Paralabrax clathratus) and California Sheephead (Semicossyphus pulcher) are economically and ecologically valuable rocky reef fishes in southern California, making them likely indicator species for evaluating resource management actions. Multiple spatial datasets, aerial and satellite photography, underwater observations and expert judgment were used to produce a comprehensive map of nearshore natural rocky reef habitat for the Santa Monica Bay region . It was then used to examine the relative contribution of individual reefs to a regional estimate of abundance and reproductive potential of the focal species. For the reefs surveyed for fishes , total abundance and annual egg production of California Sheephead were 451 thousand fish (95% CI: 369 to 533 thousand) and 203 billion eggs (95% CI: 135 to 272 billion). For Kelp Bass, estimates were 805 thousand fish (95% CI: 669 to 941thousand) and 512 billion eggs (95% CI: 414 to 610 billion). Size structure and reef area were key factors in reef-specific contributions to the regional egg production. The size structures of both species illustrated impacts from fishing, and results demonstrate the potential that relatively small increases in the proportion of large females on larger reefs could have on regional egg production. For California Sheephead, a substantial proportion of the regional egg production estimate (>30%) was produced from a relatively small proportion of the regional reef area (c. 10%). Natural nearshore rocky reefs make up only 11% of the area in the newly designated MPAs in this region, but results provide some optimism that regional fisheries could benefit through an increase in overall reproductive output, if adequate increases in size structure of targeted species are realized.Kelp Bass ( Macrocystis pyrifera) support a higher diversity and abundance of fishes than most other marine habitats in the region Stereolepis gigas) Haliotis sorenseni, H. cracherodii, H. corrugata and H. fulgens) In the nearshore marine environment of southern California, rocky reef habitats are a primary limited resource Paralabrax clathratus) and California Sheephead (Semicossyphus pulcher) are economically and ecologically valuable rocky reef fishes in southern California Kelp Bass . There was no coverage for this data in the portion of the study area from Point Dume northwest to the study area boundary, or in the central portion of the study area from Topanga to Flat Rock (Macrocystis pyrifera), the use of the data to map marine hard bottom capable of supporting kelp holdfasts introduces some error into the analysis. Three years  of data were used in an effort to account for some of the annual variation in kelp canopy. An example of this layer can be seen in Initial mapping of reef extent to create the preliminary habitat map was accomplished by combining three existing vector polygon spatial data sets. The first data set was Habitat Classification  derived from side scan sonar surveys from the Sea Floor Mapping Lab at California State University Monterey Bay  were compared with the corresponding point locations on the preliminary habitat map. All maps were then reviewed and checked for accuracy by scientific divers who have extensive experience in the area. Finally, specific reefs were delineated and named by best professional judgment as known areas of coastline of similar contiguous habitats . In ArcG2 of sea floor, abundance per transect was summed across all levels and divided by 60. Divers counted and estimated total length (TL) of small fish (<15 cm TL) to the nearest cm, and larger fish (>15 cm) to the nearest 5 cm interval. Some California Sheephead tend to follow divers, while Kelp Bass appear to be repelled by divers in some circumstances and attracted to divers in others. This was accounted for by using only highly trained divers that count each fish once, always looking forward on the transect, and not counting fish that come up from behind California Sheephead and Kelp Bass density and size structure were extracted from data collected from 2007 through 2009 following a standardized comprehensive community monitoring survey protocol; for more details on the protocol see At each site, transects are laid out in a stratified random design, with 4 transects located in each of four depth zones: inner , middle , outer , and deep . Only depth zones containing rocky reef habitat at each site are sampled, thus for a site containing all four depth zones, 16 fish transects  would be conducted on a sampling occasion. Canopy portions were only surveyed when giant kelp canopy was present and otherwise it was observed that Kelp Bass and California Sheephead were not present along the ocean surface in the absence of kelp canopy during these surveys. Additionally, for shallow (<5 m depth) inner zone transects, where midwater and canopy portions overlapped considerably, midwater portions were not surveyed (n = 48). Three meters of visibility was the minimum threshold for sampling.Annual egg production was estimated for each Kelp Bass and California Sheephead observed using a method following Tetreault and Ambrose Total numerical abundance and annual egg production  for each reef and for the region was calculated, based on a depth zone stratified sampling approach, following McCormick and Choat The influence of depth zone on abundance of each species per transect was investigated with a model selection approach using AIC http://www.dfg.ca.gov/mlpa/pdfs/sc_boundaries.pdf). We also estimated the contribution of this habitat, in each of the MPAs, to regional abundance and annual egg production. This was done by multiplying the percentage of reef area in each MPA by the total abundance or annual egg production for each reef and dividing by an estimate of total abundance or annual egg production for the region. For this calculation we estimated the regional totals including the un-surveyed reefs by assuming mean density or egg production for all un-surveyed reefs.Using ArcGIS, we calculated the total area of natural rocky reef habitat <30 m depth in each of the four MPAs in the region that were adopted by the California Fish and Game Commission on 15 December 2010, to be implemented 1 January 2012 (MPA boundaries can be found at: http://college.oxy.edu/vrg). There are approximately 2246.2 hectares of nearshore (<30 m depth) natural rocky reef habitat in the study region  comprising 82% the natural rocky reef habitat <30 m depth that was mapped in the region  of the total area of each of the four MPAs designated in Santa Monica Bay contain the rocky reef habitat <30 m depth mapped in this study . Once thThis study produced the first comprehensive map of nearshore rocky reef habitat and associated area estimates of habitat <30 m depth for the Santa Monica Bay region. Applying these measurements of reef area, with available fish density and size structure data for two important recreational fish species, provided a regional estimate of abundance and annual egg production against which contributions of individual reefs could be measured. Results demonstrated California Sheephead residing in relatively small fractions of the regional reef area (ca. 10%) can potentially produce a substantial proportion of the regional egg production (>30%). While this critical habitat makes up only a few percent of the newly designated MPAs in the region, results provide some optimism that regional fisheries could benefit through an increase in overall reproductive output, if adequate increases are realized in the size structure of targeted species.The mapping process proved effective in identifying and delimiting rocky reef habitat in areas, and at depths, where no side-scan data were available. Fine scale maps of rather complex habitats were produced for both areas where there was substantial shallow rocky reef habitat at the shoreline  and areas where there was mid-depth rocky reef along a sandy shoreline . The spatial extent of giant kelp canopy served as a useful proxy for rocky reef across most of the depth range covered in the study, while satellite imagery proved valuable for mapping shallow habitat. As high quality satellite images become freely available through sources like Google Earth™, this method is particular applicable and cost effective for delimitating the extent of general habitat types  along coastlines where other spatial habitat data is unavailable. Additionally, recent research suggests that measurement of giant kelp canopy area from satellite imagery can also serve as a good proxy for the underlying biomass of giant kelp Estimates of abundance and egg production produced for this study are likely conservative for both species. First, these estimates did not include the 18% of rocky reef habitat that was mapped but not been surveyed for fishes. The same applies for the artificial reef structures  located predominantly along the sandy bottom portion in the center of Santa Monica Bay. These include nine artificial reefs of various designs created from 1960 to 1987 which occupy 114 hectares The precision of reef-specific fish metrics, particularly egg production potential, was relatively low for some reefs as evident by the large 95% confidence intervals. However, the method we followed to make these estimates Future efforts to quantify total abundance could benefit from stratifying a greater number of random samples amongst rocky reef micro-habitats and optimally allocating effort to those according to habitat specific variance. This would likely increase accuracy, precision and sampling efficiency While the data available may have limited the precision at which estimates of some reef-specific metrics were made, combining reef area estimates with fish density, size-structure and size-fecundity relationships still provides useful insight into the potential value of specific reefs from a regional-scale perspective in terms of standing stock and reproductive output. While the smallest reefs in the region (Big Rock and Point Dume) had some of the highest density values for both species, from a regional fisheries perspective, they have limited value. On the contrary, some of the largest contiguous reefs may have lower densities, but as a whole, can contribute substantially to regional reproductive output. Therefore, it is important that the extent of appropriate habitat is considered when resource management actions are being planned and evaluated.The size structures of both focal species in this study illustrate impacts from fishing. Throughout southern California, commercial and recreational fishing pressure on California Sheephead has reduced the prevalence of larger females, as well as impacted other life history characteristics Size structure was a key factor in how much reefs contributed to the regional egg production. Further, results demonstrate the potential that relatively small increases in the proportion of females in the larger size classes on larger reefs could have on regional egg production, due to the exponential relationship between body length and annual fecundity . For CalIncreases in density, biomass and age structure of fish populations over time within the new MPAs may also result in changes to other life history characteristics that have the potential to negatively or positively impact their overall reproductive success. Observations of fish populations across spatial or temporal gradients of density and/or biomass suggest that, as density and/or biomass builds within MPAs, fishes are likely to experience some level of density dependence in somatic growth, with evidence to date being most clear for juvenile stages Given the multiple anthropogenic stressors southern California's urban reefs experience and declines in recreational and commercial fisheries


Sequence and structural elements in the 3'-untranslated region (UTR) of Japanese encephalitis virus (JEV) are known to regulate translation and replication. We previously reported an abundant accumulation of small subgenomic flaviviral RNA (sfRNA) which is collinear with the highly conserved regions of the 3'-UTR in JEV-infected cells. However, function of the sfRNA in JEV life cycle remains unknown.Trans-acting effect of sfRNA on JEV translation was studied using a reporter mRNA containing the luciferase gene fused to partial coding regions of JEV and flanked by the respective JEV UTRs. In vivo and in vitro translation revealed that sfRNA inhibited JEV translation.Northern blot and real-time RT-PCR analyses indicated that the sfRNA becomes apparent at the time point at which minus-strand RNA (antigenome) reaches a plateau suggesting a role for sfRNA in the regulation of antigenome synthesis. Transfection of minus-sense sfRNA into JEV-infected cells, in order to counter the effects of plus-sense sfRNA, resulted in higher levels of antigenome suggesting that the presence of the sfRNA inhibits antigenome synthesis. in trans.Our results indicate that sfRNA modulates viral translation and replication  Flaviviridae family, is a major zoonotic agent. Pigs and birds are the principal viremic hosts and mosquitoes are responsible for the transmission between these vertebrates to human [Japanese encephalitis virus (JEV), a member of the to human . In humato human . The JEVto human .As with all positive-sense RNA viruses, JEV RNA replication begins with the synthesis of negative-strand antigenome, which serves as a template for the synthesis of progeny positive-strand genomic RNA. The asymmetric RNA replication leading to 10- to 100-fold excess of positive strands over negative strands which was observed in Kunjin virus and dengue virus (DENV), and in JEV infected cells -7. In adcis-acting sequences required for JEV replication might be slightly different from those other flaviviruses. In addition to RNA-RNA interactions, numerous studies have been shown that 3'-UTR interacts with both viral and cellular proteins, and is required for RNA synthesis and translation [The 3'-UTR of flaviviruses has been shown to serve various important functions such as translation, replication, and encapsidation -17. Thernslation ,26-36. Inslation -39. Prevnslation .in vivo translation studies in cultured cells and a rabbit reticulocyte lysate assay system was used for in vitro translation studies. Our results indicated that the sfRNA inhibits antigenome synthesis and also down regulates viral translation in trans.The presence of such essential motifs in sfRNA and its abundance in infected cells present a compelling indication of a possible function that prompted us to elucidate its role in the viral life cycle at the cellular level. We found that high levels of sfRNA accumulates in the cytoplasm during the late stages of viral life cycle suggesting that sfRNA may inhibit either viral translation or minus-strand synthesis or both. To test this, plus- or minus-strand forms of the sfRNA were separately transfected in virus-infected cells and the effects on genome and antigenome accumulation were measured. The effect of sfRNA on JEV translation was determined by co-transfecting plus or minus sense of sfRNA with a luciferase reporter RNA for Replication of flavivirus RNA takes place mainly in the cytoplasm, however, the major replicase proteins NS3 and NS5 were also found to localize within the nucleus . To dete3 copies per cell at 24 h postinfection, and its abundance continued to increase throughout the 48-h infection period, except for a slight diminution at 44 h postinfection. The RNA levels reached to 2.91 × 105 copies per cell at 48 h postinfection. The accumulation of the antigenome followed the same trend as the genomic RNA throughout the experimental period but the amount was less by one to two orders of magnitude than that of genomic RNA. From 24 to 48 h postinfection there was a 63-fold increase in genome accumulation whereas only a 15-fold increase for the antigenome indicating that the increasing rate of genomic RNA accumulation is nearly four times faster than that of the antigenomic RNAs  of 0.01, the RNA was extracted at 4 h intervals during a 48-h postinfection period and analyzed by Northern blot. The probe used was 3JEV10950(-) oligonucleotide which detects RNA containing the very 3'-terminal 27 nts of the JEV genome. The results showed that genomic RNA was detected at 22 h and sfRNA at 28 h postinfection, and their abundance continued to increase throughout the experimental period sfRNA did not affect genomic or antigenomic RNA accumulation compared to mock transfection sfRNA with precise sequences complementary to the authentic sfRNA, we then constructed pUC18/JEV(-)10976-10454 and used for generating (-)sfRNA without extra sequences at the termini. Transfecting of the (-)sfRNA did not have significant influence on the accumulation of genome sfRNA, (-)sfRNA, or control RNA (cRNA) and effects on reporter translation were measured at 8 h posttransfection. Interestingly, transfecting either (+)sfRNA or (-)sfRNA but not control RNA inhibited luciferase translation driven by JEV 5' UTR sfRNA as well as (-)sfRNA in rabbit reticulocyte lysate sfRNA (by transfection) at 28 h postinfection countered the effects of naturally occurring sfRNA thereby increasing the accumulation of antigenome Figure  and 3F is Figure  where ths Figure  and 3C acis-acting sequences including promoters, enhancers, and repressors that aid in the in regulation of minus-strand synthesis have been reported in many plus-strand RNA viruses [in trans [trans-acting function could be one of the factors contributing to the asymmetry in JEV RNA replication.The presences of  viruses -47 and tin trans -50. Sevein trans . In addiin trans . Thus, tin vitro was also impaired in the presence of sfRNA but was restored to control levels by the addition of equal amounts antisense sfRNA into the reaction. This clearly shows that sfRNA does impair JEV translation in trans, as depicted in Figure It has also been reported that the RNA elements in the flaviviral 3'-UTR influences viral translation efficiency ,15,33,35trans-acting riboswitch) that shuts down both antigenome synthesis and JEV translation thereby promoting only genomic RNA synthesis that needs to be packaged and released for the next infectious cycle.As seen in our results Figures  and 3C, Culex tritaeniorhynchus was used in this study [Baby hamster kidney (BHK-21) cells were grown in RPMI 1640 medium supplemented with 2% fetal bovine serum (FBS) (Gibco-BRL) at 37°C. JEV strain RP9, a variant of NT109 isolated originally from is study .pGEMT/JEV3642-3821 plasmid Figure  used forin vitro transcription with DIG RNA labeling mix . Hybridization was done at 54°C for oligonucleotide probes and 68°C for riboprobes. DIG luminescent detection of the viral specific bands was done according to the manufacturer's instructions .RNA extraction and Northern analyses were done as described previously . BrieflyAF014161. 3JEV10950(-) oligonucleotide (5'-AGATCCTGTGTTCTTCCTCACCACCAG-3') detects RNA containing the very 3'-terminal 27 nts of the JEV genome. 18S rRNA(-) oligonucleotide (5'-GCACTTACTGGGAATTCCTCG-3') location corresponds to mouse 18S rRNA, GenBank accession number X00686. Mitochondria 12S rRNA(-) oligonucleotide (5'-AAGGCCAGGACCAACCT-3') was synthesized according to GenBank accession number NC_005089.JEV genome nucleotide positions correspond to those for JEV RP9, GenBank accession number in vitro transcripts of positive-sense RNA were generated from T7 transcription (Promega) of the Sal I-linearized pGEMT/JEV3642-3821 and the minus-sense transcripts were transcribed from SP6 promoter of the Nco I-linearized pGEMT/JEV3642-3821  and subjected to real-time RT-PCR using the one-step RT-PCR master mix reagent kit following the manufacturer's instructions (Applied Biosystems). Oligonucleotides JEV3642(+) and JEV3821(-) were used as primers for binding to minus and plus-strand RNA, respectively, during RT step carried out at 48°C for 30 min. The PCR amplification conditions were 95°C for 10 min, followed by 40 cycles of 95°C for 15 sec and 60°C for 1 min with primers JEV3650(+), JEV3726(-) and TaqMan probe JEV3705(-) 10976-10454 were used for making plus- or minus-strand form of the sfRNA, respectively. Before RNA transfection, cells in 6-well plates at 50 to 80% confluence (approximately 2 × 106 cells) were infected with JEV RP9 at an MOI of 0.01 by incubating cells with inoculum at 37°C for 1 h, refeeding with 2 ml of growth medium containing 5% FBS, and incubating at 37°C for 27 h. For transfection, each dish of cells was rinsed three times with RPMI and treated for 10 min at 0°C with 200 μl of Opti-MEM medium (Gibco-BRL) containing 10 μl of lipofectin (Invitrogen) and 1 μg of RNA transcripts. Cells were rinsed with 2 ml of RPMI medium three times and incubated at 37°C with 2 ml of medium containing 5% FBS until cytoplasmic RNA extraction was done at the indicated time points.For run-off transcription, Renilla luciferase (Rluc) fused in-frame to the JEV coding regions as a single ORF in the following order; the core (nt 96-158), Rluc (933 nts), E (nt 2388-2477), NS1 (nt 2478-2693), and NS5 (nt 10203-10391), and this ORF unit was flanked by the authentic JEV 5'- and 3'-UTRs . Luciferase assays were performed using extracts from transfected cells and also from the in vitro translation assay. For in vivo translation in cultured cells, 1 μg of transcribed minicon RNA, together with 1 μg of plus- or minus-strand of sfRNA were transfected into BHK-21 cells using Lipofectamin 2000 (Invitrogen). Renilla luciferase activity was measured at 8 hour posttransfection. For in vitro translation, 50 ng of RNAs were translated in nuclease-treated rabbit reticulocyte lysate (Promega) in the presence of 40 units of RNasin Ribonuclease Inhibitor (Promega), 20 μM of each amino acid, and either plus-strand, minus-strand of sfRNA, or a 445-nt control RNA composed of JEV sequences (nt 2401-2689) plus 156 nts derived from vector sequences. The reactions were incubated at 30°C for 30 min and 2.5 μl of reaction sample was measured with 20/20n Single-Tube Luminometer (Promega).JEV minicon (kindly provided by Dr. Yi-Ling Lin) contains a s Figure . The RluFor statistical analysis, one-way ANOVA Dunnet's multiple comparisons test was used to compare the control group against others .CS: conserved sequences; DAR: downstream of the AUG; DENV: dengue virus; JEV: Japanese encephalitis virus; MOI: multiplicity of infection; sfRNA: small flaviviral RNA; SLA: stemloop A; UAR: upstream of the AUG; UTR: untranslated region; WNV: West Nile virus.The authors declare that they have no competing interests.YHF carried out the translation experiments and statistical analyses. MN designed one of the translation experiments, the model, and participated in drafting of the manuscript. CCC and CCW carried out the replication studies. YTL carried out the nuclear and cytoplasmic fractionation and Northern blot analysis. RYC conceived of the study, participated in its design and coordination, and finalized the manuscript in its final form. All authors read and approved the final manuscript.


DI-RNAs have been further exploited to discover host factors that modulate Tomato bushy stunt virus replication, as well as viral RNA recombination. This review discusses the current models on generation and evolution of DI-RNAs, the roles of viral and host factors in DI-RNA replication, and the mechanisms of disease attenuation.Defective interfering (DI) RNAs are subviral RNAs produced during multiplication of RNA viruses by the error-prone viral replicase. DI-RNAs are parasitic RNAs that are derived from and associated with the parent virus, taking advantage of viral-coded protein factors for their multiplication. Recent advances in the field of DI RNA biology has led to a greater understanding about generation and evolution of DI-RNAs as well as the mechanism of symptom attenuation. Moreover, DI-RNAs are versatile tools in the hands of virologists and are used as less complex surrogate templates to understand the biology of their helper viruses. The ease of their genetic manipulation has resulted in rapid discoveries on  Intriguingly, many viruses have their own parasites, including defective interfering (DI) RNAs, as well as satellite viruses and satellite RNAs. For viruses with RNA genomes, the DI molecules consist of RNA sequences derived from the parent RNA virus, whereas the origin of satellite RNAs are not known in most cases. Both classes of subviral RNA are parasitic, since they have to use proteins coded by viruses, called helper viruses, for their replication inside the host cells. This review will focus on the DI-RNAs and their intimate relationship with their parent viruses and host cells.trans. DI-RNAs are referred as “interfering” because they can attenuate the symptoms caused by the helper virus .,29.pol Oequences .2.1.2.de novo formation of novel DI-RNAs. Due to the presence of large number of ribonucleases in host cells, this mechanism might be rather common for RNA viruses and DI-RNAs [This model is a modified version of the replicase-driven template-switching model. The model postulates that the viral replicase switches template when it encounters the 5′ end of the template, which serves as a strong stop signal for the replicase. The 5′ end could be the natural end of the DI-RNA, so that recombination leads to the generation of head-to-tail DI-RNA dimers. New 5′ ends could also be generated via cleavages by host endo- and exoribonucleases, leading to partial degradation of the DI-RNA as demonstrated for the TBSV DI-RNAs –33. Thes DI-RNAs .2.1.3.Another mechanism for RNA recombination based on RNA ligation has also been reported for Q-beta bacteriophage and poliovirus ,37. The 2.2.pol polymerase, which overlaps with p33, did not have similar affects on recombination, suggesting different roles for RPR in p33 and p92pol proteins with respect to RNA recombination. How these mutations affect recombination, or how they enhance template-switching remains to be tested.Evidence to support the roles of replication proteins in DI-RNA formation has been obtained with the helicase-like protein 1a of BMV. Mutations within the 1a protein altered the sites of RNA recombination when compared with BMV infections containing the wt 1a . In addipol and p33 replication proteins in yeast, a surrogate model host [pol was expressed at a high level in yeast. In addition, the ratio of p33 and p92pol also affected recombinant DI-RNA formation. It is possible that too high a level of p92pol RdRp makes the viral replicase complexes less precise and more prone to template switching.Most plant RNA viruses code for two or more replication proteins and the amount and the ratio of the replication proteins could be another factor affecting RNA recombination and DI-RNA formation. This was tested with the tombusvirus p92del host . High le2.3.cis-acting replication elements in RNA recombination has been proposed before in several viral systems [cis-acting replication elements likely promote the formation of DI-RNAs as well, as shown for the RIII enhancer element and for RII that is required for RNA recruitment during TBSV replication [N. benthamiana protoplasts [The role of  systems , includi systems ,23,24,44lication ,25,45,46toplasts . The recAdditional highly active “hot-spot” regions for RNA recombination that might also promote DI-RNA formation consist of AU-rich stretches or stable secondary structures ,48. InseCymbidium ringspot virus (CymRSV) [Strong RNA secondary structures in templates can also serve as recombination hot spots. These structures might form between two viral RNAs containing short complementary sequences, leading to the formation of a heteroduplex . Strong (CymRSV) .cis-acting replication elements that bind to viral or host proteins. For example, region II (RII) in TBSV is a recombination hot spot [Hairpin structures serve frequently as hot spot ,32, whichot spot . Anotherhot spot ,30,32,332.4.XRN1 5′ to 3′ exoribonuclease involved in the RNA degradation pathway, led to the discovery of partial TBSV RNA degradation products in xrn1Δ yeast and xrn4-silenced N. benthamiana plants [In vitro data using purified Xrn1p, as well as in vivo work of overexpression of this exoribonuclease demonstrated that Xrn1p works as a suppressor of TBSV RNA recombination [Arabidopsis thaliana AtXrn4p, which is an orthologue of the yeast Xrn1p in Nicotiana benthamiana, led to degradation of viral RNAs . In agreement with the yeast data, silencing of XRN4 in N. benthamiana led to higher accumulation of the DI-RNA recombinants (Jaag and Nagy 2009).In spite of significance in virus replication and possibly DI-RNA formation, our understanding of the role of the hosts in RNA recombination is rather limited. Significant progress has been made by establishing yeast as a model host for TBSV replication/recombination . Serviena plants –33. The bination . Moreovepol and p33 in yeast, which affected DI-RNA formation [de novo generation of DI-RNAs from the parent virus was not studied, instead, already generated DI-RNA was used as a model template.The screening of host genes for TBSV DI-RNA recombination was further extended to 800 essential yeast genes . The autormation ; (ii) otCryphonectria parasitica, the chestnut blight fungus. Moreover, in Δdcl-2 strain, the absence of DI-RNA indicated that replicase errors might not be the only factors affecting the generation/accumulation of DI-RNAs [Another example of the host’s role in generation of DI-RNA is the demonstrated requirement of host dicer in production of DI-RNAs . The aut DI-RNAs .2.5.Broad bean mottle virus (BBMV) infection were observed in some host plants even when infections were started with low multiplicity of inocula (MOI), whereas in other plants, high MOI for several passages were needed to generate DI-RNAs. Interestingly, the sizes of the DI-RNAs as well as the rate of their accumulation varied with the growth temperature. It is not yet known whether these changes are because of some change in selection pressures on DI-RNA accumulation (i) due to variable activities of the host defense mechanisms, such as gene silencing; (ii) due to altered RdRp activities, such as template-switching, under various conditions; or (iii) due to changes in the structure of the viral RNA. We also cannot rule out the combination of all the above factors.Apart from the roles of viral replicase proteins and the replication/recombination elements present in the viral genomic RNA, host specificity and growth conditions have also been implicated to affect the synthesis DI-RNAs . For exaN. benthamiana and pepper (Capsicum annuum) [N. benthamiana, DI-RNA accumulation was easily detectable and able to attenuate the symptoms, whereas, in pepper, even continuous virus passages failed to generate detectable levels of DI-RNA. Moreover, the pepper plants showed severe local and systemic chlorosis. Thus, the results from N. benthamiana indicates that DI-RNA formation is a way for plants to attenuate virus-induced symptoms.The important role of the host for TBSV DI-RNA accumulation was further supported by an altered rate of DI-RNA accumulation in  annuum) . In N. b3.Most RNA viruses undergo rapid evolution due to their error prone RdRps –61. Muta3.1.DI-RNAs are mainly formed via RNA recombination/genome rearrangements. It is likely that, even though they are more robust, only a fraction of DI-RNAs formed will be replication competent and able to compete in plants. The replication competent DI-RNAs have to compete with the helper virus for the viral RdRp and with other DI-RNAs, resulting in continuous DI-RNA evolution under high selection pressure. DI-RNAs with mosaic genomes, such as the TBSV-associated DI-RNAs  are form3.2.cis-element for binding to the viral p33/p92pol replicase proteins. This in turn, could result in enhanced viral RNA recruitment in case of DI-R1. A single U insertion in RII was found to make DI-B103, containing only one RII, more competitive than DI-R1 containing two wt RIIs. This insertion may have made the single RII in DI-B103 more efficient at binding p33, however this idea remains to be tested.Albeit sequence deletions and rearrangements are the most common events during DI-RNA formation, additional genetic changes also occur frequently. For example, DI-RNAs can undergo other types recombination events, such as segment duplication or nucleotide insertions that could enhance their competitiveness ,63. By ucis-acting elements required for the assembly of replicase complex. This minimal DI-RNA goes through rapid evolution in yeast and in plants by sequence duplication and even triplication. . The presence of multiple cis-acting elements may help the new DI-RNAs to increase binding to more replicase proteins cooperatively or facilitating fast recruitment for replication. It is interesting to note that too strong competition with the helper virus for the replicase complex could be counterproductive for the DI-RNA, since then this leads to reduced number of helper virus genomes per cell, followed by reduction in the synthesis of viral replication proteins. This in turn, limits further replication of the DI-RNAs. Therefore, it is likely that the best adapted DI-RNAs are capable of compromise, allowing the helper virus to replicate as well. Many RNA viruses also utilize “cis-preferential” replication [trans-replicating DI-RNAs have to “steal” the replication proteins from a pool of newly synthesized proteins.Competition for limited viral- and host resources drives the evolution of DI-RNAs at faster rates than that of their parental virus. Accordingly, we observed rapid evolution with a minimal DI-RNA carrying only a minimal set of essential lication ,64–66 whNevertheless, their small size and limited or no contribution to expression of functional proteins give DI-RNAs a major advantage over their helper virus. In many cases, this results in attenuation of disease symptoms caused by the helper virus. In order to systemically infect the host plants, the DI-RNAs also have to move from cell to cell via plasmodesmata. If viral movement proteins bind cooperatively to the viral RNA, DI-RNAs would be at a disadvantage, as they have short genomes. To circumvent this problem, DI-RNAs might generate head-to-tail dimers . In this4.cis-acting replication elements are usually excellent templates during replication due to: (i) their small sizes; (ii) being efficient RdRp substrates; (iii) and lacking competition between translation and replication processes for the RNA templates in case of translation-incompetent DI-RNAs. Therefore, in many cases the accumulation of the helper virus is inhibited by the most competitive DI-RNAs, resulting in the symptom attenuation in host plants. However, the interaction between the DI-RNA, helper virus and the host is more complex, due to the effect of DI-RNAs on induction of host defense responses or sequestering resources of the host cells by the DI-RNA. Accordingly, DI-RNAs of BBMV and TCV enhance the symptoms caused by their helper viruses [reviewed by Those DI-RNAs that maintain critical 4.1.N. benthamiana protoplasts with TBSV and DI-RNAs, showed 65% suppression of TBSV genomic RNA accumulation when equimolar amount of helper versus DI-RNAs were used [In the absence of DI-RNAs, the pool of replication proteins, host factors, nucleotides, host membranes and all other factors is utilized entirely by the helper virus for its own multiplication. Once DI-RNAs are in the picture, the above factors then have to be shared by both the viral and sub-viral RNAs. Eventually, the helper virus is out-multiplied by the more competitive DI-RNAs. This type of helper virus inhibition by DI-RNA is known to be dose-dependent. The co-infection experiments done in ere used .Additional findings, like inability to attenuate the symptoms by some DI-RNAs that accumulate to high levels , and hos4.2.N. benthamiana plants were co-infected with TBSV genomic transcripts and DI-RNAs, the reduction in viral genomic RNA replication proteins accumulation was less dramatic than the reduction in the levels of p19 suppressor of gene silencing and p22 movement proteins along with the levels of subgenomic (sg) RNA2 [Several studies on the attenuation effects of DI-RNAs on the symptoms caused by the helper virus did not fit into the simple model of competition desribed above. For example, when sg) RNA2 . Not onl4.3.RNA interference (RNAi) or posttranscriptional gene silencing (PTGS) is a eukaryotic cellular response to the presence of double-stranded (ds) RNAs. These dsRNAs can be either endogenous or parasitic in nature [reviewed by N. benthamiana plants triggered PTGS, resulting in the accumulation of siRNAs corresponding to the CymRSV sequence. They further showed that the PTGS machinery did not efficiently degrade the shorter DI-RNAs. Moreover, this defect was not associated with the RNase complex (RISC) because the system could efficiently silence the larger DI-RNAs and a different helper virus. The length of target molecule was not a deciding factor in this target activity, rather specific sequences and structures of the DI-RNAs played a major role in PTGS. Thus, DI-RNAs could trigger potent gene silencing response against the helper virus without hurting themselves from the same response. In a further attempt to characterize DI-RNA associated symptom attenuation, sequence elements of tombusvirus-associated DI-RNAs were identified [In another study, a more direct role for DI-RNA in triggering gene silencing against its own helper virus emerged. It was shown by Szittya and colleagues  that theentified ,78.The more we will learn about viral pathogenesis and the interaction and competition between DI-RNA and the helper virus, more we can focus our research to dissect DI-RNA mediated attenuation in molecular mechanistic term. It is possible that DI-RNA can modulate interaction between two or more viral proteins or affect their posttranslational modifications. It is also not unrealistic to imagine that DI-RNAs can sequester important host factor(s) away from helper virus replication and thus, reducing helper virus accumulation and moderating the symptoms associated with helper virus infections.5.5.1.The use of plant viruses for recombinant protein production is widespread in research and growing for industrial production of useful proteins. In recent years, we have seen advances in both development of first-generation (based on ‘full virus’) vectors and second-generation (‘deconstructed virus’) vectors [reviewed by 5.2.cis-acting replication elements for replication and other steps in viral multiplication cycle. Since many DI-RNAs do not code for proteins, mutational studies on DI-RNAs make the results easier to interpret due to separation of trans-acting protein factors and cis-acting RNA elements. Thus, critical cis-acting replication elements important for different steps in viral replication can be deductively discovered. These RNAs also are adapted to use trans  factors for their replication enabling a more experimental control over those factors so that a virologist can regulate them spatially and/or temporally. Indeed, using DI-RNAs combined with other modern tools, many cis-acting RNA elements for tombusviruses were deciphered [reviewed by cis-acting replication elements, viral and host factors discovered with the assistance of DI-RNA, we divide the replication cycle of the plus stranded RNA viruses further into different steps in chronological order [reviewed by DI-RNAs are versatile tools at virologists’ disposal. DI-RNAs often multiply at higher rates, are smaller than the full genome of the virus and contain the 5.3.N. benthamiana plants [in vitro [Similar to the viral genomic RNA, DI-RNAs should also be recognized selectively from the large pool of host RNAs. The selection of the viral RNA requires specific interaction with viral- or host proteins. For TBSV DI-RNAs, the selective RNA recognition is mediated by the cytosol-exposed C-terminal portion of p33 replication protein in yeast ,93. Detaa plants ,93. Thusin vitro ,94.In addition to interacting with the viral replication proteins, DI-RNA is likely bound by select host proteins. To identify the host proteins that bind viral RNA, TBSV DI-72 (+)RNA was used as a probe to screen RNA binding proteins in a yeast proteome-wide chip carrying 4,100 purified yeast proteins . Five ofin vivo was lost when DI-72 repRNA missing RIII, the target for Nsr1 binding, was used as a replicon RNA in above study. This protein may inhibit TBSV replication via specific binding to the viral RNA and, thus, resulting in inefficient repRNA recruitment for replication.Another host factor that exerts its effect on viral replication directly via binding to the viral RNA is Nsr1p . This factor was discovered during screening of the yeast knock out (YKO) library for TBSV replication . Virus rin vitro and is also associated with the replicase complex, albeit only temporarily. When Pex19p was mistargeted to mitochondrial membranes, the wt p33 also ended on the same mitochondrial membranes. Interestingly, viral replication as well as the replicase activity was inhibited as a result of Pex19p mislocalization to the mitochondrial membranes.After the RNA has been selected for replication, the viral proteins and RNA complex has to be transported to the site of replication. TBSV assembles the replicase complex on the cytosolic surface of peroxisomal membranes –99. It hAnother host protein involved in localization/transportation of the viral replication proteins is the heat shock protein 70 (Hsp70). Using a temperature-sensitive mutant of Hsp70 at nonpermissive temperature has led to cytosolic localization of p33 replication protein . Shiftinpol complex to the site of replication.Overall, the above studies revealed that both viral- and host proteins are involved in RNA template selection and the recruitment of the viral RNA/p33/p925.4.in vivo and in vitro [cis-acting elements required for the assembly of the tombusvirus replicase complex. The affinity purified viral replicase complex was active on external templates only when the full-length DI-72 repRNA was co-expressed with the p33 and p92pol replication proteins in yeast. The minimal repRNA still capable of supporting the assembly of the replicase complex consisted of RII(+)-SL hairpin, the replication silencer element and genomic promoter  in the 3′ end of (+)DI-RNA [in vitro. It is currently under investigation how this interaction is regulated and what viral- or host proteins are involved in “unsilencing” this interaction.After the assembly of the replicase complex is finished, the synthesis of the complementary (−)-strand from the plus (+)-stranded DI-RNA takes place. The newly made (−)-strand then serve as a template for synthesis of new (+)DI-RNAs. The replication process of (+)-stranded RNA viruses and associated DI-RNAs is asymmetrical, leading to 20–100-fold more copies of (+)RNAs than (−)-strand RNA. One of the major factors in regulation of DI-RNA replication is the DI-RNA carrying +)DI-RNA , a compl+)DI-RNA  and repl+)DI-RNA . An intr+)DI-RNA . This elcis-acting RNA replication elements, but host factors as well. One such host factor is a metabolic enzyme called glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which was discovered as a component of the tombusvirus replicase complex via a proteomics analysis of a purified viral replicase preparation [N. benthamiana host [The asymmetrical RNA synthesis is affected not only by paration . When TBparation . Down-reana host .5.6.After the synthesis of the new (+)DI-RNA progeny by the replicase, (+)RNA is released to the cytosol , while tin vivo and in vitro as well [in vitro phosphorylation of the p33:DI-72 RNA complex led to the release of the RNA from the complex [So far host factors and viral RNA sequences have not been discovered playing role(s) in the release of (+)RNA progeny. However, viral protein modification has been proposed to modulate this process . This is as well ,110. The complex . In contin vitro [Roles of other host proteins in viral protein modification have also been demonstrated. A host ubiquitin conjugating enzyme Cdc34p as well as Rsp5p ubiquitin ligase have been shown to ubiquitinate p33 in vitro ,111. HowAltogether, the use of DI-RNA as surrogate template contributed a great deal to our understanding of viral RNA replication and virus - host interactions. Future studies will further exploit DI-RNAs to dissect the mechanism of virus replication, recombination and the role of the host as well as compare similarities between DI-RNAs and helper viruses.6.cis-acting elements for replication were also discovered using DI-RNAs. Several of the above findings with DI-RNAs were subsequently validated with infectious TBSV in the natural plant host. These advances indicate that DI-RNAs are highly evolved molecules that possess authentic cis-acting replication elements and utilize helper viral- and host factors for their multiplication. Future experiments will likely dissect the detailed mechanisms of DI-RNA formation and evolution during viral infection. The roles of host and environmental factors will also be studied to throw light on how viruses and sub-viral particles co-evolve in the context of their environment.Existence of DI-RNAs associated with the infectious parent viruses is one of the perplexing examples of molecular diversity and parasitism. DI-RNAs have a complex relationship with the helper virus and they affect most aspects of virus biology. The relationship between the DI-RNA and the helper virus includes competition for viral- and host factors, induction/suppression of host antiviral responses, and modulation of disease symptoms caused by the helper virus. It will be interesting to explore how DI-RNAs are maintained in the natural viral infections. What advantages do the helper viruses or the host have in co-existing with the DI-RNAs? It is plausible that they might help limit the extent of damage caused by viral infections to their hosts, so that viruses do not kill their hosts as rapidly, thus prolonging the time a host can participate in spreading viruses. As importantly, DI-RNAs are used as surrogate templates in viral replication studies that have contributed to our understanding on the biology of viral infections. DI-RNAs of TBSV have also facilitated the identification of host factors affecting TBSV replication as well as RNA recombination. Many of the


The recent discovery of a significant amount of RNA in spermatozoa contradicted the previously held belief that paternal contribution was limited to one copy of the genome. Furthermore, detection of RNA in sperm raised the intriguing question of its possible role in embryonic development. The possibility that RNAs may serve as epigenetic determinants was supported by experiments showing inheritance of epigenetic traits in mice mediated by RNA. We used high-throughput, large-scale sequencing technology to analyze sperm RNA. The RNA sequences generated were diverse in terms of length and included mRNAs, rRNAs, piRNAs, and miRNAs. We studied two small noncoding RNAs enriched in mature sperm, designated sperm RNAs (spR) −12 and −13. They are both encoded in a piRNA locus on chromosome 17, but neither their length (20–21 nt), nor their sequences correspond to known piRNAs or miRNAs. They are resistant to periodate-oxidation-mediated reaction, implying that they undergo terminal post-transcriptional modification. Both were detected in sperm and ovulated unfertilized oocytes, present in one-cell embryos and maintained in preimplantation stages, but not at later differentiation stages. These findings offer a new perspective regarding a possibly important role for gamete-specific small RNAs in early embryogenesis. O-methyl groups at their 3′ ends The discovery of significant amounts of RNA in the transcriptionally inert spermatozoon led to speculation regarding its possible role in embryonic development Our results identify two novel sncRNAs in sperm, also present in zygote and maintained in the embryo exclusively at the very early stage. Their size, nucleotide sequences, expression patterns, and genetic location make these RNAs distinct from known miRNAs and piRNAs.Sequencing of the small RNA fraction prepared from mouse sperm using a 454 sequencer resulted in 359,840 RNA sequences, ranging from 13 to 248 nt. As expected from previous studies We then searched computationally for small RNAs that have stem-loop structure, and predicted 13 putative RNAs see . Nine ofBoth spRs are encoded in a region of approximately 40 kb, within a piRNA cluster on chromosome 17, and are oriented divergently, in the same way as the proximal piRNAs . Analysihttp://mirbase.org/, piRNABank: http://pirnabank.ibab.ac.in) The piRNAs were reported to generate endogenous siRNAs (∼21 nt) from the long double-stranded RNAs (dsRNAs) expressed from retrotransposons and pseudogenes in mouse oocytes In summary, we showed that two small noncoding RNAs, distinct from typical micro- and pi-RNAs, are present only in the gametes and maintained during the early stages of development. Their accumulation, zygotic transfer and maintenance during early development raise a number of intriguing questions for further studies. The event from piRNA expression to spR accumulation indicates a novel pathway for small RNA biogenesis during spermatogenesis. Maintenance of spRNAs during the pre-implantation period and their disappearance at the blastocyst stage suggest that they may exert important function(s) in early embryonic development. Future genetic and biochemical studies will hopefully help to determine the roles of small RNAs during early embryogenesis.This research involving animals was conducted according to the approval of the Animal Research Ethics Committee of RIKEN Yokohama Institute. The approved permit number for this study is “22–033”.2O). This procedure generates an essentially pure population of spermatozoa Spermatozoa were collected from 74 mouse caudae epididymides of strain C57BL/6. Motile spermatozoa were washed twice in MEM buffer  by centrifugation. Sperm pellets were resuspended in phosphate-buffered saline (PBS), followed by centrifugation. The pellets were washed twice in storage buffer , then stored at −80°C. Subsequent to storage, the samples were thawed, and washed twice in cell lysis buffer (0.1% sodium dodecyl sulfate (SDS) and 0.5% Triton X-100 in HA small RNA cDNA library was generated from ∼0.8 µg of total RNA, as previously described http://genome.gsc.riken.jp/osc/english/dataresource/). The mature and star sequences of miRNAs were downloaded from miRbase (version 15.0); RefSeq mRNA sequences were from the UCSC Table Browser; and piRNA, snoRNA, and snRNA were from NONCODE (version 2). The tRNA sequences were prepared from the genome sequence with an additional CCA at the 3′-end, which was based on RefSeq annotation of NCBI Build 37 (mouse). The remaining small RNA sequences, which did not match to the RNA sequences above, were aligned with the mouse genome sequence (NCBI Build 37 or mm9), and their genomic coordinates were compared with the repeat masker track in the UCSC genome browser database. The small RNA sequences were classified based on their matches to known RNA sequences or overlaps on the genomic coordinates.First, we aligned the small RNA sequences to known RNA sequences retrieved from public databases. We accepted only complete matches using nexAlign (version 1.3.5) (http://rna.tbi.univie.ac.at/cgi-bin/RNAfold.cgi) http://mfold.bioinfo.rpi.edu/) We aligned the sequenced 20 to 23-nucleotide small RNA sequences, which were neither miRNA nor rRNA, to the genome sequence. After screening out RNAs that mapped to two or more loci, we identified 39 loci as potential candidates. The putative secondary structures of their proximal regions were predicted by the RNAfold , testes at various ages , and embryo (15 days), and 0.7 µg of total RNA for sperm (9 weeks). The RNA was polyadenylated using a poly(A) Tailing Kit (Ambion) and used in the synthesis of small RNA cDNA with PrimeScript II RTase  and 2.5 µM of RTQ primer 7 molecules each) were denatured for 5 min at 65°C in loading buffer containing 50% formamide, separated on 12% polyacrylamide/8 M urea gels, and then transferred to Nytran N membranes  by electroblotting. The RNAs were cross-linked to the membrane by exposure to UV light. The 5′ oligonucleotide was end-labeled with [γ -32P] ATP and T4 polynucleotide kinase. The membranes were hybridized with 32P-end-labelled oligonucleotides for 18 hours at 42°C in hybridization buffer  and washed 3×10 min with 2× SSC, 0.1% SDS at 42°C and 2×10 min with 0.1% SSPE, 0.1% SDS at 42°C. The hybridization signals were visualized using a PhosphorImager (Molecular Dynamics).Total RNA (10 µg) from testis or 1 µg from sperm samples and synthetic spR-12 and -13 oligoribonucleotides  and 2.5 µl of freshly dissolved 200 mM NaIO4 and incubating for 10 min at room temperature. 2 µl of glycerol was added to quench unreacted NaIO4 and incubated for an additional 10 min at room temperature. Samples were dried by centrifugation under vacuum for 1 hour at room temperature, then dissolved in 50 µl of 1× borax buffer  and incubated for 90 min at 45°C. 20 µg of glycogen was added, the RNAs were precipitated at −70°C for 1 hour, the precipitate samples collected by centrifugation and then dissolved in the denaturing gel loading buffer and ran in 20% polyacrylamaid gel containing 7 M urea. Synthetic spR-12 and -13 oligoribonucleotides, piR-1 [23] and miR-34c were used as controls for the β-elimination reaction. Northern blot hybridization using Hybond N+ membrane  was performed with antisense spR-12, spR-13, flanking-spR-12, piR-1 and miR-34c synthetic oligonucleotides as probes  from testis or 2 µg from sperm samples were subjected to periodate-oxidation-mediated reaction and β-elimination as described s probes .Stem-loop quantitative RT-PCR reactions DNase-treated total RNA (0.5 µg) from adult testis and epididymis was reverse-transcribed using PrimeScript II RTase (Takara) with gene-specific RT-primers . The piR


Aedes albopictus and Aedes aegypti, the production of viral small RNAs that exhibit the hallmarks of ping-pong derived piwi-associated RNAs (piRNAs) after infection with positive or negative sense RNA viruses. Furthermore, these cells produce endogenous piRNAs that mapped to transposable elements. Our results show that these mosquito cells can initiate de novo piRNA production and recapitulate the ping-pong dependent piRNA pathway upon viral infection. The mechanism of viral-piRNA production is discussed.The siRNA pathway is an essential antiviral mechanism in insects. Whether other RNA interference pathways are involved in antiviral defense remains unclear. Here, we report in cells derived from the two main vectors for arboviruses,  Arboviruses are maintained in a transmission cycle between hematophagous arthropod vectors and vertebrate hosts. Within their arthropod vector, arboviruses encounter several anatomical and immunological barriers that determine the potential of the virus to be transmitted. RNA interference (RNAi) is a major antiviral defense mechanism in insects The siRNA and piRNA (piwi-interacting RNA) pathways are both gene regulatory mechanisms guided by small silencing RNAs in association with an Argonaute family member. piRNAs differ from siRNAs in several aspects 1) that are usually antisense to TE transcripts. Cleavage of complementary transposon RNA by primary piRNAs initiates the second biogenesis pathway: the ping-pong amplification cycle that involves AGO3 and aub 1 bias for aub-associated piRNAs and a bias for adenosine at the tenth position (A10) of AGO3-associated piRNAs. PIWI-associated piRNAs have a strong strand bias: AGO3 associates with sense TE piRNAs, whereas piwi and aub associate with antisense TE piRNAs Two mechanisms have been proposed for the biogenesis of piRNAs Drosophila ovarian somatic sheet cells (OSS cell line) 1 bias was observed. These cells are capable of producing primary piRNAs but are incapable of ping-pong amplification, due to the lack of aub and AGO3 expression While the siRNA pathway is well characterized as an antiviral defense mechanism in insects, the involvement of the piRNA pathway has been recently suggested. Indeed, the potential of the primary piRNA pathway to recognize and process viral RNAs was shown in Here, we show for the first time that mosquito cells infected with (+) and (-) RNA arboviruses produce viral small RNAs with the hallmarks of ping-pong amplification. These results show that mosquito tissue culture faithfully recapitulates the piRNA pathway from an exogenous trigger and may combine RNAi pathways to control a viral infection. These observations have important implications for our understanding of insect innate immunity.Aedes albopictus larvae 32P-labelled dsRNA was effectively processed into 21 nt small RNA in U4.4 cell extracts , a (+) RNA arbovirus, expressing GFP as a reporter of viral replication. Small RNAs ranging from 19 to 30 nt in length were recovered from infected cells and deep sequenced. Consistent with the Dcr-2 activity detected, the size distribution of virus-derived small RNAs displayed a sharp peak at 21 nt  and (-) RNA viruses, we next analyzed a published dataset from C6/36 cells infected with La Crosse virus (LACV) 1 bias on the genomic (−) strand and an A10 bias for the antigenomic (+) RNA strand . While this result does not rule out a germline origin of the cell line, we do note that the identification of piRNAs with a ping-pong signature in somatic tissues in flies implies that a functional PIWI pathway is present in the soma of insects To address a potential germline source of the Aag2 cells, we analyzed the expression of Drosophila and other animals were described to map to transposable elements. As the genome sequence of A. aegypti is available http://tefam.biochem.vt.edu/tefam/index.php). TE-derived small RNAs display a sharp 21 nt peak and a broader peak centering around 27 nt, which is suggestive of TE targeting by the Aedes siRNA and piRNA pathways  strongly suggests that the piRNA pathway constitutes another facet of the antiviral RNAi response in insects. Unlike the siRNA pathway, the piRNA pathway is highly enriched in the gonads where it plays a critical role in the control of transposition in the germ line. Because arboviruses can be transmitted vertically in arthropod vectors Antiviral RNAi activity in insects has thus far only been attributed to the siRNA pathway. The identification of vpiRNAs in 1 bias for primary piRNAs. In U4.4, Aag-2 and C6/36 cells however, the nucleotide bias signature is inverted, regardless the polarity of the viral genome. The vpiRNAs that derive from the (−) strand (i.e. the antigenomic strand of SINV and the genomic RNA strand of LACV) present a U1 bias, whereas those that derive from the (+) strand display an A10 bias. This disparity between OSS cells and mosquito cells is unlikely to be due to differences in piRNA biogenesis, as our results on TE piRNAs in Aag-2 and observations in Bombyx Mori BmN4 cells Contrary to the OSS cell line that only produces primary vpiRNAs 1 and secondary A10 vpiRNAs follows strand stoichiometry. According to these observations, we propose two non-mutually exclusive hypotheses for the production of vpiRNAs through a ping-pong mechanism. The first hypothesis is based on the relative amounts of (+) and (−) strands during viral replication. For primary vpiRNAs that are produced from the abundant (+) strand, the generation of secondary vpiRNAs from the (−) strand is limited due to the relative limited amount of viral (−) RNA strands. Conversely, the production of primary vpiRNAs from the (-) strand may allow the generation of abundant secondary vpiRNAs from the abundant (+) strand. According to this hypothesis, as the (+) strand is more abundant than the (−) strand, the second ping-pong mechanism supersedes the first one. As a second hypothesis, the production of primary vpiRNAs from the (−) strand may result for a differential accessibility of the viral RNAs by piRNA pathway components. We propose that the PIWI protein that is responsible for primary piRNA biogenesis can better access viral (−) RNAs, and that the PIWI proteins that are responsible for secondary piRNA biogenesis can mostly access viral (+) RNAs. This may be due to spatial restriction of piRNA pathway proteins or to a differential accessibility of PIWI proteins to the viral RNAs engaged in replication and in translation.The +/− strand ratio is uneven in ssRNA viruses. In (+) RNA viruses, the (+) strand is over-represented compared to the negative strand that serves as template for the production of progeny viral RNA. In many (−) RNA viruses, the (+) viral RNA strand that corresponds to viral transcript is over-represented compared to the genomic (−) strand, although the relative amounts of transcripts are variable. In LACV, there is a gradient of +/− strand ratio between highly (S segment) and slightly (L segment) expressed transcripts. In both (+) and (−) RNA viruses, the genome and the intermediates of replication are shielded from cytoplasmic components, contrary to viral RNAs that engage in translation. Interestingly, primary vpiRNAs are produced from the (−) strand, regardless viral genome polarity. Moreover, in most cases, the ratio between UFinally, we show that viruses trigger the piRNA and the siRNA pathways in a similar way as transposons. This suggests that the RNAi pathways only discriminate common features of parasitic nucleic acids rather than their origin.A. albopictus U4.4 cells and A. aegypti Aag2 cells . Virus-derived small RNAs were analyzed using Paparazzi Aedes aegypti transposon dataset available at TEfam (http://tefam.biochem.vt.edu/tefam/). The aligned reads were processed similarly to the virus-derived small RNA with in-house Perl scripts. Sequences were submitted to the Sequence Read Archive at the National Center for Biotechnology Information under accession number SRA047263.Small RNA libraries were prepared as described 2 flask of Aag2 cells using Isol-RNA Lysis Reagent (5 Prime) according to manufacturer's recommendations. cDNA synthesis was performed on 1 µg of DNase-I (Invitrogen) treated total RNA using an oligo-dT primer and TaqMan reverse transcriptase (Roche). PCR was performed using the following primers: F-AaeNanos, CAAACGTGAAGCGGAAGATT; R-AaeNanos, AATCAACGATGGATCGGATT; F-AaePIWI1/3a, TGTAGGGGAAGTAATGCATCG; R-AaePIWI1/3a, TCTACGGCAATGGTATCTGCT; F-AaePIWI1/3b, GGCCGTTAGCGAGTCTCAT; R-AaePIWI1/3b, GGCAGAACCTTCGTGGTAAG;; F-AaePIWI2, ATGAAAGCCGGGAAGGTC; R-AaePIWI2, CTGCTACCATTGCCATTTCC; F-AaePIWI4, TGACCGTTACTCTCAAGGGCGCTACCGT; R-AaePIWI4, GACCGTTCACGGCCACCTGCCGAT; F-AaePIWI5, GCCATACATCGGGTCAAAAT; R-AaePIWI5, TGAGGTTGTTGCTTCTGAGGT; F-AaePIWI6, TAATCCACAGGAAGGCTCCA; R-AaePIWI6, CTCCTCCATTGTCCGATCCT; F-AaePIWI7, GGAGGTCGTGGAGGTAACAA; R-AaePIWI7, CCTTCCAATCACGATTGCTT; F-AaeAgo3, TCGGTTTACCGCCAGCTGGGAGTTTTG; R-AaeAgo3, AGGTTATCTCAGCGGGAAAATCATGTCGCT.Total RNA was isolated from a confluent 75 cm


Tapping panel dryness (TPD) is one of the most serious threats to natural rubber production. Although a great deal of effort has been made to study TPD in rubber tree, the molecular mechanisms underlying TPD remain poorly understood. Identification and systematical analyses of the genes associated with TPD are the prerequisites for elucidating the molecular mechanisms involved in TPD. The present study is undertaken to generate information about the genes related to TPD in rubber tree.To identify the genes related to TPD in rubber tree, forward and reverse cDNA libraries from the latex of healthy and TPD trees were constructed using suppression subtractive hybridization (SSH) method. Among the 1106 clones obtained from the two cDNA libraries, 822 clones showed differential expression in two libraries by reverse Northern blot analyses. Sequence analyses indicated that the 822 clones represented 237 unique genes; and most of them have not been reported to be associated with TPD in rubber tree. The expression patterns of 20 differentially expressed genes were further investigated to validate the SSH data by reverse transcription PCR (RT-PCR) and real-time PCR analysis. According to the Gene Ontology convention, 237 unique genes were classified into 10 functional groups, such as stress/defense response, protein metabolism, transcription and post-transcription, rubber biosynthesis, etc. Among the genes with known function, the genes preferentially expressed were associated with stress/defense response in the reverse library, whereas metabolism and energy in the forward one.The genes associated with TPD were identified by SSH method in this research. Systematic analyses of the genes related to TPD suggest that the production and scavenging of reactive oxygen species (ROS), ubiquitin proteasome pathway, programmed cell death and rubber biosynthesis might play important roles in TPD. Therefore, our results not only enrich information about the genes related to TPD, but also provide new insights into understanding the TPD process in rubber tree. Hevea brasiliensis Muell. Arg.) is a perennial tropical tree for the production of natural rubber (NR). In the world, at least 2000 plant species are recognized for producing latex, but the rubber tree is the only economically viable source of NR. Rubber molecules are produced, aggregated and packaged in the latex vessels (laticifers) of rubber tree. The latex, a cytoplasmic component of the laticifers, expels from the laticifers upon tapping. Over the past decades, the rubber yield has been significantly increased, due to the cultivation of high-yielded clones and the utilization of ethephon (an ethylene generator). However, latex production still faces serious economic losses caused by TPD. At present, there are no effective measures to prevent or treat TPD in rubber tree. It was estimated that the losses due to TPD accounted to 12-14% of the annual rubber production . Among 237 unique genes, 162 and 75 were from the reverse and forward SSH libraries, respectively  convention . The 237To validate the genes differentially expressed between the two libraries, 10 genes from the forward library and 10 genes from the reverse one were selected to perform the expression analyses with RT-PCR and real-time PCR. These genes were associated with signal transduction, metabolism and energy, RB, unknown function, stress/defense response, transcription and post-transcription, protein metabolism, transporter and cell biogenesis. The specific primers of these 20 genes are listed in Table Hevea latex [Hevea clones, tapping systems and stages of TPD, etc. Therefore, it was necessary to identify more genes related to TPD with different experimental materials. The molecular mechanism underlying TPD could be precisely elucidated only if enough the genes related to TPD were identified. Compared with the functional classification of TPD-related genes from Venkatachalam et al. [We reported here the identification and characterization of the genes associated with TPD in rubber tree. In our research, the subtraction efficiency was validated by the expression analyses of 18s rRNA gene Figure . The cloea latex . Among tm et al. , one obvOsRac1 and SSH37 matching phospholipase C, were all upregulated in TPD tree. It was reported that the upregulation of OsRac1 and phospholipase C might induce ROS production in plants [OsRac1, was also increased in TPD tree. The CCR activity and ROS production were enhanced in the transgenic cell cultures constitutively expressing active OsRac1 [Escherichia coli overexpressing rBmTCTP in vivo was subjected to oxidative stress [ROS, as toxic molecules, are capable of injuring cells. 17 genes probably involved in producing and scavenging ROS were identified in this research  was reduced in TPD tree; the previous findings indicated that eIF5A negatively regulated programmed cell death [Arabidoposis [Besides the induction of UPP, ROS can also initiate the programmed cell death process -47. It ie stress . The gengrations -54. HbMyber tree ,21. The ber tree . A gene ll death -61. An aidoposis . A gene idoposis ,64. Threidoposis ,66. A geidoposis -69. The idoposis . Due to SPOP underwent apoptosis [Mlo gene was elevated in TPD tree. In barley, Mlo transcripts were increased during leaf senescence, suggesting that Mlo might play important roles in preventing cell death [PCD has been defined as a sequence of events controlling and organizing the cell destruction . In thispoptosis . The expll death . Chen etll death -22. In aHevea brasiliensis [HbTOM20 and small rubber particle protein also play important roles in rubber biosynthesis [HbTOM20, one small rubber particle protein and one 5-phosphomevelonate kinase were all downregulated in TPD tree  pathway in iliensis -80. Besiynthesis ,81-84. ITPD tree . AlthougAltogether, the genes associated with TPD were identified and their characterizations were further analyzed in the paper. Among 237 unique genes, 205 were firstly reported to be related to TPD in rubber tree; these genes laid the foundations for unraveling the molecular mechanisms involved in TPD. This result also demonstrates that it is necessary to identify the genes associated with TPD from different clones, tapping systems and stages of TPD. Of different functional categories, the large numbers of genes related to TPD were associated with transcription and post-transcription, metabolism and energy, protein metabolism or stress/defense response. In addition, the characterization and expression of the genes related to TPD suggested that ROS producing and scavenging, UPP, PCD and RB might play important roles in TPD occurrence, which provides new insights into understanding TPD in rubber tree.The RY8-79, a high-yielding clone but prone to TPD, was planted at the experimental farm of Chinese Academy of Tropical Agricultural Sciences. In this experiment, the latex has been harvested for the past 11 years. During the past 11 years, the rubber trees were regularly tapped with a standard tapping system . Besides, 1.0% ethephon was applied to stimulate latex yield two days before tapping, and the stimulation frequency was once three tappings. The trees affected by TPD syndrome were still tapped along with healthy trees to maintain uniform conditions until sample collection. The fresh latex samples were separately collected and pooled from five healthy and TPD trees, and immediately frozen in liquid nitrogen for total RNA extraction.Total RNA was extracted from the latex samples of healthy and TPD trees according to the method of Tang et al. . The RNA+ mRNA was purified from total RNA with an Oligotex™-dT30 mRNA Purification Kit (Takara). SSH was performed using the PCR-Select™ cDNA subtraction kit  according to the manufacturer's protocol and the method of Diatchenko et al. [+ RNA samples generated from healthy and TPD trees. The driver and tester cDNAs from forward and reverse libraries were separately digested with RsaI and then purified. The digested tester cDNA was subdivided into two portions, and each was separately ligated to different adaptors (adaptor 1 or adaptor 2R) provided by the manufacturer. After the ligation, the resulting cDNAs (tester cDNAs ligated with adaptors) were divided into two populations: one for subtraction study and the other for the evaluation of subtraction efficiency.For PCR-select cDNA subtraction, Poly(A)o et al. . For forE. coli (DH5a) cells to generate SSH libraries. The transformants were planted on LB agar plates with 100 ug/ml ampicillin, 40 ug/ml 5-Bromo-4-chloro-3-indolyl β-D-galactopyranoside  and 1 mM isopropyl-beta-D-thiogalactopyranoside (IPTG) for blue-white screening. The white colonies were picked and cultured at 37°C in LB liquid culture medium with 100 ug/ml ampicillin. The glycerol stocks of bacterial cultures were frozen in liquid nitrogen and stored at -80°C.Two hybridizations were then performed. In the first hybridization, an excess of driver was added to each tester samples, leading to the enrichment of differently expressed sequences. During the second hybridization, the two primary hybridization samples were mixed together to form new double-stranded hybrids with different ends. Fresh denatured driver cDNA was added to further enrich differentially expressed sequences. After two hybridizations, the resulting annealed material was used as the PCR template. The primary PCR was performed with the following parameters: 94°C for 25 s followed by 27 cycles of 94°C for 30 s, 68°C for 30 s and 72°C for 1.5 min, and then 72°C extension for 7 min. The primary PCR products were diluted 10-fold and used as the template in secondary PCR. The secondary PCR was performed for 14 cycles with the same parameters as the primary one. The subtraction efficiency was evaluated by PCR reaction with the primers of rubber 18s rRNA. The PCR products from subtracted samples were inserted into the pMD18-T vector (Takara) and then transferred into chemically competent All recombinant clones from the two subtracted libraries were picked and cultured overnight in LB liquid culture medium with 100 ug/ml ampicillin. The cDNA inserts were amplified with nested PCR primers (F 5'-tcgagcggccgcccgggcaggt-3' and R 5'-agcgtggtcgcggccgaggt-3'). The PCR amplifying conditions were as follows: 94°C for 5 min; 30 cycles of ; and a final extension at 72°C for 10 min. The PCR products were then electrophoresed on 1.2% agarose gel.a-32P-dCTP using Random Primer DNA Labeling Kit (Takara). The labeled probes were obtained and purified on Sephadex G50 columns. The denatured PCR products of inserts (about 10 ng) were spotted onto nylon membranes. The membranes were incubated in standard prehybridization solution at 65°C for 3 h and then hybridized with a-32P-labeled probes at 65°C for 12 h. Following hybridization and sequential washing, the radioactive membranes were exposed to x-ray film. The clones, only hybridizing with the labeled tester probes or indicating at least threefold signal with the labeled tester probes than the labeled driver ones, were selected to sequence.To identify and select the differentially expressed clones, reverse Northern bolt was performed with labeled cDNA probes. The subtracted and unsubtracted cDNAs were labeled with The differentially expressed clones in two cDNA libraries were selected and sequenced. Raw sequence trace files were performed by DNA Sequencing Analysis Software 5.1 (Applied Biosystems) to obtain base-calling with quality scores. Low quality , short (< 100 bp), vector and adaptor sequences were removed by Lucy program . All uniThe cDNA synthesis was performed using 2 ug total RNA according to the manufacture's protocol . All RT-PCR experiments described here were repeated at least three times using independent cDNA samples. In each PCR reaction, the gene-specific primers were used, and rubber 18s rRNA gene was used as the internal reference. All primers used for expression analysis are provided in Table -ΔΔCT method [For real-time PCR analysis, the RNA samples were prepared from three biological repetitions; one was used to construct SSH library; two were different from those used for SSH. The real-time PCR reactions were performed with ABI 7900 real-time PCR system. The SYBR Premix Ex Taq kit (Takara) was used according to the manufacturer's protocol. The expression level of each gene was normalized against the 18S rRNA gene. The cDNA samples were prepared with a series of 100-fold dilutions, and the amplification efficiency of each gene was adjusted to be equal to 18S rRNA. For each target gene, the PCR reactions were carried out in triplicate. The relative expression values were calculated from three biological replicates using a modified 2T method .APX: ascorbate peroxidase; BLAST: basic local alignment search tool; CAT: catalase; CCR: cinnamoyl-CoA reductase; DDRT-PCR: differential display reverse transcription PCR; EST: expressed sequence tag; GO: Gene Ontology; GLR: glutaredoxin; IPTG: isopropyl-beta-D-thiogalactopyranoside; MVA: mevalonate; GR: glutathione reductase; NAD(P)H: oxidized and reduced nicotinamide-adenine dinucleotide phosphate; RB: rubber biosynthesis; ROS: reactive oxygen species; RT-PCR: reverse-transcription PCR; SPOP: speckle-type POZ protein; SOD: superoxide dismutase; SSH: suppression subtractive hybridization; TPD: tapping panel dryness; TCTP: translationally controlled tumor protein; UPP: ubiquitin proteasome pathway; X-Gal: 5-Bromo-4-chloro-3-indolyl β-D-galactopyranoside.All the authors read and approved the final manuscript. DL supervised the experiments, and carried out RNA extraction, library construction and data analyses. In addition, he drafted and revised the manuscript. ZD prepared clones for sequencing, performed blast analyses and functional classification of ESTs, RT-PCR and real-time PCR analyses. CC collected plant materials, and performed RNA extraction, clone selection and PCR identification. ZX carried out reverse Northern analysis, PCR detection and data analyses. MW and PH collected plant materials, performed RNA extraction and library construction. SC supervised the research.The identification of non-redundant clones upregulated in TPD trees from the reverse SSH library. The file contains functional classification, SSH clone ID, cDNA insert size, GeneBank accession no., putative identify and E-value of non-redundant clones from the reverse SSH library.Click here for fileThe identification of non-redundant clones downregulated in TPD trees from the forward SSH library. The file contains functional classification, SSH clone ID, cDNA insert size, GeneBank accession no., putative identify and E-value of non-redundant clones from the forward SSH library.Click here for file


T = 1 icosahedral virus with a single-stranded RNA genome. It is widely accepted that the RNA genome plays an important structural role during assembly of the STMV virion. While the encapsidated form of the RNA has been extensively studied, less is known about the structure of the free RNA, aside from a purported tRNA-like structure at the 3′ end. Here we use selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE) analysis to examine the secondary structure of in vitro transcribed STMV RNA. The predicted secondary structure is unusual in the sense that it is highly extended, which could be significant for protecting the RNA from degradation. The SHAPE data are also consistent with the previously predicted tRNA-like fold at the 3′ end of the molecule, which is also known to hinder degradation. Our data are not consistent with the secondary structure proposed for the encapsidated RNA by Schroeder et al., suggesting that, if the Schroeder structure is correct, either the RNA is packaged as it emerges from the replication complex, or the RNA undergoes extensive refolding upon encapsidation. We also consider the alternative, i.e., that the structure of the encapsidated STMV RNA might be the same as the in vitro structure presented here, and we examine how this structure might be organized in the virus. This possibility is not rigorously ruled out by the available data, so it remains open to examination by experiment.Satellite tobacco mosaic virus (STMV) is a  T = 1 icosahedral virus with a single-stranded, positive-sense RNA genome, 1058 nucleotides in length. A capsid of 60 identical protein subunits surrounds the genome in the STMV particle. Like other satellite viruses, STMV encodes its own capsid protein but requires a helper virus for replication. For a review on the general properties of STMV, see Dodds Satellite tobacco mosaic virus (STMV) is a Efforts to characterize the RNA and its role in assembly have produced mixed results. The virus crystal structure has been solved at 1.8 Å resolution et al. used chemical probing to examine the RNA structure inside the virus. They combined these data with the assumption of co-replicational folding to produce an ensemble of models for the secondary structure Schroeder et al. have concluded that the TLS is not compatible with their chemical modification data et al. have argued that, if the tRNA-like structure and replication recognition site structure were maintained inside the virus, there would be insufficient RNA remaining to connect the stem-loop segments The capsid-free form of STMV RNA has been relatively overlooked in structural studies, in part because the secondary structure of the encapsidated RNA is believed to be different than the free RNA in vitro transcribed STMV RNA, based on chemical probing data obtained using selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE) et al. for the RNA in virio, Here we report a secondary structure model for O-adducts. Reverse transcription using fluorescently labeled primers gives cDNA fragments whose lengths are determined by locations of the 2′-O-adducts, and whose quantities can be measured by capillary electrophoresis.SHAPE in vitro transcribed STMV RNA in the presence of 250 mM Na+ using the SHAPE reagent N-methylisatoic anhydride (NMIA). Under these conditions (no Mg++), one would expect the formation of secondary structure, but not necessarily tertiary structure We first probed the a priori reason for doing so for an RNA probed in vitro.The SHAPE reactivity information was incorporated into the thermodynamic folding algorithm RNAstructure We recognize that chemical probing cannot define a single secondary structure We evaluated the agreement between the model and the data by comparing the distribution of reactivity values in single-stranded nucleotides with the distribution of reactivities in base paired nucleotides  It is not possible to make a rigorous comparison, because Schroeder’s data were obtained on the RNA in the mature virus, while our model represents the RNA free in solution. It is hard to evaluate how much the capsid might protect the RNA, and impossible to know which residues might be affected. It is also unclear to what extent encapsidation of a structure like ours might cause local structural disruptions. There appears to be a not unreasonable agreement between the Schroeder data and our model in the extended region (residues 1–730), and in the tRNA-like domain  images showed that STMV RNA that has been phenol-extracted from the intact virus exists in two temperature-dependent and reversible conformations, an open and a closed conformation 2+ is not required for their formation.To examine the effect of Mg profile , indicatThe secondary structure proposed here  raises fin vitro transcribed RNA biologically relevant? A study by Mirkov et al. suggests that it is. They demonstrated that STMV RNA transcribed in vitro was biologically active, showing a consistent ability to infect tobacco plants also infected by TMV First, is the structure of the Second, does this structure play a role in viral assembly? It appears likely that the TLS represents a recognition signal for replication et al. have proposed that the tRNA-like structure (TLS) in STMV is essential for stability of its RNA et al. showed that “viroid and satellite RNAs are significantly resistant to RNA silencing-mediated degradation, suggesting that RNA silencing is an important selection pressure shaping the evolution of the secondary structures of these pathogens” Third, if this secondary structure is not that of the packaged RNA in the mature STMV virion, then what is its function? One plausible explanation is that it protects the RNA from degradation. Felden et al. don’t give a firm answer to this question. Could the extended domain be arranged to cover the edges of the icosahedron, perhaps surrounding the tRNA-like structure in the core? Finally, is it possible that this secondary structure is maintained inside the intact virion? As argued above, the chemical probing data from Schroeder in vitro transcribed STMV RNA is highly extended, and the data support the predicted tRNA-like fold at the 3′ end of the RNA in vivo. The predicted secondary structure of the RNA transcribed in vitro is considerably different from that proposed for the genome in the intact virion The SHAPE-restrained secondary structure of If the genomic RNA is packaged co-replicationally, as originally proposed Escherichia coli, purified using the Endo-Free Plasmid Maxi kit (Qiagen), and sequenced bi-directionally (MWG Operon). This in vitro transcript runs as a single band in native gel electrophoresis (STMV DNA appended with a 5′ T7 promoter and 3′ HindIII recognition sequence was synthesized by MWG Operon and provided in a pCR 2.1-TOPO plasmid. The plasmid was cleaved with PstI (New England Biolabs), gel purified, and religated to remove an extraneous T7 promoter. The plasmid was amplified in dH5α phoresis , suggestTranscription reactions were performed by the run-off method 5′-ACAACATTCGAATTGTCACC-3′; 411, 5′-TCATTTACTGGCGGTGTTAA-3′; 668, 5′-AGGAGCGGATCGTTTAACCT-3′; 831, 5′-ACAATGGATCTATTCCATAA-3′ and 1039, 5′-TGGGCCGCTTACCCGCGGTT-3′.SHAPE probing of STMV RNA was performed as described in et al.We converted the capillary electrophoresis (CE) data traces, or electropherograms, into SHAPE reactivities using in-house Matlab code. This procedure has been described in detail in Athavale ++), the final reactivity values represent the average of nine separate datasets: three at a concentration of 3.25 mM NMIA, three at 6.5 mM NMIA, and three at 13 mM NMIA. For the SHAPE data acquired in 250 mM Na+ and 10 mM Mg2+, the final reactivity values represent the average of three separate datasets: one at a concentration of 3.25 mM NMIA, one at 6.5 mM NMIA, and one at 13 mM NMIA. As reported earlier Tetrahymena Group I ribozyme, getting results that are similar to previous reports on the same molecule For the SHAPE data acquired on the RNA in 250 mM Na+ (no MgWe folded the entire STMV RNA sequence (1058 nucleotides) using the thermodynamics-based free energy minimization algorithm in the RNAstructure software package, version 5.3 MLD〉), we first generated a random sample of 1000 suboptimal structures, drawn with probabilities equal to their Boltzmann weights, using RNAsubopt, a program in the Vienna RNA software package, version 2.0 MLD〉 as We calculated the MLD values using a C program . To compute the ensemble-average maximum ladder distance . After scaling all of the primers to one another such that the overlapping regions match up, the resulting signal decays rapidly (middle). After correcting for signal decay, the overlapping regions are in agreement (bottom).(TIF)Click here for additional data file.Figure S2Quantitative correlation between peak area data in overlapping primer reads. This demonstrates that signal decay in the regions of overlapping data is similar. Pearson’s r-values are shown.(TIF)Click here for additional data file.Figure S3Combined peak area signal after decay correction. The thick black line fitted to the corrected peak area data has a slope of zero, ensuring that intense values in the beginning, middle, and end of the signal are of uniform height.(TIF)Click here for additional data file.Figure S4Predicted secondary structures for STMV RNA. SHAPE MFE and Subopts #1–9 were predicted using the SHAPE experimental data as constraints. Default MFE was predicted without the SHAPE data. Each secondary structure is shown as an arc diagram, in which the sequence is arranged along a horizontal line and base pairs are shown as arcs connecting the corresponding bases. The structures are listed in order of ascending pseudo-energy values. Pseudo-energy is the calculated free energy that includes the SHAPE pseudo-energy terms. Also shown are the energy values evaluated using the default energy model parameters ignoring SHAPE terms. MLD is the maximum ladder distance. All structures predicted using RNAstructure version 5.3.(TIF)Click here for additional data file.Figure S5In vitro transcribed STMV RNA runs as a single band on a native gel. STMV RNA is run on a 1% agarose gel. No sample was loaded in lanes 2 or 4. Lanes 1 and 3 contain STMV RNA in SHAPE probing buffer without Mg2+  and lane 5 contains STMV RNA in 100 mM Tris-HCl pH 8.0. All samples were heated to 90°C for 2 min. Samples in lanes 1 and 5 were snap-cooled by chilling on ice, while the one in lane 3 was allowed to slow-cool to room temperature. The samples were then loaded on the gel using 6X native gel loading dye (New England Biolabs) and stained with SYBR Gold nucleic acid gel stain (Invitrogen). Lanes 1, 3 and 5 contain a single band, indicating a single dominant conformation.(TIF)Click here for additional data file.Table S1Primers used to analyze the STMV RNA.(PDF)Click here for additional data file.Methods S1(PDF)Click here for additional data file.