We found the corridor between intertidal marsh and oyster reef breakwaters supported higher abundances and different communities of fishes than control plots without oyster reef habitat. Among the fishes and mobile invertebrates that appeared to be strongly enhanced were several economically-important species. Blue crabs  were the most clearly enhanced (+297%) by the presence of breakwater reefs, while red drum (Sciaenops ocellatus) (+108%), spotted seatrout (Cynoscion nebulosus) (+88%) and flounder  (+79%) also benefited. Although the vertical relief of the breakwater reefs was reduced over the course of our study and this compromised the shoreline protection capacity, the observed habitat value demonstrates ecological justification for future, more robust shoreline protection projects.Shorelines at the interface of marine, estuarine and terrestrial biomes are among the most degraded and threatened habitats in the coastal zone because of their sensitivity to sea level rise, storms and increased human utilization. Previous efforts to protect shorelines have largely involved constructing bulkheads and seawalls which can detrimentally affect nearshore habitats. Recently, efforts have shifted towards “living shoreline” approaches that include biogenic breakwater reefs. Our study experimentally tested the efficacy of breakwater reefs constructed of oyster shell for protecting eroding coastal shorelines and their effect on nearshore fish and shellfish communities. Along two different stretches of eroding shoreline, we created replicated pairs of subtidal breakwater reefs and established unaltered reference areas as controls. At both sites we measured shoreline and bathymetric change and quantified oyster recruitment, fish and mobile macro-invertebrate abundances. Breakwater reef treatments mitigated shoreline retreat by more than 40% at one site, but overall vegetation retreat and erosion rates were high across all treatments and at both sites. Oyster settlement and subsequent survival were observed at both sites, with mean adult densities reaching more than eighty oysters m Nearshore, biogenic habitats of estuaries support a broad spectrum of marine life and serve as nursery grounds for economically-important fishes and shellfish The “engineering first” approaches, including vertical bulkheads, concrete and granite rip-rap revetments and seawalls, are often used by coastal engineers because they are viewed as permanent and non-retreating structures. Unfortunately, insufficient concern may have been given to the ecological, aesthetic or socioeconomic impacts of these hardened structures. A major concern in implementing bulkheads and seawalls for coastal property protection is that erosive wave energies are reflected back into the water body, instead of being absorbed or dampened The benthic setting adjacent to many armored shores is generally absent of complex, structured habitats Living shoreline projects often involve the planting or restoration of naturally-occurring biogenic habitats that have numerous ecological benefits, in addition to providing a buffer for wave action. In their natural setting, oyster reefs are often found seaward of salt marshes and can attenuate erosive wave energies, stabilize sediments and reduce marsh retreat, thereby making them an attractive living shoreline approach Located on the northern Gulf of Mexico, Mobile Bay is one of the best examples of a classic estuary In this study, we experimentally examined the ecological effects of constructing subtidal breakwater oyster reefs for coastal and estuarine shoreline protection. In addition to documenting changes in the physical setting near breakwaters and unaltered control treatments, we quantified the habitat value for oysters, fishes and mobile invertebrates. We focus particular attention on the potential impacts on economically-important species, as this provides insight into the economic implications of different shoreline protection alternatives. We hypothesized that the addition of breakwater reefs of oyster shell would: 1) mitigate shoreline retreat, (2) provide substrate for recruitment and survival of oysters, (3) support higher densities of small fishes, mobile macro-invertebrates and larger and transient fishes and (4) promote higher species richness and a different community structure than unaltered control areas.This study was conducted in accordance with the laws of the State of Alabama and under IACUC protocols (Permit # 05047-FSH) approved by the University of South Alabama.Spartina alterniflora) and black needlerush (Juncus roemerianus). Remnants of oysters (Crassostrea virginica) are found throughout the marsh and buried in the subtidal sediments. The second site, Alabama Port , is located along the southwestern shore of Mobile Bay, just north of the Dauphin Island bridge. The treatments at Alabama Port were located along a two kilometer stretch of eroding shoreline that has been encroached by armoring at its northern and southern extents. Small patches of Spartina alterniflora can be found at Alabama Port, but the most abundant vegetation is Phragmites sp., which is largely present in the upper intertidal zone. Both sites were selected within regions of high oyster spat settlement (40–180 spat m−2 day−1) To determine the ecological and physical effects of created breakwater oyster reefs, we conducted a manipulative field experiment at two sites in coastal Alabama that contained stretches of rapidly eroding coastlines. Study sites were selected within regions known to have adequate larval supply of oysters 2 openings) that was anchored by rebar. The purpose of the mesh covering was to help maintain the vertical relief of breakwaters until adequate recruitment of oysters cemented the loose shell in place. The initial height of each reef was slightly above MLLW (∼1 m), under the assumption that the loose oyster shell would settle below that level and eventually become subtidal. The subtidal design of the reefs allowed for maximum exposure for oyster settlement and increased available substrate for foraging by transient and larger resident fishes, while maximizing potential capacity for wave attenuation.The experimental oyster reefs were designed as subtidal wave-attenuating breakwaters, a common coastal engineering approach Mean surface water temperatures, recorded by electronic thermometer, and salinity, measured by a refractometer, were recorded during each sampling event. To observe longer term patterns in salinity, we utilized publicly available data recorded by hydrographic monitoring stations located at Cedar Point and Dauphin Island, AL. The Cedar Point station is approximately 17.5 km from Point aux Pins and 4.0 km from Alabama Port site center points. The Dauphin Island station is approximately 25.5 km from Point aux Pins and 11.0 km from Alabama Port site center points. The Cedar Point station has been active since 2008 and the Dauphin Island station since 2003. To consider the effects of wave climate and dominant wind direction and magnitude on our study setting, we reviewed historical and recently published coastal engineering studies Vegetation retreat and changes in nearshore depth profiles were monitored to evaluate the effect of the breakwaters on the nearshore setting. Bathymetry surveys were conducted at both sites during preliminary site selection and yearly following construction at Point aux Pins. Bathymetric data was collected using a Ceeducer Pro DGPS system with an integrated depth sounder mounted to a 1 m×2 m platform on pontoons. We surveyed each site manually by walking the pontoon through multiple parallel transects of the reef and control treatments. At each reef treatment, the breakwater reef footprint was delineated using the Ceeducer DGPS to measure reef spreading and consequential reduction in reef height. The width of each reef section was also measured by transect tape at reef construction and the end of the study to measure changes in reef footprint. The data collected by the Ceeducer unit was imported in ESRI's ArcView, corrected for tidal amplitude, and maps depicting depth at mean low water (MLW) were created. To measure the shoreward retreat of emergent vegetation, permanent rebar stakes were installed at 25 m intervals along the 100 m stretch of shoreline at each replicate treatment. Each 6 m rebar stake was driven into the marsh edge so that 1 m remained visible. These shoreline stakes were installed shortly after breakwater construction at both Point aux Pins and Alabama Port and were monitored periodically thereafter. During each survey, marsh retreat was measured as the distance from the rebar stake to the living vegetation line. Mean differences between vegetation retreat rates adjacent to breakwaters and controls were analyzed by repeated-measures analysis of variance (ANOVA). Because of differences in reef creation dates and sampling period, Point aux Pins and Alabama Port were analyzed separately.2 quadrat, which was haphazardly placed at three locations on each reef section (n = 9 per replicate reef). The exposed layer of shell within the quadrat was collected and placed in a large container. Juvenile (≤3 cm) and adult oysters (>3 cm) were enumerated and measured in the field, and then returned to the reef. Mortality was quantified by enumerating dead oysters, which had both valves still articulated and were absent of fouling organisms inside the shell. We sampled the breakwater reefs at Point aux Pins in July, August and November 2007, May and October 2008 and June 2009. We sampled the reefs at Alabama Port in March, June and October 2008 and June 2009. For the final sampling period of June 2009, six 0.25 m2 quadrats were sampled from each section (n = 18 per replicate reef) to account for the reef spreading and to assure a similar proportion of reef surface area was sampled.To assess the value of the breakwater reef complexes for oysters and other sessile invertebrates, we periodically collected quadrat samples. Oyster settlement, growth and survival were quantified using a 0.25 mWe used univariate one-way ANOVA to test for differences in densities of live juveniles, live adults and dead oysters among sampling events. Point aux Pins and Alabama Port recruitment data were analyzed separately because the independent variable of sampling date was different at each site. Density estimates determined by individual quadrat samples  for each reef section were averaged. The pooled values from each of the three reef sections for each of the two replicated treatments were used as replicates in a one-factor ANOVA to test the effect of sampling date. These data were tested for normality using the Kolmogorov-Smirnov test and homogeneity of variances using Bartlett's test. To meet the assumptions of ANOVA, all values were log transformed and retested. After transformation, minor violations of normality and equal variances were still present for live adults and dead oysters at both sites. Because the violations from quadrat sampling are generally minor and ANOVA is considered robust to such violation The response of fishes and mobile invertebrates was measured using a combination of gear types to target small and large individuals. Experimental gillnets (2 m×30 m) were used to capture larger species and individuals of coastal finfish species. Sampling occurred twice each month for one year following construction and monthly thereafter through all seasons, but was reduced to every other month during winter. Gillnets were deployed on adjacent sides of each reef or control treatment and perpendicular to shore. Each net was comprised of two 15 m panels (5 cm and 10 cm maximum opening) to broaden the size range and body shape of animals captured. Gillnets were fished for two hours starting one hour prior to sunrise. During winter months, low tides prevented crepuscular sampling so nets were fished for two hours starting one hour prior to sunset. Gillnets were retrieved in the same order they were deployed, and soak time was recorded as the time from when the net was first deployed until the time retrieval began. All specimens captured were placed in labeled bags and returned to the lab where they were identified, measured and their biomass recorded.To quantify smaller fishes and invertebrates, we seined adjacent to each breakwater reef and control monthly, except every other month during winter. At each treatment, a 6 m wide bag seine with 6.25 mm mesh was towed three times between the treatment and shore. All seine distances were 15 m and terminated into the shore at Point aux Pins or a 4 m wide block net at Alabama Port. All captured mobile invertebrates and fishes were placed in labeled bags and returned to the laboratory where they were identified to the lowest taxonomic level possible, measured and biomass recorded.To determine the effects of site and treatment on the communities of fishes and invertebrates, we used multivariate and univariate analyses. Differences in community structure between reef and control treatments and between Alabama Port and Point aux Pins sites were tested for each gear type using permutational analysis of variance (PERMANOVA). Multivariate PERMANOVA used Bray-Curtis similarity matrices of log (x+1) transformed abundance data with 4,999 permutations Menidia sp., Paralichthys sp.). For all tests, we considered results of p≤0.05 to be significant. The ANOVA and Wilcoxon tests were run using the R Statistical Platform Version 10.1.1 To determine the effects of breakwater reefs on the most common demersal fishes and decapods, we analyzed these taxa separately as they include many economically-important coastal species. We used Wilcoxon signed-rank tests to compare relative abundances of each species (≥1%) between the paired breakwater reef and mudflat control treatments. This approach allowed us to test for overall treatment effects, while controlling for date and site variability through the paired experimental design but ignored their interactive effects. Certain species that were closely related or difficult to distinguish were analyzed as grouped taxa  measured by digital thermometer, and salinity averaged 23.1 PSU (±8.7 SD) measured by refractometer. Mean water temperature at Alabama Port was 21.8°C (±7.8 SD) and salinity averaged 16.1 PSU (±7.4 SD). Salinity data, shown as box and whisker plots, was acquired from hydrographic monitoring stations at Cedar Point  and DaupMost wind-driven wave energies along coastal Alabama shorelines are generated by dominant south to southeasterly winds from spring through early fall and north-oriented winds from late fall throughout most of winter Changes in the nearshore and shoreline environments of reef and control sites were observed from measuring vegetation retreat and bathymetric surveys. Bathymetric surveys at Point aux Pins found that, in addition to a general trend of decreasing depth, areas inshore of breakwater reefs appeared to gain more sediments than areas inshore of control plots . The foo−2 in November 2007, but were much lower the following year with ranges between 50 and 150 m−2  and found in lower densities in June 2009 (∼20 oysters m−2)  was recorded in October 2008  between breakwater and control treatments was tested using paired Wilcoxon signed-rank tests. For 5 cm gillnet samples, six demersal species contributed ≥1% of the total catch . Of thosOur study found that breakwater reefs constructed of loose oyster shell provided substrate for oyster recruitment and harbored a more diverse community of fishes and mobile invertebrates than control areas without reefs. This habitat enhancement is uncommon among shoreline protection schemes and could be a vast improvement over traditional armoring techniques, many of which have detrimental impacts on nearshore species Roland and Douglass (2005) found that many stretches of Alabama's shoreline are faced with wave energies well above critical limits where vegetation can naturally persist and proposed breakwaters as a potential mechanism to reduce wave energies Stramonita haemastoma) and several species of crabs near the reefs. Stomach content analysis of the black drum collected in gillnets usually found oyster shell remains and dead oysters often showed signs of predation . A recent mark and recapture study of subtidal oyster reefs in coastal Alabama waters also documented drills as the most prevalent cause of mortality due to visible scarring on dead spat shells At both Alabama Port and Point aux Pins, we documented oyster recruitment and survival to reproductive size, but substantial mortality limited reef cementing and success. The high mortality recorded at both sites during October 2008 sampling appeared to be caused by predation or physical disturbance, such as wave energy. During this sampling period, very few exposed oysters were observed to be alive. In contrast, nearly all live oysters observed were found sheltered inside of dead, but still hinged oyster shells. This suggests that it is unlikely disease was the cause of mortality, since structurally-protected oysters would have no reprieve. Another factor that frequently affects oyster survival is reef height as tall reefs escape the poor water quality sometimes found near the sediment The communities of fishes and mobile invertebrates that benefit from oyster reefs have been well-described, but very few studies have examined the enhancement from oyster reefs designed for protecting shorelines. The elevated species richness and densities that we observed during our study concur with most literature describing oyster reef habitats 2).Landscape attributes, such as adjacent habitats or bathymetric features, commonly influence community composition It has proven quite challenging to predict the ecosystem services to be expected from restoring reefs at different scales or in different settings Awareness of the detrimental impacts of shoreline armoring has increased in recent years, but movement towards more ecologically-responsible methods has been limited by the lack of cost-effective alternatives. “Living shoreline” approaches, including breakwater reefs, that protect coastal uplands could provide a more ecologically-responsible alternative to traditional armoring and not only mitigate coastal erosion, but also enhance certain economically-valuable fish stocks. However, as our study demonstrated, efforts to sustainably and responsibly protect coastal shoreline habitats must balance both engineering and ecology.Table S1Results of Repeated-Measures ANOVA on Vegetation Retreat.(DOCX)Click here for additional data file.Table S2Relative Abundance of the Most Abundant Fishes and Mobile Invertebrates.(DOCX)Click here for additional data file.Table S3Results from Multivariate PERMANOVA Tests.(DOCX)Click here for additional data file.Table S4Results from Univariate PERMANOVA Tests.(DOCX)Click here for additional data file.Table S5Results from Wilcoxon Signed-Rank Tests on Single Species or Grouped Taxa.(DOCX)Click here for additional data file.


Auditory information is widely used throughout the animal kingdom in both terrestrial and aquatic environments. Some marine species are dependent on reefs for adult survival and reproduction, and are known to use reef noise to guide orientation towards suitable habitat. Many others that forage in food-rich inshore waters would, however, benefit from avoiding the high density of predators resident on reefs, but nothing is known about whether acoustic cues are used in this context. By analysing a sample of nearly 700,000 crustaceans, caught during experimental playbacks in light traps in the Great Barrier Reef lagoon, we demonstrate an auditory capability in a broad suite of previously neglected taxa, and provide the first evidence in any marine organisms that reef noise can act as a deterrent. In contrast to the larvae of species that require reef habitat for future success, which showed an attraction to broadcasted reef noise, taxa with a pelagic or nocturnally emergent lifestyle actively avoided it. Our results suggest that a far greater range of invertebrate taxa than previously thought can respond to acoustic cues, emphasising yet further the potential negative impact of globally increasing levels of underwater anthropogenic noise. Across the animal kingdom, acoustic information is frequently used in orientation, habitat selection and predator avoidance. Marine coastal habitats, for example, are characterised by a high level of biological and abiotic noise, and coral reefs are particularly noisy due to high densities of resident shrimps, urchins and fishes Many benthic marine organisms undergo an early developmental stage at sea and must settle to suitable habitat for juvenile and adult life Here we use experimental playbacks and light traps in the waters of the Great Barrier Reef lagoon to test the responses to coral reef noise of a broad suite of tropical crustaceans with a range of life-history strategies. We predict that larval stages of taxa that inhabit reefs as adults will, if they can detect the sound, be attracted to reef noise. In contrast, we predict that both pelagic taxa (those that remain in the water column throughout their lives) and nocturnally emergent taxa  will, if they are capable of detecting it, be deterred by reef noise.All work was carried out under the guidelines of the Ethics Committees of the Australian Institute of Marine Science and Lizard Island Research Station, and with permission from the Great Barrier Reef Marine Park Authority, Australia.cv , Great Barrier Reef, Australia. We sampled for 34 nights using a pair of light traps which consisted of an 8 W fluorescent light housed in a clear Perspex box with one 1×25 cm entry slit on each side Traps were deployed at dusk and retrieved at dawn. The catch was preserved in 70% ethanol and the crustaceans separated from the fish prior to categorisation and counting. To ensure that the numbers of captured crustaceans had not been modified by fish predation in the traps, we dissected 90 pelagic baitfish and 90 settlement-stage reef fish selected evenly from the two sound treatments and randomly from nine different nights. We found no fish with freshly consumed crustaceans in their mouth, throat or stomach, so rule out the possibility that differential predation drives any differences in crustacean catches.The vast majority (99.3%) of the nearly 700,000 crustaceans caught were divided into 15 reliably distinguishable categories using a dissecting microscope; the remainder were not included in analyses. Any categories for which more than 50% of nights produced no catch in both traps  were discarded prior to analysis. This criterion eliminated Euphausiacea, Palinura and Stenopodidae. Remaining categories for which the mean nightly catch was less than 200 individuals were also discarded. This criterion eliminated Isopoda, Sergestidae and Stomatopoda. We therefore had nine categories for statistical analysis: two larval developmental stages of reef-settling Brachyura , two pelagic taxa (Copepoda and Hyperiidea), and five taxa that tend to be mostly nocturnally emergent .2 distribution. In each model, we included trap pair (i.e. the two traps from the same night) nested in trap location as a random term. Statistical analyses were two-tailed and were conducted in Genstat .Data were analysed using generalised linear mixed models (GLMMs) to allow the inclusion of random as well as fixed terms and thus control for repeated measures from the same trap locations and paired trapping on the same night. For each crustacean category, we used a separate GLMM with a Poisson error distribution and a log link function to examine how sound treatment  affected number of individuals caught in the trap. Each GLMM was based on 68 catch totals from paired trapping on 34 nights at three different locations. Variance components were estimated using the Restricted Maximum Likelihood (REML) method, and random terms were retained unless the variance component was found to be zero . The significance of fixed terms was determined using the Wald statistic, which approximates the χOf the approximately 691,000 individuals analysed statistically, 9.3% were developmental stage reef-settling Brachyura , 18.9% were pelagic taxa , and 71.8% were taxa that tend to be mostly nocturnally emergent .There was no significant difference in the number of brachyuran megalops caught depending on sound treatment , but brachyuran zoea were caught in significantly higher numbers in traps playing back reef noise compared to control traps  that recruit to reef habitat As predicted, zoea, the pre-settlement larval stage of Brachyura, were attracted to reef noise. This is consistent with findings from temperate waters e.g. , but proIn addition to a diverse suite of biological noises, the soundscape in shallow water environments is influenced by local bathymetry, seabed characteristics and surface conditions in situ choice chambers Coral reef noise is heterogeneous in time and space, and these differences relate directly to habitat type There is much recent concern that natural marine soundscapes are being modified or dominated in some places by anthropogenic noise arising from, for example, shipping and small boats, drilling and mining, seismic surveys and offshore construction


How retroviruses regulate the amount of RNA genome packaged into each virion has remained a long-standing question. Our previous study showed that most HIV-1 particles contain two copies of viral RNA, indicating that the number of genomes packaged is tightly regulated. In this report, we examine the mechanism that controls the number of RNA genomes encapsidated into HIV-1 particles. We hypothesize that HIV-1 regulates genome packaging by either the mass or copy number of the viral RNA. These two distinct mechanisms predict different outcomes when the genome size deviates significantly from that of wild type. Regulation by RNA mass would result in multiple copies of a small genome or one copy of a large genome being packaged, whereas regulation by copy number would result in two copies of a genome being packaged independent of size. To distinguish between these two hypotheses, we examined the packaging of viral RNA that was larger (≈17 kb) or smaller (≈3 kb) than that of wild-type HIV-1 (≈9 kb) and found that most particles packaged two copies of the viral genome regardless of whether they were 17 kb or 3 kb. Therefore, HIV-1 regulates RNA genome encapsidation not by the mass of RNA but by packaging two copies of RNA. To further explore the mechanism that governs this regulation, we examined the packaging of viral RNAs containing two packaging signals that can form intermolecular dimers or intramolecular dimers (self-dimers) and found that one self-dimer is packaged. Therefore, HIV-1 recognizes one dimeric RNA instead of two copies of RNA. Our findings reveal that dimeric RNA recognition is the key mechanism that regulates HIV-1 genome encapsidation and provide insights into a critical step in the generation of infectious viruses. Viruses must package their genomes in particles to pass their genetic information to the next generation. Although many aspects of RNA packaging are well-studied, how retroviruses regulate the number of genomes in the particle is currently unknown. Based on the dimeric nature of retroviral genomes in particles, it was often assumed that two copies of RNA were packaged into one particle. This assumption was validated recently when we demonstrated that most HIV-1 particles contain two copies of viral RNA, which revealed that the number of genomes packaged is tightly controlled. In this report, we examined the mechanism that regulates the amount of RNAs encapsidated into HIV-1 particles. Our results showed that RNA packaging is not regulated by the mass of the viral RNA as two copies of small or large genomes are packaged. However, packaging of two copies of RNA can be perturbed; HIV-1 can encapsidate one copy of its genome when the RNA contains two packaging/dimerization signals that allow for intramolecular dimer (self-dimer) formation. These studies revealed that HIV-1 regulates genome packaging by recognizing the dimeric RNA structure, and suggest that the interaction of viral protein Gag and dimeric RNA serves as the nucleation point of virus assembly. In contrast, other aspects of RNA packaging, such as how retroviruses regulate the number of genomes packaged into virions, are poorly understood. For example, it is not known whether the viral particle would package more than two copies of RNA from a viral genome that is much smaller than wild type. The possibility that multiple smaller RNA genomes are incorporated into one virion has been suggested previously Many aspects of retroviral RNA genome encapsidation, such as the By determining the viral RNA content of individual particles, our recent study demonstrated that most HIV-1 particles contain viral RNA; furthermore, two copies of RNA are packaged in each virion Escherichia coli, which are tagged with yellow fluorescent protein (YFP) and a red fluorescent protein, mCherry, respectively. Viral particles are visualized by fluorescence microscopy as a portion of the Gag proteins are tagged with cerulean fluorescent protein (CeFP), and the packaged viral RNAs are distinguished by their YFP or mCherry signals. In the current study, we modified this system to express RNA genomes that are ≈3 kb or nearly 17 kb so that we could examine the effects of viral RNA length on RNA packaging. Our results indicate that HIV-1 packages two copies of viral genome regardless of whether the RNA is ≈3 kb or ≈17 kb. We also found that HIV-1 can package one copy of the genome when the RNA contains two dimerization/packaging signals allowing for the formation of an intramolecular dimer (self-dimer). This observation indicates that the recognition of the dimeric RNA structure is a key element in the regulation of RNA encapsidation by HIV-1.To test these two hypotheses, we examined packaging of HIV-1 genomes that are much larger or smaller than the wild-type 9-kb RNA by directly visualizing the viral RNA contents of individual particles. In this previously described single-virion analysis assay cis-acting elements required to express, export, and package HIV-1 RNA; they express functional Gag-CeFP, Tat, and Rev proteins, whereas the pol, env, vif, vpr, and vpu genes were inactivated by deletions. Additionally, Base-MSL and Base-BSL contain stem-loop sequences in the pol gene positions that are recognized by the coat protein of MS2 bacteriophage and the BglG protein of E. coli, respectively.We generated a series of constructs expressing larger RNA genomes based on the previously described HIV-1 constructs GagCeFP-MSSL and GagCeFP-BglSL We inserted viral and nonviral sequences into Base-MSL and Base-BSL to generate the HIV-1 constructs Long-MSL and Long-BSL, respectively; the full-length viral RNAs expressed from these two constructs are 13–14 kb . We then+ particles that are positive for RNA signals (mCherry+ or YFP+). Our results showed that viral RNAs were packaged efficiently (>93%) in particles derived from Base, Long, and XLong constructs . Similar results were obtained from BglG and MS2 stem-loop-containing constructs . In our previous experiments using the Base constructs, we found that ≈45% of the CeFP+ particles were YFP+mCherry+; similarly, ≈38% of the CeFP+ particles that were generated by coexpressing XLong-MSL and XLong-BSL were YFP+mCherry+  that contains intact gag-pol with an inactivating mutation (D25N) in pro. Particles derived from all of these constructs exhibit similar immature morphology   should have a reduced ratio of viral particles with two RNA colors; furthermore, the level of reduction should be similar to that of the Base vectors. In contrast, if multiple dimers are packaged into a viral particle, then the effects of reducing RNA heterodimers on the generation of particles with two RNA colors will be lessened, because many particles will contain one dimer in which both RNAs have GCGCGC and one dimer in which both RNAs have GUGCAC, thereby increasing the proportion of particles with two RNA colors (The ability of two HIV-1 RNAs to be copackaged into the same virus is strongly influenced by their DIS sequences. We have shown that RNAs from two Base vectors can be copackaged at a near-random level when both RNAs have Cherry+) . TherefoA colors .GCGCGC in their DIS, along with helper constructs; ≈25.7% of the CeFP+ particles exhibited both mCherry and YFP signals. When we coexpressed Mini-BSL with Cdis-Mini-MSL that contains GUGCAC in its DIS, we found that ≈9.6% of the CeFP+ particles exhibited both RNA signals (YFP+mCherry+), which corresponds to a 2.7-fold reduction (25.7%/9.6%) from that produced by two Mini RNAs with GCGCGC in their DIS. We also examined the effects of changing the DIS sequences of Mini constructs into two complementary nonpalindromic sequences and generated 6G-Mini-MSL and 6C-Mini-BSL that contain GGGGGG and CCCCCC in their DISs, respectively. When coexpressed with helper constructs, 6G-Mini-MSL and 6C-Mini-BSL produced ≈40.4% of heterozygous particles, which corresponds to a 1.6-fold increase from that generated by Mini-MSL and Mini-BSL , they were still encapsidated as one dimer and not as multiple dimers.The effects of altering DIS sequences in Base vectors and in Mini vectors on the production of YFPTaken together, these results show that HIV-1 particles have the capacity to accommodate HIV-1 RNA significantly larger than the 9-kb wild-type genome. However, regardless of whether the RNA was ≈17 kb or ≈3 kb, one dimer was packaged into a viral particle. These findings do not support the “RNA mass” packaging hypothesis; rather, they are consistent with the hypothesis that HIV-1 regulates its genome encapsidation by copy number.env gene a second copy of the 5′ leader sequence including the DIS, the resulting RNAs can form both intermolecular dimers and intramolecular dimers (self-dimers) Our results from constructs with large RNA or Mini RNAs are consistent with the hypothesis that HIV-1 regulates genome packaging by RNA copy number. It is possible that HIV-1 regulates genome packaging by encapsidating one RNA dimer; alternatively, HIV-1 may exert the regulation by packaging two copies of viral RNA. We sought to determine the unit  that HIV-1 uses to regulate RNA packaging. Sakuragi and colleagues previously demonstrated that by inserting into the pol gene of the Base vectors a segment of the NL4-3 genome from U5 to the first 34 nt of gag, with a mutation in the splice donor site to abolish its function We used this strategy and generated HIV-1 vectors containing two packaging signals  by inser+mCherry+ particles should remain the same as that generated by the Base vectors. In contrast, if HIV-1 regulates the virion genome by packaging one dimeric RNA, then one copy of the self-dimer should be packaged, and the ratio of YFP+mCherry+ particles should be lower than that generated by the Base vectors (GCGCGC), Bdis-BSL-Bdis and Bdis-MSL-Bdis, and found that the YFP+mCherry+ particle ratio in the viral population was reduced from ≈44% to ≈32%  binding sites that are buried in the monomeric RNA Retroviruses are known to package an assortment of cellular mRNA into their particles, especially in the absence of the viral genome Regulation of genome encapsidation has implications for multiple aspects of viral replication and evolution. Our results reveal that dimeric RNA recognition is the key element that regulates viral genome packaging into HIV-1 virions. These findings have direct implications for the dynamics of virus assembly, the potential for recombination to generate viral diversity, and the adaptive strategies employed by retroviruses for their replication.pol that was deleted in Base-BSL, then inserting into the nef gene two DNA fragments, one from pB0-Spe6C lacZ. The ouabain-resistance gene from pSVα3.6 For simplicity, previously described GagCeFP-BglSL and GagCeFP-MSSL pol gene of Base-BSL and Base-MSL, respectively. The 5′ DIS sequences of the two aforementioned plasmids were changed to GTGCAC to generate Cdis-BSL-Bdis and Cdis-MSL-Bdis. The structure of all plasmids was verified by restriction digests; PCR-amplified regions were confirmed by sequencing.Mini-MSL and Mini-BSL were derived from pKD-HIV(GFP-I-Hy) mCherry in pSynGag-mCherry cefp; pTat-1 pro.Plasmids expressing MS2-YFP and Bgl-mCherry have been reported previously Human 293T cells were maintained as previously described gag fragment 32P-labeled probe generated from an 8-kb AvaI digestion fragment that covered most of the NL4-3 sequences Viral particles were treated with RNase-free DNase to remove DNA prior to virion RNA isolation. Virion RNA isolation g for 80 or 120 min at 23°C in a Beckman SW41 Ti rotor. Collected fractions were analyzed by quantitative real-time RT-PCR. HIV-1 RNAs were detected using primers and probe sets annealed to gag as previously described Linear 15%–30% sucrose (w/v) gradients were prepared as previously described Figure S1EM analysis of HIV-1 particles produced from different viral constructs. (A) Representative images of HIV-1 particles produced from different viral constructs. NL4-3 PR*, protease-deficient NL4-3-based construct H0-PR*. (B) Diameters of HIV-1 particles produced from different viral constructs. For each sample, at least 250 particles were measured. Values represent the mean ± SD from two independent experiments.(DOC)Click here for additional data file.Table S1RNA contents of HIV-1 particles containing different genome sizes.(DOC)Click here for additional data file.Table S2Proportion of heterozygous particles generated from constructs with different genome sizes.(DOC)Click here for additional data file.Table S3Proportions of HIV-1 particles containing Mini RNA genome.(DOC)Click here for additional data file.Table S4Effects of DIS sequences on the proportion of two-RNA-color viral particles containing Mini RNAs.* Within each experiment, the percent of particles with two RNA colors for each sample was compared to the percent of particles with two RNA colors in the Mini-MSL + Mini-BSL sample.(DOC)Click here for additional data file.Table S5Effects of DIS sequences on the percent of heterozygous particles containing HIV-1 RNAs with two dimerization signals.* The percent of heterozygous particles in each sample was compared to the average percent of heterozygous particles for Base-MSL + Base-BSL samples in Supplemental table S2 that was set to 1.0.(DOC)Click here for additional data file.


In the absence of previously published data, this work aimed to assess the temporal stability of the background offset.Phase-contrast velocity images often contain a background or baseline offset error, adding an unknown offset to the measured velocities. For accurate flow measurements, this offset must be corrected or shown to be negligible. Some correction techniques depend on replicating the clinical flow acquisition using a uniform stationary phantom, in order to measure the baseline offset at the region of interest and subtract it from the clinical study. Such techniques assume that the background offset is stable over the time of a patient scan, or longer if the phantom scans are acquired later, or if the corrections are derived from pre-stored background correction images. Factors such as gradient power amplifier heating, and variations in eddy currents or their corrections are liable to influence stability. In the absence of previously published data, this work aimed to assess the temporal stability of the background offset.A fixed phase-contrast retro-gated cine acquisition at a fixed location and orientation was repeated 5 times in rapid succession, and this was performed weekly on stationary uniform phantoms using 3 different manufacturers’ scanners for 8 weeks. The largest change in the background offset within 50mm of isocenter was determined, defining a change of >=0.6cm/s as significant. The 8 week interval did not include any relevant system service visits. Any automatic background correction available was disabled since its performance in uniform phantoms is irrelevant.Over the several minutes timescale of the 5 repeats Figure , insigniOver the duration and activity of a typical patient study, background drift was insignificant. However, the combination of extended high gradient power scanning with work requiring background correction requires care to avoid drift on some machines. Over the longer term of 8 weeks, significant drift is likely, preventing accurate correction by delayed phantom correction scans or derivation from pre-stored background offset images.NIHR Cardiovascular Biomedical Research Unit funding.


Most human pre-mRNAs are spliced into linear molecules that retain the exon order defined by the genomic sequence. By deep sequencing of RNA from a variety of normal and malignant human cells, we found RNA transcripts from many human genes in which the exons were arranged in a non-canonical order. Statistical estimates and biochemical assays provided strong evidence that a substantial fraction of the spliced transcripts from hundreds of genes are circular RNAs. Our results suggest that a non-canonical mode of RNA splicing, resulting in a circular RNA isoform, is a general feature of the gene expression program in human cells. Deep sequencing of RNA from biological samples, “RNA-Seq”, is a powerful tool for discovering and cataloguing novel alterations in the expression, sequence, and structure of transcriptomes. In the present study, we used RNA-Seq in a deliberate search for transcripts that could not be accounted for by conventional splicing of primary transcripts from an unrearranged human genome. Although our initial goal was to discover cancer-specific chromosomal rearrangements by identifying the resulting fused or rearranged transcripts, we also investigated the possibility that the exon order specified by the genome sequence might be rearranged during RNA processing. We were surprised to find numerous examples of transcripts in which the exon order was a circular permutation of the order encoded in the genome. We hypothesized that these anomalous transcripts might the result of intramolecular but non-canonical splicing events that joined a splice donor to an upstream (i.e. toward the 5′ end of the transcript) splice acceptor to produce a circular RNA molecule. Indeed, for many genes, in both cancer and normal human cells, we found RNAs with circularly permuted exon orders at levels comparable to those of the canonical, linear mRNA.The first observation suggesting that eukaryotic RNAs can exist in circular form was made more than 30 years ago by electron microscopy The best-characterized circular transcripts are in rodents. The mouse SRY gene, the sex-determining gene in males, consists of a single exon. During development, the RNA exists as a linear transcript that is translated into protein. In the adult testes, the RNA exists primarily as a circular product that is predominantly localized to the cytoplasm and is apparently not translated All examples of circular transcripts reported to date in humans have been found to be expressed at low levels compared to the dominant canonical linear isoform, requiring sensitive nested PCR experiments for detection; these examples were discovered inadvertently or in an effort to characterize the structure of oncogenes. Circular RNAs have also been reported to be rare isoforms of the human Cytochrome P-450 2C18, and dystrophin transcripts Two studies have suggested that scrambled exons may occur in more than a handful of transcripts. Roughly 150 sequences representing exon scrambling were found in EST databases; in each case, the authors argued that these sequences were unlikely to derive from circular RNA molecules, but rather represented bona fide examples of exon scrambling within a linear transcript We recently reported evidence suggesting that local structural rearrangements in the human genome may be under-recognized genetic lesions contributing to the progression of human cancers This analysis identified hundreds of genes with evidence of exon scrambling: in all, more than 1232 such genes had more than one junctional read. In addition, more than 700 isoforms with scrambled exons were estimated to comprise more than 10% of all transcript isoforms for a comparable number of genes. Genes with evidence of scrambling were shown to be statistically shared between the three leukocyte populations. A complete list of scrambled isoforms is provided in Two models for the origin of the scrambled exons are diagrammed in We therefore analyzed RNA from 3 leukocyte cell types: naïve B cells (CD19+), hematopoietic stem cells (CD34+) and neutrophils from the same individual, for the presence of scrambled exons, by paired-end sequencing (80 bp per end) of ribosomal RNA-depleted (Ribo-zero) total RNA. We obtained roughly 8, 19, and 5 million reads aligning to the UCSC knowngene transcriptome (mapped as single reads) from CD19+, CD34+, and neutrophils, respectively. We found sequence evidence for scrambled exons comprising at least 10% of transcripts from each of more than 800 genes in CD19+ cells, CD34+ cells, and neutrophils, respectively. Genes with evidence of scrambling in one leukocyte cell type were significantly more likely also to have transcripts with scrambled exons in one or more of the other cell types we analyzed.We also analyzed publicly available paired-end data from poly-A selected HeLa and H9 Human embryonic stem cells The scrambled exon junctions in transcripts of C1orf58, CCDC126, FKBP8, PCMTD1, SMARCA5, and SETD2 predicted by the primary sequence data, were all directly confirmed by RT-PCR from normal leukocyte RNA, followed by Sanger sequencing. Eleven additional instances of exon scrambling in eight distinct genes in ALL or HeLa were also confirmed by RT-PCR and Sanger Sequencing see .We estimated the abundance of scrambled isoforms relative to their canonical linear counterparts by two genome-wide statistical methods. For the first estimate, we counted paired-end reads in which one read mapped to a scrambled junction (a junction between exon-X and exon-Y where X≥Y), and the other read also mapped in an orientation and position consistent with the hypothetical scrambled transcript. This number was then compared to the total number of paired-end reads consistent with the splice junctions between exon X and exon Y in the canonical order predicted by the reference genome sequence. The relative abundance of scrambled transcript isoforms as a fraction of all transcripts for a given gene is depicted for three normal leukocyte cell types in By this first method (method 1), we identified 229, 207, and 122 scrambled transcripts, representing 481 distinct genes, that were expressed at levels comparable to the canonical linear isoforms in human CD19+ leukocytes, CD34+ leukocytes and neutrophils, respectively. By the same method, we estimated that 513, 275, and 320 scrambled transcripts, representing 880 distinct genes, were at least 10% of the abundance of the canonical isoform, in CD19+ cells, CD34+ cells and neutrophils, respectively. The differences among these cell types in the apparent relative abundance of the scrambled isoform were consistent with sampling variation due to differential expression of the corresponding gene among these cell types see .Our direct observations of scrambled isoforms indicate that they comprise at least 10% of all transcripts for roughly 1–2% of genes detectably expressed in the CD19+ cells, CD34+ cells and neutrophils. Ignoring relative expression level of the scrambled isoform, we identified a scrambled isoform in roughly 10% of all detectably expressed transcripts. This is likely to be an underestimate of the prevalence of scrambled isoforms, as the pathognomonic junctional reads from inabundantly expressed genes were unlikely to be detected with this sequencing depth - more than half of the distinct transcript isoforms detected in these samples were sequenced at an average depth of one or less .The most abundant transcripts with scrambled exon order in the CD19+, CD34+, neutrophils samples, respectively, corresponded to KIAA0182 (encoding a putative subunit of the BRAF-HDAC complex), MAN1A2 , and CCDC126 (encoding a coiled-coiled containing protein of unknown function). Exon-scrambled isoforms accounted for more than half of all transcripts from each of these genes, as estimated by method 1 above. We also detected low levels of scrambled isoforms of noncoding RNAs  a subset of the scrambled exons we identified are contained in linear, poly-adenylated RNA molecules and b) a second subset of the scrambled exons we identified are contained in non-poly-adenylated, presumably circular, molecules. More specifically, the RNA samples from which these sequences derived were obtained by a double poly-A selection; the selected molecules were prepared for sequencing; the flow-through fraction from the second poly-A selection was depleted of ribosomal RNA and also sequenced Overall, the scrambled exon junctions from suspected circular RNAs were observed at tenfold higher frequency in the poly-A depleted fraction in both HeLa and H9 cells than in the poly-A selected fractions see . This reFor a small fraction of the scrambled exon junctions we found compelling RNA-Seq evidence that they derived from linear RNAs. In one case, the apparently “scrambled exons” are explained by a known exon duplication in the human genome (IFI16) which served as a convenient positive control; in another case (STAU2), the exons affected by the apparent tandem duplication correspond to a known human structural variant . The failure to detect essentially any evidence for the predicted complementary linear transcripts is circumstantial evidence that most circular transcripts are unlikely to be incidental byproducts of alternative splicing.−4) as the acceptor exon in circular RNA, and a statistically significant enrichment in median intron length among genes with circular isoforms, particularly in the intron upstream of acceptor exons and to a lesser extent in the intron downstream of donor exons . We analyzed these data using the same pipeline we had applied to the leukocyte data (using the mm9 RefSeq and UCSC annotations (downloaded 9/2011) as the reference). We found more than one thousand mouse genes with evidence of exon scrambling see .For more than 30 years, sporadic reports have described the presence of circular mRNA transcripts in mammals. Many of these circular RNAs were discovered serendipitously, and were largely disregarded as nonspecific byproducts when they were found to be expressed at low levels. In contrast to this prevailing view on the abundance of circular RNA isoforms, we found strong evidence that circular isoforms of hundreds of human transcripts, with out of order splice junctions precisely at normal exon boundaries, were present at levels comparable to their canonical linear counterparts. While the canonical linear transcripts were sensitive to RNaseR treatment in all tested cases, transcripts with scrambled exons were generally resistant to RNaseR treatment, as expected for circular RNAs, but not for RNAs with scrambled exons generated by template switching or other artifacts of reverse transcription from a canonical linear RNA. In an analysis of publicly available RNA-Seq data from HeLa and H9 human embryonic stem cells, we found that RNAs with scrambled exons were enriched in non-poly-adenylated fractions. Taken together, these findings provide strong evidence that most scrambled exon sequences we detected in human RNA were derived from circular molecules.While we have found that hundreds of human genes express circular RNA isoforms, the limitations of our experimental design may actually have led us to underestimate the prevalence of circular RNA isoforms. First, the size selection step during sequencing library preparation would miss small circular RNAs, or highly structured circular RNAs with fragmentation kinetics incompatible with our size selection. It is also possible that our size selection procedure would enrich for circular RNAs in the selected size range that failed to be fragmented. Second, although our sequencing depth was adequate to detect diagnostic junctional reads for many circular RNAs, it may not have been adequate to accurately identify and quantify rare circular isoforms, and our search for exon-exon junctions was restricted to exons annotated in RefSeq, which we know to be an incomplete catalogue of exon boundaries. Thus, we may still be underestimating the number or prevalence of circular RNAs in human cells.The previously unappreciated abundance and diversity of circular RNAs in human cells raises important questions: What is the molecular mechanism of circular splicing? Recent evidence suggests that canonical pre-mRNA splicing does not necessarily proceed in sequential order from the 5′ to 3′ end of the RNA How widespread and evolutionarily conserved is circular splicing? Our preliminary analysis of ribosomal-RNA depleted RNA from the mouse brain suggests there are hundreds of genes with scrambled isoforms in that organ; we would not be surprised to find that this phenomena is pervasive across the animal kingdom and perhaps more broadly. In addition, while we have not found evidence of differential circular isoform expression between tissues, this area should be further explored.In vitro studies have shown that circular RNAs can be translated, raising the possibility that some circular RNAs might encode proteins with functions distinct from those of their canonical counterparts What functions do circular RNAs serve and what roles might they play in normal human biology or disease? The vast majority of circular RNA molecules we detected were transcribed from a gene that is also known to encode a conventional linear mRNA. Our data suggest there are at least a handful of known noncoding RNAs with circular isoforms. A circular isoform of the noncoding RNA ANRIL has been reported to correlate with INK4/ARF expression, as well as atherosclerosis risk, suggesting the possibility that circular RNAs might have some role in human disease. An antisense transcript of the CDR1 gene, relatively abundantly expressed in mouse brain, was recently shown to be circular, and to positively regulate the corresponding sense transcript We cannot yet rule out the possibility that they are incidental to regulated splicing of a conventional linear RNA and perhaps accumulate due to their relative resistance to degradation. However, the high abundance of many circular RNA isoforms relative to their linear counterparts and lack of evidence for the predicted alternatively spliced linear RNA co-product suggest that circular RNA isoforms are not simply accidental byproducts of splicing. Further investigations of origins and activities of circular isoforms of mRNAs are likely to lead us in surprising directions.Samples were collected following procedures approved by the IRB at Stanford University.5 hyperdiploid acute lymphoblastic leukemia diagnostic bone marrow samples were obtained on a protocol approved by the Stanford Institutional Review Board. The cells were counted and frozen using Ficoll-Paque Plus  at a concentration of 0.5–10 million cells/ml. Peripheral blood was also obtained from the same patients after they were confirmed to be in remission based on the inability to detect minimal residual disease after induction therapy. The buffy coat was isolated by pipetting after centrifugation. CD19+, CD34+, and neutrophils that were isolated from the bone marrow of a single individual were purchased from All Cells Inc . Total RNA was isolated from all samples using Trizol  followed by RNEasy column  according to the manufacturer's instructions.in vitro chimeric transcripts. A second size selection was then performed for 350–600 bp to select against chimeric inserts and remove unligated adapters. Libraries were PCR amplified for 15 or 20 cycles for the leukemia and normal hematopoietic samples, respectively. Concentrations were measured using the High Sensitivity DNA Kit for the Bioanalyzer , and samples were then diluted to 10 pM. Clusters were generated using the Paired-End Cluster Generation Kit v2 with the cBot according to manufacturers instructions . The samples were then sequenced on the Illumina Genome Analyzer II using kit v5. All samples were run as a single lane on a 80 bp paired-end run. Leukemia samples were sequenced together, while the remission blood and normal bone marrow subpopulations were a separate run. Sequencing data are deposited in GEO with series number GSE33772.A modified protocol from our previous work was used to produce the sequencing libraries All mapping was performed using Bowtie version 0.12.1. Paired-end RNA-Seq data was mapped to the reference transcriptome (UCSC knowngene annotation) allowing up to three mismatches and any number of alignments. Reads uniquely aligning to the RefSeq annotation database were considered to represent gene expression. Reads failing the alignment to the UCSC knowngene annotation were mapped to a custom database of all intragenic exon-exon junctions in expressed genes with exons determined by the RefSeq database. Paired-end reads where read 1 mapped to an exon-exon junction inconsistent with the reference transcript and read 2 mapped to the same transcript, and were considered as supportive of an exon scramble. The total number of distinct paired offsets and mismatch profiles were summed, and the rate of reads supporting scrambled exons was computed per nucleotide (denoted j) and used as an estimate of the relative abundance of each scrambled isoform. Per-nucleotide gene expression (e) was estimated as the number of single reads mapping to a gene divided by gene length. The quantitative estimates of the relative expression of a scrambled transcript compared to the canonical linear isoform quoted in this paper was made using the ratio of j/e.In a separate analysis, we employed paired-end mapping ratios to further quantify the relative abundance of scrambled isoforms to canonical linear isoforms. Each gene was tiled by dividing it into even length bins of 200 base pairs. For each pair of bins, the number of reads aligning to bin A in the+orientation and bin B in the - orientation was computed. To detect scrambled and canonical linear transcripts, bin A could be up or downstream of bin B. Conditional on reads aligning to bin A, the relative ratios of reads aligning to each bin B were computed. This procedure is further detailed in In addition to the data we generated, the following public data was downloaded from the SRA and analyzed: paired-end data from H9 cells  and HeLa (SRR065529) and single end data from Total RNA reaction was reverse transcribed using the SuperScript III First-Strand Synthesis System  with random hexamers according to the manufacturer's instructions. 20 ng of cDNA was then used for each PCR validation using Platinum Blue PCR Supermix . PCRs were run for 35 cycles under standard conditions with 30 second 55 degree extension.The following procedure was used to calculate the per gene probabilities for observing evidence of a linear transcript among genes with evidence of scrambled exons.Compute the empirical length distribution of all sequenced fragments by aligning reads to the RefSeq annotation.Compute quantiles from 0–100 of empirical insert length distribution, and call this distribution F.For each gene g with an exon X to exon Y (X≥Y) junction, compute inferred circle length, sum of length of exons between Y and X on a per gene basis .0(g) = (F(L) )n.For each read 1 crossing the exon X- exon Y junction, approximate the probability that, if the scramble were contained in a linear transcript, read 2 would align to sequence upstream of exon y or downstream of exon X by 1-F(L). This is an approximation because the exact alignment offset of the junction (which can vary between 0 and 60) will influence this probability. Call this probability p. In most cases, this approximation will not be biased towards increasing or decreasing the calculated probability p. For n junctional sequences observed for a given gene, the probability of seeing 0 reads aligning to sequences outside of the exon X to exon Y interval is p0(g) over all genes, we computed the number of total genes expected to have observed reads supporting linearity under the hypothesis that scrambled exons are contained in linear, rather than circular, transcripts by computing the number of genes with p0(g) in the qth quantile and multiplying this by the average p0(g) value. The result establishes an under-representation of reads supporting the hypothesis that scrambled exons are contained in transcripts with exons upstream of exon y or downstream of exon X.Using the probabilities pth of PCR volume) was used as template in a 35-cycle PCR with Phusion polymerase , each cycle being: 94°C 5″; 68°C 15″; 72°C 2′.HeLa total RNA was isolated with TRIZOL, followed by rRNA depletion using RiboMinus (Life Technologies). Samples were then treated with DNase I  for 15 min at 37°C, then diluted 16-fold into RNase R buffer containing 0, 3,10, or 100 units of RNase R (Epicentre) per µg of RNA. We have observed some batch to batch variation in the results of our RNase R assays and suspect that some batches of RNase R may have contaminating endoribonuclease activity: we include this as a note of caution. Reactions were purified with RNA Clean & Concentrator-5 columns . Reaction product corresponding to 5 µg of input total RNA was reverse-transcribed in a 20 µl Superscript III reaction with random hexamers (Life Technologies). cDNA reaction  according to the manufacturer's instructions. Primer and probe pairs for each qPCR reaction were designed using the RealTime PCR Assay Design Tool , see 50 ng of cDNA was added to Taqman Gene Expression Master Mix  along with primer and probe at final concentrations of 0.5 and 0.25 µM, respectively. Assays were run for 40 cycles using standard conditions on the 7900HT Fast Real-Time PCR System . Ct values were generated using SDS software. Relative expression levels of linear and circular isoforms were computed as differences in Ct values, and standard errors were taken over biological replicates.Probe was generated from PCR product of MAN1A2 circular RNA. A band that had previously been confirmed to correspond to the 5-2 junction by Sanger sequencing was isolated using the QUIAquick Gel Extraction Kit . The probe was then biotinylated using the Brightstar Psoralen-Biotin Kit . Total and cytoplasmic RNA were isolated from HeLa using PARIS Kit . Labeled marker, 3.7 and 6.2 ug of total and ctyotplasmic RNA were run on a 1% agarose gel, transferred, and hybridized according to NorthernMax Kit protocol . 10 pM of probe was hybridized for 24–48 hours. Detection was performed using the BrightStar BioDetect Kit .MAN1A2 Probe (481 bp)CTATTCCCAACCTTGTAGGAATACGTGGTGGAGACCCAGAAGATAATGACATAAGAGAGAAAAGGGAAAAAATTAAAGAGATGATGAAACATGCTTGGGATAACTATAGGACATATGGGTGGGGACATAATGAACTCAGACCTATTGCAAGGAAAGGACACTCCCCTAACATATTTGGAAGTTCACAAATGGGTGCTACCATAGTAGATGCTTTGGATACCCTTTATATCATGGGACTTCATGATGAATTCCTAGATGGGCAAAGATGGATTGAAGACAACCTTGATTTCAGTGTGAATTCAGAGGTGTCTGTGTTTGAAGTCAACATTCGATTTATTGGAGGCCTACTTGCAGCATATTACCTATCAGGAGAGGAGGGAAGAGGAAGAACGTCTGAGAAATAAAATTCGAGCTGATCATGAGAAGGCCTTGGAAGAAGCAAAAGAAAAATTAAGAAAGTCAAGAGAGGAAATTCGAGCAG.Figure S1Number of Genes with Circular Isoforms by Total Gene Expression and Detection Probability. Total gene expression was computed for each of the three cell types , and genes were ranked in expression according to sequencing reads per length. Expressed genes were categorized into 100 even quantiles by cell type. The number of genes with sequencing evidence of exon scrambling was computed for all genes in each quantile (each quantile contains the same number of genes) and are depicted as colored histograms. The median number of reads per length (gene expression) for each expression quantile determines the probability of detecting a scrambled transcript if it were expressed at the same rate as the median expression quantile. This detection probability is shown by a thin black line. If the probability of having scrambled exons did not vary by expression quantile (the uniform hypothesis), this probability would be proportional to the number of genes with scrambled transcripts. For all samples, the very highest expression quantile shows relative depletion in exon scrambling events compared to its expectation and compared to the number detected in the previous quantile under the uniform hypothesis.(EPS)Click here for additional data file.Figure S2No-Template and No-RT Controls Do Not Produce PCR Products. DNase-treated total RNA or cDNA from HeLa underwent 35 cycles of PCR amplification using primers specific for each of the noted scrambled transcripts. Each of the reactions with cDNA template produced expected products (C), while those without template (A) or with RNA that had not undergone reverse transcription (B) did not produce PCR products.(EPS)Click here for additional data file.Figure S3Enrichment in Intron Length in Genes with Circular Isoforms. We investigated whether genes with exon scrambles have typical intron lengths compared to the genome-wide distribution. At left: to test the association of intron length with scrambling events, we computed the median length of intron 4  among scrambled isoforms where the donor exon was exon 3 , and found the relative quantile of this length in the length distribution of intron 4 in all human genes. We performed this analysis for all intron and exon pairs x and y where x,y≤9. At right: the analysis was repeated by stratifying scrambled junctions by acceptor exons. Several features of genes containing scrambled isoforms emerge from this analysis. First, the length of each intron of such genes are enriched compared to the whole-genome median, regardless of which donor or acceptor exon is involved in the scramble. This implies that genes with scrambled isoforms are longer than the typical gene. Secondly, there is an enrichment in the length of the intron immediately upstream of the acceptor exon , and a relative depletion in the length of the intron immediately downstream of the acceptor exon . A less striking enrichment in intron length is seen downstream of the donor exon.(EPS)Click here for additional data file.Figure S4Denaturing RNA Gel Serves as Second Control for Cytoplasmic and Nuclear Fractionation. 2 µg of total RNA from total lysate(T), as well as cytoplasmic (C) and nuclear (N) fractions were separated on 1% aagarose gel. As expected, the cytoplasmic fraction has higher concentrations of 28 s and 18 s ribosomal RNA bands. In addition, the nuclear fraction shows precursor ribosomal RNA bands that are absent in the cytoplasmic fraction.(EPS)Click here for additional data file.Table S1List of scrambled isoforms detected in the leukemia data with RefSeq exon enumeration beginning at exon 0.(TAB)Click here for additional data file.Table S2List of scrambled isoforms detected in the leukocyte data with RefSeq exon enumeration beginning at exon 0.(TAB)Click here for additional data file.Table S3Sanger sequences and primers for RT-PCR validation (see (DOC)Click here for additional data file.Table S4List of genes with reads in order inconsistent with the reference detected with RefSeq exon enumeration beginning at exon 0. Table description and statistical methods for generating the table can be found in the (CSV)Click here for additional data file.Table S5Genome-wide assessment of evidence for scrambled isoforms being circular with RefSeq exon enumeration beginning at exon 0. Table description and statistical methods for generating the table can be found in the (TAB)Click here for additional data file.Table S6Scrambled isoforms with evidence of being contained in linear RNA compiled from paired-end HeLa (poly-A selected), H9 (poly-A selected) and Leukoctye data with RefSeq exon enumeration beginning at exon 0 (see (XLS)Click here for additional data file.Table S7qPCR primer and probe pairs.(DOC)Click here for additional data file.Table S8Table of scrambled exon-exon junctions detected from the public RNA-Seq dataset of ribosomal depleted mouse brain (see (CSV)Click here for additional data file.Text S1Supplementary Methods.(DOC)Click here for additional data file.


Is it acceptable to…”) or reporting the choice of action they would make (“Would you do…”). We also investigated the influence of varying the number of people benefiting from the decision and the closeness of relationship of the decision maker with the potential victim on these two types of decision. Variations in the number of beneficiaries from the decision did not influence judgment nor choice of action. By contrast, closeness of relationship with the victim had a greater influence on the choice of action than on judgment. This differentiation between evaluative judgments and choices of action argues in favor of each of them being supported by  different psychological processes.Everyone has experienced the potential discrepancy between what one judges as morally acceptable and what one actually does when a choice between alternative behaviors is to be made. The present study explores empirically whether judgment and choice of action differ when people make decisions on dilemmas involving moral issues. Two hundred and forty participants evaluated 24 moral and non-moral dilemmas either by judging (“ Is it acceptable to…” to “Would you do…in order to….” Interestingly, these different questions may target different psychological processes , with the hypothesis that one favors unknown individuals when they are a substantial number therefore maximizing aggregate welfare  with the hypothesis that we favor the individual(s) that are socially close to us because of cognitive mechanisms at stake when human beings evolve in restricted groups , for a total of 120 moral dilemmas, were presented in eight different questionnaires. Each group of participants therefore completed one of the eight questionnaires.Versions A, B, C, and D: the independent variable was the number of people that could potentially benefit from the judgment and choice of action, ranging from less than four (versions A) to hundreds or thousands (Versions D). The figures were adapted to ensure plausibility of the scenarios. In all versions the potential victim of the decision had no affective proximity with the evaluator.Versions E, F, G, and H: the independent variable was the affective proximity between the participant who had to make a decision and the potential victim(s) of the decision. Versions E always involved a first-degree relative ; versions F a second degree relative ; versions G a close friend or relation; versions H involved unknown individuals with no affective or genetic proximity . The number of people that would potentially benefit from the decision was kept constant throughout the versions.Non-moral dilemmas were identical in all questionnaires. The order of presentation of moral and non-moral dilemmas was counterbalanced within and between questionnaires.Anonymous paper and pencil questionnaires were used. The questionnaire started with socio-demographic questions to collect anonymous data on gender, familyhood, age, level of education, work, marital status, children, religion, and mother tongue.Is it acceptable to …?” (Judgment condition) and “Would you …?” (Choice of action condition).After reading each dilemma, the participant had to answer successively two questions by Yes or No: “Participants were instructed to imagine each hypothetical situation as vividly and realistically as they could and to be as honest as possible in their decision. In addition, we insisted on the anonymous nature of the questionnaires and instructed participants not to go back to check and/or change any of their prior responses.Written informed consent was obtained from all participants. The French Law does not require approval of an ethics committee when data from a questionnaire are collected and analyzed anonymously.For non-moral dilemmas, “appropriate” responses implied the maximization of beneficence overall consequences . For moral dilemmas, response to each question was coded 1 if it favored maximizing the good of more people at the expense of very few identified individuals , and zero for the reverse situation.For the analysis of the difference of responses between conditions (Judgment-Choice of action) for each dilemma, we quoted 1 when judgment response was utilitarian but choice was not, −1 for the reverse situation and 0 when judgment and choice responses were identical.Statistical analyses were performed using SPSS software Version 17.0 .Probabilities of utilitarian responses in judgment and choice of action conditions were calculated for all dilemmas.t-tests.We compared responses between judgment and choice conditions using paired To characterize the effect of the variation of the two variables  on both the judgment and choice of action, we performed a logistic regression analysis.To statistically test the possibility that the two variables could have a different effect on the two conditions, we further performed an ordinal regression on the differences of responses between conditions for each dilemma.To account for the non-independence of within-subject responses, regression models were fitted using the generalized estimating equations method , and choice  conditions  ; 9; p < 0.p = 0.850]. It further indicates that the effect of people benefiting from the decision is identical in both conditions.As shown by OR values, variations in the number of lives saved seem to influence identically the responses in both conditions. The ordinal regression on the differences of responses between conditions for each dilemma did not yield any significant results  and choice of action  conditions ; = 1.49 1.6; 1.62; 7; = 1.49The very high number of appropriates responses for both judgment and choice when facing non-moral dilemmas illustrates that participants were able to make appropriate decisions. Furthermore, in this non-moral context responses were coherent across conditions of judgment and choice of action.In the case of moral dilemmas, we first observe a dissociation between judgment and choice of action, with responses to choice of action being overall more utilitarian than responses to judgment. Quite surprisingly, participants would thus generally accept to perform an action they have judged as morally unacceptable. Everyone has experienced the potential discrepancy between what one judges as right, from a moral point of view, and what one actually does when a choice between alternative behaviors is to be made. This is coherent with a recent study reporting that a substantial fraction of participants chose an action they judged morally wrong during moral dilemma evaluation (Kurzban’s et al., Our second observation is that the more the number of lives saved is high, the more participants tend to be utilitarian in their judgment and choice of action. This result is in line with findings from previous studies on moral judgment studying responses to a single question close to our choice of action condition (O’Neill and Petrinovich, As expected from results of previous psychological studies, participants’ decisions are generally less utilitarian when potential victims are more affectively related to them (O’Neill and Petrinovich, This suggests that choice of action becomes less utilitarian than judgment as the level of personal consequences worsen, most likely because of a greater influence of primary emotional contextual information (Greene et al., The present study provides empirical evidence that abstract judgment and hypothetical choice of action are differently influenced by variations of contextual parameters during moral dilemma evaluation. This provides an experimental evidence for a divergence between judgment and choice of action during moral evaluation, suggesting that moral judgment and moral choice may be underlied by distinct psychological mechanisms (Tassy et al., The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Sabin3-like) CVB3 strains and to investigate the effect of the Sabin3-like mutation on these potential interactions. For this purpose, “Electrophoretic Mobility Shift” assays were carried out. Data obtained did not show any RNA-RNA direct interactions between the 5′- and 3′- ends. Therefore, we can suggest that the possible mechanism by which 3′UTR enhances CVB3 IRES activity may be by bridging the 5′ to the 3′ end through RNA-protein interaction and not through RNA-RNA direct contact. However, these findings need to be confirmed by carrying out further experiments.Coxsackievirus B3 (CVB3) is a causative agent of viral myocarditis, meningitis and pancreatitis. CVB3 overcome their host cells by usurping the translation machinery to benefit viral gene expression. This is accomplished through alternative translation initiation in a cap independent manner at the viral internal ribosomal entry site. The 5′ untranslated region (5′UTR) of CVB3 genomic RNA is highly structured. It is the site of multiple RNA-protein and RNA-RNA interactions and it plays a critical role during translation initiation. Similar to the 5′UTR, CVB3 3′ untranslated region (3′UTR) also contains secondary structural elements consisting of three stem-loops followed by a poly (A) tail sequence. Long-range RNA-RNA interactions between 5′ and 3′ ends of some viral genomes have been observed. Because of their dual role in translation and replication, the 5′ and 3′UTRs represent promising candidates for the study of CVB3 cardiovirulence. Taking into account that efficient initiation of mRNA translation depends on a temporally and spatially orchestrated sequence of protein-protein, protein-RNA and RNA-RNA interactions, and that, at present, little is known about RNA-RNA interactions between CVB3 5′ and 3′UTRs, we aimed in the present study, to assess a possible RNA-RNA interaction between 5′ and 3′UTRs during the initiation of translation of a wild-type and a previously characterized mutant ( Picornaviridae, is the causative agent of virus-induced myocarditis and dilated cardiomyopathy  but the most useful of these mutations is the lacZ Delta M15 mutation which allows blue/white screening for recombinant cells.Total RNA was extracted using the Acid Guanidinium Thiocyanate Phenol-Chloroform method . After e2, 1 mM dNTP (Promega), 10 mM DiThioThreitol (DTT), 1 mM antisense primer IRES-R (5′- TTT gCT gTA TTC AAC TTA ACA ATg-3′), 1U RNasin , 50 U of Moloney Murine Leukemia Virus Reverse Transcriptase (MMLV-RT) (Promega). The reaction was carried out at 42 °C for 30 min followed by a denaturation step of 5 min at 99 °C.cDNA was synthesized in 50 μL volume containing 2 μg RNA, 10 mM Tris-HCl (pH 8.3), 75 mM KCl, 2.5 mM MgClgAA TTCTAA TAC gAC TCA CTA Tag gTA ACT TAg AAg TAA CAC A -3′) containing an EcoRI restriction site and the sequence of the T7 promotor; and 5′UTR-R (5′- TAT ggA TCC TTg CTg TAT TCA ACT TAA CAA TgA ATT gTA ATg TTT TAA -3′) including a BamHI restriction site. The amplification reaction was performed in a volume of 50 μL containing 100 ng cDNA, 25 mM Tris-HCl (pH 8), 50 mM KCl, 4 mM MgCl2, 200 μM dNTP, 40 ρmoles from each primer (5′UTR-F/5′UTR-R) and 1U Taq DNA polymerase (Promega). Distilled water was included as a negative control. Amplification was initiated by a denaturation step for 2 min at 95 °C followed by 5 cycles of 30 s denaturation at 95 °C, 30 s annealing at 48 °C and 30 s extension at 72 °C. Then, 25 cycles of 30 s at 94 °C, 30 s at 58 °C and 30 s at 72 °C were performed. Steps were followed by a final extension (10 min at 72°C). PCR products were electrophoresed in 1.5% agarose gels (Sigma) in 1X TBE buffer  in the presence of 0.5 μg/mL of Ethidium Bromide (Sigma); and visualized by using Gel Doc 2000 system (Bio-Rad). Image processing and analysis of DNA bands were performed by using Quantity One software program (Bio-Rad).5′UTRs of the two CVB3 studied strains were amplified using primers 5′UTR-F (5′- TAT GenBank database under accession number M88483 and referring to the 3′UTR secondary structure model previously proposed [gAA TTCTAA TAC gAC TCA CTA TAg TTT TAg ATT AgA GAC AAT TTg AAA TAA TTT AgA TTg gCT TAA CCC TAC TgT gCT AAC CgA ACC AgA TAA Cgg -3′) and 3′UTR-R (5′- TAT AAg CTTTTT TTT TTT TTC CgC ACC gAA TgC ggA gAA TTT ACC CCT ACT gTA CCg TTA TCT ggT TCg gTT AgC ACA gTA ggg TTA AgC - 3′) were designed for synthesis of the 3′NCR by primers- hybridization and extension method. EcoRI restriction site/T7 promoter upstream and HindIII restriction site/poly (A) tail downstream were introduced into primer’s sequences.Based on the sequence of the CVB3 complete genome published in the Primers- hybridization and extension technique was used to synthesize small DNA strands (100 to 250 nucleotides), that was the case of the 3′UTR (150 nucleotides). This technique consists briefly in reacting a pair of primers amplifying the gene of interest and designed so that an overlapping sequence of about 20 nucleotides between forward and reverse primers is incorporated. In practical terms, the reaction mixture was prepared as follows: Taq Buffer (1X) (Roche), 0.05 mM dNTPs, 100 pmol from each primer 3′NCR-F/3′NCR-R, 1U Taq polymerase (Roche) and the final volume (50 μL) was adjusted by adding sterile distilled water. Tubes were then incubated in a thermocycler (BioRad) according to the following thermal profile: 4 min at 72 °C, 6 min at 44 °C and 60 min at 70 °C. Amplification products were revealed by electrophoretic migration on an agarose gel containing Ethidium Bromide.E. coli DH5α cells. Transformants were selected in LB agar supplemented with Ampicillin (100 μg/mL), X-Gal (64 μg/mL) and IPTG (0.2 mM). Blue-white selection was used to identify white clones containing inserts, while blue clones contained undigested cloning vector. White colonies were individually cultured in LB broth.Amplicons encoding 5′UTRS of both CVB3 studied strains were digested with EcoRI and BamHI (Roche Applied Science) (restriction sites were added by PCR). Similarly, the 3′UTR DNA was digested using EcoRI and HindIII (Roche Applied Science). Digestion products were, then, purified using the “QIA quick PCR Purification Kit” (Qiagen) and inserted into the pUC19 plasmid (Invitrogen) digested with the appropriate enzymes and purified using the “Wizard SV Gel and PCR Clean-Up system kit” (Promega). Ligation products were transformed in chemocompetent To analyze the cloned sequences, randomly chosen clones were tested by PCR-colony. A single colony isolated and added into 5 μL sterile water was used as template DNA in PCR reaction containing 15 μL premix solution. The amplification program was a pre-denaturation at 94 °C for 4 min followed by 30 cycles of denaturating at 94 °C for 1 min, annealing at 50 °C for 1 min, extension at 72 °C for 1 min, and a final extension at 72 °C for 10 min. After electrophoresis, an agarose gel was stained and DNA bands were compared to the expected sizes to determine positive clones. Additionally, these clones were sequenced using an ABI Prism BigDye Terminators Sequencing Kit (Applied Biosystems). Vector primers M13-F (5′- TgT AAA ACg ACg gCC AgT -3′) and M13-R (5′- CAg gAA ACA gCT ATg ACC- 3′) (New England Biolabs) were used for sequencing.TgA TAC TTg AgC TCCCAT TTT gCT gTA TTC AAC TTA ACA ATg- 3′) as reverse primer. 15 nucleotides from the coding region and the initiating codon AUG were incorporated in the sequence of this reverse oligonucleotide. For this purpose, pUC19 recombinant plasmids (pUC19/5′UTR wild-type and pUC19/5′UTR Sabin3-like) were linearized using BamHI restriction enzyme and a PCR amplification reaction was performed using T7/5′UTR-AUG primers. The reaction mixture components were: 50 ng linearized pUC19/5′UTR, 1 μM from each primer, 0.2 mM dNTPs, Taq Buffer (1X), 5U Taq polymerase and the final volume of reaction was adjusted by adding distilled sterile water. PCR was carried out following a thermal profile consisting in a pre-denaturation step 3 min at 94 °C; 35 cycles of denaturating 1 min at 94 °C, annealing 1 min at 50 °C, extension 1 min at 72 °C, and a final extension step 10 min at 72 °C.DNA sequences corresponding to the 5′UTRs cloned into pUC19 were amplified by PCR using T7 (5′- TAA TAC gAC TCA CTA TAg- 3′) as forward primer and 5′UTR-AUG , 5 mM DTT, 4 mM NTP’s, Transcription Buffer (1X)  and 0.04U RNase Inhibitor (Applied Biosystems/Ambion) and incubated 2 h at 37 °C. During this incubation, and when a precipitate of RNA appears, 1U of Pyrophosphatase Alkaline (New England Biolabs) is added. Upon synthesis, RNAs were treated with RQ1 RNase-free DNAse (Promega) for 30 min at 37 °C.Both 5′UTRs and 3′UTR RNAs were then produced by RNAs were, then, precipitated with 2.5 M Lithium chloride (LiCl), incubated for 30 min at −20 °C, centrifuged at 13,000 rpm, 30 min at 4 °C, washed with 75% ethanol, resuspended in 50 μL of nuclease-free water and purified by size exclusion chromatography. RNA concentration was determined spectrophotometrically (NanoDrop Technology) and RNA integrity was monitored by electrophoresis on an agarose gel.Sodium periodate was used to oxidize the 3′ terminus of the RNA. The excess of the oxidant was removed by adding potassium chloride, and then the fluorescent dye, HiLyte Fluor™ 680, was added to label the 3′ terminus of RNA. The detailed procedure was as follows: the 5′UTR RNA was oxidised using sodium periodate (0.1 M) with sodium acetate  and incubated at 25 °C for 90 min. To stop the reaction, potassium chloride (250 mM) was added. The mix was incubated on ice for 10 min, then, centrifuged at 13000 rpm for 5 min at +4 °C. The supernatant was purified using Sephadex G50 column  to remove the excess of oxidant. Coupling was performed by addition of 8.6 mM HiLyte Fluor™ 680 (Eurogentec) in the presence of sodium acetate  then incubation at 25 °C for 4–17 hours in the dark. The molar ratio of the fluorescent dye to the RNA was 30:1. Fluorescent labeled RNA was precipitated by adding LiCl (7.5 M) and 2.5 vol ethanol, standing at −20 °C for 30 min, and then centrifuged  for 20 min. The precipitate was washed with 75% ethanol. Labeled RNA was resuspended in nuclease free water and the excess of fluorophore was removed by G50 Sephadex column purification (Roche Applied Science).An electrophoretic mobility shift assay or mobility shift electrophoresis, also referred as a gel shift assay, gel mobility shift assay or gel retardation assay, is a common affinity electrophoresis technique used to study protein-DNA, protein-RNA or RNA-RNA interactions. The principle behind EMSA relies on the fact that DNA-/RNA- protein complexes migrate slower than DNA or RNA alone in a native polyacrylamide or agarose gel. The speed at which different molecules move through the gel is determined by their size and charge, and to a lesser extent, their shape. This difference in electrophoretic separation of RNA-protein or RNA-RNA complexes can be visualized as a shift in migration of DNA or RNA band. For visualization purposes, the nucleic acid fragment is usually labeled with a radioactive or a fluorescent label since standard ethidium bromide staining is less sensitive than these methods and can lack the sensitivity to detect the nucleic acid if small amounts are used in these experiments.2), 1 mL acrylamide 40%, 100 μL APS 10%, 10 μL TEMED and 8 mL H2O). THEM (1X) was used as a running buffer and the gel was subsequently visualized and analyzed by Odyssey scanner . In parallel to CVB3 RNAs, the 5′ and 3′ non coding regions of HCV were used as a positive control for the shift reaction as previously described by Romero-Lòpez and Berzal-Herranz [In order to assess a possible interaction between CVB3 5′ and 3′UTRs, labeled 5′UTR wild-type and mutant RNAs and cold 3′UTR RNA were heat denatured in water for 2 min at 80 °C and slowly cooled to room temperature in Recon Buffer (2X)  for 10 min and then incubated 10 min at 4 °C. Constant amount of the labeled RNA (10 nM) was mixed with increasing concentrations of serially diluted  unlabeled 3′UTR RNA and incubated 15 min at 4 °C and, in parallel, the same assay was carried out with an incubation of samples during 30 min at 37 °C. Samples were then mixed with loading buffer (10% sucrose or 30% glycerol) and separated in a native 4% polyacrylamide gel (1 mL THEM buffer (10X) (containing 340 mM Tris, 570 mM Hepes, 1 mM EDTA, 25 mM MgClIn summary, 5′–3′ ends interactions could take place through direct RNA-RNA contacts, through protein bridges mediating RNA circularization or both. The present results support the existence of a protein-dependent 5′–3′ communication in the CVB3 genome that involves RNA elements essential for viral translation. It is tempting to speculate that one or more of these host proteins may be involved in interacting simultaneously with the 5′ and 3′UTRs, thus bringing about circularization of the mRNA. It is also possible that some of this ITAF interaction, together with long range RNA-RNA interaction, might contribute to the efficient IRES activity of CVB3 RNA. Therefore, a further investigation by functional analysis to confirm the identity and the role of these proteins in mediating cross-talk between the 5′ and 3′ ends of CVB3 RNA may provide new insights into our understanding of the role of protein-RNA and RNA-RNA interactions in the regulation of viral translation and its relationship to cardiovirulence.