Responses of coral assemblages varied after the bleaching event, including shifts of cover from massive corals (Orbicella spp.) to opportunistic (Porites astreoides) and branching corals  and/or turf algae; partial recovery of reef substrate cover by O. annularis complex; and no measurable changes in coral assemblages before and after the event.This work analyzes the mortality, recovery, and shifts in the composition of scleractinian corals from Puerto Rico one decade after the 2005 regional coral bleaching event. Temporal and spatial patterns of coral community structure were examined using a stratified, non-random sampling approach based on five permanent transects per reef at 16 reef stations. A negative correlation between percent coral cover loss and light attenuation coefficient (Kd Coral reef communities have been changing dramatically over the past four decades . NaturalCoral bleaching is the whitening of corals due to the loss of their symbiotic algae. Reductions in salinity , decreasTurbidity can be caused by either inorganic (sedimentation) or organic sources (phytoplankton biomass), each having different environmental implications. Inorganic turbidity is caused by an influx of sediments from coastal development, river runoff, and/or the resuspension of materials in the benthos. The influx of phytoplankton does occur naturally, however, anthropogenic activities influence nutrient loading in coastal environments due to activities including, but not limited to, agriculture and/or sewage leaching. In the west coast of Puerto Rico, a positive correlation has been observed between turbidity and chlorophyll-a , with weThe most severe coral bleaching event reported for Puerto Rico and the US Virgin Islands occurred in late August 2005 , and coiOrbicella annularis complex was among the most impacted of all coral that bleached in 2005 , one decade after the 2005 regional coral bleaching event and analyze the relationships between organic turbidity and coral loss and recovery.A total of 16 reef stations were included in this time series analysis: Isla Desecheo (hereafter Desecheo), Isla de Mona (hereafter Mona), Rincón, Mayagüez, Cabo Rojo, Guánica, Ponce, and Isla de Vieques (hereafter Vieques). Depth-stratified samplings were performed at Desecheo , Mona , Mayagüez , Rincón , Cabo Rojo , and Vieques . Reef stations in Desecheo, Mona, Mayagüez, and Ponce represent offshore/shelf-edge sites, whereas reefs at Rincón, Guánica, Cabo Rojo and Vieques are representative of coastal sites . Reef stAt each reef, a set of five permanent 10 m long replicate transects were non-randomly established to represent reef areas of optimal coral growth within similar depths (+/−2 m) and reef physiographic zones. Transects were permanently marked with metal rods drilled to the reef substrate at both ends and nails were placed along the length of each transect. The installation of transects varied between sites from 1999 to 2004, and some of the sites were not monitored annually. Sessile-benthic reef communities were characterized by the continuous intercept chain-link method (as modified from 490)   was used490) , and ModThe study area was 4°× 4°, from 16°to 20°N latitude and 68°to 64°W longitude. Level 2 satellite products  were selected over Level 3 products  since higher spatial resolutions are required for the study of water quality in coastal areas. The spatial resolution of SeaWiFS Level 3 data is 9 km and 4 km while for MODIS Aqua is 9 km. SeaWiFS and MODIS Level 2 data. Selection of coral reef sites’ pixel locations was made first by collecting the latitudes and longitudes (lat/lon) as well as depths of each respective site. Representative pixels were then selected to avoid the influence of land pixels and pixels influenced by ocean bottom reflectance signal. The lat/lon of the pixels used were selected along a line tangent to the shelf edge and starting at each coral reef site. The lat/lon used was based on where the ocean bottom was not visible while being as close as possible to each coral reef site.490 data files into the same Conic Lambert’s map projection output. Only data files with minimal or no pixel distortions were selected for the study. Daily Kd490 data files picked were re-visualized, using MATLAB version 7.12.0 . This included the transformation of daily data into monthly and annual averages. The reef sites’ Kd490 values were extracted from their respective lat/lon pixel values using the average annual data files.SeaDAS v6.4 software was used to perform data visualization  and to aOrbicella annularis complex (hereafter Orbicella annularis) between depths and years at each reef. Year and depth were assigned as the within subject factor for the analyses. There were some locations where depth was not a factor (Guánica and Ponce). In those instances, a One-way Repeated Measures ANOVA analysis was used to test variations of percent total coral cover and Orbicella spp. cover between years. The assumption of normality was analyzed before running any tests. Coral cover was arcsine transformed before analyses. Bonferroni post-hoc procedures were performed to examine yearly variations of percent coral cover at each site. Repeated measures ANOVA tests were run in SPSS software (version 17.0).Two-way Repeated Measures Analysis of Variance (ANOVA) procedures were run to compare percent reef substrate cover by live scleractinian corals and specifically of percent cover by A posteriori pairwise comparison tests were performed in PERMANOVA to further assess variations within the factors. SIMPER tests were run to identify the contribution of coral species to the overall differences between years. Multidimensional scaling (MDS) plots were produced to examine differences in coral assemblages between years. PERMANOVA, SIMPER, and MDS procedures were performed using PRIMER-e and PERMANOVA add-on software  tests  were persoftware .490 light attenuation coefficients from the immediate vicinity of the reef stations. Coral cover was the response variable, while Kd490 was treated as the fixed factor. To select the optimal structure of the random component, we ran two models, the first with no random term, and the second using sites as the random component. The optimal model was chosen based on the lowest Akaike’s Information Criteria (AIC) value by using the MuMln package. A smoothness selection was fitted by maximum likelihood through the Laplace approximation. All analyses were performed in R v.3.1.1 using package mgcv v1.8-0. Given the nonlinearity nature of the coral data, we used generalized additive models (GAM) to analyze the relationship between changes in coral cover at the upper reef depth surveyed (≤15 m depth) and satellite-derived KdOceanic and shelf-edge reefs had the highest percentages of live scleractinian coral cover  during the baseline surveys performed before the onset of a regional coral bleaching event in August 2005. Mean live coral cover ranged from 32% to 52% at reef stations from Desecheo (20 m and 30 m), Mona (20 m), Ponce, and Mayagüez (10 m and 20 m). Coral cover at coastal reefs in Guánica, Cabo Rojo, and Rincón ranged from 10 to 20%, while at the coastal reefs of Vieques ranged from 26 to 36% . Live coOrbicella annularis (complex) exhibited the highest percent cover by live corals at all reef sites except at Rincón 10 m, where Montastraea cavernosa was the dominant species. The relative contribution of O. annularis to the total coral cover at reefs ranged between 31.4% (Mayagüez 10 m) and 79.8 % (Vieques 10 m) and largely contributed to the similarity of coral assemblages within reef stations.490)  , Fig. 3Asurveyed , Table 3490 ranging from 0.036 m−1 at Mayagüez to 0.056 m−1 at Guánica.Minor declines of live coral cover were observed at Mayagüez 10 m (21.0%), but differences were not statistically significant . ChangesDepth related variations of live coral losses were measured from reefs surveyed at Desecheo, Mayagüez and Vieques. At Desecheo, live coral declined 60% and 62% at 15 m and 20 m, respectively, while the decline at 30 m was 43% . At MayaO. annularis. Statistically significant reductions of substrate cover by O. annularis were observed at all depths from oceanic island reefs; Desecheo and Mona, the Ponce shelf-edge reef, Mayagüez 10 m, and the coastal reefs of Vieques  shelf-edge reef.The 2005 coral bleaching event had a severe impact on the mortality of  Vieques . The rel490 was observed . Increases of coral cover were slower at shelf-edge and offshore reefs relative to coastal sites. A positive correlation between coral cover recovery (2008–2013) and Kdobserved , Fig. 3BThe magnitude of coral recovery also varied with depth. At Desecheo, coral cover increase was greater at 15 m (71.7%), but moderate increases were also measured at 20 m (37.0%) and at 30 m (52.7%). By 2017, coral cover increased by 60.8% at Ponce. Coral cover has not increased at reef sites in Vieques and Cabo Rojo 10 m. The lack of recent surveys from Cabo Rojo 5 m and Mona reef sites limit our assessment of potential recovery at these reef sites.O. annularis. Variations were dependent on reef geographic location and depth. At Desecheo 15 m, reef substrate cover by Porites astreoides and Agaricia spp. was not negatively impacted by the bleaching event and consistently increased through time (O. annularis during recent years (2013 to present), P. astreoides now stands as the main coral species in terms of substrate cover at Desecheo 15 m . Since then, a consistent recovery trend of O. annularis has been measured, reaching present levels of cover similar to those of M. auretenra and higher than those of P. porites, which has declined in mean cover at Mayagüez 10 m since 2011  did not exhibit any significant differences in coral assemblages associated with the 2005 bleaching event. Moderate declines of substrate cover by Puerto Rican coral reefs have changed significantly during the last decade, both regarding the total live coral cover and the relative composition of scleractinian coral assemblages. Localized impacts upon coral reefs associated with anthropogenic activities have been reported , but theElevated sea surface temperature (SST) and time of exposure to the SST anomaly were proposed as the main drivers of the 2005 coral bleaching event . FluctuaLight penetration decreases exponentially with depth, but is also affected by water turbidity from both inorganic and organic sources. Inorganic suspended sediments can increase turbidity in specific areas affected by riverine inputs. In this study, the main driver of water turbidity variations along the insular shelf and nearby offshore waters around Puerto Rico was chlorophyll-a concentrations, an indicator of phytoplankton biomass . Due to Contrary to the generalized paradigm that coastal reefs subjected to higher levels of environmental stressors may be more vulnerable to degradation, these were more resilient to the 2005 regional coral bleaching event than their offshore (shelf-edge) and oceanic counterparts. Oceanic and shelf-edge reefs and reefs located upstream from riverine inputs in this study were the most severely affected by the bleaching phenomena and showed slower recovery of live coral cover compared to coastal and/or downstream reefs. For example, Tourmaline Reef is located at the shelf edge off Mayagüez Bay and thereby influenced by estuarine conditions and higher water turbidity than the oceanic reefs of Desecheo and Mona on the west coast of Puerto Rico. The marked contrast of live coral loss at the 30 m depths from reef stations at Desecheo 30 m (43%) versus Mayagüez 30 m (0%), suggests that distance from shore and the inherent increase of water turbidity towards the coast may have also played a role in coral protection. Likewise, the negligible coral loss observed from the coastal reefs of the Cabo Rojo and Rincón shelf, influenced as well by estuarine conditions, is in sharp contrast to the significant mortalities observed at similar depths from reefs in Vieques, located far from the influence of major rivers. These patterns were supported by the negative relationship measured between coral cover loss and light attenuation (“sunblock effect”).Orbicella annularis (complex), the main reef building coral of Puerto Rican reefs and main contributor to overall coral cover, was severely affected by the coral-bleaching event. Drastic declines in Orbicella spp. after the 2005 bleaching event were also reported at Navassa Island  replaced by a proportional increase in cover by turf algae; (2) a shift of coral assemblages from massive to opportunistic and branching species , (3) a partial recovery by O. annularis; and (4) no measurable change in coral assemblages before and after the bleaching.The variation of resilience by different coral species to the bleaching event led to changes in coral assemblages at reef stations. Essentially, four major patterns of coral community structure have been noted since 2005 by this monitoring program; (1) a marked decline of cover by massive corals . Also, the higher plankton biomass nearshore may be influencing coral recovery. By 2015, there was an increase in coral cover at the majority of the reef sites surveyed driven by increases of reef substrate cover by O. annularis and/or overgrowth of dead massive coral sections by branching opportunistic species.The bleaching event in 2005 was one of the worst disturbance on record affecting coral reef communities in the Caribbean, with particularly pronounced impacts on 10.7717/peerj.3611/supp-1Supplemental Information 1Scleractinian coral cover at the different sites, depths and years.Click here for additional data file.10.7717/peerj.3611/supp-2Supplemental Information 2Orbicella annularis complex coral cover at the different sites, depths and years.Click here for additional data file.10.7717/peerj.3611/supp-3Supplemental Information 3The cover of the different coral species between the reef sites in Puerto Rico. Coral species listed contributed to at least 10% of the total cover.Click here for additional data file.


Functional 3D organ models such as precision-cut lung slices (PCLS) have recently captured the attention of biomedical research. To enable wider implementation in research and development, these new biologically relevant organ models are being constantly refined. A very important issue is to improve the preparation of high-quality RNA (ribonucleic acid) from PCLS for drug discovery and development of new therapies. Gene expression analysis at different levels is used as an important experimental readout. Genome-wide analysis using microarrays is mostly applied for biomarker selection in disease models or in comprehensive toxicological studies. Specific biomarker testing by reverse transcriptase quantitative polymerase chain reaction (RTqPCR) is often used in efficacy studies. Both applications require high-quality RNA as starting material for the generation of reliable data. Additionally, a small number of slices should be sufficient for satisfactory RNA isolation to allow as many experimental conditions as possible to be covered with a given tissue sample. Unfortunately, the vast amount of agarose in PCLS impedes RNA extraction according to the standard procedures.We established an optimized protocol for RNA isolation from PCLS from humans, rats, mice, marmosets, and rhesus macaques based on the separation of lysis and precipitation steps and a magnetic-bead cleanup procedure. The resulting RNA is of high purity and possesses a high degree of integrity. There are no contaminations affecting RTqPCR efficiency or any enzymatic step in sample preparation for microarray analysis.In summary, we isolated RNA from PCLS from different species that is well suited for RTqPCR and for microarray analysis as downstream applications. Functional 3D organ models are considered to have high impact and value in translational science. In lung research, tissue models such as precision-cut lung slices (PCLS), parenchymal strips, and isolated vessels and bronchi are used intensively and have been shown to be of importance, since their cellular composition includes varying cell types with many functions. PCLS, for example, contain epithelial cells, fibroblasts, smooth muscle cells, nerve fibers, and immune cells such as antigen-presenting cells, macrophages, and T-cells. Cells interact with each other, thereby reflecting the highly specialized function of the lung.Precision-cut lung slices are used primarily for basic research and non-clinical efficacy and toxicity testing of  compounds. The general perception has changed for many reasons. First of all, there have been significant improvements in the preparation and culture of ex vivo tissues, so that thin and precise sections are obtained, which can be maintained for days, weeks, and even months –3. Today260/A280 ratio close to 2.0. An A260/A280 ratio greater than 1.8 is usually considered an acceptable indicator of good RNA quality, whereas lower levels indicate protein contamination  and Balb/c mice [nulliparous and non-pregnant] were housed under conventional and certified laboratory conditions in a regular 12-h dark/light cycle at an ambient temperature of 22 ± 2 °C and a relative air humidity of 55 ± 15%. Diet and drinking water were available ad libitum. Animals were acclimated for at least 1 week and sacrificed by an i.p. overdose (~100 mg/kg body weight) of pentobarbital sodium  and rhesus macaques (Macaca mulatta) were obtained from German Primate Center . Marmosets were anesthetized using diazepam  and alfaxalone  Limited, Worcestershire, United Kindom) followed by a lethal dose of pentobarbital sodium  under deep general anesthesia. Rhesus macaques (Macaca mulatta) were deeply anesthetized using a combination of ketamine  and xylazine . The abdominal cavity was opened along the linea alba, and the abdominal aorta was cannulated for blood sampling. The aortic cannula was subsequently used to administer a lethal dose of pentobarbital sodium .Lungs of common marmosets Human lung lobes were obtained from male and female patients who underwent lung resection for cancer. Tumor free tissue was processed immediately on the day of resection as described below. The average age of patients was 60 ± 10 years, and 80% of them were smokers.l-glutamine and 15 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) without phenol red and fetal bovine serum supplied from Gibco™  supplemented with 100 units/mL penicillin and streptomycin  under standard cell culture conditions . Two PCLS were cultured together in 500 µL DMEM as described previously [Mouse lungs were filled in situ. Rat, human, and non-human primate lungs were filled ex situ and PCLS were prepared as previously described , 21, 22.® II , and 150 U/mL collagenase 3 ) and incubated for 30 min at 37 °C on an orbital shaker (650 rpm). Afterwards, slices were passed vigorously through a cut 1-mL tip and placed in a 100-µm CellTric® . Cells were rinsed with 600 µL ice-cold DMEM per slice, centrifuged for 3 min at 400×g at 4 °C, then immediately transferred into liquid nitrogen, and subsequently stored at −80 °C.After preparation each PCLS was placed in 200 µL digestion solution , 2.4 U/mL Dispase® CCL-185™) was obtained from ATCC . Cells were routinely cultured in 75-cm2 flasks in Dulbecco’s modified Eagle’s medium  supplemented with 10% fetal calf serum and 0.01% gentamicin at 37 °C in a humidified atmosphere containing 5% CO2. Cell numbers used for RNA isolation are indicated in Figs. The human lung epithelial cell line A549 , MagJET , and MagMAX™ . Disruption and homogenization of PCLS in the respective solutions was performed using an Ultra-Turrax® .In a first step, four rat PCLS per tube were used for RNA isolation. Several commercially available kits were used for this according to the suppliers’ instructions: RNeasy Mini Kit , QIAzol®. The homogenate was transferred to 1 volume of phenol/chloroform, carefully shaken for 30 s, and centrifuged for 5 min at 12,000×g. Subsequently, 1 volume of chloroform/isoamyl alcohol was added, again carefully shaken for 30 s, and centrifuged for 5 min at 12,000×g. The aqueous phase was transferred and RNA was cleaned up with MagMAX™ magnetic beads including the spin procedure step according to the supplier’s instructions. Total RNA was dissolved in RNase-free water and stored at −80 °C. For some samples an additional clean up step was performed using the RNeasy Mini Kit clean up protocol .In a second step, optimized RNA extraction was achieved as follows: two PCLS were pooled, followed by disruption and homogenization of PCLS in 400 µL RLT lysis buffer  using an Ultra-TurraxRNA from A459 cells was isolated with the commercially available RNeasy Mini Kit according to the supplier’s instructions.260) and purity (A260/A280 ratio) were measured by spectrophotometry . RNA integrity (RIN) was evaluated using an Agilent 2100 Bioanalyzer® .RNA concentration (A−(1/slope) − 1, corresponding to 100%) [Reverse transcription (RT) of RNA was performed using TATAA GrandScript cDNA Supermix . SYBR Green  was used as a fluorescent dye to determine the amplified PCR products after each cycle. The following primer pairs were used: B2M (NM_004048.2), fwd: GAGGCTATCCAGCGTACTCCA, rev: CGGCAGGCATACTCATCTTTT, 248 bp, ; MUC5AC, #qHsaCID0017663, 144 bp . qPCR conditions were as follows: 30 s 95 °C; 5 s 95 °C/30 s 60 °C, for 40 cycles; and 15 s 95 °C/1 min 60 °C/30 s 95 °C for the melting curve. At the end of each extension phase, fluorescence was recorded and at the end of a run quantification cycles (Cq) were determined for each sample. Serial dilutions of RT reactions  were prepared in triplicate and samples were analyzed by qPCR to measure the Cq values. A plot of Cq values versus the logarithm of target concentrations resulted in standard curves, which were used for efficiency calculations (10to 100%) –25. To c® Human Genome U133 Plus 2.0 Arrays, using 250 ng RNA as input. All steps were performed according to the manufacturer’s instructions for the GeneChip® platform . The steps included first-strand cDNA synthesis, second-strand cDNA synthesis, synthesis of labeled cRNA by in vitro transcription, purification of labeled cRNA, fragmentation, array hybridization, washing, staining, and final scanning of the arrays using the GeneChip® Scanner 3000 7G.Microarray analysis was performed with Affymetrix GeneChipwww.genexplain-platform.com) to perform a principal component analysis (PCA). The geneXplain platform was further used to detect differentially expressed genes for all samples with the EBarrays workflow.To examine the quality of the microarrays before and after normalization, we used the bioconductor package arrayQualityMetrics 3.24.0  under R 260/A280 ratio of around 2.0 and a RIN value above 7.0 . Unfortunately, this standard isolation procedure did not yield intact RNA Table . We assu.0 Table . Isolati.0 Table . A diffe260/A280 ratio (around 1.9) and by RIN values (around 8.0). Additionally, an incubation period of 3 days, which is commonly used in several experimental approaches with PCLS, had no negative impact on RNA quality  into a separate lysis procedure followed by conventional phenol–chloroform–isoamyl alcohol extraction see “. RNA waso around .9 and by260/A280 ratio and the RIN values met the requested criteria, with A260/A280 ratios >1.8 and RIN values >7 . For all samples tested, the A260/A280 = 2.09, A260/A230 = 2.12, RIN = 9.3) for the reference gene B2M. Several cDNA dilutions were used, starting with a cDNA equivalent to 50 ng RNA. The resulting calibration curve indicates an amplification efficiency of 95.1% for this specific A549 RNA preparation  and indicates an amplification efficiency of 100.6%, the melting curve shows a single peak, and the gel image shows a single band for each dilution .Finally, we verified wether RNA isolated from PCLS samples can be used for microarray analysis. For this evaluation, we used RNA from PCLS from three human donors 3 days after treatment with different chemicals . Here again, we used A549 RNA with an A260/A230 ratio of 1.9 as a quality standard for comparison  were identified after background correction and normalization in the section about distances between array comparisons, drawn with a false color heatmap (data not shown). Figure The large amount of agarose in PCLS impedes RNA extraction, making it difficult to obtain RNA of high quality from lung tissue ex vivo. We used several standard procedures and finally established a protocol for RNA isolation from PCLS from humans, rats, mice, marmosets, and rhesus macaques. The method is based on the separation of lysis and precipitation steps, and an additional magnetic-bead clean-up procedure. The resulting RNA is of high purity, possesses a high degree of integrity, and is well suited for RTqPCR and microarray analyses as downstream applications.


Therefore, novel methods need to be developed to allow the characterization of G4s in long functional RNAs and in a cellular context. This need has in part been met by our recent development of a method based on a comparison of RNA and 7-deaza-RNA that provides a test for identifying RNA G4s in such conditions.The roles of deoxyribonucleic acid (DNA) G-quadruplex structures in gene expression and telomere maintenance have been well characterized. Recent results suggest that such structures could also play pivotal roles in ribonucleic acid (RNA) biology, such as splicing or translation regulation. However, it has been difficult to show that RNA G-quadruplexes (G4s) exist in specific long RNA sequences, such as precursor messenger RNA, in a functional or cellular context. Most current methods for identifying G4s involve the use of short, purified RNA sequences  It may s of RNA .The functional significance of G4s that have been identified by bioinformatics studies and characterized by biophysical approaches is generally further investigated by site-directed mutagenesis of the guanines involved in G4 formation together with a functional assay. In the case of RNA, functional assays include translation using reporter genes such as luciferase ,52 or alMore than 800 small molecules have been described to bind specifically to G4 DNA and/or RNAs . An alteAlthough there have been many suggestions that RNA G4s exist and have roles in RNA processing or other cellular processes, a clear demonstration of either claim is still lacking. All the approaches used to study RNA G4s have been adapted from extensive studies of DNA G4s. However, in contrast with DNA, RNA is single-stranded and other secondary structures are also highly likely to form that can compete with G4 formation. It is therefore essential to investigate RNA G4s in the context of their physiological sequence, rather than using short RNA regions. It will be very important to find ways of generating high-throughput information derived from full-length RNA in cellular conditions. The identification of putative G4s relies mainly on bioinformatics tools that predict the probability of a query sequence to form a G4 based on pre-defined G4 consensus sequences. While such tools estimated the presence of >300 000 putative G4 in cellular RNAs, recent studies have demonstrated that, in contrast with DNA, RNA G4s can adopt more complex structures embedded within a larger and complex structural context and that the DNA consensus G4 sequence is probably not adequate to appreciate the full structural diversity of RNA G4s . It is t+ or Li+ ions, preventing the investigation of G4s in a cellular context. The best method available that can be applicable in long RNA and in physiological conditions is the use of a G4-stabilizing ligand to observe functional changes in functional assays. The major advantage is that such experiments can be done in cells. However, by using a G4 ligand, there arises another area of ambiguity; does the G4 form naturally or has the G4 ligand caused the G4 to form? This is also the case for the G4 antibody [trans-acting enhancer of RNA splicing exists as a diverse population of molecules under functional conditions, bound as a linear sequence by various sets of mutually incompatible proteins and also a G4 [The major challenge in the RNA G4 field remains the investigation of such structures in cellular environment. Using a specific G4 antibody, RNA G4s have been observed in cells, proving their existence in a cellular context . Howeverantibody , as addilso a G4 . The posWe have recently developed a novel strategy to identify G4s in long RNAs in a functional context . This st


Furthermore, we determined the half-lives of the Srp transcripts in control and CELF1 knockdown myoblasts. Our results indicate CELF1 is a destabilizer of at least five of the six Srp transcripts and that the relative abundance of the SRP proteins is out of balance when CELF1 is depleted. CELF1 knockdown myoblasts exhibit altered secretion of a luciferase reporter protein and are impaired in their ability to migrate and close a wound, consistent with a defect in the secreted extracellular matrix. Importantly, similar defects in wound healing are observed when SRP subunit imbalance is induced by over-expression of SRP68. Our studies support the existence of an RNA regulon containing Srp mRNAs that is controlled by CELF1. One implication is that altered function of CELF1 in myotonic dystrophy may contribute to changes in the extracellular matrix of affected muscle through defects in secretion.We previously identified several mRNAs encoding components of the secretory pathway, including signal recognition particle (SRP) subunit mRNAs, among transcripts associated with the RNA-binding protein CELF1. Through immunoprecipitation of RNAs crosslinked to CELF1 in myoblasts and  The concept of RNA regulons, in which functionally related genes are co-regulated by specific RNA-binding proteins (RBPs), was first proposed by Keene and Tenenbaum in 2002 . Since tCUG-binding protein, ELAV-Like Family member 1) is an RBP which regulates gene expression at multiple steps, including splicing, polyadenylation, translation and/or mRNA decay [CELF1 . The SRPSrp transcripts in response to CELF1 knockdown. This is likely directly due to loss of CELF1 as multiple experiments support direct binding of CELF1 to Srp transcript 3’UTRs. Furthermore, SRP19 and SRP68 proteins are over-expressed following CELF1 depletion. Our data support that this is sufficient to influence cellular processes dependent on the SRP as CELF1 depletion and SRP68 over-expression can each cause defects in cell migration, which relies on secretion of the ECM.In this study, we investigated the effects of CELF1 expression on accumulation and decay of mRNAs encoding the SRP proteins, which we previously identified through RNA-immunoprecipitation . We obse2 at or below 70% confluency in Dulbecco's Modified Eagle's Media (DMEM) containing 10% fetal bovine serum (FBS), penicillin (10 units/ml) and streptomycin (10 μg/ml). To create knockdown cell lines C2C12 myoblasts were infected using either a control lentivirus (LKO1) or a lentivirus encoding an shRNA against CELF1 (sh1739 Sigma MISSION ID clone NM_198683.1-1739s1c1), pools of cells were selected with puromycin (4 μg/ml) and CELF1 knockdown was then verified by western blot [C2C12 cells (ATCC CRL1772) were grown at 37°C, 5% COern blot . CELF1 euromycin  μg/ml an30–50 μg of whole protein extract prepared by lysis of cells in RIPA buffer  were separated on 10% or 15% SDS-polyacrylamide gels and blotted onto polyvinylidene difluoride (PVDF) membrane. Suppliers, catalog numbers and concentrations for primary antibodies can be found in Table A in E.coli as previously described [32P-UTP labeled transcripts were produced by in vitro transcription using SP6 or T7 RNA polymerase. Templates for transcription were obtained by PCR from murine cDNA with primers specified in Table B in Recombinant human GST-CELF1 protein was purified from escribed . α32P-UT2, 0.05% Nonidet P-40), RIPA, and High-Stringency RIPA , for 10 minutes each. Crosslinks were reversed by incubation in crosslink reversal buffer  for 40 minutes at 70°C. The RNA was isolated using TRIzol® (ThermoFisher) extraction according to the manufacturer’s recommendations. cDNA was synthesized using random hexamers and Improm II Reverse Transcriptase (Promega) and subsequently used for PCR with primers specific for the 3’ UTR of the Srp mRNAs or for Myc mRNA as a negative control  [Control and CELF1 KD myoblasts were transfected using Lipofectamineologies)  in a 96-ologies) . Approxi® (ThermoFisher) and treated with DNAse I. Total RNA was subjected to biotinylation as follows: 20–40μg of total RNA was spiked with total RNA extracted from S. cerevisiae that had been treated with 5mM 4-thiouracil for 5 min (see [20. The RNA was fractionated as follows: 50μL of RNA was bound to μMACS Streptavidin Microbeads (Miltenyi Biotech). Following two washes with high salt wash buffer (100 mM Tris-HCl (pH7.4), 10 mM EDTA, 1M NaCl and 0.1% Tween-20) the nascent (4sU-labeled) RNA was eluted from the column with 100mM DTT. All three fractions  were precipitated in the presence of glycogen, washed and resuspended in an equal volume of ddH20. 1μL of total and nascent RNA were used to make cDNA using random hexamers and Improm II reverse transcriptase (Promega). Digital PCR (dPCR) was performed using primers specific to each SRP gene as well as yeast MFA2 as a control for biotinylation and fractionation using QX200 ddPCR EvaGreen supermix (Bio-Rad) according to the manufacturer’s recommendations. Total and nascent RNA copy numbers were normalized to the recovery for each sample  to acquire abundances. Half-lives were determined using the equation t1/2 = tL x ln(2)/ln(1-R), where tL = labeling time and R = abundance in nascent RNA fraction/abundance in total RNA fraction. Labeling and fractionation were performed in triplicate, average half-lives were determined along with the standard error of the mean (SEM). A paired t-test (two-tailed) was performed to determine statistical significance. Abundances of Srp mRNAs were determined from the abundance in the total RNA fraction and normalized to levels of Runx3 mRNA which was selected as an appropriate reference gene based on expression level and lack of variation between the Control and CELF1 KD cell lines. A paired t-test was used to determine statistical significance. Primers are described in Table B in mRNA half-lives were determined as described previously  but usinmin (see  for detamin (see . BrieflyNM_146032.3) was amplified by PCR using cDNA derived from C2C12 cells as a template. The resulting PCR product was digested with EcoRI and BamH1 and cloned into the p3xFlag-CMV10 expression vector (Sigma). The primer sequences are shown in Table B in The entire open reading frame of the murine SRP68 gene . A more relaxed analysis (top 10% most enriched) suggested that Srp9 and Srp19 mRNAs were also significantly enriched in the CELF1 immunoprecipitate. In support of this, CELF1 CLIP-seq performed by others using mouse C2C12 cells identified binding sites in the 3’UTRs of Srp9, Srp14 and Srp72 mRNAs [Srp mRNAs as targets of CELF1 [Srp mRNAs in human and mouse.We previously used RNA-immunoprecipitation followed by microarray (RIP-Chip) to identify mRNAs associated with CELF1 . Amongstof CELF1 . Thus muSrp mRNAs (in vitro transcribed RNA fragments containing putative CELF1 binding site(s) derived from each of the Srp 3’UTRs. We found that all the 3’UTR fragments bound the recombinant protein with affinities ranging from 0.53 to 33.8 nM . We conin vitro results were not entirely consistent with those obtained in our RIP-Chip experiment in that CELF1 appears to recognize the Srp14 3’UTR in our gel shift assays but this transcript was not enriched in the CELF1 immunoprecipitate. One possible explanation is that the interaction was lost during the immunoprecipitation. We therefore repeated the RNA immunoprecipitation but this time using protein extracts from cells that had been cross-linked with formaldehyde prior to cell lysis to stabilize protein/RNA interactions during the isolation and immunoprecipitation. A control immunoprecipitation using normal IgG was also performed in parallel. RT-PCR was then used to detect CELF1-associated transcripts in the immunoprecipitate. As shown in Srp transcripts were readily detected in the anti-CELF1 immunoprecipitate but not in the control IgG immunoprecipitate. As expected, a control transcript that does not contain GU-rich elements, Myc, was not associated with CELF1. These results demonstrate that CELF1 can associate with all the Srp mRNAs in living cells, but binding to Srp14 mRNA may be more easily disrupted than the other associations as it was not observed without cross-linking [The Srp mRNA decay. We were able to use data generated from our previous global analysis of mRNA decay rates in C2C12 cells [Srp mRNAs. The calculated mRNA half-lives ranged from 2.7 hr for Srp68 to almost 6 hr for Srp72  while Srp9 and Srp14 were considerably more stable (17.4 and 13.5 hr respectively)  . These hFig B in .Srp68 mRNA showed significant increases in half-life in the CELF1 KD cells  that bears a signal peptide and is efficiently secreted into the media . We tranProduction of the extracellular matrix (ECM) is exquisitely dependent on the secretory pathway. We therIf the defects in wound healing in CELF1 knockdown cells are connected with SRP subunit imbalance, then increasing abundance of SRP subunits by other mechanisms should be sufficient to induce a similar phenotype. Indeed, when we transfected C2C12 cells with plasmid encoding Flag-SRP68 protein we were able to detect a reduction in cell migration  similar in vitro and in cultured myoblasts. Depletion of CELF1 resulted in stabilization of five SRP mRNAs and increased abundance of SRP19 and SRP68 proteins. Depletion of CELF1 also induced phenotypes consistent with disruption of the secretory pathway: CELF1 KD cells exhibited an increased ability to secrete a luciferase protein and slower migration in a wound healing assay. Finally, the defects in cell migration could be recapitulated by over-expression of SRP68, independent of CELF1 knockdown.In this study, we demonstrated that the CELF1 RNA-binding protein can specifically associate with sequences in the 3’UTR of each of the SRP mRNAs Srp9, Srp14, Srp19, Srp54 and Srp72) as well as the abundance of SRP19 and SRP68 proteins. Taken together, these data support that the SRP complex relies on CELF1 to maintain an appropriate balance of the individual subunits, however the overall impact of CELF1 binding may be different for each transcript. In part, this may reflect the affinity of CELF1 for the 3’UTR of each transcript—CELF1 had the highest affinity for the Srp72 3’UTR sequences Click here for additional data file.


The RNA genome of the hepatitis C virus (HCV) establishes a network of long-distance RNA-RNA interactions that direct the progression of the infective cycle. This work shows that the dimerization of the viral genome, which is initiated at the dimer linkage sequence (DLS) within the 3′UTR, is promoted by the CRE region, while the IRES is a negative regulatory partner. Using differential 2′-acylation probing (SHAPE-dif) and molecular interference (HMX) technologies, the CRE activity was found to mainly lie in the critical 5BSL3.2 domain, while the IRES-mediated effect is dependent upon conserved residues within the essential structural elements JIIIabc, JIIIef and PK2. These findings support the idea that, along with the DLS motif, the IRES and CRE are needed to control HCV genome dimerization. They also provide evidences of a novel function for these elements as chaperone-like partners that fine-tune the architecture of distant RNA domains within the HCV genome. RNA viral genomes show hierarchical structural organization. The primary structure encodes proteins, while the secondary and tertiary structures code for essential viral regulatory mechanismsper se21cis-acting replication element (CRE)Hepatitis C virus (HCV) genome is a positive ssRNA molecule that encapsidates as a single copy in the mature virion. It presents highly conserved structural elements23457911121314151718The 3′UTR has a key role in replication2829The CRE region is a critical regulatory partner in virus propagation. It functions as a negative regulatory agent during viral translation35373914041423742The present manuscript explores the molecular requirements for HCV dimerization apart from those described for the 3′X-tail, with the aim of establishing links between the interactions IRES-CRE-3′X and their potential involvement in the viral cycle. Electrophoretic mobility shift assays showed that dimer formation efficiency is regulated by both the IRES and CRE regions in an opposing manner. High-throughput structural analyses based on SHAPE technology showed specific residues located in essential regions of the IRES and the CRE (such as subdomain IIId or the 5BSL3.2 element) to be strongly involved in dimer formation and in fine-tuning the folding of the dimer. Taken together, these results show that viral dimerization is not only dependent on the formation of a kissing complex at the 3′X-tail, but also that it is governed and influenced by distant, conserved elements of the viral genomic RNA. To our knowledge, this is the first report providing evidence of the potential of the IRES and CRE regions providing an organizing centre in HCV genomic dimerization.HCV genome dimeric complex formation is initiated at the highly conserved palindromic sequence motif DLS in the 3′X-tail29293142P ≤ 0.05) in dimerization efficiency compared to the 3′X transcript (P ≤ 0.05) repressed the enhancement of dimer formation promoted by the CRE region  in the presence of the viral chaperone, confirming previously reported data2931It seemed feasible that the effect promoted by the IRES in genome dimerization might be the result of the acquisition of non-dimerizable RNA conformations. These unproductive structures might collapse further transitions towards dimerization-efficient architectures, which could then be released by the addition of chaperone proteins. It has been described that the HCV core protein promotes and directs the dimerization of the 3′UTR The secondary and tertiary structures of viral genomes are highly dependent on temperature. In retroviruses such as HIV, it is reported that the initial kissing complex formed during genomic dimer formation can evolve towards a stable duplex at 55 °CThese results suggest the existence of a long-range contact network involved in the control of dimerization. These interactions involve the IRES, the CRE and the 3′X-tail. The results also show that distant genomic RNA elements affect, either directly or indirectly, the conformation of the dimer, and more importantly, its thermodynamic properties.Regulation of HCV dimerization and dimer stability by the IRES and CRE regions is likely mediated by a conformational rearrangement, either promoted by a yet unknown direct IRES:3′X-tail interaction, or by an indirect effect derived from the contact IRES:CRE influencing the folding of the functional RNA domains required for the acquisition of the dimerizable isoform42Structural analysis of the dimeric conformer was performed using the SHAPE-dif methodA preliminary overview of the differential reactivity pattern for the dimerization competent molecule CU showed that a significant number of residues bear reagent specificity . Interes37P < 0.05; 37The I+CU transcript, which has limited dimerization capacity, showed a different reactivity profile for certain nucleotides with respect to CU, supporting the role of the IRES in tuning the structure of the 3′ end of the HCV RNA 43. In th35SHAPE-dif analysis was performed with I+U to discern whether the IRES itself plays a role in the 3′X folding. The resulting data provided an interesting conclusion: the IRES region influences the conformation of the 3′X-tail in the absence of the CRE  and 4. TTaken together, the results provide solid evidence of the role of the CRE and IRES regions in the conformational rearrangement of the 3′X-tail. Such structural modifications may be critical for regulating the acquisition of the dimerizable isoform.A modification interference strategyHMX analysis of the I+CU indicate that the required nucleotides in the 3′X-tail were dispersed throughout the entire region . Interes155556A series of mutants derived from the I+CU molecule was constructed based on the HMX results and thus determine which positions were biologically relevant in dimer formation.In a first attempt, deletions of the different stem-loop elements located in the CRE and the 3′X-tail, with the exception of the essential 3′SLII and 3′SLIII domains, were designed (Methods). This design was due to the disperse pattern of the identified nucleotides, which hindered the construction of punctual mutants. Transcripts held the dimerization yield to that shown by the I+CU molecule , with thThe role of specific positions of the IRES region in dimer complex formation was examined by generating nucleotide substitutions or deletions based on the data acquired using the HMX and/or SHAPE-dif strategies. For that aim, those regions bearing sets of nucleotides with HMX scores > 0.65 were identified as relevant structural elements and subjected to punctual mutations according to both sequence and conformational criteria, as reported13151857To determine whether the effects of IRES mutations are dependent on the presence of the CRE or directly affect the 3′X-tail, a new series of variants derived from I+U was designed. Surprisingly, all the mutations, by themselves, enhanced dimer formation from 2 to 4 fold  with resThere is growing interest in understanding the role of the HCV genomic RNA dimerization293132333440Electrophoretic mobility shift assays showed, for first time, the contribution of the distant IRES and CRE regions to dimer formation efficiency . While t35a priori, only determined by the interaction of two DLS motifs; however, the acquisition of a dimerizable conformation, the stability of the dimer, and/or the yield of the reaction, are actually regulated by distant conserved domains in the IRES and CRE regions. The present results go beyond previous proposals that the DLS motif is the only one required for dimer formation29Several hypotheses can be proposed about the mechanism by which the IRES and the CRE accomplish dimer formation control. It seems likely that the long-distance RNA-RNA interaction between the subdomain IIId and the 5BSL3.2 element29SHAPE-dif and HMX structural studies revealed the existence of a set of potential structural intermediates for the 3′X-tail  and 2′-hydroxyl molecular interference (HMX) analyses, DNA templates coding for the transcripts I+CU, CU and I+U were obtained by attaching a 45 nt cassette to their 3′ termini43RNA synthesis was accomplished using the TranscriptAid T7 High Yield Transcription Kit (ThermoFisher Scientific), following the manufacturer’s instructions. The resulting RNA products were purified as previously describedThe chimeric peptide 2BD, with the sequence PRRGPRLGVRATRKTSERSQPRGRRQPIPKVRHQTGRRGSRPNWGPNDPRRRSRNLGK, was chemically synthesized and purified by the American Peptide Company.2)2)TM (Ecogen), and quantified using Image LabTM software (Bio-Rad).Increasing amounts (0.1–5 μM) of the RNA molecules 3′X, CU, I+U and I+CU were incubated in SHAPE buffer , followed by incubation at 37 °C for 15 min. The protein:nucleotide saturation ratio was established at 1:50. These samples were subsequently treated with proteinase K (300 ng/μl) in the presence of 0.5% SDS for 30 min at 37 °C. RNA molecules were then purified by phenol extraction and resolved in 4–10% native polyacrylamide gels as described above.Melting curves were produced by incubating 1 μM of the transcripts 3′X, CU, I+U or I+CU in SHAPE buffer. The melting temperatures ranged from 30 °C to 80 °C. Reactions proceeded for 15 min; the RNA molecules were then electrophoresed as described above.The RNA dimerization capacity of the IRES and CRE mutants was essentially performed as described above, using 1 μM of each RNA construct.260). The formation of 2′-O-adducts with both NMIA and 1M6 was mapped by primer extension using fluorescently-labelled oligonucleotides. Briefly, primer extension reactions were performed with 2 pmol of modified RNA and 20 U of SuperScript III RT  using the following fluorescently-labelled primers: oligonucleotide StdSHAPE-dif assays were essentially performed as previously described260). Dimer formation was accomplished as described above by incubating 1 μM of each RNA variant in SHAPE buffer for 15 min at 37 °C. Complexes were resolved by polyacrylamide non-denaturing gel electrophoresis (4–6%) in TBM buffer and visualized by UV shadowing. The specific reaction products corresponding to the monomeric or dimeric isoforms were excised from the gel and passively eluted overnight at 4 °C. NMIA modifications were detected by primer extension and the cDNA products analyzed by capillary electrophoresis, as described above. HMX scores were calculated as previously describedHMX analysis was performed as previously describedHow to cite this article: Romero-López, C. et al. The chaperone-like activity of the hepatitis C virus IRES and CRE elements regulates genome dimerization. Sci. Rep.7, 43415; doi: 10.1038/srep43415 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Escherichia coli, the sigma factor σ70 directs RNA polymerase to transcribe growth-related genes, while σ38 directs transcription of stress response genes during stationary phase. Two molecules hypothesized to regulate RNA polymerase are the protein Rsd, which binds to σ70, and the non-coding 6S RNA which binds to the RNA polymerase-σ70 holoenzyme. Despite multiple studies, the functions of Rsd and 6S RNA remain controversial. Here we use RNA-Seq in five phases of growth to elucidate their function on a genome-wide scale. We show that Rsd and 6S RNA facilitate σ38 activity throughout bacterial growth, while 6S RNA also regulates widely different genes depending upon growth phase. We discover novel interactions between 6S RNA and Rsd and show widespread expression changes in a strain lacking both regulators. Finally, we present a mathematical model of transcription which highlights the crosstalk between Rsd and 6S RNA as a crucial factor in controlling sigma factor competition and global gene expression.In  Proteins called sigma (σ) factors bind to E forming Eσ holoenzymes, and direct it to transcribe from specific promoters. Escherichia coli has seven sigma factors (70 (RpoD), σ38 (RpoS), σ32, σ54, σ28, σ24 and σ19. Though it was previously reported that the cellular concentration of E exceeded that of sigma factors  is the housekeeping sigma factor, directing transcription of genes essential for growth. The major alternative sigma factor, σ38 (RpoS), is present in low concentration during exponential growth, but regulates several hundred genes  supplemented with 0.5% glucose and 0.1% casamino acids was used for RNA-seq and validation. During strain construction, ampicillin or kanamycin were used at final concentrations of 100 μg/ml and 50 μg/ml respectively.Luria-Bertani broth and agar (20 g/L) were used for routine growth. M9 defined medium , mid-exponential (OD600 ∼0.8), transition to stationary (OD600 ∼1.6), stationary , and late stationary  phases of growth. RNA was extracted using TRIzol (Invitrogen), following the manufacturer’s protocol. Total RNA was treated with DNase I  according to the manufacturer’s protocol. Further precipitation of RNA and ribosomal RNA cleanup was achieved using the MICROBExpress bacterial mRNA purification Kit  according to the manufacturer’s protocol. RNA concentration was determined using a Nanodrop 2000 (Thermo Scientific) and quality was checked by visualization on agarose gels.Overnight cultures in M9 glucose were inoculated in 100 mL fresh M9 glucose to a final ODSequencing libraries were prepared using TruSeq RNA sample preparation kit v2  according to the manufacturer’s guidelines, checked for quality on an Agilent 2100 Bioanalyzer, and sequenced for 50 cycles from one end on an Illumina HiSeq1000 platform at the Centre for Cellular and Molecular Platforms, Bangalore. RNA-Seq data are summarized in Table S2. RNA-Seq reads were mapped to the E. coli K-12 MG1655 genome using BWA .qRT–PCR was carried out using specific primers to selected mRNA targets . 5 ng RNA was used for each RT-PCR reaction. TAKARA One-step SYBR PrimeScript RT-PCR kit II (RR086A) was used according to the manufacturer’s protocol, on an Applied Biosystems ViiA 7 Real-Time PCR system.70 , Mouse monoclonal antibody to σ38 , Mouse monoclonal anti-FLAG antibody , Mouse monoclonal antibody to GroEL . Bands were visualized using SuperSignal West Dura Chemiluminescent Substrate  and imaged using an ImageQuant LAS 4000 system . Band intensities were quantified using ImageJ (http://imagej.nih.gov/ij).Cells were grown as for RNA-seq. For stationary phase samples, 5 ml culture was harvested by centrifugation. For mid-exponential phase, 10 ml was harvested. Lysates were prepared and protein concentration was estimated using BCA assay . Lysates containing equal amounts of protein were loaded onto an SDS-PAGE gel. Proteins were elecroblotted onto a nitrocellulose membrane and probed with mouse primary antibody against the protein of interest followed by horseradish peroxidase-conjugated anti-mouse secondary antibody. The primary antibodies were: Mouse monoclonal antibody to RpoB , Mouse monoclonal antibody to σE. coli K-12 MG1655 genome was downloaded from NCBI (NC_000913.2). Gene coordinates were taken from RegulonDB v8.0  - (4) and (8) – (9). KNS represents the dissociation constant for non-specific binding of RNA polymerase to DNA, which we assume is equal for E, Eσ70 and Eσ38. We also assume the specific binding constants of Eσ70 and Eσ38 to their target promoters to be equal in stationary phase.The model with DNA , while 6S RNA binds to the Eσ70 holoenzyme and prevents it from binding promoters. To identify their effects on gene expression, we carried out RNA-seq to identify genes regulated by Rsd and / or 6S RNA in five growth phases.E. coli K-12 MG1655 (Wild-type), Rsd knockout (Δrsd), 6S RNA knockout (ΔssrS), 6S RNA-Rsd double knockout (ΔrsdΔssrS), and σ38/RpoS knockout (ΔrpoS) were used for RNA-seq. These had similar growth rates in M9 glucose . RNA-seq was performed in five growth phases: early exponential (EE), mid-exponential (ME), transition to stationary (TS), stationary (S), and late stationary .Table S1 lists strains and plasmids used. Five strains: rsd strain showed only 16 differentially expressed genes. These included several non-coding RNAs  which were increased two- to sixfold in Δrsd during stationary phase. Notably, 6S RNA expression was altered in Δrsd; 6S RNA was 2.3 times its wild-type level in stationary phase, but in ME phase was reduced to about half its wild-type level.We defined differentially expressed genes as genes whose expression changed >=twofold in a mutant strain relative to wild-type, with FDR-adjusted p-value < 0.05. Using these criteria, the Δrsd, we looked for smaller changes. We found that in all growth phases, genes whose expression was significantly reduced  in ΔrpoS also displayed reduced expression in Δrsd. The box plots in 2 fold change in gene expression in Δrsd relative to wild-type. ∼75% of genes whose expression was significantly reduced in ΔrpoS showed reduced expression (log2 fold change < 0) in Δrsd. Similar plots for other growth phases are in Figure S2A. This reduced expression was less than twofold in magnitude and so was not seen when searching for differentially expressed genes.As very few genes were differentially expressed in ΔP < 10−15 in all growth phases except LS) decrease in expression across hundreds of rpoS regulated genes, in all five growth phases. Second, the average overlap between the set of rpoS regulated genes in successive growth phases is only 54% - so it is not a single set of genes whose expression is reduced in Δrsd, but a different set in each growth phase. Third, the Δrsd samples showed high inter-replicate correlation in all growth phases (Table S2). Fourth, this trend of decreased expression held true when only previously reported σ38 targets . On the other hand, expression of genes controlled by constitutive σ70 target promoters , we consider them important for several reasons. First, they represent a consistent and highly significant ; however, it is extremely difficult to obtain reliable measures of small differences in protein level using western blots. We cannot be certain based on this experiment whether the reduced expression of σ38 targets in Δrsd is due to reduced σ38 protein, reduced E-σ38 association, or both. We attempt to address this question theoretically later.Thus, the Rsd knockout behaved like a σ70 - sequestering 6S RNA play in gene regulation? Is its function similar to that of the σ70-sequestering Rsd? Our RNA-seq showed that the 6S RNA knockout (ΔssrS) was very different from the Rsd knockout. To begin with, it showed >=twofold differential expression of 447 genes in total.What role does the EσssrS in successive growth phases, even in the two stationary phase time points. Only a few genes were differentially expressed in ΔssrS throughout growth, particularly fau (ygfA), which is downstream of 6S RNA in the same operon. fau expression was increased when 6S RNA was overexpressed in a wild-type background , indicating that this is at least partly a regulatory effect of 6S RNA and not merely a polar effect.artM, artI, hisP, hisQ, hisJ, hisM, tdcC) and amino acid biosynthesis  was increased in ΔssrS, while expression of genes involved in stress responses  was reduced. On the other hand, in LS phase, genes involved in global transcriptional regulation  and the TCA cycle  were upregulated. Thus, the physiological function of 6S RNA appears largely growth phase-dependent; nevertheless, there are certain patterns throughout growth.During exponential phase, expression of several genes for amino acid transporters . This was not due to reduced σ38 protein, as western blots in stationary phase showed that the ΔssrS strain had higher σ38 protein than the wild-type . On the other hand, expression of genes under constitutive σ70 promoters was slightly increased in the ME, TS and LS phases .Like 70  was reduced in ΔssrS, and the magnitude of reduction increased with growth phase . For instance, expression of th phase . Reducedlymerase , consistcrl expression is increased in ΔssrS, and the magnitude of this effect increases with growth phase. As with the effect of reducing RNA polymerase synthesis to compensate for the loss of 6S RNA, increasing σ38 protein and Crl may be a means to compensate for reduced Eσ38 activity in ΔssrS bacteria.6S RNA also represses some genes in a dose-dependent manner. rsd was not differentially expressed in ΔssrS. To check if 6S RNA regulated Rsd post-transcriptionally, we added a 3xFLAG tag to the C-terminal of the Rsd protein, as in previous studies  forming holoenzymes (Eσ70 and Eσ38). Holoenzymes initiate transcription from target promoters (P70 and P38), releasing the sigma factor. The elongating RNA polymerase (Ee70 and Ee38) transcribes until released. Holoenzymes and E also bind DNA non-specifically. We focus on the steady state of this model, determined by equations (1) - (4) and (8) – (17) in Methods.70 and Eσ38 in the absence of DNA. This is represented by the shaded area in 70 and σ38. Each value of Rsd on the x-axis corresponds to a separate ’run’ of the model where we compute the steady-state for those parameter values, before moving on to the next Rsd value. We predict that as Rsd is increased, it sequesters σ70, reducing Eσ70 formation. This allows more E to bind σ38, increasing the formation of Eσ38, consistent with previous predictions of Rsd function  in the Rsd knockout. We did not include this in the model due to lack of quantitative data on oxyS; however, it is possible that a small reduction in σ38 protein levels, perhaps due to increased oxyS, may contribute to reduced σ38 activity in the Δrsd strain. However, it is difficult to confirm this.We also observed a 2.4-fold increase in 70, reducing the amount of E available to bind to both sigma factors and thus inhibiting formation of both holoenzymes. This decreases the rate of transcription by both holoenzymes . However, there is still relatively low overlap in the list of 6S RNA regulated genes (46 genes). This is likely because of differences in time points and media. Even within our dataset, there is little overlap between genes regulated by 6S RNA at different time points. It seems that the 6S RNA regulon varies greatly under different conditions. This supports a model where 6S RNA acts as a background-level regulator operating on RNA polymerase, with gene-level outcomes depending strongly upon cellular environment; this could involve DNA topology, transcription factors, and other RNA polymerase-binding factors, all of which vary with growth phase (in vitro transcription experiments testing whether the ability of 6S RNA to regulate known targets is affected by the presence of other regulators in the medium.6S RNA is more complex, regulating expression of >400 genes; however, we did not observe associations between promoter sequence and 6S RNA susceptibility as previously reported (th phase . Regulat38 during stationary phase. It was unknown whether this was a direct effect of 6S RNA, allowing σ38 to compete more effectively for E, or indirect, via a trans-acting factor important for σ38 activity. However, another study (38 globally, from early exponential to stationary phase, and show mathematically that this is extremely unlikely to be a direct effect of sequestration. We show that 6S RNA increases Rsd protein level and suggest that Rsd is primarily responsible for 6S RNA’s effect on Eσ38 activity.Previous work using individual promoters  had suggssrS strain, where the cell reduces RNA polymerase expression to compensate for 6S RNA loss, and increases σ38 and possibly Crl to compensate for reduced Eσ38 activity. Apart from these two, increased ppGpp may also have such a compensatory role during TS phase, while in LS phase, the 6S RNA knockout has higher expression of ppk, responsible for synthesis of polyphosphate - which activates transcription by Eσ38 (70 and Eσ38 (38 expression (It has been asked, given 6S RNA’s function as a global regulator, why its deletion does not cause a growth defect. Our data, along with others ( by Eσ38 . In statWe see that 6S RNA and Rsd regulate each other’s expression, and control each other’s effect on gene expression; finally, we report that 1780 genes across 5 growth phases—almost 40% of the genes in the cell—are differentially expressed in the 6S RNA-Rsd double knockout but not in the single knockouts added together. This provides direct evidence that 6S RNA and Rsd have non-additive effects on gene expression, since the single knockouts cannot explain the widespread gene expression changes observed in the double knockout. Previous studies have only examined single knockouts; this underscores the importance of studying 6S RNA and Rsd as a unit that controls global RNA polymerase activity.Given that 6S RNA homologs are widespread in bacteria, co-occurring with Rsd and with other RNA polymerase regulators such as the actinobacterial RbpA, and many species have two or three 6S RNA homologs with different expression patterns and potentially different binding partners (


Escherichia coli (E. coli) ribosome but certain differences are evident. The chloro-ribosome proteins are generally larger because of the presence of chloroplast-specific extensions in their N- and C-termini. The chloro-ribosome harbours six plastid-specific ribosomal proteins (PSRPs); four in the small subunit and two in the large subunit. Deletions and insertions occur throughout the rRNA sequence of the chloro-ribosome (except for the conserved peptidyl transferase center region) but the overall length of the rRNAs do not change significantly, compared to the E. coli. Although, recent advancements in cryo-electron microscopy (cryo-EM) have provided detailed high-resolution structures of ribosomes from many different sources, a high-resolution structure of the chloro-ribosome is still lacking. Here, we present a cryo-EM structure of the large subunit of the chloro-ribosome from spinach (Spinacia oleracea) at an average resolution of 3.5 Å. High-resolution map enabled us to localize and model chloro-ribosome proteins, chloroplast-specific protein extensions, two PSRPs (PSRP5 and 6) and three rRNA molecules present in the chloro-ribosome. Although comparable to E. coli, the polypeptide tunnel and the tunnel exit site show chloroplast-specific features.Protein synthesis in the chloroplast is mediated by the chloroplast ribosome (chloro-ribosome). Overall architecture of the chloro-ribosome is considerably similar to the  Escherichia coli (E. coli) 70S, the SSU is composed of 16S rRNA and 21 ribosomal proteins while the LSU consists of 5S and 23S rRNA along with 33 ribosomal proteins. The eukaryotic cytoplasmic ribosome (80S) is larger in size than the bacterial 70S ribosome, as it contains more proteins and rRNAsProtein translation is a universal process of amino acid polymerization mediated by the ribonucleoprotein complex called the ribosome11456ribosome 0S is larThe mitochondria and chloroplasts carry their own genome. Genes encoded by these organellar genomes are transcribed by their own transcriptional machineries and translated by the unique ribosomes present in these organelles101314151718192223242526Spinacia oleracea) chloroplast out of which 98 encodes for proteinsb6/f complex and RuBisCO (the large subunit), all of which are involved either in photosynthesis or respiration. Other examples of chloroplast-encoded proteins are RNA polymerases, NADH dehydrogenases, ribosomal proteins and translation factors312110According to the endosymbiotic theory30313234303633E. coli 70S but differences occur30E. coli 70S, two of the LSU proteins (bL25 and uL30) are absent and six new plastid-specific ribosomal proteins (PSRPs) are added: four in the SSU and two in the LSU31The overall composition of the chloro-ribosome is similar to the de novo modelling of two PSRPs, many of the protein extensions and rRNA insertions. The overall structure resembles the E. coli ribosome LSU but chloroplast-specific features are visible. PTC is conserved but noticeable differences in the structure of proteins lining the polypeptide tunnel and tunnel exit site are present. Our study aims to fill a gap in the field of high-resolution structures of the ribosome available and reveals unique features of the chloro-ribosome at near-atomic resolution.Here, we report a near-atomic resolution structure of the chloro-ribosome LSU from spinach at 3.5 Å resolution determined using cryo-EM and single particle analysis. High-resolution map allowed E. coli orthologous proteins E. coli ribosome 50S model into our map lead to the identification and localization of the densities for the E. coli orthologous proteins and rRNAs present in the spinach chloro-ribosome LSU. A combination of secondary structure prediction, homology modelling and tracing of amino acid side chains were used for model building. Details of the protein models are presented in Since the chloro-ribosome is more similar to the E. coli ribosome LSU could be identified in the spinach chloroplast LSU map with the exceptions of two proteins, namely bL25 and uL30 , the spinach chloro-ribosome LSU contains an additional rRNA molecule named as 4.8S rRNA  to as long as 97 residues (in bL21c). We have localized and partially modelled 16 of these protein extensions, de novo , L4 (39 residues), L5 (41 residues), L13 (49 residues), L15 (37 residues), L19 (46 residues), L21 (97 residues), L22 (89 residues), L23 (35 residues), L24 (40 residues), L27 (53 residues), L29 (46 residues), L31 (23 residues) and L32 (23 residues). The presence of these extensions in the chloro-ribosome proteins are mainly responsible for altered protein: rRNA ratio in the chloro-ribosome (2:3) as compared to the E. coli (1:3)31A summary of the modelled chloroplast-specific extensions is presented in es), L24  residuesE. coli orthologous proteins , chloroplast-specific protein extensions and rRNAs, extra densities at two places remain unaccounted. High-resolution map allowed us to identify the unaccounted densities as PSRP5 and PSRP6 and build de novo models for these two proteins  and basic in nature. As expected from the basic nature of PSRP5, its density is surrounded by rRNA suggesting its close association with rRNA . Densityet al.30Localization of the PSRP5 in our map does not agree with Sharma de novo modelling of the PSRP6 reveals that it is mostly disordered except for two very short stretches (residues ranging from 57 to 62 and 69 to 73) of helical turns in the N-terminal region. Interestingly, in the previous cryo-EM study of spinach chloro-ribosomePSRP6, the second of the PSRPs present in the spinach chloro-ribosome LSU, is also a conserved, nuclear-encoded, plastid-specific protein of 116 amino acids in length (mature protein consists of 69 aa) and basic in nature. However, in contrast to the PSRP5, density for the PSRP6 is mainly extended with the presence of minimal secondary structural features . The denThe fact that both bL25 and uL30 are absent from the chloro-ribosome LSU while two new proteins, namely PSRP5 and PSRP6 have been acquired, lead to the assumption that these two PSRPs might have replaced the role of bL25 and uL30 in the chloro-ribosome. However, locations and structures  of theseE. coli 50S rRNA (3024 nt). Unlike E. coli 50S, where only 5S (120 nt) and 23S rRNAs (2904 nt) are present, chloro-ribosomes contain three molecules of rRNAs named as 4.8S (106 nt), 5S (121 nt) and 23S rRNA (2810 nt).Total length of the rRNA in the spinach chloro-ribosome LSU is 3037 nucleotides, which is 13 nucleotides longer than the E. coli LSU and the spinach chloro-ribosome LSU. Nucleotide sequence alignment also reveals that insertions and deletions have occurred in the entire span of rRNA sequence in chloro-ribosome. But deletion in a position is balanced by insertion in a nearby position (and vice versa), keeping the overall structure of the chloro-ribosome rRNA comparable to the E. coli. However, analysis of our map reveals that nucleotide insertions and deletions have altered local rRNA structure significantly at five different places (E. coli 23S rRNA numbering) and the fifth one is located in the 4.8S rRNA surrounding positions 1–28. All of these alterations in the rRNA structure are located far from the conserved core (PTC forming region of domain V and VI in 23S rRNA).Global sequence alignment using EMBOSSt places . Four ofE. coli 23S rRNA secondary structure diagram obtained from http://rna.ucsc.edu/rnacenter/ribosome_images.html) and H19  of H45 due to a net deletion of 6 nucleotides in the chloro-ribosome 23S rRNA sequence.The second region with alterations in rRNA structure results due to a combined 20 nucleotides long insertion in domain II of 23S rRNA between positions 938–969 H38, . This inE. coli ribosome, H63 extends near to this region but deletion of 26 residues in equivalent position in chloroplast 23S rRNA sequence makes H63 much shorter . This insertion creates a protrusion in H58 which extends towards the solvent exposed side (E. coli numbering 129–153) of 23S rRNA and results in the deletion of H9 from the chloroplast 23S rRNA structure  creates a bulge between H100 and H101, which resides in close proximity with chloro-specific N-terminal extension of bL19c.The last region with significant alterations in rRNA structure results from a deletion and an insertion in the 4.8S rRNA . DeletioE. coli 23S rRNA. Therefore, it is possible that 4.8S rRNA might have got separated from a 23S rRNA precursor during evolution and thereafter co-existed as a separate rRNA molecule along with the 23S rRNA to facilitate rRNA remodelling by loosing H98 at this cleavage site  containing sites by Klein et al.H. marismortui 23S rRNA  while the tunnel exit site is surrounded by uL22, uL23, uL24 and uL29. Our map of the spinach chloro-ribosome LSU reveals that the overall architecture of the polypeptide tunnel and tunnel exit site is similar to the E. coli 50S. However, certain differences are visible. The density for a conserved beta hairpin loop present in the E. coli uL23, which extends to the polypeptide tunnel is absent in uL23c 54E. coli SRP (4.8S RNA) is substituted with a protein named as cpSRP43. Unlike the E. coli SRP system which functions only co-translationally, the cpSRP has been suggested to function both co-translationally and post-translationally. The alteration in the structure of uL23c raises a possibility that a different mechanism of SRP recruitment is followed by the chloro-ribosome. However, further investigations are required to confirm this hypothesis.Nascent polypeptide chains travel through a polypeptide tunnel of about 100 Å in the LSU and interact with a diverse range of protein biogenesis factors involved in protein maturation, folding and targeting at the tunnel exit site. The tunnel exit site acts as a docking platform for these protein biogenesis factors. In in uL23c . Additiocoli 50S . Moreovecoli L23 . uL23 haThe density for 15 amino acids of the chloroplast-specific N-terminal extension of uL24c is clearly visible in our map which extends towards the polypeptide tunnel suggesting a possible chloroplast-specific interaction between uL24c and nascent polypeptide . DensitySpinacia oleracea) leaves bought from the local market were deveined, washed (using distilled water) and homogenized using 0.7 M sorbitol in buffer A  using a blender (two shots of 10 sec each). The homogenized leaves were filtered through a double-layer of cheesecloth followed by one layer of Miracloth . Resultant filtrate was centrifuged at 1200 × g for 15 min. The pelleted chloroplast was resuspended in 0.4 M sorbitol in buffer A and thereafter centrifuged again at 1200 × g for 15 min. The pellet was resuspended in 2% (vol/vol) Triton X-100 in buffer A and incubated on ice for 30 min. The lysed suspension was centrifuged at 26,000 × g for 30 min to achieve clarification and thereafter crude ribosomes were pelleted by centrifugation at 86,000 × g for 17 hr through a sucrose cushion (1 M sucrose in buffer A). The pellet was washed and gently resuspended in buffer B (buffer A with 10% glycerol). The suspension (crude ribosomes) was clarified by centrifugation at 26,000 × g for 15 min and was loaded onto 10–40% sucrose gradient in buffer A (with RNase inhibitor) and centrifuged at 111,000 × g for 4 hr. Fraction corresponding to 70S chloro-ribosomes was collected and further centrifuged through a sucrose cushion  at 84,000 × g for 2.5 hr. The chloro-ribosome pellet was suspended in a grid buffer containing 20 mM Tris·HCl, pH 7.6, 100 mM KCl, 10 mM MgOAc, 100 mM sucrose, 7 mM 2-mercaptoethanol, 1 unit/ml RNase inhibitor and 0.1% protease inhibitor.Purification of chloro-ribosomes was performed as previously described31260/ml were applied onto glow-discharged 2-nm carbon coated holey grids (Quantifoil R2/2) and incubated for 30 sec. Grids were blotted for 3 sec in 100% humidity at 4 °C and flash frozen in liquid N2-cooled liquid ethane using FEI Vitrobot. Grids were loaded to an FEI Tecnai Arctica cryo-transmission electron microscope, operated at 200 kV and equipped with a back-thinned Falcon III direct electron detection device. Electron micrographs were collected automatically by using FEI’s automatic single particle acquisition software EPU. A total of 1,729 micrographs were recorded in a movie mode as a set of 7 frames  at a calibrated magnification of 109,375 resulting in a pixel size of 1.28 Å on the object scale and at defocus values from 0.2–2.5 μm.4 μl of purified chloro-ribosome at final concentrations of 4 ODE. coli 70S map2), prior to visualization.After data collection and screening for good micrographs, 1590 micrographs were selected for data processing. Whole-image drift correction was carried out using motioncorrhttp://rna.ucsc.edu/pdbrestraints/index.html) and used throughout the refinement cycles of the rRNA models using phenix.real_space_refineCoordinates for the 23S, 5S and 4.8S rRNAs were extracted from the reported coordinates of the spinach chloro-ribosome (PDB ID: 4V61)E. coli ribosome (PDB ID: 4YBB)de novo modelled using the tool ‘place helix here’ in COOTde novo using the toolsets mentioned above. Optimal fitting of individual proteins in the density was achieved by running phenix.real_space_refinephenix.real_space_refineTo model chloro-ribosome LSU proteins, sequence information for the mature proteins were obtained from UniProtThe entire structure for the spinach chloro-ribosome LSU was validated using Molprobityet al. (EMDB ID: 1829)UCSF ChimeraAccession codes: The 3.5 Å cryo-EM map of the spinach chloro-ribosome LSU has been deposited in the Electron Microscopy Data Bank with accession code EMD-9572, the coordinates of the atomic model have been deposited in the Protein Data Bank under accession code 5H1S.How to cite this article: Ahmed, T. et al. Cryo-EM structure of the large subunit of the spinach chloroplast ribosome. Sci. Rep.6, 35793; doi: 10.1038/srep35793 (2016).


In Streptococcus pyogenes, a common and potentially deadly pathogen, many sRNAs have been identified, but only a few have been studied. The goal of this study is to identify trans-acting sRNAs that can be substrates of RNase III. The endoribonuclease RNase III cleaves double stranded RNAs, which can be formed during the interaction between an sRNA and target mRNAs.Small noncoding regulatory RNAs (sRNAs) are post-transcriptional regulators, regulating mRNAs, proteins, and DNA in bacteria. One class of sRNAs, Streptococcus pyogenes and its RNA sequencing (RNA-Seq) data were analyzed and compared to that of the wild-type. First, we developed a custom script that can detect intergenic regions of the S. pyogenes genome. A differential expression analysis with Cufflinks and Stringtie was then performed to identify the intergenic regions whose expression was influenced by the RNase III gene deletion.For this study, we created an RNase III null mutant of p ≤ 0.05. Using Artemis and Bamview genome viewers, these regions were visually verified leaving 6 putative sRNAs. This study not only expanded our knowledge on novel sRNAs but would also give us new insight into sRNA degradation.This analysis yielded 12 differentially expressed regions with >|2| fold change and  S. pyogenes, also known as Group A Streptococcus (GAS), is an important human pathogen that affects 700 million people worldwide each year resulting in about 500,000 deaths due to various complications . Ci. Cicis-aTrans-sRNAs employ many different modes of action to regulate RNA expression [cis-sRNAs, trans-sRNAs are transcribed from intergenic regions (IGRs) throughout the genome  data processing has been simplified to an easy to use open source pipeline that has been widely used for alignment and observation of RNA-seq data. This process relies on many different algorithms that can yield multiple types of results, from simple alignments to complex differential expression analyses. This pipeline starts with Bowtie2 as a base which creates an index of the genomic file . The indS. pyogenes and then determine whether they are sRNAs. First, a bioinformatics approach was developed by employing two different pipelines . S. pyogenes grown on plates (solid media) was incubated in anoxic conditions using the Gas Pak EZ anaerobe containment system . Escherichia coli Top10 (Invitrogen) was used for plasmid construction. E. coli was cultured in Luria-Bertani broth (LB) at 37 °C with shaking. When appropriate, optimum concentration of antibiotics was added to the media.b strain , and hasb strain .Todd Hewrnc was created as follows. The primers of 5outRNase3IFD-KpnI (aaaggtacccaaagagttagcgcatatgacg) and 3outRNase3IFD (cagtatctttagtctgtctttcttgagc) were used to amplify a 2.02 kb DNA fragment including rnc. This amplified fragment was digested and inserted between KpnI and XbaI restriction sites in the multiple cloning site of pCRII (Invitrogen). The KpnI restriction site is located in the primer sequence of 5outRNase3IFD-KpnI, which is underlined, and the XbaI site is located near the 3′ end of the PCR-amplified product. The resulting plasmid was then used as a template in an ‘inside-out’ PCR reaction with the primers of 5inRNase3IFD-XmaI (aaacccgggattagtgagaaaggacctgccc) and 3inRNase3IFD-XmaI (aaacccgggctctgaaataatcaattgtagaacagcg). Restriction of this fragment with XmaI followed by subsequent re-ligation resulted in a nonpolar inframe deletion that replaces DNA sequence encoding Y61 – V181 of RNase III with the sequence of cccggg encoding PG. The in-frame deletion allele of rnc was then inserted between the BamHI and XbaI restriction site in the S. pyogenes-E. coli shuttle vector, pJRS233. The generated plasmid, pJRS233::rnc-IFD, was used to replace the wild type rnc with the in-frame-deleted rnc by a method that employs the temperature sensitivity of the pJRS233 replication origin [An in-frame deletion allele of n origin .S. pyogenes cell pellet from 10 ml culture was resuspended in 700 ml of the Qiazol lysis reagent (Qiagen) and transferred to a Lyse Matrix B blue cap tube . Cells were then lysed by the beadbeater, FastPrep 24  at the speed of 6.0 for 40 s twice. The remaining procedure for RNA extraction followed the manufacturer’s protocol of the miRNeasy kit. During RNA purification, RNase-Free DNase (Qiagen) was treated on column to remove residual DNA. The A260/A280 ratio of the extracted RNA was measured with Eppendorf BioSpectrometer® to determine the RNA concentration and purity (accepted if >1.8). The extracted RNA was mixed with 1 ul of RNasin , and treated with the RNAstable kit for safe transport.Next-generation sequencing, RNA-Seq.The wild type (HSC5) and the RNase III mutant (ΔRNase III) were grown in THY media to the exponential phase (OD600 of 0.4–0.5). Then, total RNA was extracted using the combination of the miRNeasy kit (Qiagen) and the FastPrep beadbeater . An The extracted RNA samples were submitted to Macrogen Corporation  for RNA-Seq assays. The quantity, integrity and purity of total RNA were assessed using Ribogreen  and Agilent Bioanalyzer 2100. RNA was subjected to rRNA depletion using the RiboZero Meta-Bacteria kit  and cDNA was generated from the rRNA depleted RNA using the NEBNext mRNA Sample Prep kit . cDNA was profiled using Agilent Bioanalyzer, and subjected to Illumina library preparation using NEB Next reagents . The quality and quantity and the size distribution of the Illumina libraries were determined using an Agilent Bioanalyzer 2100. The libraries were then submitted for Illumina HiSeq2000 sequencing according to the standard operation. Paired-end 90 or 100 nucleotide (nt) reads were generated.To detect sRNAs expressed from the RNA-seq data, raw sequencing data was aligned using TopHat  with a BOnce the reads that aligned to IGRs were detected and isolated, each SAM file was loaded into the differential expression pipeline provided by the Cufflinks package –25  and the RNase III null mutant (ΔRNase III) were sequenced. Using the raw RNA-seq data, two differential expression pipelines Cufflinks and RSEM/EBSeq were executed, adding a step after Tophat alignment Fig. . This adIn order to ascertain the validity of the potential regions identified above, we turned to an in silico visual confirmation using the Artemis software provided by the Sanger Institute. Visual confirmation detected regions involved in operon structure or regions that have low enough read support to be determined to be false positives. We proved the final decisions on the 12 identified regions, with 6 of these (~50%) being confirmed as either likely or highly likely sRNA (Table For further confirmation, we also looked for predicted rho-independent terminators within the regions that were detected by our software pipeline. The ARNold software uses two different methods for prediction. The top predictions are shown in Table The RNase III family of endoribonucleases has been well studied across all organisms, from bacterial RNase III to the Drosha/Dicer family in eukaryotes . BacteriS. pyogenes.Through the development of an automatic step to add to the typical RNA-seq processing pipeline that detects and isolates the reads that align to IGRs, our study has shown that it is effective in finding potential sRNAs. Through visual validations, we were able to estimate that our methods had a recovery rate of 50% for finding potential sRNAs for the studied samples in