methoxy and benzyloxy substituents are electron-donating groups, decreasing the rate of depurination in the monomers, as confirmed by N-glycoside bond stability assessments. Thermodynamic stability studies indicated that substitution of guanosine by 8-methoxy- or 8-benzyloxyguanosine significantly decreased the thermodynamic stability of RNA duplexes. Moreover, the presence of 8-substituted guanosine derivatives decreased mismatch discrimination. Circular dichroism spectra of modified RNA duplexes exhibited patterns typical for A-RNA geometry.Modified nucleobase analogues can serve as powerful tools for changing physicochemical and biological properties of DNA or RNA. Guanosine derivatives containing bulky substituents at 8 position are known to adopt  In biological studies, they often serve as enzyme inhibitors and are able to inactivate the replication, transcription, translation, repair and processing pathways of nucleic acids [Chemically modified nucleic acids are a quickly growing class of compounds that have various uses. They can be successfully applied in investigations of nucleic acid structure and interactions between nucleic acids and proteins. Nucleoside analogs also exhibit promising properties ic acids –3.syn conformation. The 8-substituted purine nucleosides exhibit promising anti-proliferative properties by inducing apoptosis of different cancer cell lines, e.g., multiple myeloma and other leukemias [More than two decades ago, research began to focus on purine nucleosides containing various modifications at the 8-position ,5. The peukemias . Moreoveeukemias ,11. 8-sueukemias ,13. The eukemias ,15.i.e. increase thermodynamic stability of duplexes are promising tools in allele-specific PCR, antisense therapies or in the detection of single nucleotide polymorphisms (SNPs) [i.e. decrease thermodynamic stability of duplexes allow to use of primers and probes when it is required to detect hybridization of multiple sequences simultaneously (hybridization independent of base composition) and minimize mismatch discrimination [RNA consists of canonical and non-canonical secondary structure motifs, such as internal loops, hairpins, and bulges. One of the most ubiquitous types of RNA secondary structure motifs are single mismatches . All thes (SNPs) . On the mination . These mmination .Herein, we report the chemical synthesis of 8-methoxyguanosine (mxG) and 8-benzyloxyguanosine (bxG) phosphoramidites and their incorporation into RNA using standard β-cyanoethyl chemistry and automated RNA synthesis. The hybridization properties of modified oligoribonucleotides were thermodynamically characterized by UV melting method, and the influence of complementary mxG-C and bxG-C base pairs, as well as of mxG and bxG mismatches with A, G and U, was determined. Moreover, RNA duplexes containing 8-substituted derivatives of guanosine were analyzed by circular dichroism to investigate possible alterations in helix geometry. In addition, the acidic stability of the N-glycoside bond of 8-methoxyguanosine and 8-benzyloxyguanosine was determined since the formation of abasic sites changes the RNA secondary structure.All details concerning chemical synthesis of 8-substituted guanosine derivatives are described in All oligonucleotides were synthesized on an automated RNA/DNA synthesizer using standard phosphoramidite chemistry . The 8-sThe rate of depurination process was tested for both 8-substituted guanosine derivatives and compared with the stability of guanosine. To the 30 μl of 14 mM solution of corresponding nucleoside, 240 μl of water and 30 μl of 1M HCl were added. The surface of reaction mixture was tightly covered by silicone oil and heated at 85°C. Aliquots of reaction mixture were removed after 0, 0.25, 0.5, 1, 2, 4, 6, 8, and 24 hours and the oil left-overs were removed. The hydrolysis reaction was quenched each time by addition of the excess of 30% aqueous ammonia. The relative amount of the substrate and product was determined by HPLC with UV detection at 220-300nm wavelength range.2EDTA, pH 7.0. Single-stranded oligonucleotide concentrations were calculated based on the absorbance measured above 80°C and extinction coefficients were approximated using the Oligo Calculator from RiboTask ApS website (www.ribotask.com). Modified and unmodified RNA strands with identical sequences were assumed to have identical extinction coefficients. The samples were denatured at 90°C for 2 min and then cooled to room temperature. The measurements were performed for nine different concentrations of each sample in the concentration range of 10−4–10-6 M. Melting curves were obtained using the UV melting method at 260 nm in the temperature range of 3–93°C with a heating rate of 1°C/min on a JASCO V-650 spectrophotometer equipped with a thermoprogrammer. Melting curves were analyzed and the thermodynamic parameters determined by nonlinear curve fitting using the MeltWin 3.5 software [m-1 versus ln (CT/4) plots was within 15% of that derived from averaging the fits to individual melting curves for majority of analysed duplexes  [37 = 2.36 kcal/mol) [8-methoxyguanosine was incorporated into the central position of a 9 nt oligoribonucleotide. The presence of mxG decreases RNA duplex stability by 1.87 kcal/mol , which ccal/mol)  or 8-brocal/mol) .37 = 2.74 kcal/mol), whereas the presence of an mxG-G mismatch decreased the thermodynamic stability of duplexes by 1.44 kcal/mol compared with complementary modified RNA duplex. In contrast, natural mismatches destabilized RNA duplex by 4.95 kcal/mol (G-A), 3.07 kcal/mol (G-U), and 4.18 kcal/mol (G-G). The decrease of binding specificity of RNA duplexes containing 8-methoxyguanosine compared with mismatched unmodified RNA duplexes is most likely the result of prior local distortion of helix due to the presence of a bulky modification. Hence, the presence of mismatches within modified RNA duplex causes slighter energetic loss relative to unmodified duplex.The presence of a methoxy group at the 8-position of guanosine decreases mismatch discrimination . The larm-1 versus ln (CT/4) plots and averaging the fits to individual melting curves prevented obtaining reliable thermodynamic data for those duplexes  was clearly non-two state. The large discrepancy between thermodynamic parameters derived from Tduplexes . In geneduplexes . The decCD spectra were performed for unmodified RNA duplex and two modified duplexes containing mxG-C and bxG-C base pairs in the central part of the duplex to examine possible alterations in helix geometry . All recin vitro and in vivo [in vivo. Moreover, depurinated by mutagenic compounds DNA has been suggested to play a key role in cancer initiation. 8-substituted nucleosides are usually chemically stable during oligonucleotide synthesis. However, there are reports concerning some synthetic problems with oligonucleotides modified i.e., with 8-bromoadenosine [Depurination is an important process in the chemistry of nucleic acids  in vivo ,30. Glycdenosine . Studiesdenosine , who havsyn conformation, presumably could serve as convenient modifications to increase ribozymes activity or improve antiproliferative properties of G-quadruplex based aptamers targeted towards cancer cells [The results presented herein confirm that a methoxy or benzyloxy group introduced at the 8 position of a guanosine moiety significantly increases the stability of the N-glycoside bond. At the best of our knowledge, this thermodynamic analysis is the first performed for RNA duplexes modified with 8-methoxy- or 8-benzyloxyguanosine. RNA duplexes containing 8-methoxy- or 8-benzyloxyguanosine are less stable compared with unmodified RNA due to the steric bulk of the substituents. Such modified RNA duplexes are also characterized by reduced hybridization specificity due to decreased discrimination of mismatches formed between mxG or bxG with A, G or U. However, the CD curves indicate that the presence of such bulky substituents at the 8 position of a guanosine moiety does not influence A-type geometry, which is characteristic of the RNA helix. As a consequence, oligonucleotides modified with 8-methoxy- and 8-benzyloxyguanosine might be useful tools to simultaneously detect hybridization of multiple sequences or to reverse the relative thermodynamic stability of siRNA ends, thus minimizing off target effects and increasing the rates of gene silencing ,31. Moreer cells ,33. NeveS1 FigM provides the second van't Hoff method for calculating folding thermodynamics. Moreover, the dependence of the TM on the oligonucleotide strand concentration reveals the molecularity of the transition. More details can be found in “Spectrophotometry and Spectrofluorimetry, Practical approach” edited by Michael G. Gore, chapter 13, p. 329, “The use of spectroscopic techniques in the study of DNA stability” authored by John SantaLucia Jr.for RNA duplex modified with 8-methoxyguanosine (A) and 8-benzyloxyguanosine (B). Thermodynamic parameters are calculated according to two independent methods. The first method is a van't Hoff analysis of the data by assuming a two-state model. The absorbance versus temperature profile is used to determine the temperature dependence of the equilibrium constant, allowing the calculation of ΔH° and ΔS° for the transition from a van't Hoff plot. The concentration dependence of the T(DOCX)Click here for additional data file.S1 File(DOCX)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.


BruUV-seq utilizes UV light to introduce transcription-blocking DNA lesions randomly in the genome prior to bromouridine-labeling and deep sequencing of nascent RNA. By inhibiting transcription elongation, but not initiation, pre-treatment with UV light leads to a redistribution of transcription reads resulting in the enhancement of nascent RNA signal towards the 5′-end of genes promoting the identification of transcription start sites (TSSs). Furthermore, transcripts associated with arrested RNA polymerases are protected from 3′–5′ degradation and thus, unstable transcripts such as putative enhancer RNA (eRNA) are dramatically increased. Validation of BruUV-seq against GRO-cap that identifies capped run-on transcripts showed that most BruUV-seq peaks overlapped with GRO-cap signal over both TSSs and enhancer elements. Finally, BruUV-seq identified putative enhancer elements induced by tumor necrosis factor (TNF) treatment concomitant with expression of nearby TNF-induced genes. Taken together, BruUV-seq is a powerful new approach for identifying TSSs and active enhancer elements genome-wide in intact cells. The principal steps of the transcriptional cycle are initiation, elongation and termination. Initiation occurs after recruitment of RNA polymerase II to promoter elements by transcription factors and by the aid of active enhancer elements11347in vitro assays for enhancer activity, suggesting that production of eRNA is linked to functional activityin vitro nuclear run-on assay activates paused RNA polymerases and promotes elongation, it is difficult to conclude that the measured levels of all eRNAs accurately reflect in vivo activity.RNA Pol II can generate RNA from enhancer elements, leading to the production of enhancer RNA (eRNA) whose function is unclear. eRNA is often transcribed bi-directionally but may not be polyadenylated and thus may be excluded from poly(A)RNA-based assaysHere we present BruUV-seq, an approach that complements the Bru-seq technique by enhancing nascent RNA signal around promoters and enhancers genome-wide in intact cells. UVC light (254 nm) introduces predominantly cyclobutane pyrimidine dimers and 6–4 photoproducts in DNA that are distributed more or less randomly in the genome22222 of UVC light (254 nm) and immediately labeled nascent RNA with 2 mM bromouridine (Bru) for 30 min. UVC-irradiation has been determined to induce on average 1 transcription-blocking lesion (CPD or 6-4PP) per 100 kb in the transcribed strand per each J/m22 UVC dose. Bru-labeled RNA was isolated and subjected to deep sequencing  sample were aligned by their annotated TSSs and divided into 250 bp bins. An aggregate view representing the median RPKM per bin across 1225 genes was produced . Nascent2  or 100 J2  to publicly available GRO-cap data from K562 cellsC. elegans and D. melanogaster28D. melanogaster, but are thought to be absent in humansTTTY15 and USP9Y on the Y chromosome appeared to fit this paradigm  element . InteresIn addition to a redistribution of reads from the bodies of genes to TSSs, we hypothesized that eRNA signal may be enhanced following UV-irradiation due to stabilization when associated with RNA polymerases stalled at UV-induced lesions. The basis for this is that the 3′-end of the transcripts should be protected against the 3′–5′ RNase activity of the nuclear RNA exosomeIn order to determine whether the intergenic BruUV-seq peaks observed coincided with enhancer elements, we compared our data to enhancers predicted by the combined genome segmentation annotation from ENCODE17in vitro and presumably in the absence of RNA-degrading enzymes, it is expected that unstable RNA species, such as eRNA, will be detected. Indeed, when we compare enhancer signal from BruUV-seq to previously published GRO-seq data from K562 cells, GRO-seq signal was similar in shape, but overall greater in magnitude (Because the nuclear run-on assay for GRO-seq and GRO-cap is performed agnitude . Additioagnitude . GRO-seqagnitude .2) sample). Thus, some of these GRO-cap-only enhancer segments may be explained by its higher sensitivity of these elements compared to BruUV-seq. Indeed, when we compared the intensity of the GRO-cap signal between the enhancers that are detected by both GRO-cap and BruUV-seq with enhancers detected by GRO-cap alone we found that high intensity GRO-cap signal was overrepresented in the group of enhancers detected by both methods . Furthermore, 50 genes had 1–5 induced enhancer elements while 34 genes appeared to be induced by TNF in an enhancer-independent way .We next used a hidden Markov model to identify intergenic regions with enhanced BruUV-seq signal (UVE regions). The TNF-induced change in RNA production in these UVE regions was then compared to the TNF-induced change in pre-mRNA expression of their nearest gene. A positive correlation was observed (r = 0.428), suggesting that changes in gene expression and nearby, putative eRNA production are correlated genome-wide . Not allHere we present BruUV-seq as a companion technique to Bru-seq, BruChase-seq1738BCR-ABL gene fusion by showing that this fusion gene initiated from a single TSS in the BCR gene  expressing hTERT172740442/s as measured with a UVX radiometer . Immediately following UVC irradiation, the cells were supplied with conditioned media containing 2 mM bromouridine (Bru) (Aldrich) and they were incubated for 30 minutes to label nascent RNA. Isolation of Bru-containing RNA, cDNA library preparations and deep sequencing were performed as previously described17The media of adherent cells grown on 100 mm plates was removed and 100 μl of PBS was added to keep the cells from drying out during the UV-irradiation. Suspension cells were gently pelleted and suspended in 1 ml of PBS and placed in a 100 mm plates for UV-irradiation at room temperature. Cells were irradiated in 100 mm plates without the lid on with different doses of 254 nm UVC light. The irradiation source  generated UVC light with a dose rate of 1 J/mSequenced reads  were initially mapped to human ribosomal DNA complete repeating unit (U13369.1) using Bowtie v0.12.81717et al.50A two state Hidden Markov Model (HMM), similar to the one described by Veloso The mapped reads for different Bru-seq and BruUV-seq experiments were sampled without replacement to simulate libraries with progressively fewer numbers of mapped reads. For each sequencing depth , 10 individual simulations were created by randomly choosing the desired number of reads from the previously mapped reads. Genes determined to have an active TSS according to the original data were used in this analysis. Their expression signal (RPKM) was measured for the simulated Bru-seq and BruUV-seq libraries. For Bru-seq, the whole annotated gene was used to determine the expression level, while only the first 5 kb downstream of the TSSs were used in the BruUV-seq analyses. The coefficient of variation (mean divided by standard deviation) in expression of each gene from the 10 simulations was calculated for each sequencing depth in each sample.de novo discovery of regions with BruUV-seq signal enhancement . eRNA was distinguished from genic RNA by including only intergenic UVE sites, defined as regions that do not overlap genes or their transcription units. Transcription units were determined in K562 cells by Bru-seq as previously describedTwo approaches were taken to determine genomic regions that were likely to represent enhancers. The first approach was to measure signal at enhancer segments (class E) as annotated by ENCODE’s combined genome segmentation for K562 cellsUV) determined for each bin, where each read was considered to be a Bernoulli trial with a sample-pair-dependent probability of being from the BruUV-seq sample. Bins in annotated genes with a Bru-Seq gene RPKM of at least 0.25 that were more than 20 Kb downstream of the TSS were then used to determine the cumulative fUV in the presumptive UVR portion of each gene. The mean and variance of these fUV values were used to calculate the α and β shape parameters of the binomial distribution with over dispersion of fUV, i.e. the beta binomial distribution, for the UVR state. Similar shape parameters were obtained for the UVE state by reflecting the UVR beta distribution such that bins with a high probability of being UVR had a low probability of being UVE. Emissions probabilities for the UVR and UVE states were then calculated for each bin using these shape parameters and the bin’s normalized Bru-seq and BruUV-seq read counts. The Viterbi algorithm was finally solved across all genome bins using a fixed transition probability of 0.005, with sequences of UVE bins taken as UVE peaks. Importantly, this process only used gene annotations to train the model, and so could detect a UVE peak at any genome location.Relative to Bru-seq, BruUV-seq signal is enhanced just downstream of TSSs followed by a decrease at more distal gene positions e.g. . Based ohttp://hgdownload.cse.ucsc.edu/goldenPath/hg19/encodeDCC/wgEncodeCshlLongRnaSeq. In order to carry out a direct comparison between Bru-seq, BruUV-seq and the ENCODE’s RNA-seq data, the ENCODE’s RNA-seq reads were trimmed to 52 base pairs and remapped as single-reads using our custom-built analysis pipelinehttp://genomes.ucsc.edu/)All the Bru-seq and BruUV-seq data generated in this study can be found at Gene Expression Omnibus GSE75398). Bru-seq and BruChase-seq data for HF1 was generated in our laboratory and previously published at Gene Expression Omnibus (GSE43440)98. Bru-sHow to cite this article: Magnuson, B. et al. Identifying transcription start sites and active enhancer elements using BruUV-seq. Sci. Rep.5, 17978; doi: 10.1038/srep17978 (2015).


The emergence of new technologies for characterizing transcriptome outputs, notably RNA-seq, are improving noncoding RNA identification and expression quantification. However, a major challenge is to robustly distinguish functional outputs from transcriptional noise. To establish whether annotation of existing transcriptome data has effectively captured all functional outputs, we analysed over 400 publicly available RNA-seq datasets spanning 37 different Archaea and Bacteria. Using comparative tools, we identify close to a thousand highly-expressed candidate noncoding RNAs. However, our analyses reveal that capacity to identify noncoding RNA outputs is strongly dependent on phylogenetic sampling. Surprisingly, and in stark contrast to protein-coding genes, the phylogenetic window for effective use of comparative methods is perversely narrow: aggregating public datasets only produced one phylogenetic cluster where these tools could be used to robustly separate unannotated noncoding RNAs from a null hypothesis of transcriptional noise. Our results show that for the full potential of transcriptomics data to be realized, a change in experimental design is paramount: effective transcriptomics requires phylogeny-aware sampling.Noncoding RNAs are integral to a wide range of biological processes, including translation, gene regulation, host-pathogen interactions and environmental sensing. While genomics is now a mature field, our capacity to identify noncoding RNA elements in bacterial and archaeal genomes is hampered by the difficulty of  We have analysed more than 400 public transcriptomes, generated using RNA-seq, from almost 40 strains of Bacteria and Archaea. We discovered that the capacity to identify noncoding RNA outputs from this data is strongly dependent on phylogenetic sampling. Our results show that, for the full potential of transcriptomics data as a discovery tool to be realized, a change in experimental design is critical: effective comparative transcriptomics requires phylogeny-aware sampling. We also examined how comparative transcriptomics experiments can be used to effectively identify RNA elements. We find that, for RNA element discovery, a phylogeny-informed sampling approach is more effective than analyses of individual species. Phylogeny-informed sampling reveals a narrow ‘Goldilocks Zone’ (where species are not too similar and not too divergent) for RNA identification using clusters of related species. In stark contrast to protein-coding genes, not only is the phylogenetic window for the effective use of comparative methods for noncoding RNA identification perversely narrow, but few existing datasets sit within this Goldilocks Zone: by aggregating public datasets, we were only able to create one phylogenetic cluster where comparative tools could be used to confidently separate unannotated noncoding RNAs from transcriptional noise. Genome sequencing has transformed microbiology, offering unprecedented insight into the physiology, biochemistry, and genetics of Bacteria and Archaea A further source of bias in our genome-informed view of microbes derives from a protein-centric approach to genome annotation. The majority of genome sequences deposited in public databases carry limited annotation of noncoding RNAs and cis-regulatory elements, yet it is rapidly becoming clear that RNA is essential to our understanding of molecular functioning in microbes The paucity of annotations is understandable, as RNA gene annotation is non-trivial We have therefore assessed the value of RNA-seq data for identification of unannotated non-coding and cis-regulatory RNA elements in bacterial and archaeal genomes. We show that numerous, hitherto uncharacterised, expressed RNA families are lurking in publicly available RNA-seq datasets. We find that poor sequence conservation for RNA families limits the capacity to identify evolutionarily conserved, expressed ncRNAs from existing genomic and transcriptomic data. Our results suggest that maximising phylogenetic distance, a sampling strategy effective for identification of novel protein families To assess the relative contribution of noncoding RNAs and protein-coding genes to transcriptional output, we collected all publicly-available bacterial and archaeal RNA-seq datasets (available as of August 2013), spanning 37 species/strains and 413 datasets. For all datasets, we supplemented publicly available genome annotations with screening for additional loci against the Pfam and Rfam databases We next examined the relative abundance of transcripts within each RNA-seq dataset, yielding an expression rank for individual transcripts. This analysis reveals that most transcriptomes are dominated by highly expressed non-coding RNA outputs   evolutionary conservation, 2) conservation of secondary structure, 3) evidence of expression in more than one RNA-seq dataset. For this analysis, we compared and ranked transcriptional outputs across species/strains see . In contEffective comparative analysis requires appropriate phylogenetic distances between species under investigation To assess the effect of strain selection on our capacity to identify RNA families using comparative analysis, we first generated F84 phylogenetic distances between 2562 bacterial strains and 154 archaeal strains using SSU rRNA sequences from each strain , only 11 are in the Goldilocks Zone for RNA (phylogenetic distance between 0.0118 and 0.0542) covering 9 species/strains. While five pairs of datasets are ‘too hot’ , the remaining 490 comparisons are ‘too cold’ for effective comparative RNA analysis . The datE. coli strains are extremely similar, and share 99% of their RUFs, suggesting that these strains are too similar for us to robustly separate expression of bona fide RNAs from noise. While these outputs could be genuine RNAs, these strains are in the ‘too hot’ region, meaning if everything is conserved, comparative power is lost. In contrast, only 12% of RUFs are conserved between strains/species pairs in the ‘too cold’ region  are among the most abundant transcripts. These results suggest that ncRNA may play an even greater role in the molecular workings of Bacteria and Archaea than hitherto realised.bona fide lineage-specific ncRNAs with potentially novel functions.This use of transcriptome data clearly improves our capacity to identify noncoding outputs: applying three criteria  we have identified 163 high-confidence expressed RUFs from public data . An addiOur results indicate that the greatest gain in analytical power for ncRNA discovery will come from phylogenetically-informed experimental design. Indeed, we find that this is critical to successful element identification, since the ‘Goldilocks Zone’ for optimal comparative analysis of RNA elements is surprisingly narrow. Hence, existing efforts to maximise phylogenetic coverage of genome space Given that isolation, cultivation and study of individual bacterial and archaeal strains can be extremely challenging All available bacterial and archaeal genomes were downloaded from the European Nucleotide Archive (ENA)  All genomes were re-annotated for both RNA genes and protein coding genes. Non-coding RNA genes were annotated using cmsearch (v.1rc4) From the mapped RNA-seq data, potential novel RNA genes  were picked manually by locating regions in the genomes that showed high levels of expression without overlapping annotated protein coding or RNA genes. Only RUFs of lengths 50 to 400 nucleotides were included, yielding a total of 844 RUFs in Bacteria and 78 RUFs in Archaea.Homologs of the identified RUFs were found in all the downloaded genomes using nhmmer For each strain, the available RNA-seq datasets were pooled and a list was created of transcripts showing expression in that strain in at least one experiment (defined as a transcript having a median depth of at least 10 reads in any experiment). A RUF homolog was defined as being expressed if the median read depth of the homologous region was at least 10X. Transcripts were ranked for each strain based on median expression (i.e. the most highly expressed transcript will have rank 1), which makes relative comparison across strains and datasets possible. The final set comprises 452 different Rfam families, 922 different RUFs, and 7249 different Pfam domains.For comparative analysis, if a gene was found to be expressed in more than one strain/species, the minimum rank was used (i.e. showing the relatively most abundant expression of the gene). This ensures that transcripts that are always low abundance will remain low abundance, whereas genes that are highly abundant in at least one of the sampled time points and conditions will be treated as such. The ranking is used as a measure of expression.“Family conservation” is based on SSU rRNA alignments of all Bacteria and Archaea, respectively. For each genome, the best hit to the Rfam model of SSU rRNA was extracted (RF00177 for Bacteria and RF01959 for Archaea). The sequences were aligned to the model using cmalign We ranked datasets based on the following quality control metrics  to be the number of annotated positions that are expressed; false positives (FP) to be the number of unannotated positions that are expressed; true negatives (TN) to be the number of unannotated positions that are not expressed; and false negatives (FN) to be the number of annotated positions that are not expressed. Note, not all annotated genes are expected to be expressed, and not all unannotated positions are false. Therefore, we calculate the positive predictive value (PPV): This measures the fraction of expressed positions that are annotated. We also calculate the fraction of the genome that is annotated: To make the PPV more robust, our final concordance metric normalizes PPV by ANN.Table S1The Pfam, Rfam and RUF identifiers for each entry corresponding to (XLS)Click here for additional data file.Table S2Strain/species names, genome accessions, RNA-seq data sources, Pubmed IDs, sequencing platform and notes for each dataset used for this study.(XLS)Click here for additional data file.Table S3Quality control measures computed for each RNA-seq dataset used in this study. The values are defined in detail in the Methods section.(XLS)Click here for additional data file.


Picornaviridae is a large family of positive-sense RNA viruses that contains numerous human and animal pathogens, including foot-and-mouth disease virus (FMDV). The picornavirus replication complex comprises a coordinated network of protein-protein and protein-RNA interactions involving multiple viral and host-cellular factors. Many of the proteins within the complex possess multiple roles in viral RNA replication, some of which can be provided in trans , while others are required in cis . In vitro studies have suggested that multiple copies of the RNA-dependent RNA polymerase (RdRp) 3D are involved in the viral replication complex. However, it is not clear whether all these molecules are catalytically active or what other function(s) they provide. In this study, we aimed to distinguish between catalytically active 3D molecules and those that build a replication complex. We report a novel nonenzymatic cis-acting function of 3D that is essential for viral-genome replication. Using an FMDV replicon in complementation experiments, our data demonstrate that this cis-acting role of 3D is distinct from the catalytic activity, which is predominantly trans acting. Immunofluorescence studies suggest that both cis- and trans-acting 3D molecules localize to the same cellular compartment. However, our genetic and structural data suggest that 3D interacts in cis with RNA stem-loops that are essential for viral RNA replication. This study identifies a previously undescribed aspect of picornavirus replication complex structure-function and an important methodology for probing such interactions further.The IMPORTANCE Foot-and-mouth disease virus (FMDV) is an important animal pathogen responsible for foot-and-mouth disease. The disease is endemic in many parts of the world with outbreaks within livestock resulting in major economic losses. Propagation of the viral genome occurs within replication complexes, and understanding this process can facilitate the development of novel therapeutic strategies. Many of the nonstructural proteins involved in replication possess multiple functions in the viral life cycle, some of which can be supplied to the replication complex from a separate genome  while others must originate from the template . Here, we present an analysis of cis and trans activities of the RNA-dependent RNA polymerase 3D. We demonstrate a novel cis-acting role of 3D in replication. Our data suggest that this role is distinct from its enzymatic functions and requires interaction with the viral genome. Our data further the understanding of genome replication of this important pathogen. Picornaviridae family of small, single-stranded positive-sense RNA viruses contains approximately 30 genera and includes many important human and animal pathogens. Poliovirus (PV) is the prototype of the enterovirus genus, which also includes the coxsackieviruses and the rhinoviruses. Other well-studied genera are the cardioviruses and the aphthoviruses, such as foot-and-mouth disease virus (FMDV). FMDV is the causative agent of foot-and-mouth disease, a highly infectious disease of cloven-hooved animals. Infection presents as an acute systemic disease with lesions on the feet and mouth; however, under certain circumstances the virus can adopt an asymptomatic carrier state. The virus is endemic in many regions of the world and can cause major outbreaks in domestic livestock with significant economic losses. Although vaccines are available, disease control is often complicated by high antigenic variability, transmissibility, and infectivity combined with the problem of asymptomatic carrier animals.The cis-acting replicative element” or CRE (5–The FMDV RNA genome contains a single open reading frame flanked by both 5′ and 3′ untranslated regions (UTRs) . The FMD2–” or CRE , 5–8. Thor CRE 5–, 10.pro and the P1-2A, 2BC, and P3 precursors. Lpro is self-proteolytically processed at its C-terminal boundary with P1 (–––1–3) (–pol) (1–3 (3AB in PV) and 3CD , while others are required in cis   proteins. Initial processing of the polyprotein generates four primary products: L with P1 . Release with P1 –16. Subswith P1 ––19. Inteith P1 ––. Final pith P1 –––23, thre(–––1–3) –28, the –––1–3) –, 30, and) (–pol) . Due to  and 3CD –34. In aolecule) –40.in vitro studies using FMDV or PV have elucidated some of the interactions within the replication complex. The crystal structures of 3D alone and in complex, e.g., with RNA and the primer peptide 3B  have shed light on the mechanism of RNA polymerization  were maintained in Dulbecco's modified Eagle's medium with glutamine (Sigma-Aldrich) supplemented with 10% fetal calf serum (FCS), 50 U/ml penicillin, and 50 μg/ml streptomycin as previously described . BSR-T7 The FMDV replicon plasmids pRep-ptGFP (where GFP is green fluorescent protein) and pRep-mCherry along with the equivalent polymerase knockout controls 3D-GNN have already been described . To intrDeletion of the S fragment was performed by two-step overlapping PCR mutagenesis with first-round reactions using primer pairs FMDV_Sfrag5′Eagl_rvs + FMDV_JW1_fwd and FMDV_Sfrag5′EagI_fwd + FMDV_Sfrag_3′Eagl_rvs. Products from the first round were used in second-round PCR with primers FMDV_JW1_fwd + FMDV_Sfrag3′EagI_rvs, and the PCR product was digested with NheI + SspI and cloned into NheI + SspI-digested pRep-mCherry to generate pRep-mCherry-Eagl. The S fragment was subsequently removed by digestion of pRep-mCherry-EagI with EagI before religation to generate pRep-mCherry-ΔS.A1G.To introduce the previously published replication-defective CRE point mutation  into pReHA and pRep-ptGFP-3DFLAG. Insertion of 3D point mutations was performed using the strategy described above. To replace the ptGFP reporter gene for Renilla luciferase, the Renilla luciferase reporter gene was amplified by PCR using primers RenLuc_NdeI_fwd and RenLuc_XmaI_rvs and used to replace the ptGFP reporter gene by NdeI + XmaI restriction enzyme digest. Primer sequences are available on request.Introduction of C-terminal 3D FLAG and hemagglutinin (HA) epitope tags first required introduction of a unique BspEI restriction enzyme site within the 3′ UTR to generate pRep-ptGFP-BseEI. This construct was subsequently used as the template in a two-step PCR, the first-round reactions involving primer pairs 3D_FLAG_fwd or 3D_HA_fwd with FMDV_BspEI_7643_21_rvs and 3D_FLAG_rvs or 3D_HA_rvs with FMDV_6299_6319. The second-round PCR product, generated using the two first-round PCR products with flanking primer pair FMDV_6299 + FMDV_BspEI_7643_21_rvs, was digested with BamHI + BspEI and used to replace the equivalent BamHI + BspEI fragment from pRep-ptGFP-BseEI, to generate pRep-ptGFP-3DIn vitro transcription reactions were performed as described previously , in accordance with the manufacturer's instructions. All transcripts were quantified by NanoDrop 1000 (Thermo Scientific), and RNA integrity was assessed by morpholinepropanesulfonic acid (MOPS)-formaldehyde gel electrophoresis before transfection.eviously . Briefly2 of RNA using Lipofectin transfection reagent (Life Technologies) as previously described  according to the manufacturer's instructions.For DNA transfection of BSR-T7 cells, 6.25 × 10in vitro-transcribed replicon RNA, cells were detached by trypsin and washed once in ice-cold phosphate-buffered saline (PBS), and total RNA was extracted using the RNeasy Plus RNA extraction kit (Qiagen) according to the manufacturer's protocol. Total RNA was treated on-column with RQ1-DNase, and total FMDV cDNA was amplified using Superscript II (Life Technologies) according to the manufacturer's protocol. Both mCherry and ptGFP replicon genomes were amplified separately using forward primers specific for either mCherry (mCherry_fwd) or ptGFP (ptGFP_fwd) with FMDV-specific reverse primer (FMDV_7200_7180) with Phusion High-Fidelity DNA polymerase (NEB). PCR products were blunt-end ligated into pCRBlunt. Individual colonies were isolated, and the presence of insertional mutation was determined by DNA sequencing.Following cotransfection of BHK-21 cells with mCherry and ptGFP Immunoblotting was performed as described previously  with priin vitro-produced transcripts, fixed at the indicated time in 4% paraformaldehyde, washed in PBS, and permeabilized in saponin buffer  for 1 h at 4°C. Primary antibodies rabbit anti-FLAG (Sigma-Aldrich) and mouse anti-HA (Sigma-Aldrich) were added in saponin buffer for 2 h at room temperature. Secondary antibodies used for detection were anti-rabbit-Alexa Fluor 568 and anti-mouse-Alexa Fluor 647 (Life Technologies), as appropriate. Coverslips were washed three times in saponin buffer between antibody steps. Coverslips were washed a final time in PBS before mounting in ProLong Gold with 4′,6-diamidino-2-phenylindole . Images were captured using a Zeiss LSM-880 inverted confocal microscope.BHK-21 cells seeded onto glass coverslips were cotransfected with 2, to reach equimolar proportions. Crystallization was performed by the hanging-drop vapor diffusion method at 293 K in a 24-well Greiner plate. One microliter of protein solution was mixed with 1 μl of precipitant solution; the volume reservoir was 1 ml. The best crystals appeared in 2 or 3 days from a solution containing 30% polyethylene glycol (PEG) 4000, 0.2 M magnesium acetate, 0.1 M MES (morpholineethanesulfonic acid [pH 6.0]), and 4% ϕ-butyrolactone.The GC216/7AA mutation was introduced into the His-tagged 3D expression clone pET28a-3D (as described in references The 3D-GC216/7AA data sets were collected at the Alba-Cells synchrotron light facility (Barcelona) on a Pilatus 6M Dectris detector at beamline Xaloc using a wavelength (λ) of 0.979 Å. All the data sets were recorded at 100 K. Crystals diffracted to 2.8-Å resolution . Four hu221 , after PDB 1WNE ) (Table PDB 1WNE ) and Phe5JXS.The crystallographic coordinates determined in this study have been deposited in the Protein Data Bank, with accession number trans  . In this study, we set out to investigate such differential functions of FMDV 3D.Due to the limited coding capacity of positive-strand RNA viruses, many viral proteins have multifunctional roles in RNA replication. Several studies have demonstrated that the replication of picornaviruses containing replication-defective mutations in some NS proteins can be recovered by expressing the corresponding wild-type protein in olecule) –40. Thistrans by cotransfection with wild-type “helper” replicons in a complementation assay using replicons with different reporters. We also observed that a replication-defective point mutation in motif C of 3D (3D-GNN) could be recovered using a wild-type helper replicon  exit site. Insertion mutations 3DTn-I102, 3DTn-C249, and 3DTn-S250 were located on the polymerase surface and are predicted to be involved in 3D protein-protein interactions directing the crystal packing in different 3D crystal forms , 43–45.43–The presence of small replication-defective insertional mutations in 3D acts as a genetic marker allowing the replication-defective genomes to be easily distinguished from the replication-competent “helper” genomes by PCR. Cotransfection of replication-defective with replication-competent genomes might be expected to give rise to replicative recombinants through polymerase strand switching.To investigate whether we could detect recombinants or wild-type revertants, the mCherry replicons bearing the replication-defective 3D insertional mutations (3DTn-I102 to 3DTn-S250) were cotransfected with the wild-type ptGFP helper construct, the ptGFP-3D-GNN construct, or yeast tRNA controls and RNA extracted at 8 h posttransfection. Both mCherry and ptGFP replicon genomes were amplified separately by reverse transcription-PCR using forward primers specific for mCherry or ptGFP, as appropriate. As anticipated, an mCherry-specific band of the expected size could be observed only when the 3DTn-L163, 3DTn-3DTn-A175, 3DTn-D240, and 3DTn-A246 mutant replicons were cotransfected with wild-type ptGFP helper replicon (data not shown). To isolate recombinants, this PCR product was subcloned into a plasmid vector, and the nonstructural region was sequenced from individual clones.A total of 20 clones of the 4 recoverable 3D insertions  were sequenced. The insertional mutation was clearly identifiable in 14 clones, with 6 clones having lost the insertional mutation, thus having a wild-type sequence (data not shown). No other point mutations were identified across the sequenced region. These data would suggest that the majority of genomes are not recombinants and that the recovery observed is not solely through the effect of recombination. However, recombinant genomes were clearly detected, presumably as a result of polymerase strand switching within mixed replication complexes.trans  and cis  functions of 3D. However, all of these mutations represent 5 amino acid insertions, which could cause a general disruption to protein folding and stability, as opposed to point mutations designed to abrogate a specific protein function (such as a GNN active-site point mutation). We therefore sought to replicate the phenotypes observed above with the replication-defective 3D insertions with site-directed point mutations. For this, we chose one location that was recovered and one that was nonrecoverable; these were the replication-defective mutations in motif A and the dsRNA exit site, respectively.The results above showed that approximately one-half of the replication-defective 3D insertions could be recovered by cotransfection with a wild-type helper replicon. The other half were nonrecoverable with a wild-type construct, suggesting differential Replication-defective insertions within motif A, characterized by insertional mutations 3DTn-D240 and 3DTn-A246, were located adjacent to either of the catalytic aspartic acid residues D240 and D245, shown in crystal structures to interact with the catalytic metal ion. We therefore hypothesized that these insertions abrogated the function of these aspartic acid residues, and to investigate this, we generated three new ptGFP replicon constructs bearing either a D240N or D245N point mutation or a DD240/5NN double point mutation. The two replication-defective nucleotide insertion mutations located in the dsRNA exit site, 3DTn-C217 and 3DTn-P219, were situated in close proximity to residues G216 and P219, which are thought to interact with RNA in the dsRNA exit site . HoweverThe ability of these six new ptGFP constructs to replicate was assessed in BHK-21 cells along with a wild-type positive control and polymerase active-site 3D-GNN mutation negative control . As expeNext, we determined whether these replication-defective point mutations in 3D motif A or the dsRNA exit site could be recovered in a complementation assay  were significantly recovered by cotransfection with a wild-type helper mCherry replicon . In contScmbl and 3D-239-246Scmbl, therefore maintained the amino acid sequence of 3D, but with a different RNA sequence. The replication of both scrambled replicon constructs was assessed in BHK-21 cells along with wild-type ptGFP positive control and the motif C polymerase knockout negative control (3D-GNN)  . Both reStudies with PV have indicated that transient cell chilling followed by the addition of the drug nocodazole blocked complementation by the depolymerization of microtubules and prevention of coalescence of replication complexes , 95. To Contrary to data published for PV, treatment with 5 μM nocodazole resulted in approximately 30% reduction in replicon replication (data not shown). Despite this reduction, normalization of the data revealed that treatment with nocodazole significantly inhibited recovery of the 3D-GNN construct by wild-type helper replicon . These dHaving confirmed that the replication-defective changes to 3D were a result of protein modification and not local RNA changes, we sought to identify whether such mutations affected 3D processing or stability. Directly transfecting replicon DNA into BSR-T7 cells (BHK-21 cells stably expressing T7 polymerase) allows for continuous replicon transcription and translation in the absence of replication, thus allowing for the detection of nonstructural proteins from replication-defective constructs. BSR-T7 cells were therefore transfected with replicon construct DNA containing the 3D motif A and dsRNA exit site mutations along with wild-type and 3D-GNN controls. Western blot analysis revealed no notable difference in 3D precursors between the wild-type and mutant constructs . There wIn vitro-synthesized RNA from these new epitope-labeled constructs was transfected into BHK-21 cells along with appropriate wild-type positive and polymerase knockout (3D-GNN) negative controls, and ptGFP expression was monitored over time . There was no appreciable difference in the cellular distribution of mutant and wild-type polymerases, all of which showed a diffuse punctate pattern of staining scattered throughout the cytoplasm (data not shown), albeit with the wild-type replicon displaying greater 3D expression, as would be expected.Renilla luciferase and the 3D modified to contain a C-terminal HA tag to permit dual-color immunofluorescence analysis. The new luciferase-expressing helper constructs were cotransfected into BHK-21 cells along with the ptGFP replicons carrying FLAG-labeled polymerase mutations  or wild-type control, and cells were fixed at 4 h posttransfection and costained for FLAG and HA expression  allowed us to study the localization of mutant and helper 3D in replicon-cotransfected cells. Helper constructs were generated in which the mCherry fluorescent reporter was replaced with pression  and C. Icis and so reduce its availability to recover replication-defective replicons. To test this hypothesis, a new set of replication-defective mCherry constructs were generated containing either a previously described functionally inactivating A>G point mutation in the CRE (mCherry-CREA1G)  were assayed for their ability to recover replication of the 3D-DD240/5NN, 3D-GC216/7AA, and 3D-GNN mutant constructs, in reciprocal complementation experiments , interact with both the CRE and a 5′ RNA structure known as the S fragment , 66, 96.eriments   yielded root mean square deviation (rmsd) values of only 0.51 Å. Structural comparisons also showed a subtle movement of the loop containing the mutated residues GC216/7AA, which was displaced by ∼0.74 Å toward the RNA primer  significantly recovered replication of the 3D-GNN replication-defective replicon, demonstrating an approximately 100-fold increase in ptGFP expression. In contrast, no recovery was observed when expressing 3D alone (plus 3D), compared to the yeast tRNA control . Thus, itrans has been well documented, including the NS proteins 2B, 3A, and 3D and RNA elements such as the CRE and the IRES  recombination. The majority of replication-defective genomes amplified by reverse transcription-PCR still maintained the mutagenic insertion. However, a small number of recombinant genomes were detected, presumably as a result of polymerase strand switching during RNA replication. This finding, together with our other observations, suggests that recovery occurs through the mixing of replication complexes or sharing of viral NS proteins, and it is therefore likely that genome recombination in our system is a downstream process following from successful complementation. Despite the generally good levels of recovery observed with 3D motif A and motif C mutations, ptGFP expression never achieved the levels observed with the wild-type construct. However, it is important to keep in mind that recovery of replication-defective 3D mutations relies on simultaneous cotransfection of the replication-defective construct with the helper construct. Therefore, the imperfect recovery observed in our system is likely to be largely limited by the intrinsic efficiency of RNA cotransfection, although other possibilities, such as the efficiency of delivering 3D molecules in cis versus trans viral and host cell protein-protein interactions generated in the formation of the replication complex and how 3D/3CD molecular protein interactions contribute in cis as part of a ribonucleoprotein complex and the transition from translation to genome replication.Of note, our initial insertional mutagenic screening  also ide75–cis and trans functions of the FMDV 3D RNA-dependent RNA polymerase that are essential for viral RNA replication. This includes a previously undescribed nonenzymatic cis-preferential role of 3D, which our data imply has altered RNA binding affinity and plays a role in binding structured RNA elements in the UTR. Our data advance the current understanding of picornavirus replication complex assembly.In summary, we have identified novel


During 60S biogenesis, mature 5S RNP consisting of 5S RNA, RpL5 and RpL11, assembles into a pre-60S particle, where docking relies on RpL11 interacting with helix 84 (H84) of the 25S RNA. How 5S RNP is assembled for recruitment into the pre-60S is not known. Here we report the crystal structure of a ternary symportin Syo1–RpL5-N–RpL11 complex and provide biochemical and structural insights into 5S RNP assembly. Syo1 guards the 25S RNA-binding surface on RpL11 and competes with H84 for binding. Pull-down experiments show that H84 releases RpL11 from the ternary complex, but not in the presence of 5S RNA. Crosslinking mass spectrometry visualizes structural rearrangements on incorporation of 5S RNA into the Syo1–RpL5–RpL11 complex supporting the formation of a pre-5S RNP. Our data underline the dual role of Syo1 in ribosomal protein transport and as an assembly platform for 5S RNP. Biogenesis of the 80S ribosome involves more than 200 pre-ribosomal factors, which ensure the sequential assembly of ribosomal proteins and RNAs. Here the authors show that the nuclear transport adaptor Syo1 shields the 5S RNP-docking site on RpL11 before incorporation into the pre-60S through molecular mimicry. Ribosomes catalyse protein synthesis in the cytoplasm23To date, only little data are available for the 5S RNP assembly, which seems to involve a number of factors before the complex is docked to the large ribosomal subunit assembly intermediate (pre-60S) in an early stage of 60S maturation8Here we report the crystal structure of the Syo1–RpL5–RpL11 complex, which shows that both cargos of Syo1 simultaneously bind to opposing sites of its α-solenoid structure. Syo1 chaperones 5S RNP and masks the major binding site of RpL11 for the 60S ribosomal subunit. Pull-down experiments and crosslinking mass spectrometry (XL-MS) provide insights into the 5S RNP assembly and underline a dual role of Syo1 in r-protein transport and ribosome biogenesis.Chaetomium thermophilum RpL11 (residues 15–167) in complex with RpL5-N (residues 2–30) and Syo1 . Crystallization was performed at 291 K by the sitting-drop vapour-diffusion method. Crystals belong to the orthorhombic space group P212121 with unit cell parameters of a=60.1, b=106.0 and c=147.2 Å  of the 25S RNA12At present, it is not known how 5S RNP assembly proceeds in the nucleolus and how Syo1 is involved. Association of the 5S RNA with the ternary complex does not release Syo1, but generates a tetrameric 5S RNP intermediate (pre-5S RNP) consisting of Syo1–RpL5–RpL11 and the 5S RNAS-transferase (GST)-tagged RpL11 variant in an in vitro pull-down approach , we performed the competition experiment with H84 now in the presence of the 5S RNA. In mature 5S RNP, RpL11 interacts with the 5S RNA as well as with RpL5 ref. . Therefo16To gain insights into the structural rearrangements underlying the pre-5S RNP assembly, we performed XL-MS experiments on the Syo1–RpL5–RpL11 complex with and without the 5S RNA. The purified complexes were crosslinked with the lysine-specific reagent disuccimidyl suberate and crosslinked peptides were identified by high-resolution tandem mass spectrometry using the xQuest and xProphet software2021The structure of the Syo1–RpL5-N–RpL11 complex (this study) and of the mature 5S RNP from the 80S ribosomeIn the presence of the 5S RNA, the crosslink pattern is the same overall, but shows some specific changes. The number of intralinks at the N-terminal region of Syo1 increased, indicating that the globular domain of RpL5 has moved on binding to 5S RNA and all the previously shielded lysine residues in that area are now free to be crosslinked . The relSyo1 was identified as a nuclear import adaptor, which allows for the simultaneous import of RpL5 and RpL11 ref . We haveFollowing a Kap104- and RanGTP-dependent import of the ternary complex into the nucleus, the 5S RNA can bind. The analysis of the 5S RNA interaction with the ternary complex by pull-down experiments, EMSAs and crosslinking MS shows that 5S RNA binding to RpL5 and RpL11 induces rearrangements, indicating the formation of a pre-5S RNP. These data support the idea that Syo1 serves also as an assembly platform for the 5S RNP, and we propose the following working model for 5S RNP biogenesis : Syo1 biOur data suggest that in addition to its role as an import adaptor, Syo1 acts as a molecular chaperone in the ribosome assembly pathway that masks a major binding site for the 60S subunit and prevents unspecific and/or premature RNA interactions. Syo1 copies the H84 interaction with RpL11, and thereby might ensure already in the cytosol that only properly folded RpL11 is imported for 5S RNP biogenesis, and is kept competent for docking to the pre-60S. Quality control of r-proteins before nuclear import and before entering the path of ribosome biogenesis and maturation could be an elegant mechanism to avoid wasteful intermediates.In essence, our study illustrates the complexity underlying the assembly of an important, but ‘simple' RNP with only three components. It may serve as a model for the understanding of assisted ribonucleoprotein assembly. We expect that similar quality-control mechanisms exist for other r-proteins to avoid nuclear import or recruitment of defective ribosomal components.Escherichia coli DH5α for cloning and plasmid propagation. All cloned DNA fragments generated by PCR amplification were verified by sequencing. Plasmids used in this study are listed in All recombinant DNA techniques were performed according to established procedures using 2. Crystallization was performed at 291 K by the sitting-drop vapour-diffusion method on mixing equal volumes (0.3 μl) of protein solution (23 mg ml−1) and crystallization buffer with a reservoir volume of 100 μl. Crystals appeared within 2 h in 0.1 M sodium acetate, 8% (v/v) PEG 4000.The Syo1–RpL5-N–RpL11 complex and the Syo1/RpL5/RpL11 complex were produced as describedhttp://www.pymol.org).To reduce radiation damage during X-ray data collection, crystals were flash frozen in liquid nitrogen after cryoprotection by transfer into cryosolution containing mother liquor and 20% (v/v) glycerol. Diffraction data were measured using beamline ID23eh2 at the European Synchrotron Radiation Facility , at 0.8726 Å under cryogenic conditions  using a CCD detector (MAR Research) and processed with the X-ray Detector Software packageA synthetic DNA template encoding helix 84  of yeast 25S RNA, cloned into the pEX-A vector via the EcoRI and HindIII restriction sites, was purchased from Eurofins (MWG Operon). Preparation of the 5S RNA and helix 84 was done as describedThe RNA-binding activity of the Syo1–RpL5–RpL11 complex was monitored by EMSAs. RNA was folded according to an established protocol6- tagged proteins from E. coli. SEC-purified Syo1-FLAG–(His)6-RpL5–RpL11 complex was immobilized on anti-FLAG-M2 magnetic beads (Sigma). Equimolar concentrations of folded H84 was added to the immobilized complex on the beads and incubated at 4 °C overnight. After washing with three column volumes of the binding buffer containing 20 mM HEPES-Na (pH 7.5), 150 mM NaCl, 10 mM KCl, 10 mM MgCl2, elution was performed by incubation with 0.1 mg ml−1 FLAG peptide. Coomassie-stained SDS–polyacrylamide gel electrophoresis revealed the proteins at each step and the RNA was observed by 0.8% agarose electrophoresis and ethidium bromide staining. Similar method was used for the SEC-purified Syo1-FLAG–(His)6-RpL5–RpL11–5S RNA complex. As a negative control, buffer was used in place of H84.Standard methods were used to purify (His)6–GST-RpL11 complex was immobilized on a GSTrap . Equimolar concentration of folded H84 was loaded onto the column and incubated at 4 °C overnight. After washing with binding buffer (20 mM HEPES-Na (pH 7.5), 150 mM NaCl, 10 mM KCl, 10 mM MgCl2), elution was done with elution buffer .Immobilized ion affinity chromatography-purified Syo1–RpL5(His)4HCO3 to 0.1 M for 10 min at 35 °C. Crosslinked proteins were denatured in 4 M urea and 0.1% (w/v) RapiGest Surfactant (Waters), reduced using 10 mM DTT (30 min at 37 °C) and cysteines were carbamidomethylated with 15 mM iodoacetamide (30 min in the dark). Protein digestion was performed first using 1:100 (w/w) LysC (Wako) for 4 h at 37 °C and then finalized with 1:50 (w/w) trypsin (Promega) overnight at 37 °C, after the urea concentration was diluted to 1.5 M. Samples were then acidified with 10% (v/v) trifluoroacetic acid and desalted using MicroSpin columns (Harvard Apparatus) following standard procedures. Crosslinked peptides were enriched using SEC, as described in ref. −1. Fractions eluting between 0.9 and 1.3 ml were generally pooled, evaporated to dryness and reconstituted in 20–50 μl 5% (v/v) ACN in 0.1% (v/v) FA according to 215 nm absorbance. Between 2 and 10% of the collected fractions were loaded onto a BEH300 C18  nanoAcquity UPLC column mounted on a nanoAcquity UPLC system (Waters) and stepwise eluted with a 3–85% (v/v) ACN in 0.1% (v/v) formic acid gradient connected online to an LTQ-Orbitrap Velos Pro mass spectrometer (Thermo). Data acquisition was performed using a TOP-20 strategy where survey ms scans  were acquired in the orbitrap , and up to 20 of the most abundant ions per full scan were fragmented by collision-induced dissociation  and analysed in the LTQ. To focus the acquisition on larger crosslinked peptides, charge states 1, 2 and unknown were rejected. Dynamic exclusion was enabled with repeat count=1, exclusion duration=60 s, list size=500 and mass window ±15 p.p.m. Ion target values were 1,000,000 (or 500 ms maximum fill time) for full scans and 10,000 (or 50 ms maximum fill time) for ms/ms scans. All the samples were analysed in technical duplicates.Syo1–RpL5–RpL11 complex alone and with the 5S RNA were crosslinked with 0.5 mM of H12/D12 isotope labelled disuccimidyl suberate (Creative Molecules) for 30 min at 35 °C under shaking (850 r.p.m.). To generate an XL-MS data set that can be processed as a whole with intrinsically consistent false discovery rate (FDR) estimation, we decided to rely on isotope-coded crosslinkers throughout the entire study. The reaction was quenched by the addition of NHRaw files were converted to centroid mzXML using the MassMatrix file conversion toom/z of the peptide ion identified by xQuest using a mass tolerance of ±20 p.p.m. Peaks were univocally identified based on their retention time, charge state and the presence of both light and heavy peptide ions. Peaks integration was performed using Xcalibur and manually inspected. The integrated area of each peak was then normalized to the average intensity of the sample and used for quantification. The quantification was performed on both technical replicates (repeated injection of the same sample) and the average value was used. We generally observed good reproducibility between technical replicates (average s.d. <12%). In several cases, crosslinked peptide ions were detectable above noise level only in one of the two conditions tested. Such instances were therefore unequivocally assigned only to one condition. When crosslinked peptides were quantified in both the conditions, we used an arbitrary cutoff of twofold change in normalized peak area to determine whether the crosslink was affected by the presence of 5S RNA.For crosslink quantification, peaks corresponding to crosslinked peptide ions were manually extracted from raw files using Xcalibur (Thermo). Raw data were queried using the F.R.C., G.B., K.W., M.B. and I.S. designed the experiments, analysed the data and wrote the manuscript. F.R.C., S.K., A.H. and A.O. performed the experiments. D.K. and E.H. analysed the data and discussed the experiments. All authors read and commented on the manuscript.Accession codes: Protein Data Bank: coordinates and structure factors have been deposited under the accession code: 5AFF.How to cite this article: Calviño, F. R. et al. Symportin 1 chaperones 5S RNP assembly during ribosome biogenesis by occupying an essential rRNA-binding site. Nat. Commun. 6:6510 doi: 10.1038/ncomms7510 (2015).Supplementary Figures 1-10, Supplementary Table 1 and Supplementary References


Drosophila melanogaster. The analysis of associations between editing levels and single-nucleotide polymorphisms allows us to map putative cis-regulatory regions affecting editing of 16 A-to-I editing sites . The observed changes in editing levels are validated by independent molecular technique. All identified regulatory variants are located in close proximity of modulated editing sites. Moreover, colocalized editing sites are often regulated by same loci. Similar to expression and splicing QTL studies, the characterization of edQTLs will greatly expand our understanding of cis-regulatory evolution of gene expression.RNA editing usually affects only a fraction of expressed transcripts and there is a vast amount of variation in editing levels of ADAR  targets. Here we explore natural genetic variation affecting editing levels of particular sites in 81 natural strains of  ADAR-mediated adenosine to inosine deamination (A-to-I editing) is the most widespread type of RNA editing in metazoans. Inosines are recognized by cellular machinery as guanosines, and editing may result in alteration of the encoded proteins . The discis-regulatory elements that determine the editing levels of individual sites is essential to unraveling the underlying regulatory mechanisms. Analysis of genetic variation is a powerful approach to study the regulatory mechanisms underlying various phenotypic traits. Previously, analysis of population-level transcriptomic data has revealed widespread natural variation in gene expression and splicing profiles. Genome-wide associations between this variation and whole genome sequences can be used to map the putative regulatory variants affecting these traits. This approach was successfully utilized for mapping of expression and splicing quantitative trait loci (Identification of cis-regulatory variants associated with changes in editing levels at 600 genomic loci of Drosophila melanogaster (cis-RNA editing quantitative trait loci or cis-edQTL), and found a sizeable fraction of them significant at nominal false discovery rate (FDR) level . While several strong effect edQTLs were replicated in both studies, the concordance was incomplete. Importantly, we used Sanger sequencing experiments to validate our analysis, and to address any concerns regarding the technical biases in the analysis of RNA editing based on short read data. We conclude that the identified edQTLs represent a novel type of functional genetic variation affecting the editing levels of individual target sites of ADAR.Very recently, Ramaswami and colleagues used targeted RNA sequencing to map putative D. melanogaster was downloaded from NCBI SRA . The analyzed strains represent two natural populations [Raleigh, North Carolina (The genotyping data for 216 inbred strains of Table S1.The transcriptomic data for 81 F1-hybrids are available at NCBI SRA (PRJNA281652). These hybrids were generated by crossing 81 inbred strains (the subset of 216 described above) with one common tester line (w1118). Total mRNA from adult female heads was sequenced on the Illumina HiSeq2000 platform (D. melanogaster genome (dm3/BDGP 5.72) using STAR (http://broadinstitute.github.io/picard/). For further analysis we used only uniquely and concordantly mapped reads.Paired-end RNA-Seq reads (2 × 100 bp) were mapped to the reference Drosophila strains. Additionally, we removed editing sites that were overlapping with annotated repetitive regions, and sites from intergenic and heterochromatic regions  for given editing sites in a given sample. Further analysis was performed on editing sites with estimated editing levels in at least 40 F1-hybrids. The filtered set comprised 1619 editing sites in 597 genes. The full list of editing sites is provided in Association tests between RNA editing level and proximal SNPs were performed using EMMAX  . λ was defined as ratio of medians of observed and expected uniform log P-value distributions. Statistically significant associations are provided in Table S3 (Bonferroni-corrected P < 0.05).To test for genome-wide inflation of false-positive results, we performed additional association tests for each edQTN-associated editing site. To this end, we performed association tests of given editing sites with all SNPs on the same chromosome arms. The resulting P-value (top edQTN). For Cluster 8, only one of the edQTNs was located in the same gene as the regulated site, and this SNP was considered as top edQTN.RNA editing sites that were associated with the same SNP (or block of linked SNPs) were grouped into the clusters . For eacTable S4). The validations were performed on homozygous inbred strains.Sanger sequencing was used to validate editing level estimates based on RNA-Seq data. For each edQTN-associated editing site, we designed a pair of primers to cover corresponding regions of the transcript (450–660 bp). Closely-associated editing sites were analyzed using the same amplicons. For each validated edQTN we chose two strains that carried two alternate alleles of a given SNP . The RNA editing level was calculated as the ratio of the height of the G-peak over the sum of heights of G- and A-peaks. Chromatograms are shown in Figure S2, Figure S3, Figure S4, Figure S5, Figure S6, Figure S7, Figure S8, Figure S9, Figure S10, Figure S11, Figure S12, Figure S13, Figure S14, Figure S15, and Figure S16.Total RNA was extracted from 10 adult female heads using a Direct-zol kit (Zymogen). cDNA templates were generated using Superscript III First-Strand Synthesis System (Life Technologies). Afterward, cDNA templates were amplified using the Phusion High-Fidelity PCR Kit (NEB). PCR products were confirmed, by running 2% agarose gels, purified and submitted for Sanger sequencing . The primers and specific annealing conditions are provided in Genomic and transcriptomic data analyzed in this study is available at NCBI SRA .D. melanogaster. We analyzed the set of editing sites that were identified in four recent large-scale transcriptomic studies . Hereafter, we refer to the variants associated with changes in RNA editing levels as RNA editing quantitative trait nucleotides (edQTN).These editing level estimates were used as quantitative traits to map putative regulatory variants associated with differences in RNA editing. The analyzed natural strains of The edQTNs represent associations between RNA editing levels of 16 sites, and 142 proximal SNPs. In eight cases, editing sites were associated with two or more SNPs in linkage disequilibrium (LD) blocks. In three cases, clusters of colocalized editing sites were jointly associated with the same edQTNs . In one case, the cluster of three editing sites was associated with a large LD block spanning several hundred kilobases .rtp, which was associated with a large LD block  plot for observed and expected distributions of association P-values are shown in Figure S2, Figure S3, Figure S4, Figure S5, Figure S6, Figure S7, Figure S8, Figure S9, Figure S10, Figure S11, Figure S12, Figure S13, Figure S14, Figure S15, and Figure S16.One representative case is an editing site that represents a protein-recoding event in the gene stj . Editingstj . To contWe used the difference between median editing level in F1-hybrids that carried two alternate alleles of the edQTN as a measure of effect size ∆REL, . ∆REL–vaThe analysis of RNA editing using short reads can be easily confounded with various technical artifacts . Overall, the results of Sanger sequencing were in good agreement with estimates based on RNA-Seq data (Table S4). In 15 of 16 cases the direction of edQTN-associated changes in editing observed by Sanger sequencing agreed with estimates based on RNA-Seq data , the correlation was 0.98 .Similar validation experiments were performed for all detected editing site/edQTN associations (Seq data . Note thP = 0.0001). These sites are codon-recoding events that lead to changes in the protein sequences of four genes. These genes were involved in eye functions [prom . In addition, we applied less stringent significance threshold for association P-values of P < 10−5 (which corresponds to genome-wide FDR of 5%), and generated an extended list of significant associations. The extended list of putative edQTNs consists of associations between 78 RNA editing sites and 539 SNPs (Table S7), and 15 of these associations were also significant in Table S6). This comparison was complicated by the fact that P-values in our analysis . With all these differences, we are pleased to note that the overall results of these two studies obtained using different techniques and approaches complement and confirm each other.In a recent study, Ramaswami and colleagues applied a targeted mmPCR-Seq assay to measure RNA editing at 789 A-to-I sites in 131 P < 10−8). We observed that a single locus frequently affects editing of several clustered sites. The low level of LD in fruit flies allowed us to map most of the putative regulatory variants at single nucleotide resolution. This revealed that causal variants affecting editing are most commonly located in the same genic regions as the editing sites. This may be useful to account in future studies of organisms with more complex haplotype structures. Similar to expression and splicing QTL studies, the identification of edQTLs will greatly expand our understanding of gene regulation. The increasing availability of population-level transcriptome data will make similar studies possible in humans and other organisms.In summary, we were able to map and validate 11 edQTLs associated with changes in editing levels of 16 A-to-I sites (


KSHV ORF57 (MTA) promotes RNA stability of ORF59, a viral DNA polymerase processivity factor. Here, we show that the integrity of both ORF59 RNA ends is necessary for ORF57-mediated ORF59 expression and deletion of both 5’ and 3’ regions, or one end region with a central region, of ORF59 RNA prevents ORF57-mediated translation of ORF59. The ORF59 sequence between nt 96633 and 96559 resembles other known MTA-responsive elements (MREs). ORF57 specifically binds to a stem-loop region from nt 96596–96572 of the MRE, which also binds cellular RBM15. Internal deletion of the MRE from ORF59 led to poor export, but accumulation of nuclear ORF59 RNA in the presence of ORF57 or RBM15. Despite of being translatable in the presence of ORF57, this deletion mutant exhibits translational defect in the presence of RBM15. Together, our results provide novel insight into the roles of ORF57 and RBM15 in ORF59 RNA accumulation and protein translation. Following primary infection, KSHV establishes a lifelong latency in which the KSHV episome is replicated by the host cell replication machinery. Upon reactivation to lytic cycle, the replication of KSHV genome depends on several viral proteins, including DNA polymerase Pol-8, ORF9), processivity factor of DNA polymerase PF-8, ORF59), helicase HEL, ORF44), primase PRI, ORF56), primase-associated factor ORF40/41), single-strand DNA binding protein , replication and transcription activator (ORF50 or RTA), and replication-associated protein  , single-,3. ORF594, primas9, helica, primase, ORF9, poriLyt) . RTA alsoriLyt) . Lytic roriLyt) , but prooriLyt) . ORF57 poriLyt) ,14,15,16oriLyt) . ORF57 boriLyt) ,19,20. WoriLyt) ,18 and ppVM7 for FLAG-tagged ORF57, pVM68 for 3 × FLAG-tagged ORF57, pVM18 for FLAG-tagged ORF59 and RBM15-FLAG were described in previous reports ,21,22. T5/well) plated in a six-well plate. Twenty four hrs after seeding the cells were co-transfected with 1 µg of individual ORF59 expression vector together with 0.2 µg of ORF57 (pVM7 or pVM68) or RBM15 vectors. The cells cotransfected with an empty pFLAG-CMV-5.1 vector were used as a negative control. All transfections were carried out with Lipofectamine 2000  as recommended. Protein samples were obtained by direct cell lysis with 0.5 mL of 2× SDS protein loading solution  supplemented with 5% 2-mercaptoethanol and analyzed by Western blotting with following antibodies: rabbit polyclonal anti-ORF57 antibody [32P-labeled antisense oligo probes: oVM11 for ORF57, oVM158 and oJM65 for ORF59, oZMZ270 for GAPDH and oST197 for U6 small nuclear RNA . All co-transfection assays were carried out in HEK293 cells (5 × 10antibody  (1:3000)antibody . The exp6 of TREx BCBL1–RTA or –vector cells in RIPA buffer as described [KSHV-infected, engineered BCBL1 cells, TREx BCBL1-vector and TREx BCBL1-RTA cells , were cuescribed . RNA-proescribed ,26 usingescribed . The proORF59 RNA decay was analyzed in HEK293 cells transiently transfected with an ORF59 expression vector in the presence or absence of ORF57 as described previously ,19. The In an attempt to delineate ORF59 sequences responsible for regulation by ORF57, we constructed a series of progressive 5’ to 3’ and 3’ to 5’ truncations of ORF59 A and evaIt has previously been shown that ORF57 is capable of enhancing translation of viral mRNAs by recruiting PYM, a cellular protein capable of facilitating the assembly of the 48S pre-initiation complex onto viral intronless mRNAs . As all Previous anti-ORF57 CLIP assays  identifivs. V in Our previous studies have shown that ORF57 affects ORF59 accumulation by interacting with RBM15, an RNA binding export cofactor . TherefoTo evaluate the relevance and functionality of the ORF59 5’ MRE sequence, we analyzed the MRE truncation mutants pJM22 and pJM23 A furtherKSHV ORF57 interacts with RNA export factor Aly/REF and NXF1 ,30 and cWe also analyzed RBM15's ability to accumulate RNA from other progressive 3’- and 5’-truncation mutants of ORF59. All of the mutants analyzed showed higher levels of ORF59 RNA expression in the presence of RBM15 A. In agrIn summary, this study has demonstrated that KSHV ORF59 contains multiple regions or motifs responsible for viral ORF57 and cellular RBM15. ORF57 promotes protein translation from ORF59 RNA and requires two terminal regions or one terminal region plus a central region of ORF59 for this function. The MRE identified by anti-ORF57 CLIP binds ORF57 protein and RBM15 and appears to be important for RNA export and stability of ORF59. However, this MRE motif is also important for RBM15-mediated protein translation of ORF59. Together, these studies provide further insight into how ORF57 promotes ORF59 expression by balanced interplay with RBM15.


Carcharhinus amblyrhynchos) can be one of the numerically dominant high order predators on pristine coral reefs, yet their numbers have declined even in the highly regulated Australian Great Barrier Reef (GBR) Marine Park. Knowledge of both large scale and fine scale genetic connectivity of grey reef sharks is essential for their effective management, but no genetic data are yet available. We investigated grey reef shark genetic structure in the GBR across a 1200 km latitudinal gradient, comparing empirical data with models simulating different levels of migration. The empirical data did not reveal any genetic structuring along the entire latitudinal gradient sampled, suggesting regular widespread dispersal and gene flow of the species throughout most of the GBR. Our simulated datasets indicate that even with substantial migrations  both large scale genetic structure and genotypic spatial autocorrelation at the reef scale were maintained. We suggest that present migration rates therefore exceed this level. These findings have important implications regarding the effectiveness of networks of spatially discontinuous Marine Protected Areas to protect reef sharks.Grey reef sharks ( Carcharhinus amblyrhynchos, the whitetip reef shark, Triaenodon obesus and the blacktip reef shark, C. melanopterus are abundant mesopredators that occupy the highest trophic level of permanent food webs in coral reefs7910The conservation of higher order predators is important for the maintenance of marine biodiversity, due to their potentially pivotal role in shaping communities2Reef sharks exhibit a combination of life history strategies and habitat specialization that make them particularly susceptible to anthropogenic pressure. They are slow growing, have delayed sexual maturity, and low reproductive output121514222314The grey reef shark is the most abundant reef shark in the Indo-Pacific, accounting for up to 50% of higher order predator biomass on healthy reefs14232426To be effective in increasing local biomass of target species, MPAs must meet two requirements: 1) they need to protect the target species by encompassing a large proportion of its home range; and 2) they need to be at least partially self-seeding 28r) ranged from 5.6 to 6.93, and observed heterozygosity over all loci ranged from 0.79 to 0.8 (Fis) were small or negative (h) ranged from 0.61 to 0.8  and did not significantly differ from 0 (p > 0.27). Similarly, all measures of genetic differentiation obtained from the microsatellite dataset were not significantly different from 0  and non-significant (p > 0.3). The results from the PCA analysis  were very close to 0 and their 95% confidence interval always overlapped with 0 and fell well within the expectations of the null model. None of the correlograms were statistically significant according to the non-parametric heterogeneity test developed by Smouse, et al.Spatial autocorrelation analyses did not reveal any spatial structure regardless of whether the dataset was analysed as a whole, or split according to sex . AutocorF’st range from 0.49 (1% migration) to 0.08 (25% migration), were all significant (p < 0.05) and their confidence interval did not overlap with the F’st value for the observed data. Positive spatial autocorrelation was detected in all simulations, and the extent of spatial autocorrelation was largely dependent on migration rates  between neighbouring reefs. With respect to temporal scales relevant to ecological processes, we found no genotypic spatial autocorrelation within reefs. Structuring of allele frequencies via genetic drift may be effectively prevented by a small number of migrants per generation in large populations, and therefore may only become evident across large spatial and temporal scalesCarcharhinus melanopterus there are clear patterns of genetic structure between reefs separated by as little as 50 km within French Polynesia36(Triaenodon obesus), also revealed very strong genetic structure (Φst = 0.25) between North and Central GBRet al.Our results have important implications for the management of this species, and are in sharp contrast with the findings of several previous studies on other reef associated sharks363738et al.et al.et al.Grey reef sharks are often described as strict habitat specialists, associated exclusively with coral reef habitats1517402323et al.This interpretation, however, relies on the assumption that grey reef sharks within each reef represent a closed populationet al.et al.et al.Acanthaster planci) outbreaksHeupel, et al.et al.et al.Carcharhinus albimarginatus), and especially large males, have wider home ranges that encompass multiple reefs. Given that larger sharks can traverse greater distances for comparable energetic expenditureC. melanopterus) has been shown to exhibit ontogenetic dispersal between inshore nursery and offshore reefsThe lack of genetic structure in this study may be the result of either high juvenile or adult dispersal, or both, albeit available movement data point to adults representing the dispersal stage. In two acoustic tagging studies conducted on the GBR, adult grey reef sharks were found to exhibit limited reef fidelity and move repeatedly between multiple reefs. Espinoza, et al.et al.26Espinoza, et al.et al.The widespread gene flow documented in this study, along with the findings of recent acoustic telemetry studies on grey reef shark movement suggest that while MPAs play an important role in protecting grey reef sharks, they are no panacea for reef shark conservation. Complementary management strategies appear necessary. The shortcomings of the current model of discontinuous networks of MPAs for the protection of coral reef organisms with adult dispersal have recently been highlighted by Momigliano, 2 was taken before the animals were released. Sampling was conducted in accordance with Macquarie University’s approved guidelines and the sampling protocol was approved by Macquarie University’s Animal Ethics Committee (AEC reference No.: 2012/044). Samples were collected under permit G13/35796.1 issued by the Great Barrier Reef Marine Park Authority (GBRMPA). Samples were stored in 95% ethanol at −20 °C. Details of sampling, including geographic coordinates of all sampling locations, are given in the Fin clips from 121 grey reef sharks were obtained along a latitudinal gradient spanning more than 1200 km and 9° of latitude  and nucleotide diversity (π) were estimated in DNAspFst between regions based on ND4 gene sequences were calculated in Arlequin 3.5Mitochondrial DNA sequences were aligned using BioeditMicrosatellites were scored using GeneMapper (Version 4), and alleles were binned using the software TANDEM following the method outlined by Matschiner and SalzburgerFstG’stDF’stAllele frequencies and measures of genetic differentiation were estimated in the R statistical environment using the package diveRsityet al.et al.A Principal Component Analysis (PCA), using region as the grouping factor, was performed using the package Adegenetr)71r for each distance class. The analysis was performed first for all sampled individuals, and then separately for males and females to detect possible sex-bias in spatial autocorrelation. The overall significance of each correlogram was tested using the non-parametric heterogeneity test outlined in Smouse, et al.To assess for the presence of spatial structure, we estimated genotypic spatial autocorrelation across three distance classes; 1) within single reefs 2) within the same geographic region 3) across the entire sampling area. We calculated individual based genetic distances in GenAlEx 6.5, using the codom-genotipic genetic distance option  to census size (Nc) ratio of 1:10To determine whether the statistical analyses performed were capable of detecting genetic structuring at different levels of between-reef migration, we carried out a range of simulations using the software EASYPOPHow to cite this article: Momigliano, P. et al. Connectivity in grey reef sharks (Carcharhinus amblyrhynchos) determined using empirical and simulated genetic data. Sci. Rep.5, 13229; doi: 10.1038/srep13229 (2015).