The virulent Newcastle disease virus (NDV) strain NA-1 (genotype VII) was isolated from an epizootic involving a flock of geese in Jilin Province, Northeast China, in 1999. Compared with the classical NDV strains, which have a genome size of 15,186 bp, the more recently isolated NDV strains, including that involved in the goose outbreak, have an extra six nucleotides in the 5′-untranslated region (UTR) of the nucleoprotein (NP) gene. This extra sequence, TCCCAC, is highly conserved and has been found in multiple NDV strains, including ZJ-1, WF00G, JSG0210, and NA-1. In the current study, an infectious clone from strain NA-1 was isolated and designated rNA-1. Subsequently, strain rNA-1 was mutated to delete the six-nucleotide insertion, producing strain rNA-1(−). Virulence of the recombinant virus was then assayed in chickens and geese.The recombinant virus rNA-1(−) showed similar biological characteristics to the parental NA-1 strain in DF-1 chicken fibroblast cells. However, the virulence of rNA-1(−) in 9-day-old embryonated chicken eggs and 1-day-old specific pathogen-free (SPF) chickens was decreased compared with the rNA-1 control. Furthermore, the virulence of the recombinant strain was slightly decreased in 1-day-old SPF chickens when compared with that in 1-day-old geese.Following deletion of six nucleotides in the 5′-UTR of the NP gene of NDV strain NA-1, the virulence of the rNA-1(−) recombinant strain was decreased in both chickens and geese. However, rNA-1(−) was more virulent in chickens than geese, as seen by the higher intracerebral pathogenicity index value. Avulavirus, within the Paramyxoviridae family [Newcastle disease (ND), caused by Newcastle disease virus (NDV), is one of the most serious avian diseases worldwide. NDV, also known as avian paramyxovirus serotype-1 (APMV-1), is a member of the genus e family , and is e family . The NDVe family . RNA edie family . The gene family . Althouge family , there ie family ,8.Based on standard pathotyping assays, NDV strains are classified into three main phenotypes: velogenic, mesogenic, and lentogenic . For decGenome size in all NDV strains follows the ‘rule of six’ , which pThe current study aimed to develop a rescue system that would allow genetic modification of the virulent NA-1 strain. The system was then used to examine the effects of mutations within the 5′-UTR of the NP gene. This rescue system, based on the intracellular synthesis of T7 RNA polymerase transcripts corresponding to the NDV genome, not only serves as a technical platform for the investigation of NA-1 gene function, but may also provide an opportunity to develop a live-attenuated vaccine candidate for geese.The genotype VII NDV virus designated NA-1 was isolated from geese in Jilin Province, Northeast China, in 1999. This virus has previously been sequenced  and the ad libitum.All birds were housed in individual ventilated cages supplied with filtered air under a 12-h light/dark cycle. SPF hygiene status was approved by Harbin Veterinary Research Institute. Food and water were available RNA from NA-1 was extracted from allantoic fluid using TRIzol (Invitrogen) according to the manufacturer’s instructions. cDNA was prepared using a Superscript II reverse transcriptase kit (Invitrogen) and random hexamers, as per the manufacturer’s instructions. Six pairs of specific primers (sequences available on request) were designed to amplify the complete genomic cDNA complement. Resulting PCR amplicons, named A, B, C, D, E, and F, were purified and cloned into pMD18-T vector (TaKaRa). A full-length cDNA copy of the NA-1 genome was constructed using a modified pBR322 cloning vector  and 6FMP4 (5′-CACATTGACACAGTCATTCGGTCtGTGATTTAC-3′). Another pair of primers, 6FP1 (5′-CGCGTCGACTCAGAGAACGAGAGGACAGAGATAC-3′) and 6FP2 (5′-CGCCGGTCCGACCAAACAGAGATTTGGTGAATG-3′), were designed to amplify the F segment of the NA-1 genome in a two-step process. The first part of the F segment was amplified using 6FP1 and 6FMP3 at 54°C. Then, primers 6FMP4 and 6FP2 were used to amplify the second part at 55°C.There is one RsrII site in the F protein segment of the NA-1 genome. To differentiate rescue virus from wild-type, the interior RsrII site was synonymously mutated from cggtcThe 5′-UTR of the NP gene was located in PCR fragment A described above. Using the plasmid constructed from segment A and the pMD18-T vector (named T-A), the six additional nucleotides (nucleotides 1648–1653) in the 5′-UTR of the NP gene were deleted using PCR. Primer pair 6AMP1 (5′-TCCGCCCAAAACCCACTCCCCGACCC-3′) and 6AMP2 (5′-GGGTCGGGGAGTGGGTTTTGGGCGGA-3′) was designed to amplify T-A and delete the six nucleotides. The resulting PCR product was digested using DpnI. Plasmids were transformed into competent cells DH5α, and clones containing the 6-bp deletion (named T-A(−)) were screened by PCR. To construct a full-length cDNA clone, T-A(−) was used to replace the same segment in pAF using SalI and AgeI. The resulting product was named pAF(−). Six segments of rNA-1 were amplified from pAF(−) and sequenced.BHK-21 cells were grown overnight to 70–80% confluence in six-well culture dishes. These were cotransfected with 0.25 μg of pAF or pAF(−), 0.4 μg of pCI-NP, 0.2 μg of pCI-P, and 0.2 μg of pCI-L, using 8 μL of X-tremeGENE HP DNA Transfection Reagent (Roche). pAF, pAF(−), pCI-NP, pCI-P, and pCI-L were transfected into BHK-21 cells using liposome method separately as controls. Following incubation for 6 h, the culture medium was replaced with complete DMEM medium containing 5% allantoic fluid. After 5 days, the supernatant was harvested, passed through a 0.22 μm filter, and inoculated into the allantoic cavities of 10-day-old embryonated SPF eggs. Allantoic fluid was harvested after 96 h and hemagglutination assays (HA) were performed according to the World Organization for Animal Health’s standard method . Viral R2. At 8-h intervals, the supernatant was collected to determine viral titers in a 50% tissue culture infection dose (TCID50) per milliliter by end-point titration. All titrations were performed in triplicate.To generate a growth curve, replication of the parental and rescued viruses was examined in DF-1 cells infected at a multiplicity of infection of 10 under one-step growth conditions. After adsorption for 1 h, the DF-1 cell monolayer was incubated in DMEM supplemented with 5% fetal calf serum at 37°C and 5% COin vivo. After 3 days, the allantoic fluids were examined by HA assay [9 HA titer, was diluted by 0.001% every passage and the inoculated into the allantoic cavity of three 10-day-old embryonated SPF eggs at 37°C. Each virus generation was identified using reverse-transcription polymerase chain reaction (RT-PCR), and the 56–1789 nucleotide region of the NP gene was sequenced after each passage of NA-1. After 10 passages, the mean time until death (MDT) and intracerebral pathogenicity index (ICPI) were determined in embryonated SPF eggs or in SPF chickens [The rescued virus was passaged in 9-day-old SPF eggs, obtained from the Harbin Veterinary Research Institute, to assess the genetic stability and pathogenicity of the rescued virus HA assay  to confichickens .-ΔΔCT method [The total RNA of each of the virus strains was extracted using TRIZOL reagent (Invitrogen), and cDNA was subsequently synthesized using a random hexamers primer, and then standardized to 1000 ng of total RNA per sample. To assess the transcription level of the NP gene, gene-specific primers were designed. The primers used for these mRNA analyses were: 5′-TGTACCACCAACTGCTTG-3′ and 5′-ATCACGCCACAGTTTCC-3′ for GAPDH, 5′-GGCAGCATGGACATTCCT-3′ and 5′- CCTTGCGGCTTGTTTGAT-3′ for NP. Each real-time PCR reaction (20 μL volume) contained 10 μL SYBR Green real-time PCR master mix (TAKARA), 0.25 μM gene-specific primers, and 1 μL standardized template cDNA. Amplification conditions were as follows: 95°C for 5 min, followed by 35 cycles of 95°C for 30 s, 56°C for 30 s, and 72°C for 30 s. After a final extension at 72°C for 10 min, a melting-curve analysis was performed to ensure specificity of the products. NP gene transcription levels were normalized to that of GAPDH, and transcription relative to the sample with the lowest expression was calculated using the 2T method .g for 10 min and used for western blot analysis. Samples of the cell lysate were mixed with Laemmli sample buffer and boiled for 3 min prior to electrophoresis. The boiled samples were separated by SDS-polyacrylamide gel electrophoresis on a 12% gel, and the resolved proteins were transferred to nitrocellulose membrane and incubated using a primary monoclonal antibody against the NDV NP protein, or GADPH protein as a loading control. The membranes were washed with PBS-T, and then incubated with the second horseradish peroxidase-conjugated goat anti-rabbit IgG antibody. Bands were visualized using SuperSignal West Pico Substrate (Thermo Scientific). This experiment was performed in triplicate.To assess the effects of deletions in the UTR of the NP gene on translation, NP protein expression by the NDV mutant was analyzed by western blotting . DF-1 ce9, was diluted 1/10 in sterile isotonic saline, and 0.05 mL of the diluted virus was injected intracerebrally into each of 10 geese in group 1. Groups 2 and 3 were infected with rNA-1 (HA titer 29) and rNA-1(−) (HA titer 29), respectively, using the same method. The birds were observed for clinical symptoms and mortality daily for 8 days. At each time point the birds were scored as follows: 0, no clinical symptoms; 1, showing signs of illness, and 2, deceased. The intracerebral pathogenicity index (ICPI) results represent the mean score per bird per observation over the 8-day period.One-day-old Huoyan geese, obtained from in Jilin Province, Northeast China, were negative for both NDV antigen and maternal antibody. Thirty 1-day-old geese were divided into three equal groups. Purified NA-1 virus, with a hemagglutination assay (HA) titer of 2in vitro experiments. We used analysis of variance, as calculated by GraphPad Prism 5, to establish the statistical significance of differences between the experimental groups. Individual inter-group comparisons were made using a two-tailed unpaired t-test with Welch’s correction. Differences were considered significant at P < 0.05 (*).All values are the mean ± SEM of at least three individual samples for the Animals were treated humanely and with regard for alleviation of suffering. All animal experiments were carried out according to the experimental practices and standards approved by the Animal Welfare and Research Ethics Committee at Jilin University .To determine whether the cDNA segments combined in our modified pBR322 vector (pAF from NA-1 and pAF(−) from NA-1 minus the additional nucleotides in the NP 5′-UTR) were full-length copies of the viral genome, six segments of pAF and pAF(−) were amplified and sequenced. Sequencing showed that pAF and pAF(−) were full-length cDNA copies of the parental virus. pAF(−) was identical to pAF except for the 6-bp deletion in the 5′-UTR of the NP gene at nucleotides 1648–1653.4 and 23, respectively.To generate infectious NDV from the cDNA of pAF and pAF(−), plasmids were cotransfected with BHK-21 cells and incubated for 5 days. On day 6, the supernatants from the experimental and control groups were inoculated into the allantoic cavities of SPF 10-day-old embryonated eggs. Allantoic fluid was harvested 96 h later and virus identified via HA assay. The viruses rescued from pAF and pAF(−) were named rNA-1 and rNA-1(−), respectively, and had identical genome sequences apart from the 6-bp deletion in the 5′-UTR of the NP gene. The rescued rNA-1 and rNA-1(−) viruses had HA titers of 2F segments of rNA-1 and NA-1 that were amplified by 6FP1 and 6FP2, respectively, and digested with RsrII showed that rNA-1 can be differentiated from parental virus NA-1 by restriction fragment length polymorphism , and that there was no significant difference in expression levels between NA-1 and rNA-1 (P > 0.05) , but that the ratio in rNA-1(−) was significantly lower than that of rNA-1 or NA-1 (P < 0.05)  than in rNA-1 and NA-1  Figure . Western) Figure . This reth passage were 59 h and 1.62, respectively. The MDT and ICPI values for rNA-1(−) from the 10th passage were 62 h and 1.52, respectively (Table in vivo. Although the virulence of rNA-1(−) did not change significantly in chickens, it was decreased compared with NA-1 and rNA-1.Growth curves showed that rNA-1(−) replicated more slowly than rNA-1, but that the two strains have similar growth characteristics Figure . SequencThe pathogenicity of the viruses in geese was examined, and the results were calculated by the ICPI method. At 10 days post-infection, all animals from the three groups were dead. The ICPI results for NA-1 and rNA-1 were 1.60, while the value for rNA-1(−) was 1.35. This indicated that rNA-1(−) was less pathogenic than rNA-1 in geese.th century. The most recent pandemic was caused by genotypes VII and VIII in the late 1980s and 1990s [Highly pathogenic NDV causes a rapidly spreading illness in poultry, with four worldwide pandemics since its emergence in the early 20nd 1990s ,29. For nd 1990s ,31. It iVirulent NDV strains are still isolated frequently from vaccinated birds, demonstrating that NDV remains a sustained threat to commercial flocks. Nowadays, the prevailing NDV strain of geese in China is genotype VII, while vaccine strains such as B1, Clone30, La Sota, and V4, which are used to protect geese from NDV infection, belong to different genotypes. The antigenic differences between the prevailing and vaccine strains might explain current ND outbreaks in vaccinated poultry flocks ,32,33.Methods for controlling and preventing ND may be the same as those used for avian influenza virus, with a significant need for epidemiological surveillance. The best control method is the development of homologous vaccines with high efficiency and safety. However, no current commercial vaccine is available for geese for protection against virulent genotype VII NDV. Therefore, we set out to establish a reverse genetics system that allows the genetic manipulation of a virulent strain, which could be used as a vaccine candidate or a viral vector to express foreign genes in our ongoing studies.4 and 23, respectively, in the current study. A correlation between virulence and the efficiency of viral replication has been observed for many viruses [The amino acid sequence at the F protein cleavage site is a major determinant of NDV virulence ,35, alth viruses . The lowThe results of this study demonstrate that the virulence of the rNA-1(−) strain is still at the edge of the velogenic pathotype, and is not significantly different from that of the rNA-1 strain. However, the pathogenicity of rNA-1(−) towards geese is lower than that of rNA-1. Therefore, the deletion of the six nucleotides from the 5′-UTR of the NP gene could contribute to the decrease in virulence of the mutant NA-1 strain.


Aristotelia chilensis) is a native Chilean species that produces berries that are exceptionally rich in anthocyanins and natural antioxidants. These natural compounds provide an array of health benefits for humans, making them very desirable in a fruit. At the same time, these substances also interfere with nucleic acid preparations, making RNA extraction from Maqui berry a major challenge. Our group established a method for RNA extraction of Maqui berry with a high quality RNA . This procedure is based on the adapted CTAB method using high concentrations of PVP (4 %) and β-mercaptoethanol (4 %) and spermidine in the extraction buffer. These reagents help to remove contaminants such as polysaccharides, proteins, phenols and also prevent the oxidation of phenolic compounds. The high quality of RNA isolated through this method allowed its uses with success in molecular applications for this endemic Chilean fruit, such as differential expression analysis of RNA-Seq data using next generation sequencing (NGS). Furthermore, we consider that our method could potentially be used for other plant species with extremely high levels of antioxidants and anthocyanins.Maqui berry ( RNA isolation has always been a critical first step in plant molecular biology. The structure of RNA contains a ribose sugars with a 2′-hydroxyl group attached that makes it more sensitive to degradation. This inherent instability together with the high levels of secondary metabolites found in plants hinders the RNA extraction and most of the times the RNA obtained has low concentration and poor quality  is a high-throughput sequencing technology, extremely sensitive to contamination and requires high quality of nucleic acids  has been singled out as an exceptionally rich source of anthocyanins and natural antioxidants , blueberries (Vaccinium spp.), raspberries (Rubus idaeus L.) and strawberries (Fragaria × ananassa L.) , including the one developed by Jaakola et al. To overcome these problems and to get cleaner samples, many authors have proposed the addition of contaminant absorbents like PVP in combination with a powerful reductant (β-mercaptoethanol) into the extraction buffer  and PVP (4 %) present in the extraction buffer prevented the oxidation of phenolic compounds, which were subsequently removed using organic solvents. The concentration of the absorbent component (PVP) was higher than those reported for other berries   in phenol–chloroform extraction were done to specifically remove DNA. These steps allowed the recovery of RNA with low levels of DNA contamination. The 70 % ethanol wash was essential to ensure RNA purity and, with ripe fruits, this step had to be repeated twice or three times, successfully eliminating the colorful anthocyanin pigments, which can interfere with subsequent steps.A summary of the steps of RNA extraction method it is shown in Table Table RNA integrity from our Maqui berry preps was evaluated with the 2100 Bioanalyzer instrument using the Agilent RNA 6000 Pico Assay Kit (Agilent Technologies). The RNA profiles obtained by this method allow an accurate assessment of RNA integrity by ribosomal integrity number (RIN) values Fig. . Our fig®). As this kit is optimized for 0.1–1 μg total RNA, the concentrations obtained with our method were enough for library preparation. The quality of these libraries was assessed with a DNA 1000 kit in a 2100 Bioanalyzer (Agilent Technologies). As shown in Fig. Transcriptomics using next-generation sequencing requires high concentration and quality as clean RNA material. In order to test if the RNA obtained by our new method is suitable for this technology, we performed a transcriptome analysis for different fruit tissues. Three Maqui berry libraries  were prepared using the TruSeq™ RNA Sample Preparation Kits v2 . As shown in Fig. All the solutions were prepared in 0.1 % diethylpyrocarbonate (DEPC)-treated water and autoclaved or freshly used. The extraction buffer contained the following reagents: 2 % CTAB, 4 % polyvinylpyrrolidone (PVP-40), 100 mM Tris–HCl (pH 8.0), 25 mM EDTA and 2.0 M NaCl. Mix and sterilize in an autoclave. Filter-sterilized spermidine and β-mercaptoethanol should be added to a final concentration of 0.5 g/l and 4 %, respectively, just before use. SSTE buffer: 1.0 M NaCl, 0.5 % SDS, 10 mM Tris–HCl (pH 8.0), 1 mM EDTA (pH 8.0). Mix and do not autoclave. Other solutions and reagents used in RNA isolation were the following: Chloroform-isoamyl alcohol ; phenol/chloroform-isosmylalcohol ; pH 4.3-equilibrated phenol solution ; 10 M lithium chloride; absolute ethanol; 70 % ethanol; diethylpyrocarbonate-treated and autoclaved distilled water. All non-disposable plastic materials were treated with diethylpyrocarbonate and autoclaved. Glass material, the mortar and pestle were treated for 180 °C for at least 4 h.Our RNA extraction protocol from Maqui berry was adapted from Jaakola et al. . Before g (RCF) for 10 min at 4 °C. The supernatants were removed and added to new pre-chilled Eppendorf tubes. An equal volume of chloroform:IAA was added, mixed and centrifuged at 15,700×g (RCF) for 10 min at 4 °C, in order to separate the phases. The supernatant was recovered, 1/4 volume of 10 M LiCl was added and mixed gently. RNA was precipitated at −80 °C overnight and subsequently centrifuged at 18,000×g (RCF) for 20 min at 4 °C. The supernatant was decanted by pressing the tubes against a paper towel. The pellet was washed with 500 µL of 70 % ice-cold ethanol. Then, the tubes were centrifuged briefly and the ethanol was decanted. For ripe fruits, this washing step was repeated at least twice, until the dark color disappeared.1.5 g of fresh tissue were ground to fine powder with a mortar and pestle, and liquid nitrogen. The ground material was quickly transferred to 10 sterile 1.5 ml microcentrifuge tubes. The tubes were kept on ice until the next step. 750 μl preheated extraction buffer were added to each sample and completely mixed by inversion. The tubes were incubated for 10 min at 65 °C. The samples were agitated with a vortex for a few seconds during the incubation time. The tubes were subsequently centrifuged at 10,000×g (RCF) for 20 min at 4 °C. The supernatant was carefully transferred to a new tube and an equal volume of chloroform:IAA was added. Then, samples were centrifuged as described above. The upper aqueous phase was collected carefully to a new tube and precipitated by adding an equal volume of absolute ethanol at −20 °C for 2 h or at −80 °C for 30 min. Samples were centrifuged at 18,000×g (RCF) for 15 min at 4 °C, the supernatants were discarded and the resulting pellets were carefully washed twice with chilled 70 % ethanol. The final pellets were air dried and then dissolved in 200 μl DEPC-treated water. In order to eliminate any genomic DNA contamination that could interfere with gene expression studies, the total RNA obtained by our method was subjected to an exhaustive DNase I digestion (Ambion), according to the manufacturer's instructions. 2 μl RNA samples were used for quality assessment. The rest was stored at −80 °C.The final RNA pellet was dissolved in 100 µl SSTE buffer. The 10 dissolved RNA samples were pooled into two Eppendorf tubes. For samples dissolved slowly, the tubes were briefly heated at 65 °C. An equal volume of phenol:chloroform:IAA (25:24:1) was added to the sample and shaken vigorously, then incubated on ice for 5 min and centrifuged at 18,000×® 200 NanoQuant spectrophotometer (Tecan). The RNA was diluted in autoclaved double distilled water to make dilution factor of 10, i.e. 1 µl RNA sample +9 µl autoclaved double distilled water. The quantification of RNA was done in triplicates. The O.D values were taken 260 and 280 nm. RNA concentration and the 260/280 ratios were delivered by the instrument.RNA concentration and purity was assessed by spectrophotometer. The concentration of RNA was quantified in the InfiniteRNA integrity was reported as the RIN based on the entire electrophoretic RNA sample trace, produced using the Agilent 2100 Bioanalyzer. The concentration and integrity of total RNA were determined by assaying 1 µl of total RNA using Agilent’s 2100 bioanalyzer with the Plant RNA Pico chip assay in accordance with the manufacturer’s instructions . The Bioanalyzer uses electrophoretic technology on a chip to separate RNA fragments by size, which are read by laser induced fluorescence and translated into gel-like bands and peaks (electropherograms) showing the distribution and relative amounts of RNA of different sizes. Concentration (ng/µl) of RNA was determined by comparing the sample with a standard. It measured RNA integrity using two metrics: the ratio of the large (26S) to small (18S) ribosomal RNA subunits (26S/18S) and the RIN.®), according to the manufacturer’s instructions. Briefly, 1 μg of total RNA was used as a starting material in order to isolate mRNA using Oligo(dT) beads. The poly(A) RNA was subsequently fragmented with divalent cations under elevated temperatures. These short fragments were used as templates for first-strand cDNA synthesis, producing single-stranded DNA copies from the fragmented RNA. After second-strand cDNA synthesis, the double-stranded DNA was subjected to end repair, and the 3′ends were adenylated. Finally, universal adapters were ligated to the cDNA fragments, and enrichment of those DNA fragments was performed using 15 cycles of PCR to produce the final sequencing library. Subsequently, the libraries were validated using DNA 1000 chip . The samples were pooled together in equal concentrations in one pool and running on an Illumina MiSeq™ System for a 300 cycles of paired-end sequencing.Three Maqui berry libraries  were prepared using the TruSeqTM RNA Sample Preparation Kits v2 (Illumina


This resource utilizes fixed-backbone design to optimize the sequence identity and nucleobase conformations of an RNA to match a desired backbone, analogous to fundamental tools that underlie rational protein engineering. The resulting sequences suggest thermostabilizing mutations that can be experimentally verified. Further, sequence preferences that differ between natural and computationally designed sequences can suggest whether natural sequences possess functional constraints besides folding stability, such as cofactor binding or conformational switching. Finally, for biochemical studies, the designed sequences can suggest experimental tests of 3D models, including concomitant mutation of base triples. In addition to the designs generated, detailed graphical analysis is presented through an integrated and user-friendly environment.RNA is rising in importance as a design medium for interrogating fundamental biology and for developing therapeutic and bioengineering applications. While there are several online servers for design of RNA secondary structure, there are no tools available for the rational design of 3D RNA structure. Here we present RNA-Redesign ( Engineered RNA and RNA/protein complexes are offering new routes for deciphering genetic regulation and for the eventual rational design of diagnostics and therapeutics –5. Thesehttp://rnaredesign.stanford.edu) utilizes the rna_design application in the Rosetta 3 framework to optimize the primary sequence of an RNA molecule to match a given phosphate-sugar backbone, analogous to the fixed-backbone design tools that underlie most approaches to rational protein engineering  stabilizing an existing structure relative to natural counterparts, (ii) highlighting differences in natural and computationally preferred sequences that might suggest functional constraints and (iii) designing mutations that allow tests of a predicted fold via compensatory rescue. The application returns sequence designs within minutes for RNA structures with lengths under 20 residues. Long input structures (up to 100 nt) are also accepted but require longer computational times; for these cases, the users may wish to provide an e-mail for notification of job completion. Examples of each application are presented below.syn rotamers for χ appear in natural RNA molecules and are therefore also sampled. The placement of the 2′-OH hydrogen is also simultaneously optimized with the base rotamers; the torsion angle defined by the C3′-C2′-O2′-HO2′ atoms are sampled at six torsion angles . The optimization of the nucleotide side-chain conformation and identity is carried out simultaneously at all residues by simulated anneling; the packing energy computations are accelerated by precomputation of all rotamer–rotamer pairwise energies. To sample a more detailed landscape of the sequence preference at each nucleotide position, the algorithm performs several independent separate nucleobase packing runs .RNA-Redesign takes an RNA 3D structure in the form of a PDB-formatted file. The file must contain backbone and sugar heavy atoms and may contain nucleobase atoms (which will be replaced during design). RNA-Redesign uses the Rosetta packer algorithm, well-developed for protein design  to samplhttp://www.cherrypy.org/) framework based on Python (https://www.python.org/) for basic web services and data management. For client-side programming, we coded the web pages in the HTML5 (http://www.w3.org/TR/html5/) standard, with jQuery (http://jquery.com) for interactive user-interface components and Bootstrap (http://getbootstrap.com/) for styling. On the server side, each job is generated with an ID that is assigned to a static link to the wait/results page in the form http://redesign.stanford.edu/result/JOB_ID. The data stay on the server for one month. The results are parsed by Python and rendered onto the web page, as shown in Figure The Redesign server uses the CherryPy (http://www.pymol.org/) using an in-house PyMOL coloring and graphics scheme (freely available at https://github.com/DasLab/pymol_daslab). Next, using the WebLogo3 python package (https://pypi.python.org/pypi/weblogo), a weblogo is generated to display the sequence propensity at each sequence location. Coloring is static with guanosine, cytidine, adenosine, uridine as red, green, gold and blue respectively. Finally, the native sequence recovery is plotted using CanvasJS (http://canvasjs.com) an interactive plotting interface in JavaScript. This is the likelihood of recovering the native sequence at each position. This plot can be informative because a nucleotide that is rarely recovered through RNA-Redesign but is naturally conserved may not be critical for structural stability and is therefore a candidate for other functional roles such as protein binding or catalysis. At the bottom, there is an interactive scatter plot, comparing Rosetta energy to sequence recovery. Users interested in examining specific designs can use this plot as a guide to see which designs are most favorable and which give the highest sequence recovery.The results page Figure  has fourTetrahymena ribozyme (PDB ID: 1GID), which forms contacts in the ribozyme's core  domain IV RNA (PDB ID: 1LNT), which has a conserved set of functional A–C base pairs . Utilizie's core . J4/5 ise's core . RNA-Rede's core , additio , an online server for RNA 3D design. RNA-Redesign uses the Rosetta 3 framework to design and to optimize the primary sequence of RNA with a given 3D backbone structure. These results can identify functional constraints and can suggest mutations that may stabilize or test the structures of a specific 3D RNA motif. It is our hope that RNA-Redesign can contribute to the growing development of RNA-based technology for biochemical and bioengineering applications.We developed RNA-Redesign (freely available at


We also show that cross-shelf coral cover declines with distance from the coast (R2 = 0.596). Identified coral taxa (21 genera) exhibited clear depth-stratification, corresponding closely to light attenuation and seafloor topography, with reefal development restricted to submarine antecedent bedforms. Data from this first assessment of nearshore reef occurrence and ecology measured across meaningful spatial scales suggests that these coral communities may exhibit an unexpected capacity to tolerate documented declines in water quality. Indeed, these shallow-water nearshore reefs may share many characteristics with their deep-water (>30 m) mesophotic equivalents and may have similar potential as refugia from large-scale disturbances.Mean coral cover has reportedly declined by over 15% during the last 30 years across the central Great Barrier Reef (GBR). Here, we present new data that documents widespread reef development within the more poorly studied turbid nearshore areas (<10 m depth), and show that coral cover on these reefs averages 38% (twice that reported on mid- and outer-shelf reefs). Of the surveyed seafloor area, 11% had distinct reef or coral community cover. Although the survey area represents a small subset of the nearshore zone (15.5 km Coral reefs worldwide are in serious decline, with adverse changes in coral cover17111213Despite the potential significance of such reef-building settings, empirical data on the structure, community composition and diversity of contemporary reefs within nearshore light-limited environments  remains sparse. This is largely due to difficult field working conditions especially from poor visibility. Although reef core records have provided clear evidence of prolonged  phases of reef-building under these conditions1516182 area of central Halifax Bay; 2) establish the antecedent topographic controls on reef development and community structure; and 3) use these parameters to classify patterns of habitat zonation within these turbid environments. Our findings contest conventional views regarding the threshold conditions for coral recruitment and long-term reef-building in nearshore areas. In particular, our results highlight the capacity for naturally marginal marine settings to support productive and diverse reefal habitats, and provide a framework for testing hypotheses about the apparent resilience of these reefs to some of the drivers of coral community degradation associated within seemingly more optimum  conditions on mid- and outer shelf areas of the central GBR.Here, in the largest examination of nearshore coral growth and reef development on the GBR to date, we use data to: 1) define the extent and composition of the nearshore reefs and coral communities within a 15 km2 (2). Highest mean cover (40 ± 36%) was recorded in shallow waters , and decreased rapidly with depth to 4 m below LAT, closely following light attenuation under low-turbidity scenarios , Acropora spp. (11 ± 15%), Turbinaria spp. (8 ± 17%) and Porites spp. (4 ± 8%)  encompassed an area of 15.5 km2 , 11% of (4 ± 8%) .p < 0.001; Acropora spp. and Montipora spp. dominate shallow water assemblages  increases considerably towards sea level, whereas branching and tabular Acropora spp. were found to occupy a normally-distributed range between 0.5–2.5 m below LAT (maximum relative cover of 22% at 1 m below LAT). Massive Porites spp. and large stands of foliose Turbinaria spp.  on the seafloor were recorded in very low relative abundances (<5%) at depth  topography , rangingPorites rus (24 ± 15%) on submerged reef slopes; (2) terrigenous sand/mud (91 ± 13%) covering inter-reef seafloor areas; (3) large Turbinaria spp. stands (80 ± 15%), or “coral carpets” (sensu)Montipora spp. (55 ± 19%) and Acropora spp. framework (16 ± 16%); (5) rubble-dominated (74 ± 12%) emergent reef areas, supporting low coral cover (13 ± 10%) of mainly foliose/encrusting Montipora spp. (13 ± 11%) and branching Acropora spp. (19 ± 22%); and (6) low coral cover (11 ± 10%) seafloor areas with submassive corals . A seventh habitat type (Goniastrea-dominated emergent reef flats) was identified from existing ecological assessments of Paluma Shoals North (PSN) and South (PSS)Montipora spp. (15%), Acropora spp. (6%) and Turbinaria spp. (5%) for within coral associations. Between the two coral framework-producing habitats (4 & 5 above), there was a mean cumulative dissimilarity of 55% differentiated by the relative proportion of coral rubble (24%), hard coral cover (20%), and the relative abundance of Montipora spp. (24%).Clustering analysis of benthic substrate types and coral datasets collected from across our study area differentiated six major habitat types with each comprising different benthic associations . These hramework  ± 16%; (Acropora spp. and Montipora spp.) indicating recruitment is occurring within these areas. Previous reef core data from a number of locations along the GBR have shown that reef-building has occurred in isolated nearshore locations throughout the mid- to late HoloceneTurbinaria spp. resembling those at PSRC have been reported elsewhere within a turbid high-latitude setting in Hervey Bay, Queensland, AustraliaCoral reefs growing within the nearshore zone of the central GBR experience chronic high turbidity as accumulated fine sediments are resuspended by waves27A key implication of this is that these low-light adapted coral communities may be better acclimated to cope with the stresses that have driven the major declines in coral cover observed on mid-outer shelf reefs since the mid-1980’s1630Specifically, our surveys show high coral cover (mean: 38 ± 24%) within nearshore areas of central Halifax Bay. We note that mean hard coral cover is more than twice that of the average for the central GBR shelf Acropora (A. pulchra and A. hyacinthus) are common across our sites  in depths <2 m LAT  and located relatively close to human-modified catchments, have hard coral cover levels much greater than those measured at many ‘clear-water’ mid- and outer-shelf sites on the central GBR in recent years. Our data shows that mean cover declines rapidly with distance from the mainland coast 347Turbinaria mesenterina)36Turbinaria spp., to withstand sediment loads an order of magnitude higher than maximum background conditionsA critical aspect to the apparent success of these nearshore corals must therefore lie in their ability to survive the low light conditions associated with high-turbidity. High suspended sediment concentrations, particularly those dark in colour typical of Halifax Bay, significantly reduce PAR to coralsA strong topographic control on coral development within the PSRC is also evident in our data , and is Underlying sedimentary structures built by terrigenoclastic sediments thus ironically appear fundamental in determining the location and extent of nearshore reef growth within the PSRC. Reefs rise across a depth range of 0.5 m at the shore-attached Paluma Shoals, to 4 m water depth (OPSB) and occur along a continuum of reef evolutionary states defined by their current position relative to lowest astronomical tide (LAT). These range from deeper incipient coral communities , through to juvenile (OPSC & OPSD), late-juvenile (OPSA & OPSB), mature (OPS) and senile reefs , that are fully emplaced at sea level and emergent under low tidal conditions (based on evolutionary classifications of clear-water GBR reefs from)402 of potential reef structure between Townsville and Lucinda) at sites north of the PSRC with similar geometries to those identified hereHowever, the broader distribution of reefs in the most nearshore areas of the GBR shelf still remains relatively unknown. The widespread cover of hard corals within the mapped area of Halifax Bay suggests that it is highly unlikely that such reef communities are restricted to the PSRC alone. Indeed, preliminary analysis of satellite imagery shows numerous near-surface features  of more resistant coral types to reseed degraded reefs.Our study focused on the wider Paluma Shoals Reef Complex (PSRC), located within Halifax Bay on the inner-shelf (<3 km from the coast) of the central Great Barrier Reef, Australia . Here, s15−1, with 40 days per year exceeding 88 mg l−128). As a result these nearshore reefs are extremely light-limited due to sediment laden waters, and thus we classify the PSRC as a series of shallow-water mesophotic reefs. The term “mesophotic” is conventionally used to describe coral ecosystems surviving in deep-water (>30 m) low-light conditions45The PSRC is located landwards of the shore-detached inshore sediment prism (ISP), a wedge of terrigenoclastic sands that were reworked onshore during the postglacial transgression, and fine material that has accumulated at the shoreline since sea level stabilised in the mid-Quaternary highstand2) to establish the extent and morphology of reefal structures. Because of the shallow nature of the survey area (<8 m depth) bathymetry data was collected using a single-beam echo sounder (Ceeducer Ceestar 200 kHz), coupled with a Real-Time Kinematic Global Positioning System (RTK-GPS). Data were acquired along a suite of closely spaced (every 100 m) north-south parallel lines across the location of known shoals (observed in Landsat imagery), and a series of 500 m spaced shore-perpendicular lines to provide broader context for the shoals  datum using tide gauge data at the Port of Townsville. A digital elevation model of seafloor bathymetry was constructed in Surfer 12 using kriging (10 × 10 m grid size).We conducted a high-resolution survey of seafloor bathymetry within the PSRC area  were extracted from the video at automated 8 sec intervals (n = 4420) and a digital 9-point grid overlay was added for analysis of benthic cover. Corals were classified by genera and growth morphology. Reef rugosity within each frame was assessed visually using a modified rugosity classification schemeTowed video surveys (July 2013/2014) of the seafloor were undertaken using a drop-down video system (SeaViewer with Sea-Track™ GPS overlay), as along with poor visibility due to high turbidity, the presence of saltwater crocodiles and Irukandji jellyfish make long SCUBA transects impractical. The camera was towed 1 m above the seafloor along multiple 300 m transect lines see  to delinBenthic data was classified into different habitat types using hierarchical agglomerative clustering with group-average sorting484948How to cite this article: Morgan, K. M. et al. Evidence of extensive reef development and high coral cover in nearshore environments: implications for understanding coral adaptation in turbid settings. Sci. Rep.6, 29616; doi: 10.1038/srep29616 (2016).


More recently, RNA sequencing has enabled an increased pace of fusion gene identification. However, RNA-Seq is inefficient for the identification of fusion genes due to the high number of sequencing reads needed to detect the small number of fusion transcripts present in cells of interest. Here we describe a method, Single Primer Enrichment Technology (SPET), for targeted RNA sequencing that is customizable to any target genes, is simple to use, and efficiently detects gene fusions. Using SPET to target 5701 exons of 401 known cancer fusion genes for sequencing, we were able to identify known and previously unreported gene fusions from both fresh-frozen and formalin-fixed paraffin-embedded (FFPE) tissue RNA in both normal tissue and cancer cells.Fusion genes are known to be key drivers of tumor growth in several types of cancer. Traditionally, detecting fusion genes has been a difficult task based on fluorescent  Cancer cells frequently contain chromosomal rearrangements that lead to the formation of fusion genes expressed in the cell . These fin situ hybridization (FISH), a technique in which selected regions of chromosomes are fluorescently labeled through the hybridization of specific oligonucleotide probes. Aclinician must then visually identify two chromosomal regions that have rearranged in a known pattern. FISH has many problems as a technique including being a difficult, low throughput procedure, required knowledge of both gene fusion partners, low spatial resolution, the availability of the necessary fluorescent probes and the need for highly trained personnel to decide if a fusion event has taken place [Gene fusions are typically identified in cells by fluorescent en place . These dhttp://cancergenome.nih.gov/)  has been used to identify gene fusions that are transcribed into RNAs within various cells . SoftwarIn order to test the ability of the Ovation Fusion Panel Target Enrichment System assay to effectively target RNAs of interest, we ran the assay using 100 ng of total RNA from the H2228, lung adenocarcinoma cell line to see what percentage of sequencing reads were derived from the targeted genes. The resulting library was sequenced on an Illumina MiSeq with paired end 75bp reads. 92.5% of reads mapped back to the genome with 96% of those reads mapping to the targeted RNAs. These results indicate a strong selection for targeted RNAs by the Ovation Fusion Panel Target Enrichment System. Comfortable that we were selecting almost exclusively for the sequences we were interested in, we next looked to identify gene fusions.ALK gene that is fused to EML4 and the other allele is fused to PTPN3. In our targeting panel we have probes targeting ALK and EML4, but not PTPN3 so this served as a good test for identifying fusion transcripts for which only one of the genes was directly targeted. The sequencing data were analyzed for the presence of gene fusions , which only requires the hybridization of a single primer in order to obtain sequencing information from the downstream RNA. H2228 cells carry one allele of the ions see  and we wBCR-ABL and NUP214-XKR3. 100 ng of UHR total RNA was used to prepare sequencing libraries with Ovation Fusion Panel Target Enrichment System. Libraries were sequenced on either the Illumina MiSeq or HiSeq and analyzed with SOAPFuse [BCR-ABL and NUP214-XKR3 fusion transcripts. In addition, we also identified an FGFR1-WHSC1L1 gene fusion, which has been reported as a recurring fusion in breast cancer [MLLT10 fused to PICALM. Having identified a set of gene fusions in UHR RNA, we compared the number of fusion reads in these genes detected by SOAPFuse in our datasets to those of a publically available RNA-Seq dataset that consists of 125M paired end sequencing reads [BCR-ABL fusion. The 1.6M targeted sequencing reads has fewer reads for NUP214-XKR3, which is expected because our targeting panel only contains probes for NUP214 and not XKR3. The standard RNA-Seq does not contain any detectable reads for FGFR1-WHSC1L1 nor any reads for MLLT10-PICALM, while the targeted sequencing approach described here identified both of these fusion events. Both of these previously undescribed fusions were verified by RT-PCR and Sanger sequencing. In summary, by using a targeted sequencing approach, we were able to identify known gene fusion events in UHR RNA while using only 1.3% of the sequencing reads necessary to identify approximately the same number of fusion reads in a standard RNA sequencing experiment. In addition we were able to identify previously undocumented gene fusion events using the targeted approach that were not identified in standard RNA sequencing, even though the targeted sequencing had 14–78X fewer sequencing reads. The inability of standard RNA sequencing to identify fusion transcripts in a cell line that has been studied in detail suggests that even samples for which deep RNA sequencing data already exists should be tested again specifically for the presence of fusion transcripts.We next wanted to compare our results to those of standard RNA-Seq experiments to test how many sequencing reads were necessary to identify gene fusion events when using the Ovation Fusion Panel Target Enrichment System relative to a non-targeted RNA-Seq approach. Universal Human Reference RNA (UHR) contains RNA from ten different cancer cell lines. Many standard RNA-Seq experiments have been performed using UHR RNA and these experiments have identified a number of gene fusion events . Our 401SOAPFuse . Our init cancer , but hasATP5L-MLL and EIF4E3-FOXP1. The EIF4E3-FOXP1 fusion . This common procedure is known to fragment nucleic acids making it difficult to retrieve sequence data from them. We extracted RNA from HCC1937 primary breast ductal carcinoma cells that had been formalin-fixed and paraffin-embedded . Briefly, utilizing the contents of the Ovation Fusion Panel Target Enrichment System kit, 50 or 100 ng of total RNA was reverse transcribed into cDNA. The unpurified cDNA was then made double stranded by the addition of a DNA polymerase. The double stranded cDNA (ds-cDNA) underwent end repair to blunt the ends followed by barcoded adapter ligation. Ligated samples were bead purified and hybridizated with targeting probes starting at 95°C followed by a slow ramp down to 60°C overnight. Following probe annealing, a DNA polymerase was added to the mixture which was heated to 72°C to extend the probes through the target region  was a gift from Susana Ortiz, M.D., Ph.D. . Universal Human Reference RNA (UHR) (Agilent Technologies) and Human Brain RNA (Ambion) were purchased and used without modification. IRB approval was not required as the human RNA samples used were reference samples obtained from cadavers and purchased from a commercial source. No identifying patient information was obtained by the authors. FFPE sections of cell line HCC1937 were purchased from Alamak Bioscience. 10 micron sections were cut and RNA was extracted from these samples with the Recoverall Total Nucleic Acid Extraction Kit (Ambion). Total RNA was used as input into the Ovation Fusion Panel Target Enrichment System to generate targeted sequencing libraries following the manufacturer’s recommended protocol in the User Guide and described in gion see . UnextenResulting sequences were analyzed in the following ways: 1. To determine the reads that were derived from targeted RNA sequences, forward reads were mapped to the Hg19 reference genome using STAR v2.4.0d  only allFor publically available UHR RNA-Seq data, we downloaded SRX333353  and for Selected putative gene fusions were verified by designing PCR primers around the predicted fusion site. Reverse transcription PCR was used to amplify the predicted fusion gene junctions from the same starting RNA material as was used for sequencing. Resulting PCR products were purified and Sanger sequenced to verify the fusion junction. 200 ng of total RNA input was used in the Ovation cDNA Module For Target Enrichment to generate the cDNA used for the PCR reaction (included as part of the Ovation Fusion Panel Target Enrichment kit). PCR was performed in 50 ul reactions using standard Taq buffer, 0.5mM dNTPs and standard Taq Polymerase (NEB) with 0.4 uM each primer. The PCR reaction was carried out with the following program: 95°C, 3 minutes, followed by 30 cycles of 95°C, 15 seconds, 57°C, 20 seconds and 72°C, 10 seconds. See 401 genes were selected for inclusion in the initial gene fusion targeting panel. All refseq exons were input into the proprietary probe selection software with the goal of designing at least two probes targeting each exon. For exons smaller than 300 bases probes were designed so that the 3’ end of the probe would hybridize as near as possible to the known exon boundary, with one probe designed to hybridize to each strand of the double stranded cDNA. For exons larger than 300 bases, additional probes were tiled across the exon at 300 base intervals in order to maximize coverage across these exons. Placing probes near the exon boundaries ensures that extension products will cross at least one exon-exon junction, thus increasing the probability of extending across a gene fusion junction. These design parameters resulted in the generation of 17,999 unique targeting probes across 5769 exons.S1 Table(TXT)Click here for additional data file.S2 TableForward and reverse primer sequences used in RT-PCR validation. For Sanger sequences, | denotes the breakpoint between sequence aligning to one gene versus the other in the fusion pair.(TXT)Click here for additional data file.


C9ORF72 represent the most common genetic variant of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia. We and others have proposed that RNA transcribed from the repeat sequence is toxic via sequestration of RNA-binding factors. Both GGGGCC-repeat (sense) and CCCCGG-repeat (antisense) molecules are detectable by fluorescence in situ hybridisation as RNA foci, but their relative expression pattern within the CNS and contribution to disease has not been determined. Blinded examination of CNS biosamples from ALS patients with a repeat expansion of C9ORF72 showed that antisense foci are present at a significantly higher frequency in cerebellar Purkinje neurons and motor neurons, whereas sense foci are present at a significantly higher frequency in cerebellar granule neurons. Consistent with this, inclusions containing sense or antisense derived dipeptide repeat proteins were present at significantly higher frequency in cerebellar granule neurons or motor neurons, respectively. Immunohistochemistry and UV-crosslinking studies showed that sense and antisense RNA molecules share similar interactions with SRSF2, hnRNP K, hnRNP A1, ALYREF, and hnRNP H/F. Together these data suggest that, although sense and antisense RNA molecules might be expected to be equally toxic via their shared protein binding partners, distinct patterns of expression in various CNS neuronal populations could lead to relative differences in their contribution to the pathogenesis of neuronal injury. Moreover in motor neurons, which are the primary target of pathology in ALS, the presence of antisense foci  but not sense foci  correlated with mislocalisation of TDP-43, which is the hallmark of ALS neurodegeneration. This has implications for translational approaches to C9ORF72 disease, and furthermore interacting RNA-processing factors and transcriptional activators responsible for antisense versus sense transcription might represent novel therapeutic targets.GGGGCC repeat expansions of The online version of this article (doi:10.1007/s00401-015-1429-9) contains supplementary material, which is available to authorized users. C9ORF72 represent the most common genetic variant of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) -ATP using T4 polynucleotide kinase (Fermentas). Reaction mixes were made up in RNA binding buffer  with 50 ng radiolabelled RNA and 2 µg purified recombinant protein. Mixes were incubated for 10 min at room temperature before being UV irradiated on ice at full power. Complexes were analysed by SDS/PAGE and stained with Coomassie blue before being vacuum dried and exposed on a phosphoimage screen.(GGCCCC)The following antibodies were used for IHC anti-TDP-43 (Proteintech 10782-2-AP), anti-hnRNP H/F (Abcam ab10689), anti-hnRNP A1 , anti-SRSF2 Abcam ab11826), anti-ALYREF , anti-nucleolin (Proteintech 10556-1-AP), and anti-hnRNP K (Abcam ab52600). Poly-GA was detected with anti-GA antibodies  as previously described [26, anti-C9ORF72-ALS and are characteristic of both motor (motor neurons) and extra-motor (cerebellum and hippocampus) pathology [C9ORF72-ALS cases, non-C9ORF72 ALS cases, and controls were examined for RNA foci in a blinded manner. No RNA foci were observed in tissue from controls and non-C9ORF72 ALS cases. Forty Purkinje neurons, forty motor neurons, >200 granule neurons, >150 dentate gyrus neurons, and >100 CA4 subfield neurons were evaluated from four C9ORF72+ ALS and/or FTD cases. The average frequency of sense and antisense foci per cell is shown in Table p < 0.05) and motor neurons (likelihood-ratio test p < 0.05), but significantly lower in cerebellar granule neurons (likelihood-ratio test p < 0.05). The fact that sense and antisense foci were relatively more abundant in different neuronal populations is against an artefact caused by differences in affinity of RNA FISH probes.The frequency of sense and antisense RNA foci was determined in five neuronal populations: Purkinje and granule neurons in the cerebellum, motor neurons of the spinal cord ventral horn, and neurons of the hippocampal dentate gyrus and CA4 subfield. These neuronal populations were chosen as they all exhibit neurodegeneration in As reported for sense RNA foci , we obseC9ORF72-ALS cases. More than 1000 granule neurons and approximately 50 motor neurons were examined in a blinded experiment. Neuronal inclusions containing sense RNA derived DPRs were only observed in granule neurons, whereas inclusions containing antisense RNA derived DPRs were only observed in motor neurons  and the frequency of poly-PA inclusions was significantly higher in motor neurons (likelihood-ratio test p < 0.01). The average frequencies of inclusions containing poly-GA and poly-PA protein are shown in Table As a further validation, and to extend, the conclusions of this study to a larger number of cases, dual staining of poly-GA, and poly-PA protein were examined in a further blinded experiment. Approximately 1000 granule neurons and 50 motor neurons were studied from six C9ORF72+ ALS and/or FTD cases including three cases not utilized in the earlier analysis. In each case, the correct protein was determined based on the frequency of observed inclusions . As for the sense species, there was no significant correlation between the two observations .In our previous study  we showe4 repeat RNA [We used confocal microscopy to validate in vivo some of the RRM-containing proteins that were found to interact with (CCCCGG)peat RNA . We and peat RNA , 17. We peat RNA .C9ORF72-ALS cases. Simultaneous co-staining was carried out in parallel in non-C9ORF72 ALS cases and neurologically normal controls. For ALYREF, hnRNP A1, SRSF2, hnRNP H/F, and hnRNP K, the overall cellular distribution was not specifically altered in C9ORF72+ cases except for nuclear areas where colocalisation was demonstrated 5 RNA oligonucleotides, and purified recombinant proteins synthesized in E.coli. Unlike IHC, this allows determination of direct and specific RNA:protein interactions via the formation of covalent bonds under UV light exposure. Both sense and antisense repeat RNA were observed to directly interact with hnRNP F, hnRNP A1, ALYREF, and SRSF2 proteins although the RNA-binding activity was not equal in all cases and for hnRNP A1 was relatively low  [C9ORF72-ALS cases (Supplementary Table 5). Unlike sense RNA foci, the presence of antisense foci was significantly associated with nuclear loss of TDP-43. Seventy-seven percent of antisense foci+ motor neurons displayed loss of nuclear TDP-43 compared to 13 % of motor neurons without observable antisense foci  . The smf TDP-43 . We haveWe have demonstrated colocalisation of antisense RNA foci with SRSF2, hnRNP A1, hnRNP H/F, ALYREF, and hnRNP K, but not nucleolin. This is consistent with the work of Haeusler et al. . UV-crosC9ORF72-mediated neurodegeneration. If so, our results would predict that both species of RNA foci should be equally toxic. This is consistent with observed toxicity of sense foci in various model systems [Both the IHC and the UV-crosslinking studies in this report suggest that the binding partners of sense and antisense RNA foci are not significantly different. This is also reported by others . Many of systems , 21, 28.In-vitro studies have suggested that both sense and antisense RNA sequences form complex secondary structures including G-quadruplexes and hairpin loops [In all cases, the relative frequency of sense and antisense foci varied consistently and significantly between neuronal populations. Importantly in motor neurons, the primary target of pathology in ALS, antisense foci are more abundant than sense foci. Therefore, we suggest that the key event determining toxicity leading to TDP-43 mislocalisation, of antisense as opposed to sense RNA foci, might be a propensity to produce antisense foci mediated by cell-specific transcriptional regulation. Alternatively sense RNA foci might be degraded at a higher rate than antisense RNA foci. in loops . These sIt is interesting that two populations of relatively large neurons, motor neurons and cerebellar Purkinje neurons, exhibited antisense RNA foci at a higher frequency than sense RNA foci, in contrast to the smaller cerebellar granule neurons. This suggests that our observations may be related to some property correlated with neuronal size. However, in the hippocampus, neither the larger CA4 subfield neurons nor the smaller dentate granule neurons exhibited either species of RNA foci at a consistently higher frequency.C9ORF72-mediated neurodegeneration [We observed antisense foci in the cytoplasm of motor neurons, which is consistent with aberrant nuclear export and may be a key step in the facilitation of proposed repeat associated non-ATG translation to produce DPR species . We madeneration , 20, 22 We have demonstrated that the frequency of sense and antisense foci is usually correlated i.e. a patient with more sense foci will also have more antisense foci. This is particularly interesting in case 1 from our analysis Table  who disphGle1, a mRNA export adaptor, have recently been shown to cause selective death of motor neurons [Finally, varying frequency of the expression of sense and antisense repeat RNA has implications for the formation of specific DPRs. Our observations of all five DPRs are consistent with our conclusions relating to the expression of sense and antisense RNA foci. In cerebellar granule neurons, where sense RNA foci are more abundant, there is a higher frequency of sense-RNA derived DPR inclusions; and in motor neurons where antisense RNA foci are more abundant, there is a higher frequency of antisense RNA-derived DPR inclusions. Therefore, we suggest that, at least in these neuronal populations, translation of the sense and antisense derived proteins occurs in different quantities depending of the relative availability of RNA repeat molecules. The results of the present study potentially explain the observations of others that inclusions containing poly-GA protein are much more abundant in certain neuronal populations including cerebellar granule neurons . However neurons .C9ORF72 expansion then the motor neuron population will express higher levels of antisense RNA derived foci and DPRs, but not necessarily within the same individual cells.In our previous study we showed that there was no significant correlation between the presence or absence of nuclear sense RNA foci in cerebellar granule neurons and whether or not those cells contain a cytoplasmic inclusion positive for poly-GA . SimilarC9ORF72-ALS should consider the presence of antisense RNA foci in motor neurons. An antisense oligonucleotide approach has been proposed as a therapeutic option in C9ORF72-disease [C9ORF72-mediated pathology in vivo, targeting the antisense foci may be even more important than targeting sense foci. A limitation of our study is the reliance on post-mortem tissue which represents end-stage disease and may exclude the most vulnerable cells which have already been lost; as such we await validation of our findings in model systems.Our work highlights that any therapeutic approach to -disease , 28. We -disease . Indeed,C9ORF72-ALS cases by TDP-43 IHC and RNA FISHSupplementary material 1 (PDF 103 kb) Supplementary Table C9ORF72+ casesSupplementary material 2 (XLS 58 kb) Supplementary Table C9ORF72+ casesSupplementary material 3 (XLS 29 kb) Supplementary Table Supplementary material 4 (XLS 24 kb) Supplementary Table 4: Plasmids used in this studySupplementary material 5 (XLS 28 kb)


Corynebacterium glutamicum, an actinobacterium of high industrial relevance and model organism for medically important Corynebacterianeae, such as C. diphtheriae and Mycobacterium tuberculosis.Recent discoveries on bacterial transcriptomes gave evidence that small RNAs (sRNAs) have important regulatory roles in prokaryotic cells. Modern high-throughput sequencing approaches (RNA-Seq) enable the most detailed view on transcriptomes offering an unmatched comprehensiveness and single-base resolution. Whole transcriptome data obtained by RNA-Seq can be used to detect and characterize all transcript species, including small RNAs. Here, we describe an RNA-Seq approach for comprehensive detection and characterization of small RNAs from C. glutamicum ATCC 13032 was prepared from cultures grown in minimal medium at exponential growth or challenged by physical  or by chemical stresses  at this time point. Total RNA samples were pooled and sequencing libraries were prepared from the isolated small RNA fraction. High throughput short read sequencing and mapping yielded over 800 sRNA genes. By determining their 5′- and 3′-ends and inspection of their locations, these potential sRNA genes were classified into UTRs of mRNAs (316), cis-antisense sRNAs (543), and trans-encoded sRNAs (262). For 77 of trans-encoded sRNAs significant sequence and secondary structure conservation was found by a computational approach using a whole genome alignment with the closely related species C. efficiens YS-314 and C. diphtheriae NCTC 13129. Three selected trans-encoded sRNAs were characterized by Northern blot analysis and stress-specific transcript patterns were found.In our RNA-Seq approach, total RNA from C. glutamicum and provide a sound basis for further studies concerning the functions of these sRNAs.The study showed comparable numbers of sRNAs known from genome-wide surveys in other bacteria. In detail, our results give deep insight into the comprehensive equipment of sRNAs in The online version of this article (doi:10.1186/1471-2164-14-714) contains supplementary material, which is available to authorized users. Corynebacterium glutamicum is a non-pathogenic and non-sporulating gram-positive soil bacterium which belongs to the order Actinomycetales. This microorganism has a long history of applications in the production of various amino acids and other industrially relevant compounds [C. diphtheriae or Mycobacterium tuberculosis. The genome sequence established a decade ago [C. glutamicum has been studied extensively [C. glutamicum lacks a sequence homologue of the RNA chaperone Hfq, similar to other Actinomycetales[C. glutamicum (ArnA) was detected upstream of the GntR-Regulator cg1935 and is located in antisense direction [ompounds , 2. Furtcade ago , 4 comprensively  and reveensively . Howevermycetales. So far,irection .trans-encoded sRNA genes, these databases also include RNA motifs from mRNA leader transcripts of protein-coding genes, some of which regulate translation initiation or cause transcriptional attenuation. Elements such as RNA thermometers are structures sensitive to temperature shifts and control the accessibility of the Shine-Dalgarno sequence of the mRNA leader  is often applied synonymously. All hitherto identified RNA families are collected in the RNA-families (Rfam) online database , fRNAdb iewed in ). The cliewed in ) as wellE. coli[trans-encoded sRNAs works through imperfect basepairing with target mRNAs (reviewed in [cis-antisense sRNAs (asRNA) genes are located directly in the antisense direction with respect to their target genes and thus show full complementarity . These iewed in ).E. coli, a number of sRNAs have been predicted by computational methods . Such i species , as well species . The RfaExperimental strategies for the discovery of sRNAs in bacteria started with systematic genome-wide screens by shotgun cloning and sequencing of cDNA  or by usC. glutamicum. Sequencing libraries were created by the “differential” RNA-sequencing (dRNA-Seq) approach [2O2 and diamide stress to gain a broad spectrum of transcription of potential sRNA genes in response to these conditions. Supported by promoter searches, RNA-Seq data were analyzed and led to the detection of novel sRNA genes in C. glutamicum ATCC 13032. In addition, sRNA genes were classified and compared with bioinformatic sRNA predictions based on secondary structure stability and sequence conservation.Here, we present the first deep sequencing study of sRNAs in approach  with RNAC. glutamicum, we isolated total RNA from C. glutamicum cells grown to exponential phase and from cells after a variety of stress treatments and pooled the total RNA samples , diamide stress (2 mM), and salt stress (1.5 M NaCl). To enrich small RNA for a transcriptome sequencing (RNA-Seq), the pool of total RNAs was size-selected for transcripts smaller than 250 nucleotides (nt) by precipitation and further depleted of ribosomal RNAs (rRNA) using a hybridization procedure that selectively binds rRNA species with biotinylated probes . The cis-antisense reads made up 3% and less in both libraries. Interestingly, the majority of read mappings belong to regions with no annotated features, particularly in the library enriched for primary transcripts (59%), indicating a high number of potential sRNAs in C. glutamicum.Transcription of sRNAs in bacteria is highly variable under different environmental conditions , 29. Hens Figure . The strt stress .5 M NaClt stress .5 M NaClt stress .5 M NaClcis-antisense to CDS and 1595 stacks in regions lacking annotated features). As a further filtering step, the 5′-upstream sequences of the assumed TSS were analyzed for promoters. Using the tool Improbizer [cis-antisense to CDS and 736 SigA-dependent promoters in regions lacking annotated features). The search for SigH promoter sequences was successful in case of 44 TSS (11 cis-antisense to CDS and 33 in regions lacking annotated features). For 14 transcripts, promoters of both types were detected, indicating sRNA variants with different 5′-ends.After filtering of mappings to tRNA and rRNA genes and to putative mRNAs , the potential sRNA fraction from the library 1 (primary transcripts enriched) was utilized for the definition of sRNA transcript starts Figure a. A numbprobizer , 32, we probizer  or the sprobizer , which iprobizer  and heatprobizer . Thus, 1cis-antisense to CDS and 65 in regions lacking annotated features).To determine the 3′-ends of sRNAs, we mapped reads from the two libraries and followed each of the initial stacks up to a point where the number of read starts fell below the chosen cut-off of 10 reads and defined 3′-ends from these data Figure c. In addIn 136 cases, the predicted TSS were within close distance to each other, indicating multiple promoters. Proposed multiple starts located within 100 bp at the 5′-end and with the same 3′-end were merged to a single region and annotated as such.mraW motif (at cg2377), the cspA motif (at cg0215), and nine predicted riboswitches which are part of 5′-UTRs longer than 100 nt , ThiM (cg1655), and ThiE (cg2236). Two TPP-riboswitches were found upstream of cg0825 (putative beta-ketoacyl acyl carrier protein reductase) and cg1227 (putative membrane protein). One flavin mononucleotide (FMN)-dependent riboswitch was detected at the putative nicotinamide mononucleotide uptake permease (cg0083) and one S-adenosyl methionine (SAM)-dependent type IV riboswitch upstream of cg1478 . Together with the two riboswitch related RNA-motifs (both yybP-ykoY) [In order to identify putative untranslated regions (UTRs) of mRNAs that are included in the set of potential sRNA genes, these were then grouped according to their position and direction relative to an adjacent CDS Figure . In totatif at cg377, the bP-ykoY)  and sevecis-antisense RNAs (asRNA) that start in antisense orientation within an opposing CDS, (ii) transcripts antisense to a 5′-UTR, starting within 100 nt from the 5′-end of an opposing CDS (as5′-UTR) and (iii) transcripts antisense to a 3′-UTR, starting within 60 nt from the 3′-end of an opposing CDS (as3′-UTR). This analysis defined 464 sRNA regions as asRNAs, 63 as as5′-UTRs, and 16 as as3′-UTRs. Eight as5′-UTRs were also counted as3′-UTRs and 48 mRNA leader were also counted as5′-UTRs due to special arrangements of CDS. All remaining 262 regions were designated as trans-encoded sRNAs . The last digit of the number is used to number novel features in between of old features.The class “antisense transcripts” comprises 543 regions  and found conservation in seven cases  in C. glutamicum. Beside the peptide encoded by tmRNA, only one other peptide, cgb_08775 (cg4014), was detected to be conserved in species beyond Corynebacterium. The smallest of all conserved peptides with 15 amino acids length was found to be cgb_14345 (cg4016). This peptide is already known as valine-containing leader peptide in front of the ilvBNC operon [cgb_33575 (cg4012) located upstream of trpE of the tryptophan operon, at cgb_03035 (cg4015) in front of the leuA gene (cg0303), encoding isopropylmalate synthase, the first step in leucine biosynthesis, and at aroF (cg1129). Supporting their functional assignment is the occurrence of three consecutive tryptophan residues in the putative leader peptide upstream of the tryptophan operon, four consecutive leucine codons in the presumed leuA leader peptide and the amino acids phenylalanine-tyrosine-phenylalanine in the case of the aroF leader peptide.Some of the sRNAs might actually represent mRNAs and encode small proteins. We applied the “ORFfinder” online tool to extract ORFs from sequences in multiple FASTA format and subsequently searched for ribosomal binding sites (RBSs) upstream of the extracted ORF sequences with RBSfinder  using a C operon . FurthersecY (cg0647) encoding a preprotein translocase subunit of the Sec secretion system and tatC (cg1684) encoding a twin-arginine (Tat) secretion translocase subunit. The tatC antisense RNA (cgb_16835) is apparently transcribed from a SigA-dependent promoter, whereat the secY asRNA (cgb_06475) seems to be transcribed from a SigH-controlled promoter.In our study, more than half of the sRNA regions 543 of 807) fall into the class “antisense transcripts”  encode a signal peptide for secretion or at least one transmembrane helix.In this context, we tested also the proportions of asRNAs at genes encoding transmembrane helices or signal peptides for secretion. By bioinformatic search 165 of 464 asRNAs (~35%) were detected at the corresponding genes. Statistically, this proportion is not significantly different from a normal distribution since 974 genes  and cgtS6 (cg3060) that sense a specific environmental stimulus at the membrane and the corresponding response regulators genes cgtR4 (cg2888) and cgtR6 (cg3061) that mediate the transcriptional regulation by binding to operators [cgtSR4 genes are involved in phosphate starvation [cgtR4 seems to be essential [cysR (cg0156) and sufR (cg1756) which are involved in assimilatory sulphate reduction [acnR gene (cg1738) [cg2686; cg1308). The following transcriptional regulators were also identified to have antisense transcription, SugR (cg2115) as regulator of the PEP:sugar phosphotransferase system genes [cg2112) the regulator of deoxyribonucleotide reduction [cg2627) the transcriptional activator of the ketoadipate metabolism genes [cg3202) a transcriptional regulator involved in nitrogen metabolism [cg1392; cg2040).The occurrence of asRNAs includes also two of the 13 two-component regulatory systems in perators . The cgtarvation  and cgtRssential . Furthereduction and thiolcgtS4 cg03 and cgt(cg1738)  and otheem genes , 51, Ndreduction , PcaO  overlap with 45 trans-encoded sRNA genes detected with RNA-Seq (Table trans-encoded sRNAs matching to loci predicted by RNAz (~ 30%) , M1 RNA (cgb_24535), and 4.5S RNA (cgb_02933) were predicted at positions very similar to those of the Rfam database entries that were calculated by covariance models [cgb_03605), which is known to be present in many Actinomycetales genera [C. glutamicum the 6C RNA has two stretches of eight cytosines. However, the function of these cytosine homopolymers is not known and therefore the relevance of this difference is unclear.In bacteria sRNAs often have characteristic structures that are conserved stronger in evolution than their primary sequences. Hence, structure conservation analysis is integrated in a number of sRNA prediction tools. In our approach, we used the RNAz tool  to detecs genera . The 6C trans-encoded sRNAs we retrieved a mean length of 90 nt   as an example of a sRNA with strong secondary structure conservation as shown above (Table cgb_20715 (downstream of cg2071) as an example lacking secondary structure conservation .For the total set of detected cgb_00105 appeared to be absent under heat stress conditions . The length of 5′-UTRs of coding sequences is variable, zero for leaderless transcripts and especially long for genes regulated by  studies , 60. Intoelicolor.Cis-antisense sRNAs are abundant in C. glutamicum and located in ~15% of all annotated protein-coding genes. High-resolution tiling arrays and RNA sequencing led to the discovery of extensive antisense transcription in several other bacteria icobacter. The firof cg1935, therebyucleases –66. Furtucleases . Other sucleases ) or can ucleases .trans-encoded sRNAs, can be done by sequence and structural analysis. The comparison of candidates predicted by the RNAz tool [Actinomycetales (6C RNA) or within the closely related species C. efficiens and C. diphtheriae. However, more than the half of sRNAs seems to be specific for C. glutamicum. At this point we want to note that our study did not detect two widely conserved elements known to be involved in bacterial sRNA: 6S RNA [Corynebacterium species (CRISPRdB) [Further classification, especially of NAz tool  and by tNAz tool  with seq: 6S RNA  and shor: 6S RNA ). IntereRISPRdB) , 73.in vivo expression of sRNAs with bioinformatic prediction results often revealed only little correspondence [trans-encoded sRNAs detected by our sequencing approach are not followed by a Rho-independent terminator and especially the number of asRNAs with Rho-independent terminators is marginal. A similar observation was obtained within in a search for sRNAs in Vibrio splendidus[cgb_00105) at a site without an obvious terminator structure was proven by Northern blot analysis.Many bioinformatic prediction tools were developed for sRNA research during the last decade. The comparison of the actual pondence , 74, 75.pondence  which wapondence , 38, 76 plendidus. The corcgb_00105: under heat shock we detected no transcription of this sRNA whereas cold shock and chemically induced stresses had no influence on the amount of transcript. Heat shock condition was also observed to trigger a different transcription start site for ArnA cis-antisense RNA [cgb_20715, two transcripts of different length are detected in each case. Here, rather sRNA maturation or degradation by endo- or exoribonucleases is likely. For 6C RNA no change by one of the chosen stress treatments was observed. At this point, there is no hint for the function of 6C RNA in C. glutamicum. Currently, the 6C RNA was reported to be involved in the GlxR regulatory network in C. glutamicum[Streptomyces coelicolor, 6C RNA showed an increased transcription during sporulation [RNA-Seq analyses deliver an unmatched single nucleotide resolution. However, confirmatory methods are required, such as Northern blotting and are used in the present study, to look at stress-specific transcription. An example is presented with ense RNA . In the lutamicum. GlxR isrulation .C. glutamicum corresponds well to known sequences of a wide phylogenetic spectrum [In bacteria there is an additional group of transcripts, comprising RNAs that act as both, regulatory RNAs and mRNAs. RNA with dual properties is exemplified by tmRNA, which combines the features of a tRNA and an mRNA. This housekeeping RNA recycles stalled ribosomes by adding a proteolysis-inducing tag to unfinished polypeptides . Our resspectrum .cis-regulatory motives in the Rfam database. Transcriptional attenuation was first described for the tryptophan (trp) operon in E. coli[Trp (RF00513). Accordingly, it has been observed for the ilvBNC operon in C. glutamicum[trpE of the tryptophan operon, in front of the leuA gene (cg0303), encoding isopropylmalate synthase, the first step in leucine biosynthesis, and at aroF (cg1129), encoding one of the two DAHP Synthases [C. glutamicum, responsible for the first step of shikimate pathway in the biosynthesis of aromatic amino acids. Since C. glutamicum is a well-known industrial producer of amino acids, these findings might become relevant for future engineering of amino acid producer strains.Short peptides encoded within 5′-UTRs of mRNA sequences are known as characteristic feature in a mechanism called transcriptional attenuation. In our study, we detected attenuator transcripts at different genes and operons involved in amino acid synthesis, each encoding a suitable leader peptide. Such RNAs are also included as n E. coli where telutamicum and difflutamicum. With ouynthases  in C. glC. glutamicum ATCC 13032. In C. glutamicum, more than half of all small RNAs genes was classified as antisense transcripts. Cis-antisense sRNA genes were detected at CDS with various functions. However, CDS specifying proteins from the functional classes 'cell envelope biogenesis’ and 'secretion processes’ appear to be overrepresented. Trans-encoded sRNA genes were found distributed over the entire genome and showed secondary structure conservation among corynebacteria in about 30%. The 6C RNA, already known from other Actinomycetales genera showed strong transcription at unstressed exponential growth and all tested stress conditions. The 6S RNA, highly conserved in bacteria, was not found in C. glutamicum. Additionally, we detected riboswitches, transcriptional attenuators and other cis-regulatory motives, demonstrating the potential of our study for unraveling novel regulatory processes by small RNAs in C. glutamicum.Our present study represents the first comprehensive screening for small RNAs in Corynebacteriaceae, a family that comprises important bacteria of industrial and medical relevance. High-throughput sequencing techniques are often applied for the search and investigation of sRNAs in bacterial genomes. Similar to sRNA studies in other bacteria, we detected hundreds of sRNA genes in C. glutamicum ATCC 13032 was grown in CGXII minimal medium at 30°C until exponential phase (OD600 10). Cells were treated in five different stress experiments by heat (50°C), cold (4°C), diamide , NaCl (1.5 M), and H2O2 (0.33 M ) for 15 minutes. After harvesting 2 mL bacterial culture, pellets were resuspended in 1 mL TRIzol® reagent  followed by ethanol precipitation. Afterwards, crude RNA samples were treated with DNase I . After purification using phenol/chloroform/isoamyl alcohol (ratio 25:24:1), RNA was precipitated with 0.3 M sodium acetate. Purified total RNA pellets were dissolved in 50 μL RNase-free ddH2O. Afterwards, the purified total RNA was qualified by Agilent RNA Nano 6000 Kit on Agilent 2100 Bioanalyzer .IIx . Each sample was sequenced on one separate lane and obtained 35 bases long single reads from the 5′-ends. Data analysis and base calling were accomplished using the Illumina instrument software.The purified total-RNA samples were pooled in equal parts (each condition 16 μg) and precipitated for sRNAs < 250 nt with . Afterwards, rRNAs were depleted by Ribo-Zero for Gram-Positive Bacteria . The sRNA-pool was then divided into two samples (each 5 μg). One sample (library 1) was enriched for primary transcripts by enzymatic treatment with Terminator 5′-Phosphate-Dependent Exonuclease and RNA-5′-Polyphosphatase  while the second sample was prepared as whole small transcript library (library 2). The two sequencing libraries were then prepared according to the manufacturer’s instructions of TruSeq, Small RNA Kit . Single-stranded cDNAs were created with SuperScriptII Reverse Transciptase . Following this, double-stranded cDNAs were generated by PCR using adapter specific primers. Afterwards, the purified libraries were quantified and qualified by Agilent High Sensitivity DNA Kit on Agilent 2100 Bioanalyzer . The sequencing of the libraries was carried out at the Center for Biotechnology, Bielefeld University, utilizing the Cluster Station and the Genome Analyzer C. glutamicum ATCC 13032 genome sequence [Read Explorer  was used. The Read Explorer software enables the import and visualization of a reference sequence and appropriate mapping data as so-called tracks. It is possible to scroll through the reference genome, to zoom in at each position and to look at the mapped reads at base pair level.Reads were mapped to the i, the number of read starts xi on that strand at this position exceeded a background threshold T and the ratio xi/xi-1 at this position had to exceed a threshold R. After manual inspection of TSS, T was set to 19 and R to 5 as these parameters were found to result in a good signal to noise ratio.To automatically and systematically detect TSS, the mapping data of the library 1 enriched for primary transcripts was analyzed. First, for each strand and position of the genome, all mappings starting at the given position were counted. As possible TSS all positions on a strand were taken into account that satisfied the following criteria: for a position C. glutamicum, SigA promoters are also indicated if the well conserved extended -10 region is calculated greater than or equal the maximum of determined background significance scores, regardless of the score of the poorly conserved -35 region.Relevant stacks for promoter search were obtained from the primary transcript enriched sequencing library. For promoter search, the tool Improbizer , 32) wasIf more than one stack with indicated promoter occurred within 3 following nucleotides (278 instances), the genomic position with the strongest relative read count was selected as TSS.C. glutamicum ATCC 13032 was performed with the tool TransTermHP [The search for Rho-independent terminators in nsTermHP  at standilvB transcriptional attenuator [http://www.gen-info.osaka-u.ac.jp). In every case of a predicted ORF, except for leaderless transcripts, we looked for ribosome-binding sites using RBSfinder [ORF search was set at a minimum protein length of 48 nt, which is known from the leader peptide of tenuator , up to 2BSfinder  applyingC. glutamicum ATCC 13032, C. efficiens YS-314 and C. diphteriae NCTC 13129 was created by MAUVE [At first, a whole genome alignment of by MAUVE . The seaby MAUVE  in five C. glutamicum ATCC 13032 genome were taken into account at bits scores > 90. WebLogos were created as frequency plots with the online-tool at http://weblogo.berkeley.edu. All data tables were processed with Microsoft Excel 2010, box plot diagrams were created with Origin 8.5Pro.RNA secondary structure analysis was performed with RNAShapes . Rfam daNorthern Blot analysis was performed with the total RNA isolated with TRIzol® reagent  obtained from different growth conditions as described above. For detection of transcripts, digoxigenin (DIG)-labeled RNA probes were produced as described in . The RNAList of mRNA leader transcripts. (XLS 64 KB)Additional file 1: List of Rfam predictedcis-regulative motifs inC. glutamicum. (XLS 25 KB)Additional file 2: List of cis-antisense RNAs (asRNAs and as3′-UTRs/as5′-UTRs). (XLS 125 KB)Additional file 3: List oftrans-encoded sRNAs. (XLS 71 KB)Additional file 4: List of potential small mRNAs. (XLS 34 KB)Additional file 5: Comparison of length distribution for asRNAs,trans-encoded sRNAs and mRNA leader. The box plots display the mean (little square) and the medium (cross line) values for sRNA length. Bottom and the top of the boxes represent the 25th and 75th percentile, respectively, and the whiskers represent outliers. (PNG 74 KB)Additional file 6: Primers for Northern Blotting. (XLS 24 KB)Additional file 7: