Siderastrea siderea inhabiting the forereef, backreef, and nearshore reefs of the Mesoamerican Barrier Reef System (MBRS) in the western Caribbean Sea.Natural and anthropogenic stressors are predicted to have increasingly negative impacts on coral reefs. Understanding how these environmental stressors have impacted coral skeletal growth should improve our ability to predict how they may affect coral reefs in the future. We investigated century-scale variations in skeletal extension for the slow-growing massive scleractinian coral S. siderea cores were extracted, slabbed, and X-rayed. Annual skeletal extension was estimated from adjacent low- and high-density growth bands. Since the early 1900s, forereef S. siderea colonies have shifted from exhibiting the fastest to the slowest average annual skeletal extension, while values for backreef and nearshore colonies have remained relatively constant. The rates of change in annual skeletal extension were −0.020±0.005, 0.011±0.006, and −0.008±0.006 mm yr−1 per year [mean±SE] for forereef, backreef, and nearshore colonies respectively. These values for forereef and nearshore S. siderea were significantly lower by 0.031±0.008 and by 0.019±0.009 mm yr−1 per year, respectively, than for backreef colonies. However, only forereef S. siderea exhibited a statistically significant decline in annual skeletal extension over the last century.Thirteen S. siderea colonies are more susceptible to environmental stress than backreef and nearshore counterparts, which may have historically been exposed to higher natural baseline stressors. Alternatively, sediment plumes, nutrients, and pollution originating from watersheds of Guatemala and Honduras may disproportionately impact the forereef environment of the MBRS. We are presently reconstructing the history of environmental stressors that have impacted the MBRS to constrain the cause(s) of the observed reductions in coral skeletal growth. This should improve our ability to predict and potentially mitigate the effects of future environmental stressors on coral reef ecosystems.Our results suggest that forereef  Coral reefs around the world are threatened by global warming Porites throughout the recent past Coral skeletal extension estimated from the width of coupled high and low density annual growth bands within coral cores have been interpreted as an indicator of coral health and ecological success Siderastrea siderea is one of the primary reef-building corals of the Caribbean Sea S. siderea is well-suited for investigating long-term trends in skeletal extension because it grows relatively slowly and because colonies are generally long-lived S. siderea is also a hardy species capable of surviving severe environmental stress, which results in relatively uninterrupted growth histories within cores extracted from these corals S. siderea for coral coring studies, as well as its important reef-building role on many Caribbean reefs, S. siderea has been relatively underutilized as an archive of environmental change and coral health.The massive zooxanthellate scleractinian coral S. siderea colonies from across an inshore-offshore gradient of the MBRS in southern Belize. Specifically, we quantified average annual skeletal extension and rates of change in annual skeletal extension for forereef, backreef, and nearshore S. siderea colonies, and examined whether rates of change in annual skeletal extension differed amongst these three reef zones, as well as from zero, over the last century. Establishing and comparing the history of skeletal extension for S. siderea across this inshore-offshore gradient enables us to infer whether colonies of S. siderea from different reef zones of the MBRS exhibit differing vulnerabilities to environmental stressors. This should improve our understanding of how corals from different regions of the MBRS responded to environmental stressors over the last century and how they are likely to respond in the future.In this study, we investigate variations in the skeletal extension of In general, shallow forereef corals (located on the oceanic side of the reef crest) are exposed to high wave activity S. siderea was investigated in the present study because it is well represented in each of these three sub-environments of the MBRS. Because nearshore and backreef colonies have been historically exposed to more regular and more intense environmental stressors, they are potentially more acclimatized and/or adapted to these stressors than forereef colonies S. siderea have exhibited greater resistance to increasing environmental stress over the past century than forereef colonies. We evaluate this hypothesis by reconstructing and comparing the history of skeletal extension of S. siderea corals inhabiting these three reef zones of the MBRS. Identifying systematic differences in temporal patterns of coral skeletal extension amongst these three reef zones should improve our ability to assess which regions of the MBRS, and potentially other reef systems, are most vulnerable to future increases in natural and anthropogenic stress.The coral S. siderea from the forereef, backreef, and nearshore reef zones of the MBRS in southern Belize. Forereef and backreef coral cores were obtained from the Sapodilla Cayes Marine Reserve, on the seaward and shoreward side of the reef crest, respectively. Nearshore coral cores were obtained from within 10 km of the coast in the Port Honduras Marine Reserve  affixed with a hollow extension rod  and a wet diamond core bit . Compressed air from SCUBA cylinders located on a boat was used to power the pneumatic drill. Drilling each core required a total of 5 to 8 standard size SCUBA cylinders and approximately 30–45 minutes of continuous drilling. Coral cores, approximately 20 to 100 cm in length, and 5 cm in diameter, were collected from the center of each coral colony parallel to the coral's vertical growth axis. Cores were extracted from colonies that appeared healthy and were without any obvious abnormalities, scarring, bleaching, or disease. Coral samples were collected from colonies between depths of 4 and 5 m within each of the three reef zones. After extracting each core, a concrete plug was inserted into the drilled holes and sealed with Z-spar underwater epoxy.A total of thirteen cores were extracted from thirteen different colonies of colonies , Table 1Water Pik®). The top portions of the cores were thoroughly rinsed with 95% ethanol to eliminate any remaining coral tissue. Six-mm thick slabs were cut vertically from the center of each core using a water-cooled trim saw.Newly extracted coral cores were rinsed with 95% ethanol in the field, stored in 5-cm-diameter PVC tubes and transported to the University of North Carolina at Chapel Hill for analysis. Coral tissue from the surface of the cores was removed with a water pick  radiography system at the UNC-Chapel Hill Campus Health Services Radiology Department to reveal annual cycles in skeletal density.Coral slabs were air dried and X-rayed from a source-to-object distance of 100 cm at 6.0 mA sVidar VXR film digitizer . Digital X-radiographs were transferred into Coral X-radiograph Densitometry System S. siderea deposits low density bands approximately during the dry season (December – May) and high density bands approximately during the wet season (June – November) Coral XDS to identify annual cycles in skeletal extension. This mode requires input of the coral X-radiograph image and the number of pixels per centimeter in the image X-rayed films were digitized using a The chronologies of the cores were established by identifying the most recent high density band deposited during the wet season of 2008, and counting backwards in time. We used visual cross-dating techniques to examine annual skeletal extension for cores within the same reef zone, since it is likely that these corals will share major growth patterns . Cores were visually cross-dated by identifying signature years of narrow growth to prevent dating errors associated with partial, missing, or double rings—a procedure commonly used in dendrochronology As an initial exploration of the data we grouped observations from each core amongst the three reef zones into 15-year increments and estimated average annual skeletal extension using a random intercepts model to account for observational heterogeneity . Rather nlme packageR 2.9.0Proc Mixed of SAS/STAT ® software version 9.1 of SAS System for WindowsS. siderea varied throughout the last century and amongst the forereef, backreef, and nearshore colonies. A sequence of models was fit to determine how best to describe the structural form of the data and to test the hypotheses of interest. Several models were tested, including (1) an ordinary regression model, (2) a random intercepts model with no predictors , (3) a random intercepts model that includes time as a predictor, (4) a random slopes and intercepts model in which the intercept and coefficient of time (slope) were allowed to be random, (5) a more complex version of model 3 and 4 that included level-2 predictors such as reef zone, (6) a version of model 5 that included a level-1  correlation structure, and (7) a version of model 6 that possessed a residual correlation structure without additional random effects.Statistical analyses were carried out using the The variable year was ‘centered’ using a centering constant of 1967 because this minimized correlation between the random slopes and intercepts. In general, centering enhances model interpretability and improves numerical stability by increasing the likelihood that the optimization algorithm converges on the correct solution. The estimate of the slope is unchanged by centering, but the intercept will estimate the mean value of the response variable in year 1967 (rather than in year zero of the uncentered model). The role of centering in mixed effects models is discussed in greater detail in O'Connor et al. (2007) Akaike Information Criterion (AIC) was used to identify the best-fit model i" references the core and "j" denotes an individual annual observation within that core. "I" is an indicator variable that takes on the value "1" if the condition in parentheses (Reef zonei  =  "XX") is true, and "0" if the condition is false. The construct "Reef zone" has been incorporated into the regression model as two dummy variables with "backreef" serving as the "reference group". The term "ui0" denotes the random intercept. It is constant for observations coming from the same core but differs for observations coming from different cores. Thus, equation (1) translates into three different fixed effects regression equations, one for each reef zone in equation (2) below, and thirteen different mixed effects equations, one for each core.Here "β2 and β3 measure the difference in rate of change in annual skeletal extension between the backreef coral colonies and the forereef and nearshore colonies. To investigate whether skeletal extension varied over time , estimates of the rates of change in skeletal extension derived from the ARMA  random intercepts model along with the 50% and 95% confidence intervals were displayed in a plot popularized by Gelman and Hill (2006) Here Although index master chronologies were not used in developing the model presented here , standardized annual skeletal extension has been displayed to facilitate comparison with coral skeletal extension reported elsewhere in the literature. Following standard protocol developed for dendrochronology S. siderea colonies have remained relatively consistent over this interval  random intercepts model. Thus, the rates of change in annual skeletal extension for forereef (−0.020±0.005 mm yr−1 per year) and nearshore (−0.008±0.006 mm yr−1 per year) S. siderea colonies were compared with that of the backreef colonies  mm yr−1 per year less than that for backreef S. siderea . The rate of change in annual skeletal extension for nearshore S. siderea was 0.019 (±0.009) mm yr−1 per year less than that for backreef S. siderea  over approximately the last century. The rates of change in annual skeletal extension were not significantly different from zero for either backreef  or nearshore  S. siderea colonies over this interval  and within the more thermally variable and heat-stressed backreef environment (where restricted circulation supports elevated seawater temperatures), yet decreased for corals from the cooler and more thermally stable forereef environment, suggests that acclimatization Coral skeletal extension reflects changes in environmental conditions S. siderea growth trends in the present study support this hypothesis, there are important caveats to consider. First, because all three reef zones are located within marine protected areas, the number of S. siderea cores collected from each reef zone was relatively low . Thus, sample size should be considered in the interpretation of these results. Yet despite the limited number of cores obtained, skeletal growth trends within each reef zone were relatively consistent amongst cores  of this decline poses some interesting challenges. In particular, there are numerous confounding factors associated with global climate change and increased anthropogenic stress that must be considered in order to better understand the ecophysiological and/or genetic basis for differential stress responses amongst corals inhabiting different reef zones of the MBRS. These factors will be briefly explored in the sections that follow.S. siderea inhabiting the three reef zones of the MBRS : (1) increasing seawater temperature resulting from global warming; (2) differences in light attenuation; (3) changing hydraulic regime; (4) increasing sedimentation; (5) eutrophication; (6) increasing pollution; and (7) the combined effects of multiple stressors.Several confounding factors may contribute to the observed differences in the rate of change in annual skeletal extension amongst S. siderea across this inshore-offshore gradient over the past century may have arisen either from differing temperature regimes amongst these three reef zones and/or from differences in the corals' ability to tolerate thermal stress amongst these three reef zones.Seawater temperature differs markedly amongst forereef, backreef, and nearshore reef environments within the MBRS. Backreef and nearshore reefs are generally exposed to warmer and more variable thermal conditions than forereef habitats Porites spp. were examined between 1988 and 2003 on the Great Barrier Reef −1 to 1.28 cm yr−1, equivalent to a decline of 0.016 cm yr−1 per year. The observed decline in skeletal extension was accompanied by an increase in SST over the studied interval. Another recent study compared skeletal extension of P. lutea from eight sites off South Thailand between December 1984 and November 1986 and between December 2003 and November 2005 Several recent studies have examined the effects of rising seawater temperature and associated thermal stress on coral skeletal extension M. annularis has also been reported to decrease with increasing SST in both the Gulf of Mexico (lower average SST) and the Caribbean Sea (higher average SST) S. siderea corals inhabiting the cooler forereef waters of the MBRS were more negative than for corals inhabiting the warmer nearshore reef and backreef zones  S. siderea may be more acclimatized and/or adapted to future warming, as well. Conversely, forereef corals, which typically experience cooler more stable diurnal, seasonal, and annual temperature patterns Annual skeletal extension of the species ef zones , 5, 6. IS. siderea was not correlated with measured environmental variables, including seawater temperature S. siderea was not correlated with SST S. siderea, it is possible that the effect of seawater temperature was masked by the corals' response to other anthropogenic stressors induced by the construction of the Panama Canal  S. siderea colonies near the southern coast of Belize are exposed to significantly less anthropogenic stress S. siderea along the sparsely-populated Belize coast may be more obvious than for corals inhabiting areas exposed to greater land-derived anthropogenic stressors.However, not all studies have revealed that coral skeletal extension is negatively impacted by rising seawater temperatures. A study performed on the fringing reefs of the central Caribbean coast of Panama found that annual skeletal extension for Symbiodinium) from the coral host S. siderea cores examined exhibited evidence of tissue necrosis  would be consistent with differences in light availability amongst these three reef zones. Personal observation by one of the authors  of the studied portion of the MBRS suggest that visibility and related light availability for corals is generally higher in the forereef and backreef environments relative to the nearshore environment, except for short intervals following major storm events when the forereef receives substantially more fluvial input from the watersheds of Guatemala and Honduras. Long-term in situ light monitoring along transects across the southern MBRS is needed to resolve this uncertainty.Light levels are also known to affect skeletal extension in corals P. lutea colonies growing in high wave energy regions had lower skeletal extension than those inhabiting lower wave energy reef environments Hydraulic regime has also been shown to affect coral skeletal extension M. faveolata from the MBRS found that rates of skeletal extension for corals at southern sites remained suppressed for longer intervals of time following stress events than for corals at northern sites M. faveolata from northern and southern localities of the MBRS.A recent study conducted on Sea-viewing Wide Field-of-view Sensor (SeaWIFS) images and model outputs suggest that most of the terrestrial sediments delivered to the MBRS originate from Guatemala and Honduras S. siderea exhibit more negative trend in annual skeletal extension than backreef corals  over the past century, is consistent with sedimentation being a factor differentiating skeletal growth patterns for this species amongst reef zones. This relationship between sedimentation and skeletal extension for S. siderea is consistent with earlier work on M. faveolata colonies on the southern MBRS S. siderea across a 30-year interval was not affected by increased sedimentation resulting from urbanization and river discharge along the coast of Puerto Rico On the Belize portion of the MBRS, sedimentation generally decreases seaward throughout most of the year as sediment-laden terrestrial runoff becomes diluted by open-ocean seawater containing less sediment S. siderea amongst the three reef zones investigated in the present study, it is unclear whether sedimentation was the primary factor responsible for these observed differences. Corroboration of modeled sediment outputs with long-term in situ instrumental monitoring of sedimentation across the Belize, Guatemala, and Honduras portions of the MBRS are needed to fully assess the role that increased sedimentation plays in suppressing skeletal extension rates of corals within the MBRS.Although sedimentation can result in reduced skeletal extension for some coral species S. siderea amongst the reef zones. Model outputs of terrestrial runoff into the Gulf of Honduras suggest that buoyant matter concentrations , suggesting that eutrophication did not substantially mitigate the effects of increased sedimentation and/or that other environmental stressors, such as increasing seawater temperatures, may have had a greater impact on skeletal extension patterns for this species.It has also been shown that in regions where eutrophication and increased sedimentation co-occur, such as along the forereefs of the southern MBRS, nutrient enrichment may enhance photosynthesis within coral symbionts to compensate for the reduced light associated with increased sedimentation S. siderea inhabiting the forereef environment of the southern MBRS.Eutrophication is also known to negatively affect coral skeletal extension by promoting the growth of algae, which prevent light and food from reaching the coral and which compete with corals for space on the reef S. siderea colonies, which are more proximal to the larger and more densely populated watersheds of the Honduran and Guatemalan coast—the primary source of the pollution to this region M. faveolata cores obtained along a north-south transect of the MBRS, which reported that corals bordering the Sapodilla Cayes  and temperature was a better predictor of coral skeletal extension than temperature alone. A companion study constructed century-scale records of trace-metal/calcium ratios  over time for M. faveolata cores collected from sites along the MBRS M. faveolata in the southern portions of the MBRS, compared to the northern portions.The compounding effects of multiple stressors on coral skeletal extension may be more important than the effect of any single stressor S. siderea observed in the present study resulted from a combination of anthropogenic stressors, similar to that which is thought to have caused the decline in skeletal extension for M. faveolata in this region over a similar temporal interval S. siderea, which were generally exposed to lower anthropogenic stress because of their proximity to the less-densely populated southern Belize coast over this interval. However, it is also possible that historically greater baseline levels of environmental stress  within the backreef and nearshore environments of the MBRS may have pre-conditioned S. siderea corals within these reef zones, effectively increasing their resistance/resilience to recent anthropogenic stress. However, as industrialization proceeds and global populations continue to expand, corals will be exposed to even greater anthropogenic stress . Skeletal extension within the apparently more resistant/resilient backreef and nearshore S. siderea colonies of the southern MBRS may commence a more precipitous decline once their stress-tolerance thresholds are exceeded.It is plausible that the decline in skeletal extension for forereef S. siderea corals of the MBRS have been more negative than for nearshore corals, which have been more negative than for backreef corals. However, only rates of change in annual skeletal extension for forereef S. siderea (negative) were significantly different from zero; rates of changes in annual skeletal extension for nearshore and backreef S. siderea corals were not significantly different from zero. Furthermore, since the early 1900s, average annual skeletal extension for backreef S. siderea colonies has been consistently higher than for nearshore colonies, while forereef colonies have transitioned from exhibiting the fastest to the slowest average annual skeletal extension.Rates of change in annual skeletal extension over the past century for forereef S. siderea corals differ amongst these three reef zones over the studied interval are not well constrained by the available data. However, differential thermal stress  and/or differential acclimation/adaptation to thermal stress amongst the three zones may be driving these disparate trends amongst reef zones. Increasing local anthropogenic stress  may also contribute to the differential responses of S. siderea amongst these three reef zones, especially as these stressors  appear to be greatest for forereef environments that are more proximal to the larger and more densely populated coastal watersheds of Honduras and Guatemala. It should also be noted here that these putative relationships between skeletal extension and natural/anthropogenic stressors may be species dependent. S. siderea is known to be a particularly hardy species that is able to survive harsh environmental conditions The reasons that rates of change in annual skeletal extension of S. siderea within the Gulf of Honduras may be more susceptible to environmental and anthropogenic stress, including future ocean warming, than their backreef and nearshore counterparts. Coral reef managers within the MBRS should consider potential differences in the sensitivity of reef zones when allocating resources to the protection and maintenance of reef ecosystems. Additional research is required to investigate the precise nature of the relationship between environmental and anthropogenic stressors and skeletal extension of S. siderea across the various reef zones of the MBRS, and to determine whether the pattern observed in the present study is exhibited by other species of reef-building scleractinian corals within the MBRS, and within other reef systems around the world.The results presented here suggest that forereef colonies of Table S1Population of countries along the Mesoamerican Barrier Reef System, excluding Mexico.(0.03 MB DOC)Click here for additional data file.


Here, we studied the role of the −1 and +1 nucleotide in wheat cox2 editing site recognition using an in organello approach. We found that four different recognition patterns can be distinguished: (a) +1 dependency, (b) −1 dependency, (c) +1/−1 dependency, and (d) no dependency on nearest neighbor residues. A striking observation was that whereas a 23 nt cis region is necessary for editing, some mutants affect the editing efficiency of unmodified distant sites. As a rule, mutations or pre-edited variants of the transcript have an impact on the complete set of editing targets. When some Cs were changed into Us, the remaining editing sites presented a higher efficiency of C-to-U conversion than in wild type mRNA. Our data suggest that the complex response observed for cox2 mRNA may be a consequence of the fate of the transcript during mitochondrial gene expression.The mitochondrial transcriptome from land plants undergoes hundreds of specific C-to-U changes by RNA editing. These events are important since most of them occur in the coding region of mRNAs. One challenging question is to understand the mechanism of recognition of a selected C residue (editing sites) on the transcript. It has been reported that a short region surrounding the target C forms the  RNA editing challenges the central dogma of molecular biology as it changes the information encoded for by the gene at the RNA level. This process affects a wide variety of organisms through nucleotide insertion/deletion or nucleotide conversion cis-elements on the transcriptome. Based on in vitro and in organello experiments, it has been reported that the cis-acting elements necessary and sufficient for recognition reside in a short sequence of fewer than 30 nucleotides encompassing the C target One major issue is to understand how the editing machinery can recognize specific C residues on the transcript, since there is no obvious consensus sequence when comparing the different trans-elements constituting the RNA editing machinery in plants. Organellar RNA editing trans factors were first discovered for the chloroplast trans acting factors involved in mitochondrial RNA editing cis elements is thought to recruit the catalytic element, the cytosine deaminase. However, to date, specific binding to the cis element has only been reported for two PPR trans factor, CRR4 in the chloroplast Physcomitrella patens_cis-acting elements of these sites present no similarity, the mechanism allowing the recognition of different sites by trans-acting factors remains an important question.Little is known about the cis recognition elements on the editing capacity of cox2 editing sites. Extensive mutagenesis analyses of C77 and C259 editing sites from wheat cox2 demonstrated that although the extent of cis-elements is similar, the role of each residue is different in the recognition of these targets in vitro and in silico analyses trans-acting factors. In this case, selected residues in the cis-element should make it possible to discriminate between different editing sites on the transcript. Bioinformatic analyses suggest that trans acting factors may be able to distinguish purines from pyrimidines and, at particular positions, one specific nucleotide To gain insight into the RNA editing process, we studied the consequences of point mutants of cox2 introduced into purified mitochondria. This approach allows the coincidental analysis of all editing sites on the same transcript. Our study reveals at least four different recognition patterns for C-to-U editing based on the importance of the contiguous residue. Furthermore, our findings suggest that RNA editing in plant mitochondria is not isolated but is rather part of a more general maturation process.We present here the editing status of mitochondrial mRNAs from single and multiple mutants of cob gene (Ir-cob) (accession no. AF337547). This sequence combined with the 23 bp upstream insert sequence served to specifically amplify transgenic products with nested PCR.All plasmids used in this study are based on the pCOX2Ta vector previously described Pfu DNA polymerase (Stratagene) in a final volume of 50 µl, according to the supplier's protocol. Parameters for amplification were 95°C for 2 min, 20 cycles at 95°C for 30 s, 48°C for 30 s and 68°C for 14 min, and finally 68°C for 10 min. After amplification, 10 units of DpnI endonuclease (Promega) were directly added to the PCR reaction for 3 hours at 37°C to eliminate the original DNA template. Ten microliters of the digestion reaction were used to transform competent E. coli DH5α. The sequence of selected mutants was verified before use.The list of the oligonucleotides used is given in Mitochondria were prepared from 20 grams of wheat embryos essentially as described 2, 12 mM Tricine (pH 7.2), 5 mM KH2PO4, 1.2 mM EGTA, 10 mM sodium succinate, 1 mM GTP, 2 mM ADP, 0.15 mM (each) CTP and UTP, 2 mM dithiothreitol. Mitochondria were recovered by centrifugation at 15000× g for 15 min at 4°C. RNA was purified with 800 µl Trizol® reagent (Invitrogen) according to the supplier's protocol.Electroporation was carried out with 1 mg of mitochondrial proteins in 50 µl of 0.33 M sucrose and 2 µg of recombinant plasmid in the conditions previously described One microgram of nucleic acids obtained after Trizol® treatment was digested with 2 U of DNase I Amplification grade (Invitrogen) for 15 min at 25°C. cDNA synthesis was performed with 200 units of Superscript II RT (Invitrogen) using 100 ng of random primers hexamers as primers (Promega). PCR amplifications were performed with primers 1a and 1b using the Advantage 2 polymerase mix (Clontech) as follows: 95°C for 2 min, 20 cycles at 95°C for 30 s, 64°C for 1 min and 68°C for 2 min, and finally 68°C for 10 min. Primers 2a and 2b were used for nested PCR on 2 µl of PCR1. Parameters were the same for PCR2, except for annealing temperature and cycling, which were 55°C and 20 cycles respectively.GCGGTGCAGTCATACAGATCTGC1a: TCCCGCGGGAAGCGGAAAGC1b: GAGCAGAGCTGAAAAAGATG2a: TATCCAGATTTGGTACCAAA2b: To determine the profile and the rate of C-to-U conversion in RT-PCR products, PCR bands corresponding to the mature transcripts were excised from the agarose electrophoresis gel and purified with the GFX PCR DNA and Gel Band Purification Kit . The purified fragments were ligated into pGEM-T easy vector as recommended by the manufacturer (Promega). Cloned PCR products were sequenced with the BigDye® Terminator Cycle Sequencing Kit v 1.1 (Applied Biosystems). Sequences analyses were performed at the Genotyping and Sequencing Facility of Université Bordeaux Segalen. Editing efficiency was determined by sequencing at least 16 cDNA clones from each electroporation experiment as described cox2 construct and their mutant derivatives are formed by two exons of 388 and 392 bp, split by a 1223 bp intron. The cox2 transcript possesses 17 editing sites, 6 in the first and 11 in the second exon respectively T. aestivum mitochondria, the mRNA was analyzed by RT-PCR and the identity of spliced molecules was verified by sequence analysis. The editing efficiency of wild-type and mutant cox2 transcripts was determined by sequencing at least 16 individual RT-PCR clones from each electroporation experiment. The results presented correspond to the percentage of C-to-U conversion for every editing site in the population of cDNA analyzed. The editing efficiency of site C259, which has been extensively studied The wheat cox2 transcripts is known to be affected by mutations at the −1 and +1 residues, respectively cox2 editing sites. The A residues were replaced by T and vice versa; the C residues were replaced by G, and the G residues were replaced by T. Site C385 belonging to the IBS1 region of exon1 was not modified.RNA editing of sites C77 and C259 on The editing response was defined as “severely affected” when the editing efficiency was reduced to less than 25% of the wild type, and “slightly affected” when the editing efficiency was comprised between 75% and 25% of the wild type. Individual mutants showed that residues C167, C169, C467, C550, C563 and C620 were severely affected when the neighbor +1 residue was changed, whereas sites C449 and C587 were slightly affected. Residues C30, C466, C682 and C704 were not affected by the +1 mutation . When thThese results make it possible to define four different recognition patterns based on the role of the +1/−1 nucleotide: (a) +1 dependency for sites C169, C449, C467, C550, and C259, (b) −1 dependency for sites C30, C682 and C77, (c) −1/+1 dependency for sites C167, C563 and C620, (d) no dependency on the nearest neighbor residues for sites C466, C587 and C704.As previously described, sites C482 and C638 are not considered in this analysis because they were found unedited in electroporation experiments cox2 transcripts from single −1 and +1 mutants . Notably, sites C77, C167, C169, C482, C638 and C682 were more efficiently edited in pre-edited than in wild type mRNAs. The editing efficiency of sites C30, C385, C449, C563 and C620 showed minor differences or none at all. Interestingly, site C587 behaves differently as it was less edited in the pre-edited context than in the wild type one .cis-recognition sequence of the neighboring one. The contiguous C167 and C169 editing sites allow the reciprocal influence between editing sites to be studied. For this purpose, different constructs containing either −1 or +1 mutations were expressed in isolated mitochondria. In the case of contiguous editing sites, the C targets are also part of the in organello experiments, it was demonstrated that a region formed by nucleotides −16 to +6 and encompassing the target C constitutes the cis element necessary and sufficient to perform the editing reaction trans-acting factors are involved in their recognition. The length of cis-elements and the importance of the nearest residue in RNA editing were confirmed by other approaches RNA editing is a major posttranscriptional process in plant mitochondria where hundreds of particular cytosine residues are changed into uracil in mRNAs to produce functional proteins. Based on cox2 gene and observed the effect on the editing reaction  depreaction ; (c) depcox2 mRNA could be an indication of the diversity of trans acting factors involved in RNA editing. The discovery of several genes characterized as trans-acting factors and involved in plant mitochondrial RNA editing is in agreement with this observation cis-acting elements trans-acting proteins.The different recognition patterns found for cox2 mRNAs but are unedited in electroporation experiments with the wild type construct  neighboring residues from a majority of C targets were changed on a single construct, most sites were edited with similar efficiency to that found for single mutants . Such a Structural constraints during RNA processing may indeed explain the variation in editing efficiency observed. A survey of the editing status of the complete set of editing sites for each single mutant either at the −1 or +1 position clearly shows that each construct presents different editing patterns Click here for additional data file.Table S1Oligonucleotide sequences used in the study.(DOC)Click here for additional data file.


Translational readthrough of stop codons by ribosomes is a recoding event used by a variety of viruses, including plus-strand RNA tombusviruses. Translation of the viral RNA-dependent RNA polymerase (RdRp) in tombusviruses is mediated using this strategy and we have investigated this process using a variety of in vitro and in vivo approaches. Our results indicate that readthrough generating the RdRp requires a novel long-range RNA-RNA interaction, spanning a distance of ∼3.5 kb, which occurs between a large RNA stem-loop located 3'-proximal to the stop codon and an RNA replication structure termed RIV at the 3'-end of the viral genome. Interestingly, this long-distance RNA-RNA interaction is modulated by mutually-exclusive RNA structures in RIV that represent a type of RNA switch. Moreover, a different long-range RNA-RNA interaction that was previously shown to be necessary for viral RNA replicase assembly was also required for efficient readthrough production of the RdRp. Accordingly, multiple replication-associated RNA elements are involved in modulating the readthrough event in tombusviruses and we propose an integrated mechanistic model to describe how this regulatory network could be advantageous by (i) providing a quality control system for culling truncated viral genomes at an early stage in the replication process, (ii) mediating cis-preferential replication of viral genomes, and (iii) coordinating translational readthrough of the RdRp with viral genome replication. Based on comparative sequence analysis and experimental data, basic elements of this regulatory model extend to other members of Tombusviridae, as well as to viruses outside of this family. Viruses use many different strategies to produce their proteins and some viral proteins are made with terminal extensions that confer unique properties. The polymerase that replicates the RNA genomes of tombusviruses is an extended version of another viral protein and is generated by a process called translational readthrough. We have determined the regulatory mechanism that modulates the production of this viral polymerase. Our results show that control of the readthrough process is complex and involves both local structures and long-range interactions within the viral genome. This system is also integrated with viral RNA replication elements and this allows the virus to coordinate polymerase production with genome replication. This regulatory scheme appears to represent a common tactic used by a variety of viruses. RNA viruses utilize a vast array of coding and gene expression strategies to maximize the utility and regulation of their genomes e.g. Tobacco mosaic virus (TMV) RNA sequences and/or structures next to recoded stop codons have been shown to be important for RT Tombusvirus is the type genus of the family Tombusviridae, which currently includes eight genera Within Tombusviridae, members of the genus Tombusvirus, including the species Carnation Italian ringspot virus (CIRV) In the present study, we used CIRV as a model tombusvirus to study the regulation of RT. Our results revealed that this process is modulated by a novel RNA-based system that includes two long-range RNA-RNA interactions and an RNA switch. The data show a direct link between RT and viral RNA replication, which we propose is important for regulating and coordinating these two processes. Importantly, components of this mechanism are conserved in other members of Tombusviridae and related viruses.An in vitro wheat germ extract (wge) translation system and subsequent sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) were utilized to assess translational RT leading to production of p95 from the CIRV RNA genome. Translation of the wt CIRV genome resulted in the abundant accumulation of p36 and a small amount of a larger product with an estimated molecular mass consistent with that of p95 , lane 2.We next investigated whether the 3'UTR contributed to RT levels in vitro. Relative RT efficiency for each genome was calculated as a ratio of p95/p36 accumulation, with that for wt CIRV set at 100%. This approach also provided an integrated correction for potential differences in general message decay and/or translation initiation rates, because each RT value was calculated relative to its cognate p36 level. When both the 3'CITE and RIV were deleted in mutant CIRV(Eco), the RT efficiency fell to ∼6% , lane 3.selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) Sequences and structures 3'-adjacent to RT sites are often instrumental in facilitating the RT process Two sets of CIRV genomic compensatory mutants designed to disrupt and then restore the putative PRTE-DRTE interaction were generated. The first set targeted a single pair in the proposed interaction , while the second set targeted three pairs simultaneously  . ImportaTo gain additional support for the proposed RNA-based interaction, physical studies were performed. The first was carried out in the context of the wt and mutant CIRV genomes and examined the flexibility of the PRTE region using SHAPE . If the To complement the results above, RNA-RNA electrophoretic mobility shift assays were performed using wt and mutant forms of SL-PRTE and RIV. When wt versions of the two RNA fragments were combined, there was a notable upward shift in mobility and smearing of the SL-PRTE band when compared to SL-PRTE alone , CAAUUA, was inserted just downstream of the p36 stop codon in RT-defective B2 and BS2 (that contained disrupted PRTE-DRTE interactions), thereby creating B2T and BS2T, respectively . IntroduBased on the preceding results indicating a possible role for the long-range interaction in modulating additional viral processes and the fact that the interaction involved a previously characterized RNA replication element, RIV Data from the preceding section suggested that the PRTE-DRTE interaction can modulate viral RNA replication. The known functions of RIV include mediating assembly of the viral replicase Next, to assess the effects of the same mutations on RNA replication that was independent of RT, the same sets of mutations were introduced into DI-7 . When coWhen viral genomes harboring the modifications were assessed for replication in protoplasts, only those containing substitutions predicted to allow for the formation of both SL-T and SL-2 were able to accumulate . OverallTo test the possibility that regulation of RT in the cases above was linked to control over the formation of the PRTE-DRTE interaction, SHAPE analysis in the PRTE region of mutant genome pairs SL-2B, SL-2B-R and SL-TD, SL-TD-R was performed. The results indicated nucleotide reactivities that were in general agreement with the notion of PRTE-DRTE involvement . SpecifiOur inability to demonstrate RT using a dual luciferase reporter mRNA with relevant viral regulatory regions (data not shown) suggested to us that the viral genomic context may be important for RT activity. Indeed, the multiple functional long-range interactions that occur in tombusviruses suggest that proper global folding of the viral genome is critical for proper operation of many viral processes To determine if a similar defect also occurred in vivo, an approach akin to that described in Having established a role for the PRTE-DRTE interaction in the tombusvirus CIRV, we wondered whether this phenomenon also extended to other genera in Tombusviridae. Sequence and structural analysis of the six other genera that utilize RT to express their RdRp revealed the potential for formation of extended SL structures just 3' to their RT sites that contained bulges with sequences that were complementary to 3'-terminal sequences in their viral genomes. Examples of these sequences and predicted structures for the best-studied members of these six genera are presented in To provide some experimental support for this proposal, we selected one virus genus to test the concept. The carmovirus TCV was selected as: carmovirus represents the largest genera in Tombusviridae; previous studies attempted, unsuccessfully, to define the RT elements in TCV Diverse arrays of viruses utilize translational RT in their gene expression strategy, including the majority of the members of the large virus family Tombusviridae i.e. ≥ ∼1 kb) RNA-RNA interaction to be identified in tombusviruses (i.e. an RNA switch). Such RNA switches have been implicated in the regulation of a variety of processes in viruses We have identified and characterized SL-PRTE as a structure located 3'-proximal to the CIRV RT site that is necessary for RT-based translation of p95 . Howeversviruses . Interessviruses . As suchsviruses . Additiosviruses . Anothersviruses . Such a sviruses , and sinIn addition to the long-range interaction and RNA switch, our study uncovered an additional level of control. Efficient RT also requires the UL-DL long-distance interaction that is important for RNA replication. This represents a second link between RT and RNA replication. The specific role of the UL-DL in facilitating RT will be investigated in detail in subsequent studies, however its general proximity to the SL-PRTE and RIV suggests that it could act to bring together these RNA elements in the folded genome . This coThe production of RdRp is a critical step in the replication cycle of CIRV and other plus-strand RNA viruses An obvious question regarding RT in CIRV is: why is an essential RNA element for RT located at the 3'-end of the virus genome? Several possible explanations, which are not necessarily mutually exclusive, come to mind. First, the terminal location of the DRTE may provide a quality control function during virus reproduction. That is, little or no RdRp would be produced from viral genomes that are missing their 3'-termini due to incomplete replication or ribonuclease attack. Such a safety check for genome completeness would be very sensitive for cases like TCV, where the DRTE is located at the extreme end of the viral genome .A second possible role for the distal location of the DRTE could be related to promoting cis-preferential replication, where the RdRp acts on the genome from which it is translated. This phenomenon has been reported for Red clover necrotic mosaic virus  A third reason for the 3'-terminal location of the DRTE, which is related mechanistically to that just described, could be to coordinate translation with RNA replication. Initially, in the RT mode, the inactivation of the replication function of RIV would help to prevent viral replicase initiating RNA synthesis in the 3'-to-5' direction on the genome during translation of the RdRp  that inhibits translation release factor function or promotes suppressor tRNA utilization. Further studies are ongoing to investigate such prospects.This is the first report demonstrating the requirement for a long-range RNA-RNA interaction (spanning ∼3.5 kb) for RT in a plus-strand virus. However, based on sequence analysis  and expeUAGGGGUGU) UGAC In terms of the relative timing of appearance of the long-range interactions during virus evolution, we envision the initial RT signal as being local. Subsequently, through random events and “testing” of various alternative long-range interactions, the original local structure would then morph into one incorporating a distal element, due to associated fitness gains. The long-range RNA-RNA interaction proposed for RT of the coat protein in Barley yellow dwarf virus (BYDV) Interestingly, functional long-distance RNA-RNA interactions spanning ∼4 kb and ∼2.6 kb, respectively, have been described for the frameshifting leading to RdRp production in the Luteovirus BYDV (family Luteoviridae) The full-length infectious clone of CIRV has been described previously The construction of DI-7 was based on the sequence of a naturally-occurring CIRV DI RNA Construction of TCV mutants was performed using the genomic construct T1D1 The genome sequences of AMCV (X62493), CBLV (NC_004725), CIRV (NC_003500), CuNV (NC_001469), CymRSV (NC_003532), GALV (AY830918), PLV (NC_004723), PNSV (AJ607402), TBSVc (NC_001554), TBSVnf (AY579432), TBSVp (U80935), and TBSVs (AJ249740) were obtained from the NCBI GenBank database. RNA secondary structures were predicted at 37°C using Mfold version 3.5 5 cucumber protoplasts were transfected with RNA transcripts  and incubated at either 22°C  or 26°C  for 22 hr. A minimum of three trials were conducted for each experiment. Total nucleic acid was isolated post-incubation and Northern blot analyses were conducted to detect plus-strand viral RNAs as described previously 32P-labeled DNA probes complementary to the 3'UTR of the genomes were used for viral detection and bands on membranes were quantified using a PharosFX Plus Molecular Imager (BioRad) and QuantityOne software (BioRad).Uncapped RNA transcripts were generated from DNA plasmids linearized with SmaI  or PCR templates  with a T7 promoter as described previously 35S-Met using nuclease-treated wheat germ extract (Promega) was performed as described previously Translation of sub-saturating concentrations of viral in vitro transcripts (0.5 pmol) in the presence of 2, 3.8% glycerol) in a total volume of 8 µL. Samples were subsequently cooled at 4°C for 10 min, mixed with loading buffer , and separated at 4°C in a nondenaturing 15% polyacrylamide gel containing 6 mM MgCl2 using 1X TBE running buffer containing 6 mM MgCl2. Gels were then stained with ethidium bromide to visualize bands and the negative image is shown in RNA-RNA electrophoretic mobility shift assays were performed as described previously 2, 333 mM HEPES pH 8.0) was added, and samples were incubated at 37°C for 30 min. 45 µL of refolded RNA was treated with 5 µL of 50 mM 1-methyl-7-nitroisatoic anhydride (1M7) in dimethylsulfoxide (DMSO) at 37°C for 4 min. As a negative control, an additional 45 µL of refolded RNA was treated with 5 µL of DMSO under the same conditions. RNA was recovered by ethanol precipitation  and the precipitated RNA was resuspended in 50 µL of 0.5X TE. To achieve detection of 2’-O-adducts by primer extension, a procedure modified from Wilkinson et al. 2, 167 mM Tris-HCl pH 8.3, 1.67 mM each dATP, dCTP, dITP, dTTP placed at 50°C for 2 min) and SuperScript III  were added and extension was carried out for 30 min at 50°C. For sequencing purposes, 20 µL of refolded RNA was combined with primer (6 µL containing 4 µM WellRED D2 or 4 µM IRDye 800), enzyme mix (6 µL), ddGTP  or ddTTP , respectively, SuperScript III  and primer extension was carried out. One set of primers were used that are complementary to CIRV positions 1320-1339 (5’-CCACAACCTACCAAAGGAGC). To terminate reactions, 3 M NaOAc pH 5.2 (4 µL) was added to each reaction and all reactions were combined and precipitated at -80°C for 15 min with ethanol (320 µL) and glycogen (2 µL). Pellets were washed twice with 70% ethanol, dried for 10 min at 37°C, and resuspended in 45 µL of deionized formamide. Raw fluorescence intensity versus elution time profiles were determined as described previously SHAPE analysis was performed on in vitro transcribed CIRV genomes and DI RNAs essentially as described previously Figure S1SHAPE analysis of SL-PRTE RNA. SHAPE analysis was performed on the wt CIRV genome and the results were mapped onto the mfold-predicted RNA structure for SL-PRTE. Relative reactivity of each residue is indicated by the color-coded key, with higher values corresponding to increased flexibility.(TIF)Click here for additional data file.Figure S2Comparative RNA sequence and secondary structure analysis of SL-PRTE and the PRTE-DRTE interaction. (A) The CIRV SL-PRTE secondary structure and (B) CIRV PRTE-DRTE interaction are shown along with sequence variations found in other tombusviruses. Nucleotides involved in the PRTE-DRTE interaction are shown in green and the p36 stop codon is in bold and underlined. Gray shading indicates CIRV nucleotides that are substituted by different nucleotides in other tombusviruses, with the corresponding identity of the virus indicated in brackets. The virus acronyms are as follows: AMCV, Artichoke mottled crinkle virus: CBLV, Cucumber Bulgarian latent virus; CuNV, Cucumber necrosis virus; CymRSV, Cymbidium ring spot virus; GALV, Grapevine Algerian latent virus; LNV, Lisianthus necrosis virus; PLV, Pear latent virus; PNSV, Pelargonium necrotic spot virus; TBSVc, Tomato bushy stunt virus (cherry isolate); TBSVnf, Tomato bushy stunt virus (nipple fruit isolate); TBSVp, Tomato bushy stunt virus (pepper isolate); TBSVst, Tomato bushy stunt virus (statice isolate).(TIF)Click here for additional data file.Figure S3RdRp activity is required to complement RT-deficient mutants. Northern blot analysis and quantification of DI-7 accumulation when cotransfected with non-replicating CIRV genomes in plant protoplasts. The CIRV genomes cotransfected with DI-7 are indicated below each bar in the graph. The mutants tested are described in (TIF)Click here for additional data file.Figure S4Importance of the DRTE for DI-7 replication. (A) Predicted secondary structure for RIV with the DRTE shown in green. The substitutions and deletion made in the DRTE are shown below the structure. (B) Northern blot analysis and quantification of wt and mutant DI-7 RNA accumulation when cotransfected with wt CIRV in plant protoplasts. DI-7 RNA levels were analyzed and measured by Northern blot analysis 22 hr post-transfection of plant protoplasts. The relative values shown in the bar graph below the lanes correspond to means (± standard error) from three independent experiments.(TIF)Click here for additional data file.Figure S5RT-defective genomes are not rescued by p95 supplied in trans. Northern blot analysis and quantification of CIRV genomes analyzed 22 hr post-transfection of plant protoplasts. The identities of the viral genomes are shown above the lanes. Cotransfection of CIRV genome mutants B2 or BS2 with non-replicating RTRd (providing p95) did not lead to rescue of genome replication. The relative values for genome accumulation shown below the lanes correspond to means (± standard error) from three independent experiments.(TIF)Click here for additional data file.Figure S6Analysis of CIRV genome containing an engineered MluI restriction. (A) SDS-10%PAGE analysis of p36 stop codon RT from wt CIRV and CIRV containing an MluI restriction site (CIRV MluI). The p95:p36 ratio was determined for each lane, and the relative RT percentages below each lane correspond to means (± standard error) from three independent experiments. (B) Northern blot analysis and quantification of plus-strand accumulation of viral RNAs from CIRV WT and CIRV MluI transfections of plant protoplasts. The positions of the genomic (g) and subgenomic mRNAs (sg1 and sg2) are indicated to the right of the blot. Viral RNAs were analyzed 22 hr post-transfection and the relative values for genomic accumulation below the lanes correspond to means (± standard error) from three independent experiments.(TIF)Click here for additional data file.


As our appreciation increases for the pervasive nature of transcription in the cell, so too has our appreciation for the major role of RNA decay/stability in regulating both the quantity and the quality of gene expression. As soon as viral RNAs appear in the cell, they must be prepared to combat or avoid cellular RNA decay pathways. This review describes the myriad ways that viruses deal with the general host RNA decay machinery that is active in the cell immediately upon viral infection—turning what, at first, appears to be very hostile territory for a foreign transcript into a sort of “promised land” for viral gene expression. It is interesting to note that cells likely try to adapt to this viral interference with the general RNA decay machinery by inducing a variety of novel RNases as part of a molecular arms race.The cellular RNA decay machinery constantly monitors transcripts, from the time they are synthesized in the nucleus until the end of their lifespan in the cytoplasm. Aberrant products of transcription initiation (e.g. PROMPTS), capping, and termination are quickly degraded by nuclear RNA quality control surveillance complexes. Misfolded, “mis”-translated (e.g. mRNAs with a premature termination codon), and mispackaged mRNAs are also quickly degraded in the cytoplasm. In addition to removing aberrant mRNAs, up to 50% of cellular gene expression may be controlled by changes in mRNA stability. When a typical cellular mRNA is targeted for decay, it initially undergoes deadenylation—the removal of the 3′ poly(A) tail. The mRNA is then subject to processive exonucleolytic degradation in either the 3′-5′ direction by the exosome or Dis3L2, or it is marked by the LSm1-7/Pat1 complex for decapping by Dcp1/2 and degraded in the 5′-3′ direction by Xrn1 When the transcripts of cytoplasmic viruses are generated, they must actively avoid or overcome the assault by these aggressive cellular mRNA decay complexes in order to be translated and effectively generate virions. It should be easy for the cellular RNA decay machinery to recognize these foreign transcripts—typical host mRNAs, for example, are assembled into characteristic ribonucleoprotein complexes in the nucleus, but the RNAs of cytoplasmic viruses never have this opportunity. In addition, some viral RNAs do not have 5′ caps or poly(A) tails, and some have multiple open reading frames or long 3′ UTRs, which should target them for nonsense-mediated decay. Yet, the transcripts generated by cytoplasmic viruses survive and flourish in this hostile cytoplasmic environment. Interestingly, viruses do more than simply mimic host mechanisms like polyadenylation, triple helix structures, and 5′ capping to protect their transcripts from host exonucleases Several cytoplasmic viruses directly repress key aspects of the cellular RNA decay machinery to promote viral RNA stability. Picornaviruses use an aggressive mechanism for suppression of host RNA decay factors. Xrn1, Dcp1, Dcp2, Pan3 (a deadenylase), and AUF1 (a factor that targets RNAs for decay) are rapidly degraded during poliovirus or human rhinovirus infections by viral proteases and/or the host cell proteasome Paradoxically, several cytoplasmic viruses even turn a host RNA decay factor into a stabilizing factor. Several viruses have developed a way to steal the host LSm1-7 complex that normally marks deadenylated transcripts for 5′-3′ degradation. Brome Mosaic virus genomic RNA has internal poly(A) tracts and tRNA-like structures in the 3′ UTR that facilitate LSm1-7 binding to promote viral translation and replication Arenaviridae, Bunyaviridae, and the nuclear Orthomyxoviridae families steal the 5′ capped ends of host mRNAs to incorporate this cis-acting stability element into their own transcripts trans-acting factors that stabilize host transcripts are also purloined by thieving viral RNAs.It has been known for some time that members of the The cellular HuR protein is a well-characterized shuttling factor that promotes the stability of mRNAs by interacting with U-rich elements. Alphaviruses contain highly conserved U-rich elements or other high-affinity HuR binding sites in the 3′ UTR of their RNAs that bind HuR during infection to promote viral RNA stability and efficient virus production Virally encoded endonucleases are important for many aspects of viral replication, including the fine-tuning of viral gene expression by rapidly depleting old viral mRNAs to enhance the expression of newly transcribed mRNAs Considering the importance of RNA stability in regulating transcript abundance, the inactivation or commandeering of cellular RNA decay factors by viruses is likely to significantly alter host gene expression. How might changes in host mRNA stability contribute to virus-induced pathology during infection ? One exaViral RNAs have evolved a wide variety of mechanisms to successfully interface with the host RNA decay machinery. In fact, some of the most important questions in this field have yet to be answered. What are the consequences of viral inactivation of decay factors like Xrn1 in terms of disease? Can viruses also influence host transcription by manipulating RNA decay pathways to short-circuit feedback regulatory mechanisms? How do virus-induced changes in RNA decay pathways interface with potential changes in innate immune responses?Virus families often use conserved strategies to evade the cellular RNA decay machinery. Therefore, perhaps researchers can develop effective, broad-spectrum antivirals to disarm these strategies and destabilize viral RNAs. Future research in this burgeoning field will likely uncover novel mechanisms of the regulation of host and viral gene expression and facilitate new methods for treating viral diseases.


Human blood platelets are essential to maintaining normal hemostasis, and platelet dysfunction often causes bleeding or thrombosis. Estimates of genome-wide platelet RNA expression using microarrays have provided insights to the platelet transcriptome but were limited by the number of known transcripts. The goal of this effort was to deep-sequence RNA from leukocyte-depleted platelets to capture the complex profile of all expressed transcripts.From each of four healthy individuals we generated long RNA (≥40 nucleotides) profiles from total and ribosomal-RNA depleted RNA preparations, as well as short RNA (<40 nucleotides) profiles. Analysis of ~1 billion reads revealed that coding and non-coding platelet transcripts span a very wide dynamic range (≥16 PCR cycles beyond β-actin), a result we validated through qRT-PCR on many dozens of platelet messenger RNAs. Surprisingly, ribosomal-RNA depletion significantly and adversely affected estimates of the relative abundance of transcripts. Of the known protein-coding loci, ~9,500 are present in human platelets. We observed a strong correlation between mRNAs identified by RNA-seq and microarray for well-expressed mRNAs, but RNASeq identified many more transcripts of lower abundance and permitted discovery of novel transcripts.http://cm.jefferson.edu/platelets_2012/.Our analyses revealed diverse classes of non-coding RNAs, including: pervasive antisense transcripts to protein-coding loci; numerous, previously unreported and abundant microRNAs; retrotransposons; and thousands of novel un-annotated long and short intronic transcripts, an intriguing finding considering the anucleate nature of platelets. The data are available through a local mirror of the UCSC genome browser and can be accessed at: Platelets that exhibit functional extremes convey a commensurate increased risk for bleeding or thrombosis. Notably, the propensity for such extremes has been shown to be heritable-3. Nonetregation,5, but tThe platelet transcriptome is a reflection of the megakaryocyte RNA content at the time of (pro)-platelet release, subsequent splicing events, selective packaging and platelet RNA stability, and can provide important insights into platelet biology. PlateleThe emerging important roles that non-coding RNAs (ncRNAs) play in a cell, their ihttp://cm.jefferson.edu/platelets_2012/.Because the content and properties of nuclear and cytoplasmic transcripts vary-29, the We carried out transcriptome sequencing of total RNA isolated from leukocyte-depleted platelet (LDP) preparations from four healthy adults . LDPs were prepared by density centrifugation of citrated whole blood followed by immunodepletion of CD45+ leukocytes. This prThe reads from each of the 12 generated datasets were mapped separately on each chromosome and strand of the human genome (assembly hg19) using the BWA program and the within a read-set and to compare expression levels across read-sets. This β-actin transcript was quite abundant in platelets, present at approximately 15.0 ± 1.5 cycles of PCR containing the equivalent of 10 ng of total RNA, and shows the least amount of variation (± ~3%) across the analyzed samples . It will be important for future platelet RNAseq studies with larger numbers of subjects to confirm these observations pertaining to the isoforms of these two commonly utilized platelet “normalizers.”We devised a scheme (see Methods) for estimating the expression levels of protein-coding transcripts from RNA-seq reads. To estimate transcript abundance, we normalized for transcript length and scaled using the expression levels of the β-actin isoform with ENSEMBL identifier ENST00000331789. This scheme was very effective (see below) and provided us the ability to appropriately scale expression We used the most abundant isoform among those derived from an individual protein-coding gene to represent the gene. Figure We sought to determine the correlation between our RNA-seq normalization approach and qRT-PCR on the same RNA samples. We queried 2 collections of genes: 1) 10 transcripts that exhibited a broad range  of normalized read counts, 6 of which are well-studied in platelets; and, 2) 89 transcripts for GPCR signaling proteins from a commercial platform, 19 of which were detected using both RNA-seq and qRT-PCR. Figure We also compared protein-coding transcripts from RNA-seq data with previously published microarray datasets of the platelet protein-coding transcriptome,17,32. T-10 times that of β-actin, and kept only those whose absolute ratio value was ≥ 2× between the two preparations. Unexpectedly, the number of affected protein-coding genes was high, ranging from 745 (sample 4N4) to 2,341 (sample 2N2) genes  of the ENSEMBL database. We analyzed each of the four samples and three preparations  separately. For the long RNA read sets, we considered intronic real estate if and only if platelet reads covered a minimum of 100 consecutive nucleotides and the covered region had an estimated abundance relatively to ACTB of 1:1024 . For the short RNA read sets, we only considered platelet reads mapping to intronic real estate if they were at least 30 nucleotides long and had an estimated abundance relatively to SNORD44 of 1:64 . Given the high stringencies of length and abundance, we accepted such a region if at least one of the sequenced samples showed evidence for it. Across the four samples and two long RNA preparations, we found a total of 6,992 bona fide intronic regions that give rise to currently uncharacterized long RNA transcripts satisfying the above constraints. We also found an additional 1,236 bona fide intronic regions that give rise to currently uncharacterized short RNA transcripts satisfying the above constraints. Notably, these two collections of intronic regions had only 18 members in common suggesting that the two novel populations of (long and short) uncharacterized bona fide intronic transcripts originate from distinct genomic loci. Additional fileOur work uncovered extensive evidence for the existence of transcripts that originate in the introns of known protein-coding genes. This is of particular significance considering that platelets lack a nucleus. For such an analysis it is imperative to distinguish hsa-miR-33b, hsa-miR-101, hsa-miR-191, hsa-miR-219-2, hsa-miR-374b, hsa-miR-486, hsa-miR-625, hsa-miR-766, hsa-miR-3135b, and hsa-miR-4433. The short platelet RNAs we observed had lengths typical of a miRNA and were transcribed from the strand opposite of that of the known miRNA precursor. Each of the loci listed above generated one or two distinct antisense transcripts, presumably a mature miRNA and its “star” miRNA. There was also a high prevalence of transcripts that were antisense to known protein-coding regions of the genome. Table ′UTRs, 3′UTRs or full-length exonic space of known protein-coding transcripts. Enrichment values are notable, independently of whether we computed them in terms of span (which ignores the number of reads sequenced from a genetic locus) or in terms of support (which takes into account the number of reads sequenced from a genetic locus). Unexpectedly, our analyses revealed notable enrichment in both long and short platelet RNAs that were antisense to several known repeat families. Table Our analysis also revealed the presence of a substantial number of long and short platelet transcripts that were antisense to known miRNAs, known protein-coding exons, and notably, to known repeat element families. For the miRNA analysis, we processed separately the four read sets from the short RNA preparation. For the protein-coding transcript analysis, we processed separately the eight read sets from the total and rRNA-depleted preparations. For the repeat element analysis, we processed all read sets separately for each of the four sequenced individuals. The following are the 10 miRNA precursors with previously unreported antisense transcripts: We use the characterization ‘orphan’ to refer to those RNA-seq reads that could not be mapped on the human genome using our default parameter settings. To ensure that we exhausted the possibilities, and in an effort to address the potential identities of unmapped transcripts, we conducted additional read mapping with alternative computational settings and using curated datasets.First, and in light of recent reports of extensive editing of RNA transcripts we used Since we used the full genome’s sequence to map the sequenced reads the formal possibility remains that perhaps a significant portion of the orphan reads originate from the exon-exon junctions of spliced protein-coding transcripts. Thus, our subsequent investigation used the 598,379 exons listed in Release 63 of ENSEMBL to combinatorially enumerate all possible exon-exon junctions using the known, non-overlapping exons of all 51,055 protein-coding and non-protein-coding genes contained in the Release. This gave rise to 12,382,819 junctions on which we attempted to map the orphan reads. Across all read sets that were sequenced from the total RNA preparations, an average of 185,026 reads were mapped onto the exon-exon junction set. The corresponding number for the sets obtained from the rRNA-depleted preparations was 191,736 reads. In both cases, only a very small fraction of the reads mapped to exon-exon junctions.Lastly, we examined the possibility that the orphan reads originate from the highly polymorphic human leukocyte antigen (HLA) region of chromosome 6. To this end, we used the 6,944 sequences contained in Release 3.5 of the IMGT/HLA database,42 and shttp://cm.jefferson.edu/platelets_2012/.Results have been embedded in a local mirror of the UCSC genome browser and can be examined interactively athttp://www.ncbi.nlm.nih.gov/sra. The data sets supporting the results of this article are included within the article and its additional files.The data set supporting the results of this article is available in the NCBI/GEO repository, accession number SRA062032,A prominent lesson that has emerged from the 1000 Genomes Project is the greater genetic variation in the population than previously appreciated. Transcriptomics is rapidly assuming a more prominent role in the understanding of basic molecular mechanisms accounting for variation within the normal population and inherited disease. We have sequenced RNA from the leukocyte-depleted platelets of four healthy individuals and report our findings from the analysis of the long and short RNA transcript populations. In the case of long RNAs, we carried out sequencing of both total and rRNA-depleted RNA. The generated data, accompanying genome browser, and data repository detail the totality of RNA species present in the anucleate human platelet. We are unaware of prior efforts that have provided as comprehensive a transcriptome evaluation of any human cell as offered in our report. Our approach serves as a roadmap for future transcriptome analyses and the findings have important implications for the understanding of the transcriptome and the role of platelets in health and disease.bona fide intronic transcripts, novel short and long RNAs, transcripts antisense to exons and antisense to miRNAs. Our data are publicly available and can be explored interactively through our local mirror of the UCSC genome browser athttp://cm.jefferson.edu/platelets_2012/.We utilized a distinct approach to the elucidation of the platelet transcriptome that, as we discovered, exhibits an extraordinary complexity. Features of our approach include: 1) the use of the anucleate platelet that decouples the nuclear and cytoplasmic transcriptomes; 2) the use of total RNA and not poly-A enriched RNA; 3) the use of a next generation sequencing platform (AB/LT SOLiD) that generated the high enough read numbers needed to provide the required resolution power; 4) the explicit evaluation of the impact of the ribosomal RNA depletion step prior to sequencing; 5) an enhanced mapping protocol that ensured exhaustive mapping of the sequenced reads on the un-masked human genome and the exclusion of reads that could not be mapped uniquely; and, 6) the explicit search for the presence or absence of RNA species that either have not been previously discussed in the context of platelet biology or that are not currently annotated in the public databases. Findings from our analyses reveal a much more diverse platelet transcriptome than previously appreciated, and include pseudogenes, repeat elements, Blood platelets originate from bone marrow precursor megakaryocytes. As such, most platelet RNA results from the transcription of nuclear DNA in the megakaryocyte, and thus reflects the status of the megakaryocyte at the time of platelet release into the circulation. Notably, megakaryocytes from human bone marrow are neither routinely nor easily accessible for biological studies. Megakaryocyte gene transcription responds to numerous normal physiologic and pathologic stimuli. Additionally, anucleate platelets are known to engage in both post-transcriptional processing of RNA and translation of mRNA into protein, in response to external factors,43. Consknown miRNAs or limited sets of known mRNAs. Of course, any RNA transcriptome analysis must be considered in the context of potential differences with megakaryocytes. Recently, platelet RNA-seq successfully revealed abnormal splicing events in 1) NBEAL2, thus identifying the gene responsible for the Gray Platelet Syndrome[RBM8A, thus uncovering the gene responsible for the TAR (=thrombocytopenia and absent radii) syndrome[Compared to other RNA-evaluating technologies, the current limitations of RNA-seq in general and as applied to platelets are the expense and the need for sophisticated computational analyses that have not yet been standardized or made widely available. As experience with the method progresses and prices drop, these limitations will be offset by the advantages of superior dynamic range, the discovery of novel transcripts, and the simultaneous assessment of expression levels, sequence variants and splice variants, none of which can be achieved using conventional probe-based transcript analysis. A direct digital detection technology (referred to as “Nanostring”) offers tSyndrome, and, 2)syndrome. Our datRNA-seq is highly sensitive and capable of detecting variability between samples caused by biological differences, technical variation, or environmental influence during sample handling. The samples in our study were processed using a methodology with excellent reproducibility that minIt is difficult to compare and contrast our study with that of Rowley et al. because Comparison of our data to microarray results, both ours and those in the public databases, showed RNA-seq to have significant correlation with microarray for the subset of abundant protein-coding RNAs. GO analyses indicated that the expressed mRNAs were enriched in terms such as coagulation, platelet degranulation, secretion, cytoskeletal dynamics, receptor binding and G-protein signaling. These analyses validate and support RNA-seq and our analytic approach as appropriate for assessing the platelet transcriptome.In this work we confirm and, more importantly, extend earlier platelet transcriptome studies by us and others-12,15 inDepletion of ribosomal RNA is considered a standard approach in RNA-seq studies of nucleated cells. Driving the choice is the observation that rRNA makes up ~75-80% of the total amount of cellular RNA. To the best of our knowledge, the impact of rRNA depletion has not been previously studied, certainly not in the context of platelet transcriptome analyses. Importantly, we found that rRNA depletion strongly and adversely impacts the characterization of platelet protein coding transcripts. Indeed, rRNA depletion resulted in variations in abundance estimates that confounded meaningful analyses across samples. Neither we nor others in the field have ascertained the underlying mechanism by which rRNA depletion alters relative mRNA abundances. Our finding does not appear to be a non-specific artifact: not only is the dynamic range of the observed impact very significant but the number of the affected mRNAs is large. In previous work, several authors noted that platelets dock mRNAs to ribosomes and that this process can be selective for features of specific mRNAs,51, so ishort RNAs that are antisense to many different repeat families. Naturally occurring antisense transcripts are important regulators of gene expression via interference with translation, RNA masking, etc.[Our analyses unexpectedly revealed the existence of numerous transcripts that are antisense to previously annotated genomic regions. In particular, we discovered consistent enrichment in RNAs that are antisense to the exons of known protein-coding loci across the four healthy donors. We also found enrichment in long RNAs that are antisense to known repeat families. Notably, we found even more pronounced enrichment in ng, etc., and ourWe also discovered a larger number of platelet miRNAs than previously reported, and separately characterized and reported on the two potential products that can be transcribed from a microRNA precursor. Importantly, for 10 miRNA loci, we found and report evidence of transcription of short, miRNA-like-in-length RNAs that are antisense to the known miRNA or its “star” species. Not only are these miRNA antisense species not currently contained in miRBase, but to the best of our knowledge, they have not been reported previously in the platelet context.intronic DNA genomic regions and are not currently annotated in the public repositories as known non-protein-coding transcripts from protein-coding loci, miRNAs, ribosomal RNAs, tRNAs, repeat elements, etc. In particular, the intronic loci that give rise to the sequenced long platelet RNAs are distinct from the intronic loci that give rise to the sequenced short platelet RNAs. It is important to stress that we required a minimum span of 100 nucleotides for those intronic regions that gave rise to the un-annotated long RNAs and a minimum span of 30 nucleotides for those intronic regions that gave rise to the un-annotated short RNAs. Such strict criteria suggest that the actual sources of un-annotated intronic platelet transcripts are more numerous. Although it is possible that intergenic RNAs or transcripts with retained introns are not functional in platelets, there is accumulating evidence that intronic regions likely play rather involved and functionally significant roles in a cell[An additional intriguing finding pertains to our identification of both short and long RNA transcripts that originate from thousands of n a cell-58. Thesn a cell,46 suggeper se is not new and has been reported previously, e.g.[One of the unexpected findings that emerged from our work and analyses is the pervasive presence of long and short RNAs that are both sense and antisense to the genomic locations of many families of repeat elements, and nearly all known pseudogenes. The presence of expressed repeat elements ly, e.g.-61. Addily, e.g.-66. Howely, e.g.,68; the ly, e.g.,70; the ly, e.g.-22,71,72ly, e.g.-75; etc.Our work has revealed a highly complex transcriptional landscape for the anucleate human platelet. The richness and diversity of the present RNA molecules suggests a context where platelet biology transcends protein- and miRNA-centric descriptions. By making available our findings we aim to facilitate the elucidation of previously unappreciated molecular species and molecular interactions. This will eventually permit an improved understanding of the molecular mechanisms that regulate platelet physiology and contribute to serious disorders of thrombosis, hemostasis, and inflammation.®  and RNA quality was assessed by the Agilent bio-analyzer PICO chip. In some experiments, 2 μg of total RNA was depleted of large 18S and 28S rRNA as well as 5S and 5.8S using the RiboMinus Eukaryotic Kit (Invitrogen), which uses biotinylated probes designed against these rRNAs (following the manufacturer‘s protocol). Total RNA and rRNA-depleted RNA were fragmented using RNAse III digestion for 13 min in a 10 μl reaction containing 1 μl of 10X RNAse III buffer and 20 U of RNAse III. After incubation the RNA was purified using the RiboMinus Concentration Module (Invitrogen), and the size and yield of RNA was determined using the Agilent bio-analyzer PICO chip. Library construction, emulsion PCR, workflow analysis and sequencing runs were performed following standard AB/Life Technologies protocols. A typical sequence run generated ~100 million reads of 50 nt each for long RNA and 30 nt long for short RNA, with the “strandedness” of the read on genomic DNA preserved.The study was approved by the Institutional Review Board of Thomas Jefferson University, and informed consent was obtained from all participants. Highly purified, leukocyte-depleted platelet (LDP) preparations were obtained as previously described. RNA extcutadapt utility[http://cm.jefferson.edu/platelets_2012/ by navigating to the genomic locus of interest; reads mapped to the forward strand are shown in blue, and reads mapped to the reverse strand in red.Sequenced reads were mapped onto the human genome assembly hg19 using the Burrows-Wheeler Alignment (BWA) algorithm. Reads s utility. During T, we determined the number of reads that uniquely mapped to T’s exons; distinct sequenced reads that mapped to the same genomic/exonic location of T were counted multiply. We defined the normalized expression (neT) of T by the ratio (cT/LT)/(cβ-actin/L-actinβ), where cT was the read count for T, cβ-actin was the read count for the β-actin transcript, and LT and Lβ-actin were the respective lengths of each transcript. For genes with multiple known protein-coding transcripts, the gene was assigned the neT value of its most abundant transcript. As described in the Results, we established that β-actin mRNA is abundantly and consistently expressed across samples. Throughout this study, we used the β-actin transcript with ENSEMBL identifier ENST00000331789 and the relationship of neT to that of neENST00000331789 to determine the presence or absence of protein-coding transcript T and, by extension, of the expression of the parent gene.For each protein-coding transcript SNORD44 as reference. This choice was informed by the abundance and apparent stability of SNORD44’s expression across many tissues and cell lines[For non-coding transcripts, we used the same approach but instead of β-actin we used the levels of the small nucleolar RNA ll lines.-ΔΔCT method normalized to β-actin[One microgram total RNA was reverse transcribed and 1% of the resulting cDNA  was used in the PCR. Quantitative reverse transcriptase PCR (qRT-PCR) results using primers specific for known platelet genes and for a panel of 89 genes encoding G-protein-coupled receptors are described in the Supplement. mRNA levels were assessed by the 2 β-actin.2-normalized expression of each long total RNA transcript across the 4 samples was ranked by transcript abundance and compared to published platelet transcript profiles obtained on Affymetrix GeneChip[The average logGeneChip and IlluGeneChip,17. A SpTo characterize the human platelet transcriptome with regard to possible over-representation of transcripts of a specific type, enrichment analysis was performed using the coordinates of those RNA-seq reads from both long and short total platelet RNA transcriptomes that could be mapped on the genome and the genomic coordinates of categories of transcripts as these are reported in the ENSEMBL database.The authors’ declare that they have no competing interests.PFB, SEM, PF, and IR conceived of the study, and participated in its design and coordination. IR, PFB, PF, SEM, and LCE wrote the manuscript. LCE participated in the design and coordination, and acquired and analyzed the data. IR, H-WC, EL, PL, AE, and YJ acquired and analyzed the data. SN, KD, and JK participated in the design and acquired data. All authors read and approved the final manuscript.Figure S2. The importance of distinguishing between the two products of a miRNA precursor. Table S1. Optimal choice of transcript for normalizing protein-coding expression. Table S2. Protein-coding transcripts in each of the four samples. Title and legend only Table S3. Impact of ribosomal RNA depletion on the estimate of mRNA abundance. Title and legend only. Table S4. MicroRNA transcripts in each of the four samples. Title and legened only. Table S5. Pseudogene expression in platelets. Table S6. Long platelet transcripts from repeat element regions. Table S7. Short platelet transcripts from repeat element regions. Table S8. Coordinates of protein-coding intronic regions that are not accounted for by the available annotations in the public databases. Table S9. Long platelet transcripts antisense to repeat element regions. Table S10. Mapping statistics for very lenient settings.Gene Ontology (GO) analysis of the platelet transcriptome from total RNA by RNASeq. Click here for fileProtein-coding transcripts in each of the four samples. List of the protein all coding transcripts whose expression is supported by the collected RNA-seq data. List for the four total RNA samples. In an effort to be conservative, and based on the data in Figures 1 and 2, we only considered protein-coding transcripts with an estimated abundance that was ≥ 2-10 times that of β-actin, and kept only those whose absolute ratio value was ≥ 2x between the two preparations.Click here for fileProtein-coding transcripts in each of the four samples. List of the protein all coding transcripts whose expression is supported by the collected RNA-seq data. List for the four rRNA-depleted samples. In an effort to be conservative, and based on the data in Figures 1 and 2, we only considered protein-coding transcripts with an estimated abundance that was ≥ 2-10 times that of β-actin, and kept only those whose absolute ratio value was ≥ 2x between the two preparations.Click here for file-10 times that of β-actin (ENST00000331789) and whose normalized expression differs by ≥ 2x (i.e. at least one log2 unit) between the preparations are listed.Impact of ribosomal RNA depletion on the estimate of mRNA abundance. The table shows the base-2 logarithm of the ratio of the normalized expression between the total and rRNA-depleted preparation, and for each of the four individuals that provided the sequenced RNA. Only transcripts with an estimated abundance that is ≥ 2Click here for fileMicroRNA transcripts in each of the four samples. List of all microRNAs whose expression is supported by the collected RNA-seq data.Click here for fileSupplemental Materials. Genomic coordinates and sequences of unannotated intronic regions that are found transcribed in human platelets. These are culled from both the long and short RNA-seq profiles.Click here for file