To characterise the performance of the ScreenTape® platform for RNA analysis and determine the robustness of the SDV metric, a panel of intentionally degraded RNA samples was prepared. These samples were used to evaluate the ScreenTape® platform against an alternative approach for measuring RNA integrity . The samples were also subjected to microarray analysis and the resulting data correlated to the RNA integrity metrics.Microarray data interpretation can be affected by sample RNA integrity. The ScreenTape Degradation Value (SDV) is a novel RNA integrity metric specific to the ScreenTapeMeasurement of SDV for a panel of intentionally degraded RNA samples ranged from 0 for intact RNA to 37 for degraded RNA, with corresponding RIN values ranging from 10 to 4 for the same set of samples. SDV and RIN scales both demonstrated comparable discrimination between differently treated samples , with the SDV exhibiting better discrimination at higher degradation levels. Increasing SDV values correlated with a decrease in microarray sample labelling efficiency and an increase in numbers of differentially expressed genes.® platform is comparable to the Bioanalyzer platform in terms of reproducibility and discrimination between different levels of RNA degradation. The robust nature of the SDV metric qualifies it as an alternative metric for RNA sample quality control, and a useful predictor of downstream microarray performance.The ScreenTape The use of microarray technology has revolutionised the fields of molecular biology and genetics. However, concerns have been raised over the numerous potential sources of variation that can affect assay consistency and data quality ,2. PreviTo date, no single RNA integrity metric has been adopted universally by the research community. RNA quality is commonly determined by several different techniques, including the ribosomal peak ratio (eukaryotic 28s/18s rRNA peak intensity ratio) , RIN 7]7], 5'/3'RNA purity is assessed routinely by measuring the OD260 nm/OD280 nm ratio ,11 of a ® platform that employs precast multilane gels and microfluidics enabling semi-automated operation, simplifying sample handling and reducing assay times. In this study we have compared the performance characteristics of the ScreenTape® R6K platform (Lab901) and corresponding RNA quality metric, SDV [More recently, Lab901 have developed a novel electrophoretic ScreenTaperic, SDV .We report here on the broad correlation observed for these quality metric values and associated microarray data.2. Cells were then exposed for 24 h to EMEM exposure media supplemented with 0.5% (v/v) DMSO vehicle (Sigma Aldrich) and 4 mM ACAP . Following treatment, the cells were washed with an excess of 1 × Phosphate Buffered Saline (Gibco) before being lysed in situ by the application of ice cold TRIzol® LS Reagent (Invitrogen). Total RNA was then isolated according to manufacturer's instructions (Invitrogen). RNA quantity was determined using a NanoDrop 1000 spectrophotometer.HepG2 cells (passage = 84) were grown to confluence in T175 vented culture flasks, using Eagles Minimum Essential Medium  plus 10% Foetal Calf Serum  in a humid 37°C incubator supplemented with 5% CO® (Lab901) was used in conjunction with ScreenTape® R6K, and the 2100 Bioanalyzer (Agilent Technologies) with the RNA 6000 series II Nano LabChip analysis kit. Total RNA samples were prepared for analysis according to manufacturer's recommendations. Results were compared between the platforms for six levels of RNA sample integrity and at a single RNA concentration of 25 ng/μl.For comparative analysis of RNA integrity, the TapeStationRNA from the treated HepG2 cells was diluted to a concentration of 1 μg/μl with nuclease-free water (Ambion) and aliquoted into volumes of 50 μl in 0.2 ml thin wall PCR tubes. The tubes were placed in the block of an MJ Research PTC-200 DNA Engine Thermal Cycler PCR machine which was then heated to 90°C. Tubes were removed in batches of three at 3-minute intervals. The RNA was then diluted to 25 ng/μl with nuclease-free water, and RNA integrity for each sample determined on both platforms.Homo sapiens 4 × 44K whole genome gene expression arrays according to manufacturer's instructions. The hybridised arrays were scanned using an Agilent G2505B Scanner and expression data extracted using Feature Extraction software, version 10.5 (Agilent Technologies). Data was exported to the Genespring GX (Agilent) software package, normalised to the 75th percentile of the data set and base-lined to the median signal intensity of all chips. The data were quality controlled by filtering on flags , before the number of differentially expressed genes (DEG) were determined using the combination of an analysis of variance (ANOVA) and relative fold change (FC = > 1.5).A single-colour labelling approach was adopted for the microarray hybridisation scheme. Duplicate biological samples of those employed for the Lab901/Bioanalyser platform evaluation were used, with two technical replicates (arrays) for each sample. Samples were hybridized to Agilent All statistical analysis used R version 2.9.2 .® system, which generates SDV values, and the Agilent Bioanalyzer, which generates RIN values. Replicate samples were run on three separate chips or tapes, depending on the platform, and across triplicate lanes per chip or tape. An overlay of chromatograms generated by the ScreenTape® and Bioanalyzer systems for intact and degraded RNA is presented in Figure RNA integrity was assessed for RNA samples with increasing levels of degradation using the Lab901 ScreenTapeRIN is an incremental scale which spans from 0 to 10, with increasing RNA integrity correlating with increasing RIN value. In contrast, SDV is an unconstrained metric which employs a scale of measurement spanning from 0 to infinity with higher values corresponding to increased degradation. A direct comparison of SDV to RIN was carried out to evaluate the performance of the SDV metric. Preliminary inspection of the data for both platforms Figure  illustra(i) Both RIN and SDV show good discrimination between samples in general.(ii) SDV appears to show generally better within-group precision.(iii) There is evidence to suggest that RIN and SDV do not share a linear relationship.(iv) The within-group dispersion does not appear to be constant across treatment groups for either of the metrics.The assessment of relative platform performance was determined by the use of three different statistical indicators, namely rank order correlation, intraclass correlation and classification performanceρ) and Kendall (τ) rank correlations between response and RNA integrity level are listed in Table Spearman  and linear discrimination analysis (LDA)) were applied to the data sets and the resulting groupings compared with the expected groupings. The significance for differences in the proportion of misclassifications between the RIN and SDV metrics were then tested for each of the classification methods employed. Table It can be seen that the relative performance of the two RNA degradation metrics is consistent across all four classification methods. However, more misclassifications were observed when using the RIN metric. In addition, the difference in misclassification was found to be significant at the 95% level for two of the four classification methods. This would indicate that a significant difference in performance exists between the SDV and RIN metrics.Gene expression studies are potentially sensitive to the effect of RNA integrity. To determine the broad impact of RNA integrity on microarray data, and to determine the value of the SDV metric in highlighting potential problems, microarray labelling data was generated for the degraded RNA samples used in this study. The procedure recommended by Agilent for the amplification and labelling of mRNA samples employs a 3' mediated Eberwine amplification protocol  that intIn terms of microarray assay performance, reduced sample labelling efficiency may have an impact on the robustness of microarray measurement and data reliability. RNA integrity measurement, whether by SDV or RIN, may provide a valuable tool for early prediction of RNA labelling efficiency and ultimately of overall microarray performance.Assay sensitivity (the rate of detection of true positives) and specificity (the rate of detection of true negatives) are important performance indicators for microarray experiments. To gain insight into the broad impact of RNA integrity, as determined by SDV, a preliminary gene expression experiment was performed using the samples from this study. For each data set generated using different levels of RNA integrity, the number of differentially expressed genes (DEGs) was determined by comparing the paracetamol-treated HepG2 cells to intact RNA extracted from HepG2 cells treated with vehicle control only (DMSO). To assign a measure of assay specificity to the data, a value for the degree of additional gene discovery  at each level of RNA integrity was derived by subtracting the median number of Differentially Expressed Genes (DEGs) for the technical replicates of the intact sample from the median number of DEGs determined for each of the degraded samples Table .Differential expression was defined as those genes which demonstrated a statistically significant difference of expression (p ≤ 0.05) while employing a Benjamini and Hochberg  false diin vitro transcription [It has been reported previously that gene expression profiling using Affymetrix GeneChip arrays is relatively tolerant to moderate RNA degradation as well as to 5' truncation occurring as a consequence of successive rounds of cription . Howevercription . Compariin vivo [in vivo [The measurement of gene expression is based on the assumption that an analysed RNA sample accurately represents the population of transcripts present in vivo . Many tr[in vivo , raising® platform with that of the Agilent 2100 Bioanalyzer and also compared the SDV metric with the RIN metric for the determination of RNA integrity when applied to microarray data analysis.In this paper we have compared both the performance of the novel Lab901 ScreenTapeBoth metrics performed well when using the samples employed in this study, with the data highlighting the difficulty associated with unambiguously assigning samples to a definitive integrity level when they have ether a RIN value ≤ 6 or an SDV value ≥ 15.® system was demonstrated to be a reliable and robust means of determining RNA integrity with SDV estimates, correlating well with the RIN values generated by the Agilent Bioanalyzer platform and with a better classification performance in this study. In addition, the RIN and SDV metrics both performed well in terms of distinguishing different levels of RNA degradation treatment. For microarray data, Rank correlations with treatment and intraclass correlations are high. Classification methods show that the majority of observations were classified into appropriate treatment groups by both RIN and SDV, with the slightly better classification performance of the SDV metric being significant at the P ≤ 0.05 level for two out of the four classification methods used.In conclusion, the ScreenTapeThe ScreenTape platform and SDV therefore offer an alternative to currently available systems for RNA integrity analysis and provide a performance comparable to that of the Bioanalyzer 2100. The rapid assay time and medium through put capacity may favour its use in laboratories which process large numbers of samples. Future studies with this platform could enable the development of an SDV based RNA quality threshold to be established for use with downstream applications.The authors declare that they have no competing interests.TW participated in the design of the study, carried out the platform evaluations and drafted the manuscript, AD carried out the RNA degradation and SE performed the statistical analyses. CF conceived of the study and participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.


Some of most used indicators in marine ecology are nucleic acid-derived indices. They can be divided by target levels in three groups: 1) at the organism level as ecophysiologic indicators, indicators such as RNA:DNA ratios, DNA:dry weight and RNA:protein, 2) at the population level, indicators such as growth rate, starvation incidence or fisheries impact indicators, and 3) at the community level, indicators such as trophic interactions, exergy indices and prey identification. The nucleic acids derived indices, especially RNA:DNA ratio, have been applied with success as indicators of nutritional condition, well been and growth in marine organisms. They are also useful as indicators of natural or anthropogenic impacts in marine population and communities, such as upwelling or dredge fisheries, respectively. They can help in understanding important issues of marine ecology such as trophic interactions in marine environment, fish and invertebrate recruitment failure and biodiversity changes, without laborious work of counting, measuring and identification of small marine organisms. Besides the objective of integrate nucleic acid derived indices across levels of organization, the paper will also include a general characterization of most used nucleic acid derived indices in marine ecology and also advantages and limitations of them. We can conclude that using indicators, such RNA:DNA ratios and other nucleic acids derived indices concomitantly with organism and ecosystems measures of responses to climate change  will allow for the development of more rigorous and realistic predictions of the effects of anthropogenic climate change on marine systems. Therefore, the developments of new direct methods are necessary to gain a better understanding of physiology, trophic interactions and changes in composition and structure in aquatic ecosystems. Measurements of metabolic activity have been especially valuable as indicators of condition in studies of marine organisms, groups for which accurate determination of field metabolic rates is difficult [In marine ecology the determination of the ifficult . Moleculifficult  and the ifficult . Many coifficult . One of ifficult  it has bifficult –10. Thesifficult  and in iifficult –16. All ifficult , 18. AlsThis was due to much progress been made in improving the sensitivity of analytical methods, with the application of fluorometric techniques allowing measurement at the individual level, of small marine organisms such as marine larvae and copepods. In fact, these are among taxa where these indices have been more applied, especially the RNA:DNA ratio , 19, 20.This paper characterizes the most used nucleic acid derived indices in marine ecology: 1) Organism level indices, such as RNA:DNA ratios, DNA/dry weight RNA/protein, 2) Population level as growth rate, starvation incidence and impact studies and 3) Community level such as trophic interactions, exergy indices and prey identification. The advantages and limitations of them will be also discussed.To date the most widely-used biochemical index to determine marine organisms condition is the RNA:DNA ratio, which is an eco-physiological index of activity  under a given environmental condition . This inet al. study [Some studies have advocated caution in the use of RNA:DNA ratios on the basis of techniques . Neverthl. study  of the il. study , 10 propOthers authors argue that the RNA:DNA ratios change with age in fishes , 34. To Sardina pilchardus Chicharo et al. [Sciaenops ocellatus, larvae. This could result from an endogenous rhythm, i.e. circadian periodicities in endocrine activity induced by the light/dark regimen that raises the concentration of RNA in fish larvae during certain hours [Ruditapes decussatus and Cerastoderma edule [The analysis of the day variation for RNA:DNA, RNA residuals and RNA content detected in some fish larvae also shown that this aspect needed to be in consideration when sampling. In o et al.  suggesteo et al.  also fouin hours . In factma edule  , crustaceans (Crangon crangon) (Ruditapes decussatus).The gender effect on the nucleic acids derived indices also is needed to be considered. The study of Chícharo et al.  aimed tocrangon) , and bivThere were significant differences in indices based on nucleic acid concentrations between males and females of all three species during the spawning season. RNA:DNA ratios were greater in females than in males, because of a greater content of RNA per unit dry weight in females and because of a greater content of DNA per unit dry weight in males. Sexual dimorphism and physiological and behavioural differences between males and females may explain these results. RNA:DNA ratios of adult organisms should be interpreted with caution because the effect of gender on nucleic acid concentrations may bias results if the sex ratio in the sample from which the results were derived is not representative of the sample. The RNA:DNA ratio may be underestimated if males are over-represented in the sample and overestimated if females are over-represented.Special caution is also advised when selected selecting different kinds of tissue to determine the nucleic acids concentrations and ratios, since different tissues or body parts can have different RNA-or DNA-tissue relationships . In someA high variability of RNA content, and thus RNA:DNA ratio, was revealed by estimates at the individual level and seems to be more related to feeding condition in late larval or juvenile stages than in yolk-sac and first-feeding larvae . AccordiOver the past decades, RNA:DNA ratios have been used widely as an index of nutritional condition (see above section) and, to a lesser extent, to estimate growth rate. Nevertheless the earliest studies on the topic suggested that growth rates are related to RNA concentration in several species of copepods and other crustaceans , 23, andet al. [There are only a few widely-used approaches to estimating growth rates in marine organisms, among them are longitudinal sampling of a cohort, otolith microstructure analysis and RNA:DNA ratio. In the field, cohort analysis is most often a difficult task to accomplish and in the laboratory measurements of otolith microstructure, is a very laborious technique, but can provide offer a a detail history growth of each larva. Since Hovenkamp  demonstret al.  the smalet al. . In diffet al. [According to Pepin et al.  if surviet al. . In factet al. [et al. [Besides the usually pattern of increasing RNA:DNA ratio with size is registered , 34, 42,et al. , Buckleyet al.  and Math [et al. . AccordiIf for a certain life stage the relationship between RNA:DNA, temperature and growth is fixed, then if growth decreases with size and age it follows that RNA/DNA ratio would have to also decrease with size/age.As late larvae decrease growth rates with age which is why it is very important not to directly compare growth rates or RNA:DNA ratio values of individuals with very different ages. A slower growing older larvae or juvenile may be in equally good condition to a faster growing younger larvae and a decrease in RNA:DNA ratio with age most likely reflects a decrease in growth rate but not necessarily a decrease in condition. The increased rate of chemical reactions at higher temperatures means that a higher growth rate can be achieved with the same or lower RNA concentration, and also that the RNA concentration associated with maximal growth rates declines with increasing temperature. Temperature is the dominant growth factor when larvae have adequate food supplies. Conversely, when the temperature range is narrow, food availability becomes the predominant factor for growth and condition .In recent work, RNA:DNA ratios were also related to egg production rates of copepods . RNA conet al. [Carcinus maenas. The authors argue that this is happens because protein deposition is dependent of protein synthesis but also of breakdown. Aditionally crustacean growth is also different from that of fishes. For those authors, RNA:protein is more adequate to detect changes in growth rate, because this index is more closely related to protein deposition. The RNA:protein is also very useful to detected starvation in fishes, since proteins forms an important energy source during long term food deprivation [Clupea harengus using Buckley’s general model by setting protein growth rate to zero and solving for RNA:DNA ratio at the temperature which they captured the larvae. Rooker and Holt [Houlihan et al.  have querivation . To asserivation  and is brivation . Robinsorivation  defined and Holt  suggesteSardina pilchardus from a field experiment . According to Clemmesen [A solution to this problem is to calculate for each species, under controlled conditions, the mean RNA:DNA ratios of larvae deprived of food. This kind of calibration is usually done only with fed and starved laboratory reared larvae , 19, 28.lemmesen  there isThe knowledge of the minimum values of RNA:DNA ratio needed for survival can also be useful in fisheries stress studies. Few investigations have focused on the influences of fishing methods on the health status of the target population. Nowadays there is a need to include the indirect mortality incurred during or post-escape from fishing gear. This issue is difficult to study, especially with organisms like fish that are very sensitive to handling. The condition indices, such as the nucleic acids derived indices referred to herein have been used to assess indirect mortality in bivalve dredge fisheries. Fishing by dredging is a source of acute and chronic stress for bivalve populations, especially to under-sized clams, which represent the population reservoir . UsuallyRuditapes decussatus juveniles in Ria Formosa , survival was not guaranteed when the RNA:DNA ratio was lower than 1. This value was, recently used for Spisula solida and Donax trunculus in a laboratory simulation of dredge stress, but after several hours of mechanical stress, this low value was never reached [Some of these stress indices use biochemical ratios or metabolic rates that integrate important part of life history of the individual. Chícharo and Chícharo  establis reached . This agOne of the most compelling applications of biochemical indices in ecological studies has been in testing models that predict changes in the structure of ecological communities—species distribution, species abundance, and the strength of interactions between competitors or between predators and prey—along environmental gradients in the rocky intertidal region. Two classes of models have been developed and tested using biochemical indicators of metabolism and stress: nutrient productivity models (N/P models) and environmental stress models or ESMs , 58. Thein situ physiological status for key communities in the sea, not only for benthic but also for plankton is vital for understanding the mechanisms structuring marine ecosystems. Zooplankton also plays a central role in the marine food web as selective predators and nutrient regenerators, and as mediators of energy to higher trophic levels [The ability to obtain information about c levels . Microzoc levels . DNA conc levels . Measurec levels . Nonethec levels . The datin situ zooplankton feeding on all different prey types. Therefore, the development of a new nonintrusive direct method is necessary to gain a better understanding of the trophic interactions in aquatic ecosystems. Molecular methods based on the polymerase chain reaction have recently become an important tool to study predation by arthropods, particularly insects [et al. [E. huxleyi was unambiguously detected in whole DNA extracts from copepods and from their fecal pellets. These results also suggest that prey DNA may be quantified for determination of prey-specific zooplankton feeding rates, and to trophic interactions between individual predators and all their prey in the complex natural plankton can be determined.Mesozooplankton feed on a wide range of prey, and there are presently no methods available to directly quantify  insects . Accordi [et al.  using a Pi) of producing organic matter (detritus) and the probability of ‘selecting’ its corresponding ‘genetic information’ , for each component, as follows:Pi=P1 · Pi,a and Pi,a=20g−700 , where R is the gas constant, T the absolute temperature, ci the concentration in the ecosystem of the component i and 700·g stands for an average value for the number of encoded amino-acids. With reasonable approximations, it can be computed as:ci is the biomass concentration of species i and βi is a weighing factor expressing the ‘quantity of information’ embedded in the biomass [At a community level, other potential use of nucleic acids derived indices, such as DNA concentrations is for the empirical determination of exergy. In disturbed aquatic ecosystems a useful indicator of the anthropogenic impacts is the ecological exergy . Ecologi biomass . This exIn the absence of these data, calculations are done based on rough estimates, obtained with flow cytometry . The exeIn situ calibration of indices minimum and maximum values under differing temperature regimes, and in depth analysis of how does their distribution change in response to variations in nutritional environmental condition or predation pressure, are also needed. Using indicators, such RNA:DNA ratios and other nucleic acids derived indices concomitantly with organism and ecosystems measures of responses to climate change  will allow for the development of more realistic predictions of the effects of global change on marine systems.The nucleic acid derived indices reviewed in this study showed how useful they are in understanding structure and processes in marine ecosystems, without laborious work of counting, measuring and identification of small marine organisms. However, significantly more research under controlled laboratory and field conditions will be required in future. There is a need for baseline, species-specific validation studies before the generalized application of nucleic acid-based indices to ecological issues, such as ecophysiology of individuals, growth rate, fish and invertebrate recruitment failure, trophic interactions in marine environment, detecting anthropogenic impacts and natural changes.


Although viral RNA constitutes the majority of nucleic acids packaged in virions, a late occurring step of reverse transcription leads to the presence of infectious viral cDNA in foamy virus particles. This peculiarity distinguishes them from the rest of the retroviral family.To evaluate the respective contribution of these viral nucleic acids in the replication of foamy viruses, their fate was studied by real-time PCR and RT-PCR early after infection, in the presence or in the absence of AZT. We found that an early reverse transcription step, which occurs during the first hours post-entry, is absolutely required for productive infection. Remarkably, sensitivity to AZT can be counteracted by increasing the multiplicity of infection (moi). We also show that 2-LTR circular viral DNA, which appears as soon as four hours post-infection, is transcriptionally competent.Taken together, our data demonstrate that an early reverse transcription process, which takes place soon after viral entry, is indispensable for infectivity of FVs at low moi, when the amount of DNA-containing particles is not sufficient to lead to a productive infection. This study demonstrates a key role of the packaged viral RNA in the foamy virus infection, suggesting that the replication of this virus can be achieved by involving either viral DNA or RNA genome, depending on the condition of infection. Foamy viruses (FVs) are complex animal retroviruses sharing homologies in comparison to the conventional replication pathway of retroviruses such as HIV-1. For example, it has been reported that primate foamy virus (PFV-1) integrase, which is responsible for the indispensable integration step of the viral genome into the host cell DNA, displays a new specific restriction-like cleavage activity on palindromic sequences. An analogous function has also been demonstrated for Human immunodeficiency virus type 1 (HIV-1) integrase Here, the fate of viral DNA and RNA was studied during the early stages of PFV infection, in the presence or absence of AZT. Using real-time PCR and RT-PCR, we clearly demonstrated that an early reverse transcription step is absolutely required for productive PFV infection. Furthermore, we also reported that 2-LTR circles, which are described as dead-end products of retroviral replication, are actively transcribed as already shown for HIV-1 gag-specific primers which gave the same results in comparison to those performed with primers and probes Spuma A, Spuma S and Spuma TM (data not shown). This observation suggests that the viral RNA extracted from purified FVs viral particles is a full-length RNA coding for the entire genome. This hypothesis has been reinforced by the functional role of this viral RNA (see next section). A very weak signal was obtained when the reverse transcription step was omitted, testifying the absence of viral DNA following RNA extraction   . The U5-ral RNA) . Followiral RNA) . The overal RNA)  suggestiAbsence of proviral integration and DNA synthesis under AZT treatment demonstrated total inhibition of FV replication. This finding, which confirmed our previous observations In this report, using real time RT-PCR and PCR assays, we confirmed the presence of a majority of viral RNA in extracellular FVs particles. Moreover about 6% of virions released from acutely infected cells contained viral DNA, including 2-LTR circles. Studying the fate of incoming viral nucleic acids in PFV-infected cells, we confirmed the existence of an early viral DNA synthesis step, which was abrogated under AZT treatment. Remarkably, the kinetics of viral DNA synthesis correlated with the decrease in viral RNA content, demonstrating for the first time the importance of an early reverse transcription step for FV replication.al. demonstrated, in the case of BHK-21 infection, that the titer of pcHSRV2 virus was 3.63×103/ml in the absence and 9.5×101/ml in the presence of AZT, representing a decrease of 36-fold decrease of infectivity in the presence of the inhibitor Previous reports demonstrated that FV reverse transcription occurred late in the replication cycle Under our settings, we demonstrate that, even a high concentration of AZT, viral genome is detected even 6 days post-infection. Unintegrated viral DNA is described to have a short half life in infected cells but quite stable in quiescent cells U373-MG, FAG and FAB cells . Then, RNA was recovered according to the manufacturer's instructions in 50 µl RNase-free water.Cell-free supernatant from acutely infected FAB cells was mixed with NTE buffer  placed on a 20% sucrose cushion and centrifuged  using a SW41 rotor. Pellets were resuspended in PBS and aliquots were treated, or not, with 450 units of DNAse  for 5 hours at 37°C or with Rnase A (40 µg/ml) during two hours at 37°C. DNA was extracted using the DNA Blood Mini kit  and RNA was extracted using the RNeasy mini kit (Qiagen), according to the manufacturer's instructions. For analysis of RNA contained in virions, 5×10gag gene described previously Alu-LTR nested PCR as described previously Alu-LTR sequences, derived from integrated proviruses, were amplified in a 20 µl reaction mixture containing 1x Light Cycler Fast Start DNA Master Hybridation probes (Roche), 4 mM MgCl2, 100 nM of forward primer LambdaT-SpA and 300 nM of primers Alu 1 and Alu 2. Nested PCR was performed on 1/10 of the first-round PCR products in 1x Light Cycler Fast Start DNA Master Hybridisation probes, 4 mM MgCl2, 300 nM of forward primer LambdaT, 300 nM of primer Nested R and 200 nM of each hybridisation probes Sp FL and Sp LC  using commercial available materials . All PCR conditions are given in Amplification was performed by real-time PCR using the Light Cycler Instrument  and measurements were performed using the Light Cycler quantification software version 3.5 (Roche Diagnostics). Sequences of all primers used for DNA and RNA quantifications are given in nd Sp LC . To quan2 and 500 nM of each primers and 200 nM of probes. RT-PCR cycle conditions are given in in vitro transcribed RNA  containing matching sequences. Normalization of results was performed measuring Cyclophilin A RNA. RT-PCR protocol was performed by hybridation probes technology using a standard curve generated by serial dilutions of RNA prepared by an in vitro transcribed RNA.Quantifications of viral and cellular RNA were performed by using real-time RT-PCR protocol. Amplification of viral RNA was achieved by using the TaqMan technology . Two-LTRTable S1Primer, probe sequences and PCR and RT-PCR cycle conditions.(0.26 MB PDF)Click here for additional data file.Figure S1Real-time PCR and RT-PCR strategies for the PFV-1 RNA and DNA quantification. Primers used to quantify total viral DNA (A) are located in the gag gene. Primers spanning the LTR-LTR junction were used to detect 2-LTR circles (B). Integrated DNA (C) is measured by two rounds of PCR as described previously (1.27 MB EPS)Click here for additional data file.


Non-small cell lung cancer (NSCLC) is the foremost cause of cancer-related death in Western countries, which is due partly to the propensity of NSCLC cells to metastasize. The biologic basis for NSCLC metastasis is not well understood.G12DK-ras and R172Hp53. We identified 2,209 genes that were differentially expressed in distant metastases relative to matched lung tumors. Mining of publicly available data bases revealed this expression signature in a subset of NSCLC patients who had a poorer prognosis than those without the signature.Here we addressed this deficiency by transcriptionally profiling tumors from a genetic mouse model of human lung adenocarcinoma that develops metastatic disease owing to the expression of G12DK-ras; R172Hp53 mice recapitulate features of human NSCLC metastasis and will provide a useful platform on which to study the biologic basis for lung adenocarcinoma metastasis and its prevention by novel agents.These findings provide evidence that  As a result, we understand much more about cancer cell-autonomous genetic and epigenetic changes than about the role of the supportive microenvironment. To address this need, we and other investigators have developed mouse models in which lung adenocarcinomas arise spontaneously owing to mutant K-ras alleles expressed inducibly, conditionally, or somatically Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related death in the United States and other western countries. Approximately two thirds of patients are diagnosed at an advanced stage, and of the remaining patients who undergo curative surgery, 30–50% have a recurrence with metastatic disease. Thus, a better understanding of the biologic underpinnings of metastatic disease is of paramount importance. Metastasis research in lung cancer has been hampered by the lack of good animal models and the difficulty in studying disease progression and metastasis in patients. This has resulted in a reliance on p53 missense mutation, R175H, found in Li-Fraumeni syndrome patients and in a subset of NSCLC patients, is a structural mutation that exhibits loss of function owing to inactivation of p53 transcriptional activity p53 has been previously introduced into the mouse as a knock-in allele. To evaluate the importance of p53R172H as a contributing event in lung tumorigenesis, p53R172HΔG mice were previously mated with KrasLA1 mice, which develop lung adenocarcinomas owing to somatic activation of a latent KrasG12D allele, but rarely metastasize LA1 alone, p53R172HΔG alone, or both alleles K-ras, lung adenocarcinomas were rare (13% of p53R172HΔG/+ mice). Although the presence or absence of the mutant p53 allele did not affect the frequency of lung adenocarcinomas in KrasLA1 mice , metastases were much more frequent in those with p53 mutations than in those without (36.5% versus 4.5%). In the KrasLA1/+; p53R172HΔG/+ mice, metastases were found at sites frequently observed in NSCLC patients, including the mediastinal lymph nodes, heart, parietal pleura, diaphragm, liver, adrenal gland, kidney, mesentery, pancreas, and subcutaneous tissues. The remaining wild-type p53 allele was deleted in approximately 50% of the murine tumors, which mimics the wild-type p53 allelic deletion observed in tumors from Li-Fraumeni patients. Collectively, these findings suggest that KrasLA1/+; p53R172HΔG/+ mice are a useful model for the study of metastasis in NSCLC patients.A LA1/+; p53R172HΔG/+ mice. Tumors from the lung and distant metastatic sites were transcriptionally profiled, from which we derived a metastasis signature defined as those genes that were differentially expressed in the metastases relative to paired primary lung tumors. Data mining of publicly-available expression profiles revealed this signature in a subset of primary tumors from NSCLC patients who had poor prognosis. We conclude that KrasLA1/+; p53R172HΔG/+ mice are a useful tool for the study of lung adenocarcinoma metastasis.In this study, we sought to better understand the biologic basis for metastasis in KrasR172HΔg/+ K-rasLA1/+p53 mice.We followed the guidelines set forth by the Institutional Animal Care and Use Committee of The University of Texas, M. D. Anderson Cancer Center for husbandry of R172HΔg/+ K-rasLA1/+p53 mice were isolated, carefully dissected to remove the adjacent tissue, snap-frozen in liquid nitrogen and stored at −80° until use Primary lung adenocarcinomas and metastases from R172HΔg/+ K-rasLA1/+p53 tumors was extracted by Trizol (Invitrogen) and purified with an RNeasy kit (Qiagen). RNA quality and quantity were evaluated on an Agilent Bioanalyzer following the manufacture's recommendations (Agilent Technologies). Synthesis of cRNA and hybridization to Mouse Expression Array 430A 2.0 chips were performed following Affymetrix protocols .Total RNA from the t-tests using log-transformed data determined significant differences in mean gene mRNA levels between groups of paired samples. Fold changes between groups were estimated by taking the averages of the metastasis/primary log ratios. Expression values were visualized as color maps using the Java TreeView software After scanning and low-level quantification using Microarray Suite (Affymetrix), DNA Chip (dChip) analyzer In order to score each human lung tumor within a set for similarity to our gene signature of spontaneous metastases , we deriLA1/+; p53R172HΔG/+ mice would recapitulate those in a subset of NSCLC patients. To test this, the transcriptome of tumors from KrasLA1/+; p53R172HΔG/+ mice were profiled, and the derived metastasis signature, which was defined as those genes that were differentially expressed in the metastases relative to paired primary lung tumors, was compared to previously published expression profiles from lung adenocarcinomas from several patient cohorts We postulated that the biologic processes mediating metastasis in KrasEnrichment analysis (Fisher's exact test using Gene Ontology terms) of those genes with increased expression revealed highly significant enrichment in genes with the terms ‘cell cycle’ , ‘kinetochore’ , ‘pericentric chromosome-binding’ , ‘DNA replication’ , and ‘DNA-binding’ , whereas analysis of the genes that were decreased revealed enrichment in genes with the terms ‘membrane-binding’ , ‘integral-to-membrane’ , ‘lysosomal’ , and ‘golgi apparatus’  . An overall analysis of the four datasets combined did demonstrate significant prognostic ability in the human tumors by the murine metastasis signature .To compare the metastasis signatures in KrasLA1/+Kras; R172HΔG/+p53 mice to that of NSCLC patients by performing transcriptional profiling studies. We identified a metastasis expression signature in LA1/+Kras; R172HΔG/+p53 mice that was present in primary tumors from NSCLC patients who had poor prognosis. Based upon the ability of the murine metastasis signature to discriminate patient outcome, we conclude that the murine tumors recapitulated features of human lung adenocarcinoma. We do not mean to imply that this signature is clinically useful in a prognostic or predictive fashion, but simply interpret it as evidence of the potential usefulness and relevance of the model for studying the biology of human lung adenocarcinoma metastasis.In this study, we sought to examine the fidelity of the metastatic process in K-ras mutational status, which was reported by Bhattacharjee et al. We examined whether the murine metastasis signature overlapped with genes identified from five NSCLC patient cohorts reported in two studies LA1/+Kras; R172HΔG/+p53 mice was not related simply to their proliferative potential and that other genes involved in biologic processes relevant to metastasis, such as tumor cell invasive potential, might have contributed. Of note, in that regard, were genes in the decreased-expression gene set that control cell polarity  and cell-cell attachments (Ccam1 and Cask2). Loss of polarity and cell-cell contacts are features of epithelial cells that have undergone epithelial-to-mesenchymal transition (EMT), a phenotypic change associated with enhanced invasive and metastatic properties in tumor cells Vim and Cdh2, were more highly expressed in the metastases than in the primary lung tumors that arose in LA/+ p53R172HΔg/+K-ras mice. Whether these changes reflect a phenotypic change that contributed to the metastatic capacity of these cells is currently under investigation and will be reported on separately.The increased-expression gene set in the metastasis signature was enriched in cell cycle genes, but the prognostic power of the metastasis signature was not diminished by the removal of cell cycle genes, suggesting that the metastatic capacity of lung tumor cells in LA1/+Kras; R172HΔGp53 mice will provide a useful platform to better understand the basic biologic processes that underlie metastasis, to identify biologic targets for the prevention and treatment of metastasis, and to test the efficacy of novel agents directed against those targets in preclinical studies. Such studies could have a tremendous impact on global health given that NSCLC is the most common cause of cancer-related death in Western countries, and metastasis is the most common cause of death in NSCLC patients.We conclude that Figure S1Excel spreadsheet with entire list of genes.(0.74 MB XLS)Click here for additional data file.Figure S2Excel spreadsheet with list of GO terms(1.03 MB XLS)Click here for additional data file.Figure S3PDF describing “Impact of cell cycle genes on progression-free survival and overall survival”(0.08 MB PDF)Click here for additional data file.Figure S4PDF describing “Comparison of mouse metastasis signature with other metastasis signatures”(0.27 MB PDF)Click here for additional data file.


S. pombe, plants and Drosophila. Moreover, both in Drosophila and mammals, dosage compensation requires the contribution of specific non-coding RNAs. However, whether RNA itself plays a direct structural role in chromatin is not known. Here, we report results that indicate a general structural role for RNA in eukaryotic chromatin. RNA is found associated to purified chromatin prepared from chicken liver, or cultured Drosophila S2 cells, and treatment with RNase A alters the structural properties of chromatin. Our results indicate that chromatin-associated RNAs, which account for 2%–5% of total chromatin-associated nucleic acids, are polyA− and show a size similar to that of the DNA contained in the corresponding chromatin fragments. Chromatin-associated RNA(s) are not likely to correspond to nascent transcripts as they are also found bound to chromatin when cells are treated with α-amanitin. After treatment with RNase A, chromatin fragments of molecular weight >3.000 bp of DNA showed reduced sedimentation through sucrose gradients and increased sensitivity to micrococcal nuclease digestion. This structural transition, which is observed both at euchromatic and heterochromatic regions, proceeds without loss of histone H1 or any significant change in core-histone composition and integrity.Chromatin structure is influenced by multiples factors, such as pH, temperature, nature and concentration of counterions, post-translational modifications of histones and binding of structural non-histone proteins. RNA is also known to contribute to the regulation of chromatin structure as chromatin-induced gene silencing was shown to depend on the RNAi machinery in  Density gradient centrifugation is a powerful method to separate and analyze macromolecules Here, we report results indicating that RNA plays a general structural role in eukaryotic chromatin. Our results indicate that purified chromatin contains significant amounts of RNA . Whether this chromatin-associated RNA(s) contributes to chromatin structure was addressed by analyzing the sedimentation behavior through linear sucrose gradients of native oligonucleosomal fragments, purified from chicken liver or cultured −.As shown in In the experiments described above, sucrose gradient centrifugation was performed at low ionic strength . RNA, however, remains associated to chromatin at high ionic strength . In thes2 and NaCl, and the pellets were dissolved, loaded and electrophoresed in a PAGE-SDS gel. No significant change in histone composition or integrity is detected after treatment with RNase A , Actin 5C and RP-49 were determined by RT-PCR (To address the question of whether chromatin-associated RNA(s) corresponds to nascent RNA transcripts, we analysed the effects of blocking transcription by treatment with α-amanitin y RT-PCR . In all y RT-PCR . These rDrosophila S2 cells , which is responsible for silencing of the X-chromosome Air (>100 kb long), which silences the paternal Igf2r/Slc22a2/Slc22a3 gene Here, we report evidence supporting the physical association of RNA to chromatin purified by sucrose gradient centrifugation. Sucrose gradient centrifugation is used for the purification of discrete molecular entities from complex mixtures of macromolecules. Purification is achieved on the basis of the differential sedimentation rates of the various components, which depend on their mass, density and shape − , indicats). As s depends on molecular weight, density and shape, the analysis of the sedimentation behavior could yield ambiguous information. But, when two of these parameters are constant, the change in the sedimentation rate actually reflects the variation in one particular property; for particles of similar mass and density, the value of s depends only on their shape via DNA. On the other hand, several indications suggest that RNA might stabilize binding of structural non-histone proteins to chromatin. For instance, binding of HP1 to heterochromatin is sensitive to digestion with RNase A in vitroTreatment with RNase A alters the sedimentation behavior of chromatin through sucrose gradients . In densDrosophila S2 cells were grew by conventional methods. Nuclei were isolated by Dounce homogenization of minced material (when liver) or spun cells (when S2) in 0,5 M sucrose, 0,5% Triton X-100, 100 µg/ml Phenyl Methane Sulfonyl Fluoride (PMSF), TES buffer . The homogenate was diluted with three volumes of 2,3 M sucrose in TES and layered over a 5 ml cushion of 2,3 M sucrose in TES. After centrifugation in a Beckman SW28 rotor , pelleted nuclei were washed twice with cold TES, precipitated in a Beckman JA-25.50 rotor  and resuspended in the same buffer. The DNA concentration was measured at 260 nm and the nuclei, usually at 0,5–1 mg/ml, were frozen at −80°C after the addition of Dimethyl sulfoxide (DMSO) to a 10% final concentration.All plasticware, glassware and solutions were sterilized by autoclaving. Fresh chicken livers were purchased on the public food market. 2 during different times and the reaction stopped by addition of EDTA . After pelleting in an Eppendorf tabletop centrifuge , the nuclei were disrupted by pipetting in 0,2 mM EDTA and spun again; the soluble chromatin was recovered from the supernatant and DNA concentration was measured at 260 nm. On average, up to 30–40% of the starting material was recovered as soluble chromatin.Native chromatin was obtained by mild digestion with micrococcal nuclease (MNase) of isolated nuclei in 3 mM CaClTreatment with RNaseA (DNase and protease-free) (Roche) was performed using 0,5 to 1 µg of enzyme. When indicated, RNaseA activity was inhibited by the addition of 100 to 200 units of anti-RNase (Ambion) following manufacturer instructions.RNA was extracted using Ultraspec™ RNA Isolation System (Biotecx) according to manufacturer instructions. Glyoxal-agarose gels were performed on a NaOH-treated electrophoresis cell. To minimize RNA degradation, agarose solution, electrophoresis and loading buffers were autoclaved. When the amount of RNA associated to purified chromatin was determined, chromatin purified by sucrose gradient sedimentation was deproteinized overnight at 37°C with 0,2% SDS, 0,5 mg/ml proteinase K, followed by phenol extraction and ethanol precipitation. The precipitated sample was resuspended in sterile water and total nucleic acid content determined at 260 nm using a quartz cuvette extensively treated with 2N NaOH and sterile water. The sample was, then, divided into three aliquots, containing 6 µg of total nucleic acid, which were treated at 37°C for 2 h with 20 µg of DNAse I (RNase-free), with 20 µg of RNase A (DNase and protease-free) or not treated. After these treatments, nucleic acid content of serial 10-fold dilutions of each sample was determined with RiboGreen® RNA Quantitation Reagent (Molecular Probes) in a fluorescence microplate reader  in comparison to both DNA and RNA standards. In parallel, another aliquot of deproteinized chromatin was extracted with Ultraspec™ RNA Isolation System and the purified material was divided in two portions, incubated for 2 h at 37°C with 20 µg of RNase A (DNase and protease-free) or not, and processed as before.Linear sucrose gradients were performed with a linear gradient maker. Prior to their use, the mixing chambers and polyvinyl tubing were treated during 1 h with a 2N NaOH solution and then washed with sterile water. To prevent proteases activities, PMSF was added to a 100 µg/ml final concentration to the sucrose solutions. 5%–30% sucrose  preparative gradients containing 1 to 5 mg of soluble native chromatin were centrifuged on Beckman SW28 rotor at 22.000 rpm during 14 h at 4°C. Analytical gradients containing 100–200 µg were run in a Beckman SW41 rotor .The polyadenylated status of purified chromatin-associated RNA(s) was analysed by affinity chromatography using an oligo-dT resin . Bound (poly A+) and unbound (poly A-) samples were purified and electrophoresed on glyoxal-agarose denaturant gel.Drosophila S2 cells were grew until confluence and divided in two equal portions of 300 ml of suspended cells; one of them was incubated with α-amanitin (Sigma)  during 36 h at 24°C. An aliquot (5 ml) of each cellular culture was processed for total RNA isolation and their nucleic acid content determined at 260 nm using a quartz cuvette extensively treated with 2N NaOH and sterile water. Native chromatin isolation and sedimentation analysis of samples incubated or not with α-amanitin was performed as described before. Efficiency of transcriptional inhibition by α-amanitin was measured by retrotranscription using Omniscript® RT Kit (QIAGEN). RT-reactions were performed with different amounts of RNA templates using a mixture of hexanucleotides as primers. After RT, samples were phenolized, precipitated with ethanol at room temperature and, then, subjected to PCR with Taq polymerase (Biotools) using specific primers derived from intron and adjacent exon sequences for actin 5C, Trl and RP-49 genes. Primers used were: for actin 5C, 5′-TATCACTACCGTTTGAGTTC-3′, corresponding to the first exon (positions +2 to +22) and 5′-CGTGACACGCCCACATCAGC-3′, corresponding to the first intron (positions +567 to +587); for Trl, 5′-TGGATCTAAGACTTCGGTCC-3′, corresponding to the second exon (positions +3208 to +3228) and 5′- AGCAACTCATTCCTTCCTTG-3′, corresponding to the second intron (positions +3850 to +3870) and, for RP-49, 5′- CATGTTATCAATGGTGCTGC-3′, corresponding to the first exon (positions +20 to +40) and 5′-GAATTATGCATTAGTGGGAC-3′, corresponding to the second intron (positions +702 to +722). The size of the expected fragments are 585 bp, 662 bp and 702 bp for actin 5C, Trl and RP-49, respectively.


Flow cytofluorimetric techniques, using acridine orange fluorescence to measure RNA and DNA simultaneously in EMT6 cells, have been employed to discriminate between three diploid DNA populations in vivo on the basis of their RNA content. Cells with the lowest RNA levels seem to be in the process of disintegration. Cells with the highest RNA levels correspond to those with the highest plating efficiency, and those with intermediate RNA levels are those with the lowest plating efficiency. In vitro studies have shown that log-phase cells have higher RNA levels than cells in the late plateau phase of growth.


While many molecules involved in axon guidance have been identified, the cellular and molecular mechanisms by which these molecules regulate growth cone morphology during axon outgrowth remain to be elucidated. The actin cytoskeleton of the growth cone underlies the formation of lamellipodia and filopodia that control growth cone outgrowth and guidance. The role of the Arp2/3 complex in growth cone filopodia formation has been controversial, and other mechanisms of growth cone filopodia formation remain to be described.arx genes) caused defects in axon guidance. Analysis of developing growth cones in vivo showed that arx mutants displayed defects in filopodia and reduced growth cone size. Time-lapse analysis of growth cones in living animals indicated that arx mutants affected the rate of growth cone filopodia formation but not filopodia stability or length. Two other actin modulatory proteins, UNC-115/abLIM and UNC-34/Enabled, that had been shown previously to affect axon guidance had overlapping roles with Arp2/3 in axon guidance and also affected the rate of filopodia initiation but not stability or length.Here we show that mutations in genes encoding the Arp2/3 complex (in vivo, multiple actin modulatory pathways including the Arp2/3 complex contribute to growth cone filopodia formation during growth cone outgrowth.Our results indicate that the Arp2/3 complex is required cell-autonomously for axon guidance and growth cone filopodia initiation. Furthermore, they show that two other actin-binding proteins, UNC-115/abLIM and UNC-34/Enabled, also control growth cone filopodia formation, possibly in parallel to Arp2/3. These studies indicate that,  The growth cone of a developing axon senses and responds to extracellular cues, resulting in the migration of the growth cone and thus axon to its correct target region in the nervous system ,2. Growtin vivo. In Drosophila, the Arp2/3 complex and its regulators WAVE/Scar and Kette are required for proper axon pathfinding , mig-2(mu28); I: arx-7(ok1118), hT2 [bli-4(e937) let-?(q782) qIs48] , lqIs40 [gcy-32::gfp]; II: juIs76 [unc-25::gfp]; III: arx-4(ok1093), sC1 [dpy-1(s2170)]; IV: ced-10(n1993), lqIs3 [osm-6::gfp], nT1 [qIs51] ; V: arx-2(ok1269), unc-34(e951). The chromosomal locations of lqIs49 [gcy-32::gfp] and lqIs75 [gcy-32::arx-7::mCherry] were not determined. The arx-2(+), arx-4(+), and arx-7(+) whole-gene regions were maintained as extrachromosomal arrays. Extrachromosomal arrays were generated by germ line microinjection and integrated into the genome by standard techniques . In. Inarx hd Figure . arx-2. arx-4 is the downstream gene in a C. elegans operon, and the entire operon region composed of the genes K02F3.1, K02F3.12, and Y6D11A.1 was included in the fragment. The arx-2(+) and arx-4(+) transgenes rescued the slow growth, sterility and pVul phenotype of arx-2(ok1269) and arx-4(ok1093), respectively, and transgenic animals were viable and fertile. Furthermore, PDE axon defects were completely rescued  and 34% to 1% for arx-4(ok1093); P < 0.0001 for each). The arx-7(+) fragment rescued the slow growth and pVul components of the arx-7(ok1118) phenotype as well as PDE axon pathfinding defects ; Ex [arx-7(+)] animals were still sterile. The arx-7(ok1118) deletion also affected the downstream gene Y54E10BR.1, which was not included in the rescuing fragment. The sterility of arx-7(ok118); Ex [arx-7(+)] transgenic animals might be due to the effects of ok1118 on Y54E10BR.1. These data indicate that the PDE axon guidance defects observed in arx mutants are due to mutation of the arx genes.To ensure that the phenotype observed in arx-4 acts in neurons to control axon pathfinding, a transgene was constructed consisting of the osm-6 promoter upstream of the arx-4 coding region. The osm-6 promoter is active exclusively in ciliated sensory neurons including PDE and has no apparent activity in other tissues [arx-4(ok1093M+) animals harboring the Posm-6::arx-4(+) transgene were slow-growing, Pvl, and sterile, similar to arx-4(ok1093) homozygotes alone, confirming that the Posm-6::arx-4(+) transgene was not broadly active. However, PDE axon guidance defects were rescued in these animals  and mig-2(mu28) enhanced the PDE axon pathfinding defects of arx-2(ok1269M+), arx-4(ok1093M+), and arx-7(ok1118M+) ; arx-4(ok1093M+) displayed 77% PDE axon guidance defects compared to 34% for arx-4(ok1093M+) alone; and mig-2(mu28); arx-7(ok1118M+) displayed 68% compared to 30% for arx-7(ok1118M+) alone . Double mutants did not exhibit ectopic axon formation as observed in ced-10; mig-2 double mutants [arx mutation was enhanced by both ced-10(n1993) and mig-2(mu28) suggests that the Arp2/3 complex might act in both the CED-10 Rac and MIG-2 RhoG pathways in PDE axon guidance. Possibly, both WVE-1/WAVE and WSP-1/WASP pathways converge on regulation of Arp2/3 in axon guidance.Previous results indicated that the Rac-like molecules CED-10/Rac  and MIG-) Figure . For exaunc-115 mutations enhanced PDE guidance defects of arx mutants ; arx-2(ok1269M+) displayed 83% PDE axon guidance defects compared to 15% for arx-2(ok1269M+) alone . Double mutants did not display enhanced ectopic axon formation as observed in double mutants of unc-115 with ced-10 and mig-2. That unc-115 mutations enhanced PDE axon guidance defects of arx mutations indicates that UNC-115/abLIM acts in parallel to Arp2/3 in PDE axon guidance, a result consistent with the idea that Rac GTPases employ both Arp2/3 and UNC-115/abLIM.Previous studies showed that the actin-binding protein UNC-115/abLIM acts downstream of Rac signaling in axon guidance [s Figure . For exaunc-34(e951) mutants displayed 8% PDE guidance defects. unc-34(e951); arx-4(ok1093) and unc-34(e951); arx-7(ok1118M+) displayed 91% and 97% PDE guidance defects compared to 35% and 35% for the arx mutants alone (P < 0.0001 for each comparison). Together, these data indicate that Arp2/3, UNC-115/abLIM, and UNC-34/Enabled all act redundantly to control PDE axon guidance.Previous studies showed that UNC-115/abLIM and UNC-34/Enabled had redundant roles in PDE axon guidance . As predC. elegans genome encodes one molecule similar to ezrin, radixin, and moesin called ERM-1. erm-1(tm677) is an out-of-frame deletion of the locus  animals displayed weak but significant defects in PDE axon guidance  with unc-115(ky275) and unc-34(e951) displayed PDE axon guidance defects that were not significantly different from the singles alone. These results indicate that ERM-1 has a role in PDE axon guidance but does not act redundantly with UNC-115 and UNC-34. Thus, the synergistic enhancement of arx mutants by unc-34 and unc-115 is not likely due to general actin cytoskeletal disruption. Rather, these results suggest that the genetic interaction is specific and that Arp2/3, UNC-115, and UNC-34 might act in a common process in axon guidance not affected by ERM-1.Ezrin/radixin/moesin (ERM) proteins are regulators of actin organization and interaction with the plasma membrane. The ut lumen ,36. We farx genes caused axon guidance defects, but the effect of arx mutations on the growth cone during neurite extension was unclear. Developing neurites in arx mutants were analyzed. Transgenes used to assay PDE axon morphology were not visibly expressed early in PDE development during axon extension, including osm-6::gfp [cat-1::gfp [gcy-32::gfp reporter [Mutations in m-6::gfp  and cat-t-1::gfp . Thereforeporter  was usedreporter .gcy-32::gfp expression was evident, revealing a large, wedge-shaped cell body with multiple finger-like filopodial protrusions emanating from around the cell body  mutants at 7 to 8 h post-hatching, the average number of finger-like extensions on PQR dendrites (defined as having a thickness of 0.5 μm or less) was reduced significantly compared to wild-type . That these finger-like protrusions were affected by unc-34/Enabled suggests that they are bona fide filopodia.The finger-like extensions on PQR growth cones resembled filopodia. In many systems, including cultured cells and neurons and  neurons ,41, Enabarx-2(ok1269M+) and arx-7(ok1118M+) living larvae expressing gcy-32::gfp. These larvae exhibited a general growth delay, and timing of PQR dendritic events were slowed by approximately one-half hour. At 7 h, initial wedge-like cell shape with filopodia appeared normal in arx-2 and arx-7 mutants  growth cones).Posterior PQR neurite extension was assessed in s Figure . From 7 e Figure . Neuritea Figure . Furtherarx-2(ok1269M+), and arx-7(ok1118M+) (7.5-8.5 h) were counted  displayed 2.27 and arx-7(ok1118M+) 1.78 , arx mutants often appeared less robust and smaller in size than wild type. To quantify this defect, the perimeters of growth cones (excluding filopodia) from wild type and mutant backgrounds were traced .As mentioned previously, the growth cones of d Figure . arx-7 darx mutants. However, PQR dendrites did not extend directly to the posterior as in wild type . These data indicate that arx-7(ok1118M+) affects the guidance of the PQR dendrite.To determine if these growth cone defects resulted in neurite guidance defects, we scored the final morphology of the PQR dendrites after outgrowth. Initial posterior protrusion, axon consolidation, and outgrowth of the neurite to its normal position in the tail were generally normal in e Figure . Rather,gcy-32 promoter driving arx-7 tagged with coding region for GFP was constructed. This transgene was predicted to express full-length ARX-7 with GFP at the carboxyl terminus. Animals harboring this transgene showed ARX-7::GFP fluorescence in the URX, AQR, and PQR neurons and nowhere else (data not shown). arx-7(ok1118M+) animals harboring this transgene displayed the gross phenotype of arx-7(ok1118M+) alone , indicating that arx-7(+) activity was not widely provided by the Pgcy-32::arx-7::gfp transgene. However, this transgene rescued filopodia formation to wild-type levels in arx-7(ok1118M+) PQR neurites  alone (P < 0.0001). arx-7 rescue was not significantly different than wild type . These data indicate that arx-7 acts cell-autonomously in the regulation of growth cone filopodia.A transgene consisting of the s Figure : an averunc-115 mutations enhanced PDE axon guidance defects of arx-2, arx-4, and arx-7, indicating redundancy of function in PDE axon guidance. The effect of unc-115 on PQR posterior neurite extension was examined. Alone, unc-115(ky275), a null allele, displayed no defects in PQR posterior neurite filopodia formation  enhanced filopodia defects of arx-2(ok1269M+) and arx-7(ok1118M+). unc-115(ky275); arx-2(ok1269M+) displayed 1.22 filopodia per neurite compared to 2.27 for arx-2(ok1269M+) alone (P = 0.003), and unc-115(ky275); arx-7(ok1118M+) displayed 1.04 compared to 1.78 for arx-7(ok1118M+) alone (P = 0.007).As reported above, unc-115(ky275) mutants also displayed a reduced growth cone perimeter , indicating that UNC-115 might be involved in growth cone lamellipodium regulation. unc-115(ky275) displayed 41% PQR dendrite guidance defects (the undulating dendrite), compared to 19% for wild type  had no affect on filopodia, suggesting that unc-115 PQR dendrite guidance defects were due to affects on lamellipodia and not filopodia.e Figure . Alone, unc-115; arx double mutants displayed a defect not seen in unc-115, arx-2, or arx-7 single mutants, a rudimentary or absent posterior PQR neurite ; arx-2(ok1269M+) and 9 of 75 unc-115(ky275); arx-7(ok1118M+) animals lacked a significant posterior protrusion. Some animals showed a rudimentary posterior protrusion  mutants displayed severely reduced filopodia-like protrusions on the PQR dendritic neurite . That unc-34 affected these thin, filopodia-like protrusions supports the notion that they are bona fide filopodia, as Enabled molecules have been shown to control filopodia formation in many systems, including C. elegans neurons [arx-7(ok1118M+); unc-34(e951) displayed significantly fewer filopodia than each single alone, but an additive effect of the two mutations combined could not be excluded ; unc-34(e951) double mutants lacked a strong posterior neurite from PQR (2 of 17), similar to arx; unc-115 double mutants, suggesting that UNC-34/Enabled and the Arp2/3 complex might control the ability of a neurite to form or to extend. unc-34 mutants did not display a reduction in PQR growth cone perimeter . Variability in translocation rate in each genotype, including wild type, was very high, making it difficult to assign a direct effect on growth cone morphology to translocation rate. For example, unc-115 affected filopodia to the same extent as arx-7 but did not affect translocation rate. It is possible, however, that these mutants have distinct effects on growth cones aside from filopodia that were not assayed in these studies.We also determined the average translocation rates of wild-type and mutant growth cones  of animals , but occurred at a significantly higher rate in arx-7 (P < 0.0001). While the penetrance of axon guidance defects of arx-7, unc-115, and unc-34 were similar . However, it is possible that there is reduced nucleation of lamellipodial actin filaments in arx-7 mutant growth cones, resulting in less availability of lamellipodial actin filaments to contribute to filopodial bundles. This idea is consistent with the time-lapse analysis of growth cone filopodia, which showed that arx mutants had reduced filopodial initiation rates compared to wild type . This could be due to the action of the remaining few filopodia on arx mutant growth cones or could be due to lamellipodial guidance. Indeed, unc-34/Enabled mutants lack most or all growth cone filopodia but have relatively weak effects on axon guidance , and promotes the formation of lamellipodia and filopodia in roblasts . UNC-115roblasts , but thiunc-34 mutant growth cones had reduced filopodia numbers confirm this role of Enabled. In time-lapse studies, we found that unc-34 affected the rate of filopodia formation, suggesting that Enabled controls filopodia initiation similar to Arp2/3 and UNC-115/abLIM. This is similar to previous results in cultured neurons in response to netrin [unc-34 had no effect on filopodia stability or length once a rare filopodium formed, indicating that UNC-34 has no role in filopodia outgrowth or stability. This is in contrast to cultured neurons, where Ena/VASP inhibition also resulted in filopodia extension defects [Enabled has been extensively implicated in filopodia formation, including on growth cones, and our results that o netrin . We foun defects .arx-7 and unc-115 mutants, unc-34 mutant PQR growth cones, while nearly devoid of filopodia, displayed no significant reduction in size. While all three mutations caused reduced filopodia initiation, this difference might hint at distinct mechanisms of action of these molecules. Possibly, Arp2/3 and UNC-115/abLIM primarily affect lamellipodial actin organization such that filaments are less available for filopodia formation in mutants. In contrast, UNC-34/Enabled had a filopodia-specific effect, consistent with the role of Ena/VASP molecules as actin anti-capping proteins. It is surprising that unc-34 had little or no effect on stability or length of the few filopodia that did form in these mutants. Possibly, the anti-capping activity of UNC-34/Enabled is required only during initiation and other proteins can serve its function during elongation. It is also possible that filopodial initiation requires other actin-interacting properties of Enabled that have been described recently  [In contrast to undling) ,22.arx with unc-115 and unc-34 displayed greatly enhanced PDE axon guidance defects, and previous studies showed that unc-34 and unc-115 had overlapping roles in PDE axon guidance. Possibly, perturbing multiple actin modulating molecules had general, nonspecific effects on the cytoskeleton, resulting in increased defects. We think this is not the case as these interactions were specific. erm-1 encodes the only member of the ezrin/radixin/moesin family of actin-membrane linking proteins in the C. elegans genome. erm-1 mutation caused weak guidance defects on its own, indicating that ERM-1 is required for axon guidance. However, erm-1 did not enhance unc-115 or unc-34, suggesting that the enhancement of arx, unc-115, and unc-34 is specific to these genes and is not the result of general cytoskeletal disruption.Our results indicate that Arp2/3, UNC-115/abLIM, and UNC-34/Enabled are required for growth cone filopodia formation. However, the effects of these mutations on PDE and VD/DD axon guidance and PQR dendritic guidance were incompletely penetrant. Strikingly, double mutants of unc-115 and unc-34 enhanced the PQR filopodia defects of arx-7, although the enhancement by unc-34 might simply be an additive effect because unc-34 mutants alone display severely reduced filopodia numbers. It is also possible that all three molecules affect filopodia formation and have redundant roles in other aspects of growth cone morphology. Indeed, this seems to be the case, as both unc-115; arx and unc-34; arx double mutants displayed severe PQR neurite outgrowth and formation defects. This is surprising for unc-34/Enabled, as Ena/VASP molecules were traditionally considered to be anti-capping proteins with specific effects on filopodia. However, recent studies have suggested that Ena/VASP molecules have other effects on actin, including bundling and nucleation [unc-34; arx double mutants.Together, our data indicate that Arp2/3, UNC-115/abLIM, and UNC34/Enabled might act in a common process in axon guidance, namely in the initiation of growth cone filopodia  and distinct roles (lamellipodia formation) in the growth cone during axon outgrowth.Many molecules have been implicated in axon guidance through genetic, molecular, and biochemical studies. The effects of these molecules on the behavior and dynamics of the growth cone during axon guidance are unclear in many cases. Through a combination of genetic analysis and time-lapse imaging of growth cones during outgrowth, we show that the Arp2/3 complex acts cell autonomously in the initiation of new growth cone filopodia during growth cone outgrowth, possibly explaining the axon guidance defects observed in ERM: Ezrin/radixin/moesin; GFP: green fluorescent protein; Pvl: protruding vulva; VNC: ventral nerve cord.The authors declare that they have no competing interests.arx-4. EAL, with help from ADN and JOC, wrote the paper.ADN, JOC, and EAL conceived of and designed the experiments, EAL and ADN performed the experiments, and JOC performed the cell-autonomous rescue of Time-lapse movie of a wild-type VD growth cone migrating between ventral and dorsal muscle quadrants with typical dynamic appearance. Images captured every 120 s, total movie duration 36 minutes. The scale bar in the first frame represents 5 μm.Click here for filearx-7(ok1118M+) growth coneTime-lapse movie of an . Images captured every 120 s, total movie duration 30 minutes. The scale bar in the first frame represents 5 μm.Click here for fileunc-115(ky275) growth coneTime-lapse movie of an . Images captured every 120 s, total movie duration 24 minutes. The scale bar in the first frame represents 5 μm.Click here for fileunc-34(e951) growth coneTime-lapse movie of . Images captured every 120 s, total movie duration 54 minutes. The scale bar in the first frame represents 5 μm.Click here for file


The recent deluge of new RNA structures, including complete atomic-resolution viewsof both subunits of the ribosome, has on the one hand literally overwhelmed ourindividual abilities to comprehend the diversity of RNA structure, and on the otherhand presented us with new opportunities for comprehensive use of RNA sequencesfor comparative genetic, evolutionary and phylogenetic studies. Two concepts are keyto understanding RNA structure: hierarchical organization of global structure andisostericity of local interactions. Global structure changes extremely slowly, as it relieson conserved long-range tertiary interactions. Tertiary RNA–RNA and quaternaryRNA–protein interactions are mediated by RNA motifs, defined as recurrent andordered arrays of non-Watson–Crick base-pairs. A single RNA motif comprises afamily of sequences, all of which can fold into the same three-dimensional structureand can mediate the same interaction(s). The chemistry and geometry of base pairingconstrain the evolution of motifs in such a way that random mutations that occurwithin motifs are accepted or rejected insofar as they can mediate a similar orderedarray of interactions. The steps involved in the analysis and annotation of RNAmotifs in 3D structures are: (a) decomposition of each motif into non-Watson–Crickbase-pairs; (b) geometric classification of each basepair; (c) identification of isostericsubstitutions for each basepair by comparison to isostericity matrices; (d) alignmentof homologous sequences using the isostericity matrices to identify correspondingpositions in the crystal structure; (e) acceptance or rejection of the null hypothesisthat the motif is conserved.


Therapeutic targeting of HIV latency requires an understanding of the basic mechanisms regulating viral quiescence and activation.in situ hybridization, we found that chromatin imposes silencing in a highly specific spatial and temporal pattern. We also identified several cellular factors that interact with viral RNA by a proteomic approach, pointing to post-transcriptional processes that have been greatly overlooked as mechanisms of HIV post-integrative latency.We identified several levels of transcriptional and post-transcriptional control of viral genes expression. By looking at the organization of chromatin at the site of integration by chromatin conformation capture and HIV-1 can integrate within active genes at the periphery of the nucleus, and silencing may involve repression from a peri-centromeric region in trans. Hence, a mechanism of active spatial reorganization of chromatin at the site of proviral integration may be ultimately responsible for virus silencing. Particularly relevant to flushing therapies of the viral reservoir is the observation that the silent state of an integrated provirus in a clonal population of activated lymphocytes is not homogeneous, with a small number of cells carrying a provirus embedded into heterochromatin, compared with the majority of cells where the provirus is already poised for transcription.nrb that have been previously in nuclear retention of RNA. We demonstrated that PSF/p54nrb binds nascent HIV RNA at the transcription site, but MATR3 defines a novel sub-nuclear compartment where this viral RNA is retained. Rev is able to associate with unspliced HIV RNA co-transcriptionally directing its nuclear export. These observations lead to a model for a novel cellular pathway of RNA retention that is hijacked by the virus.Proteomic analysis revealed several novel cellular factors, including PSF, MATR3 and p54Both the control imposed by the organization of the nucleus and by post-transcriptional mechanisms is relevant for the control of HIV-1 post-integrative latency.


Saccharomyces cerevisiae. Surprisingly, more than half of these proteins represent previously known enzymes, many of them acting in metabolism, providing opportunities to directly connect intermediary metabolism with posttranscriptional gene regulation. We mapped the RNA targets for 13 proteins identified in this screen and found that they were associated with distinct groups of mRNAs, some of them coding for functionally related proteins. We also found that overexpression of the enzyme Map1 negatively affects the expression of experimentally defined mRNA targets. Our results suggest that many proteins may associate with mRNAs and possibly control their fates, providing dense connections between different layers of cellular regulation.Hundreds of RNA-binding proteins (RBPs) control diverse aspects of post-transcriptional gene regulation. To identify novel and unconventional RBPs, we probed high-density protein microarrays with fluorescently labeled RNA and selected 200 proteins that reproducibly interacted with different types of RNA from budding yeast  Immediately when RNA is synthesized by RNA polymerases, RNA binding proteins (RBPs) assemble on the nascent transcript forming ribonucleoprotein (RNP) complexes, which tightly control all of the further steps in a RNA's life. On one hand, RBPs assist the processing and assembly of non-coding (nc) RNAs into RNP complexes, which mediate essential cellular functions such as splicing and translation e.g.The availability of genomic tools now allows the systematic identification of RNA targets for RBPs to obtain a global view of their gene regulatory potential. One of the main approaches include the immunopurification of RNP complexes followed by the analysis of the associated RNAs with DNA microarrays, a method referred to as RNA-immunopurification-microarray (RIP-Chip). Numerous studies applying these genomic tools revealed that many RBPs associate with distinct RNA target sets comprised of a few up to several hundred RNAs, which are often enriched for specific sequence/structural elements that define RBP binding sites. The sets of bound RNAs were often related containing mRNAs coding for functionally or cytotopically related proteins e.g.. These fSaccharomyces cerevisiae, more than 500 proteins are predicted to function as RBPs RBPs comprise 3 to 11% of the proteomes in bacteria, archaea and eukaryotes underlining the importance of RNA regulation for cell function The vast number of protein motifs linked to RNA regulation and the ancient origin of RNA regulation, which is possibly the most evolutionary conserved component of a cell's physiology, proposes that many proteins implicated in other cellular processes could have retained RNA-binding capacity. For instance, several metabolic enzymes in mammals have been shown to bind to and regulate mRNA expression , a tRNA guanylyltransferase (Thg1), and a single-stranded DNA-binding protein (Rim1) also strongly and reproducibly interacted with E2Bmin (SD>3.5 in replicates). Whether these E2Bmin binders may be implicated in the regulation of Ash1 mRNA in vivo remains to be elucidated. At least, these experiments show that specific RNA-protein interactions can be detected with our experimental set-up.We used functional protein microarrays to screen for proteins that interact with RNA . ProteinTo screen for proteins that interact either with total RNA or mRNAs, we basically used the same experimental set-up as applied for the E2Bmin experiments. We probed the protein microarrays with Cy3 labeled ‘total RNA’, which was isolated from yeast cells grown in different carbon sources, and with Cy5 labeled mRNAs isolated from total RNA via oligo-dT columns see . Becausep<10−3, We categorized the selected 180 proteins that either interacted with total RNA or mRNA based on Gene Ontologies (GO) retrieved from the Princeton GO server. 132 out of the 180 proteins (73%) had at least one known function annotated with GO. 28 proteins were annotated with the GO term ‘RNA-binding’, which is therefore over-represented among the group of all 180 selected proteins p<10−3, . Furtherp<0.003) (p<0.007) or lipid oxidation (p<0.008). Moreover, 25 of these enzymes can be mapped to the yeast metabolic network p<0.016). In agreement with this bias for enzymes, most of the herein identified potential RBPs are cytoplasmic , membrane-associated (p<0.003), and some of them located to peroxisomes (p<2×10−5) . Whereasp<2×10−5). These rS. cerevisiae were annotated with 6,119 domains in Pfam, and we analyzed whether some of these domains were over-represented among the 150 proteins (out of 180) that contained at least one Pfam domain  and occur in proteins devoid of other known RNA-binding motifs. This includes the ubiquitin motif or the weakly conserved repeat module PC_rep, which are found in several proteins involved in protein degradation control We further analyzed our experimentally defined set of 180 RBPs for the occurrence of protein domains annotated by the Pfam database S. cerevisiae from the M3D database We next asked how the expression of our selected RNA-binders varies across different growth conditions to see whether our selection is biased to any kind of expression characteristics. We therefore compiled a large collection of microarray data available for a wide range of experimental conditions for p<0.64, Wilcoxon test). However, our herein identified RBPs are generally higher expressed than non-RBPs ; an observation that has been made previously for conventional RBPs as well We further compared the expression levels of the potential RBPs identified in this study with previously annotated RBPs see . We founin vivo, we purified endogenously expressed tandem-affinity purification (TAP)-tagged proteins from cells grown in rich media, and identified co-purifying RNAs with yeast DNA oligo arrays. From our list of 180 RBPs, we selected 13 proteins that are expressed at different levels, and for which respective mRNA expression patterns are different across a variety of conditions, providing a representative sample of differentially expressed putative RBPs , further substantiating that the observed associations are selective and not merely driven by expression.We performed three independent affinity isolations with each of the 13 potential RBPs and five independent mock isolations with untagged control cells ( = mock isolates). To identify RNA that were significantly associated with the proteins we selected those features that were on average at least 3-fold enriched in the affinity isolates compared to the mock controls with a 0.01 see . This an0.01 see . NotablyFour of the 13 proteins (30%) were associated with their own mRNA . Binding to the own mRNA offers the possibility for auto-regulation through the formation of positive or negative feedback loops p<2×10−4). Therefore, it appears that although some GO terms were enriched with more than one of the proteins, it is not because these proteins bound to a common set of messages that connects to one particular GO term, but rather that they were associated with different messages that belong to the same functional class.Because many RBPs bind to mRNAs coding for functionally related proteins, we searched for common themes among the messages that were associated with the 13 proteins. For six proteins  we found significantly enriched GO groups among associated messages, offering the potential to coordinate expression of functionally related groups of messages or ‘RNA regulons’ . NoteworMAP1 compared to control cells with DNA microarrays. Map1p is a methionine aminopeptidase (MetAP) that catalyzes the co-translational removal of N-terminal methionine from nascent polypeptides, and it is functionally redundant with Map2p MAP1 under the control of galactose inducible promoter, and control cells containing an empty plasmid, were grown to mid-log phase and expression was induced with 2% galactose for 1.5 hours. Noteworthy, inducible short-time overexpression of RBPs could be beneficial to measure direct effects of proteins on gene expression by minimizing secondary effects that may raise after prolonged alterations of expression levels . We obtained mRNA expression profiles for 6,851 features representing 5,889 yeast genes  but significantly decreased compared to all non-targets  (p<0.05 (one sample t-test); and seven Map1p targets were overrepresented among the 36 down-regulated messages . The same analysis with microarray data obtained from cells overexpressing GIS2 (another ZnF protein among the selected RNA-binders) did not reveal reduced expression of Map1p targets , indicating that the observed shift in the distribution of Map1p targets was not a general effect due to protein overexpression. In conclusion, these results suggest that Map1p could negatively affect mRNA expression of selected mRNA targets.To investigate how one of the selected candidate enzymes could affect gene expression of targets, we measured the relative changes of mRNA levels of cells overexpressing  U test) . Of notebona fide RBPs. This was further initially demonstrated for Map1p, for which we observed subtle coordinated down-regulation of target mRNAs upon MAP1 overexpression, indicating that Map1p preferentially negatively affects gene expression of target messages. This study therefore expands our understanding of post-transcriptional gene networks suggesting regulatory functions to a variety of proteins not connected to gene expression regulation so far.Protein microarrays have been applied to detect protein-protein, protein-lipid, protein-DNA and protein-viral RNA interactions +) could differentially regulate the activity of responding RBPs, either through impacting RNA-binding or modulating interaction with other RNP components. Therefore, careful evaluation of the redox state and of the substrate availability will be of further need to decipher the molecular roles of enzymatic RBPs. In addition, modulation of RNA-binding function may result from direct competition between RNA and substrates/cofactors as seen with mammalian IRPs, TS and DHFR Our observations that many proteins with enzymatic activities bind to RNAs and potentially participate in RNA regulation are reminiscent to previous observations stating RNA-binding functions for several mammalian enzymes . Moreover, our analysis for the enrichment of Pfam domains among our selection of RNA-binders revealed several unexpected domains to be associated with proteins identified in our screen, namely protein-protein interaction domains such as the tetratricopeptide repeat superfamily, which includes the PC_rep and TPR1_domains in vivoNevertheless, the RNA-binding site may also be distinct from the enzymatic site. Diverse examples for shuffling of enzymatic domains next to RNA-binding domains are known such as adenosine deaminases acting on RNA or RNA helicases Several mammalian metabolic enzymes are thought to control the translation or stability of their own mRNAs In conclusion, various instances of enzymes that also act in RNA-metabolism have been previously reported. Our findings put these specific examples into a more general context indicating that RNA regulation by enzymes may be far more common than previously anticipated. A good fraction of (metabolic) enzymes may therefore have a “moonlighting” role in regulating RNA metabolism, which could allow establishing various direct connections between metabolic status and post-transcriptional gene regulation ahis3Δ1 leu2Δ0 met15Δ0 ura3Δ0), as well as plasmid pBG1805-Map1 TAP-tagged strains 20 pmol of forward and reverse complementary oligonucleotides encoding the E2Bmin sequence RNA was fluorescently labeled with either Cy3 or Cy5 using the MICROMAX ASAP RNA labeling Kit (Perkin Elmer Cat# MPS544) according to the manufacturer's protocol. Labeled RNA was purified with the RNeasy Micro kit (Qiagen) to remove unincorporated dyes and immediately used for array analysis.http://www.invitrogen.com). The frozen arrays were thawed at 4°C for 15 min and blocked for 2 hours at 4°C in phosphate-buffered-saline pH 7.4  supplemented with 1% nuclease/protease-free BSA (Equitech-Bio), 1 mM DTT, 50 µg/ml E. coli tRNA (Roche), and 50 µg/ml heparin. The arrays were dried by centrifugation at 300 g for 1 min at 4°C and immediately probed with fluorescently labeled RNAs. Therefore, Cy3 labeled total RNA (5–10 µg) were combined with either Cy5 labeled mRNAs ( = poly(A)+ RNA; 2 µg) or E2Bmin RNA (1.5 µg) and mixed in 60 µl of RNA-binding buffer  supplemented with 6 U of RNaseOUT  and applied on the protein microarray, which was covered with a lifterslip . The arrays were put into a sealed hybridization chamber to prevent drying-out, and incubated for 90 min at room temperature in the dark. The slides were washed twice for 10 min at 4°C with 25 ml of RBB buffer supplemented with 10 U/ml RNaseOUT, and twice with 1× RBB buffer lacking tRNA. The arrays were dried by centrifugation at 300 g for 5 min and immediately scanned with an Axon Scanner 4200 (Molecular Devices). Data was collected with GenePix Pro 5.1 (Molecular Devices) and imported into Acuity 4.0, which averages data for duplicated spots (Molecular Devices). For data analysis, we removed features representing non-yeast control proteins (e.g. GST) and spots with irregular shapes (FLAG> = 0). Protein microarray raw data have been deposited at ArrayExpress via http://www.ebi.ac.uk/miamexpress/ .We used commercially available protein microarrays containing duplicate probes of 4,088 yeast proteins and additional control proteins spotted on a modified glass slide  probed with E2Bmin RNA. Proteins, for which the signal intensities were at least 3.5 standard deviations (Z score>3.5) above the median of all averaged signals from replicate arrays, were considered as RNA binders  were grown in SR-Ura media at 30°C to an ODMAP1 overexpression were obtained by comparative microarray analysis of Cy3 labeled cDNAs derived from cells expressing the empty vector (pBG1805) and of Cy5 labeled cDNAs from MAP1 (pBG1805-Map1) expressing cells. Microarrays were scanned with an Axon Scanner 4200A (Molecular Devices) and analyzed with GenePix Pro 5.1 (Molecular Devices). Arrays were deposited and computer normalized at the Stanford Microarray Database www.ncbi.nlm.nih.gov/geo (accession nos. GSE21850 and GSE21864).70-mer oligo arrays representing features for all annotated nuclear yeast genes , the mitochondrial genome and various control spots were produced at the Center for Integrative Genomics, University of Lausanne. Arrays were processed and hybridized with fluorescently labeled cDNAs as described previously 2 median ratios from three independent RBP affinity isolations and five mock control isolations were retrieved from SMD and exported into Microsoft Excel after filtering for signal over background >1.8 in the channel measuring total RNA derived from the extract. We used the web interface for Cyber-T (http://cybert.microarray.ics.uci.edu/) to employ statistical analyses based on regularized t-tests that use a Bayesian estimate of the variance among gene measurements within an experiment p-value of less than 0.01 in protein affinity isolates compared to mock controls were considered as potential RNA targets . DNA microarray raw data are available at the Stanford Microarray Database (SMD) or at the Gene Expression Omnibus at www.ncbi.nlm.nih.gov/geo (accession nos. GSE21850 and GSE21864). Microarray data is compliant with MIAME protocol.Protein microarray raw data are available at the ArrayExpress database at http://go.princeton.edu/cgi-bin/GOTermFinder, Saccharomyces cerevisiae Genome Database (SGD). Commonly enriched GO terms among mRNAs associated with selected proteins were retrieved with the GO Term Finder that uses a hypergeometric distribution with Multiple Hypothesis Correction to calculate p-values . Thereby, we used 6,336 features representing ORF probes for which microarray data was obtained as the background gene set, and only terms with p<0.01 (Bonferroni corrected) were considered. Domain annotations for all S. cerevisiae proteins were retrieved from the Pfam database (Pfam 24.0) at http://pfam.sanger.ac.uk/Significantly shared GO terms among the selected proteins from the Protoarray screen were identified with the Generic Gene Ontology (GO) Term Finder at the Lewis-Sigler Institute at Princeton University  in the form of Robust Multi Array (RMA) normalized profiles were retrieved from the M3D database Figure S1Heatmap of expression profiles for 164 potential RBPs across 247 conditions. RBPs are clustered with k-means (10 groups) by employing the Euclidean distance as the distance metric to group similarly expressed genes across conditions : YORF, gene name, GO annotations, protein interacted with respective RNA on protein microarrays, classified as metabolic enzyme, number of proteins per cell Click here for additional data file.Table S2Significantly enriched GO terms among proteins interacting with total RNA or mRNA. (XLS)Click here for additional data file.Table S3List of conditions for microarray data retrieved from the M3D database. (XLS)Click here for additional data file.Table S4A selection of significantly enriched GO terms among messages associated with proteins. Terms that belong to the GO category ‘Process’ are written in black; GO terms for ‘Function’ are in red, and the ones for ‘Compartment’ are in blue. (XLS)Click here for additional data file.Dataset S1Raw data for protein arrays probed with E2Bmin RNA. Columns indicate the following (from left to right): YORF, gene name, Z-scores of two protein arrays probed with E2Bmin, mean of Z-scores, Z-scores of heat-treated protein microarray, GO process, function, S. cerevisiae Genome Database Identifier. (XLS)Click here for additional data file.Dataset S2Raw data of protein arrays probed with total RNA and mRNA. Background substracted median fluorescent signals are shown for both channel, and the percentile ranks and selected proteins preferentially interacting either with mRNA or total RNA are also shown. A key describes different worksheets. (XLS)Click here for additional data file.Dataset S3RIP-Chip data for 13 potential RBPs. Features/ORFs that were at least 3-fold enriched compared to mock isolates with p<0.01 are indicated in a separate worksheets. Therein, fold changes (log2 ratios) are in black, p-values are shown in red. (XLS)Click here for additional data file.Dataset S4Microarray raw data of MAP1 overexpressing compared to control cells. Columns indicate the following (from left to right): Spot ID (SMD); YORF; Gene name; log2 ratio for triplicate experiments (MAP1/control); average log2 ratio; average fold-change; standard deviation; p-value; Map1 target . (XLS)Click here for additional data file.


Overnight incubation of human eosinophils (Eøs) with the glucocorticoid hormone dexamethasone  resulted in lower expression of the CD11b, but not CD49d, antigen on their plasma membrane, as assessed by flow cytometry. DEX produced a consistent inhibitory effect (ranging from 16% to 20%) when tested at a concentration of 0.1 μM. Eø stimulation with 100 ng/ml eotaxin produced an increase in CD11b (+26%), but not CD11c, levels and concomitantly a reduction (–25%) on CD62L expression. The inhibition exerted by DEX upon CD11b levels was also evident following eotaxin upregulation, with a degree of inhibition similar to that seen on basal levels. These data highlight a novel mechanism of action by which glucocorticoid hormones may be effective in reducing Eø accumulation during allergic inflammation in man.