The reliability of gene expression profiling-based technologies to detect transcriptional differences representative of the original samples is affected by the quality of the extracted RNA. It strictly depends upon the technique that has been employed. Hence, the present study aimed at systematically comparing silica-gel column (SGC) and guanidine isothiocyanate (GTC) techniques of RNA isolation to answer the question which technique is preferable when frozen, long-term stored or fresh lung tissues have to be evaluated for the downstream molecular analysis.Frozen lungs (n = 3) were prepared by long-term storage (2.5 yrs) in -80°C while fresh lungs (n = 3) were harvested and processed immediately. The purity and quantification of RNA was determined with a spectrophotometer whereas the total amounted copy numbers of target sequences were determined with iCycler detection system for assessment of RNA intactness (28S and 18S) and fragment sizes, i.e. short (GAPDH-3' UTR), medium (GAPDH), and long (PBGD) with 200 bp, 700 bp, and 1400 bp distance to the 3'ends of mRNA motif, respectively.Total yield of RNA was higher with GTC than SGC technique in frozen as well as fresh tissues while the purity of RNA remained comparable. The quantitative reverse transcriptase-polymerase chain reaction data revealed that higher mean copy numbers of 28S and a longer fragment (1400 bp) were obtained from RNA isolated with SGC than GTC technique using fresh as well as frozen tissues. Additionally, a high mean copy number of 18S and medium fragment (700 bp) were obtained in RNA isolated with SGC technique from fresh tissues, only. For the shorter fragment, no significant differences between both techniques were noticed.Our data demonstrated that although the GTC technique has yielded a higher amount of RNA, the SGC technique was much more superior with respect to the reliable generation of an intact RNA and effectively amplified longer products in fresh as well as in frozen tissues. DNA-microarray and quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) are two powerful techniques widely used in functional genomics for the analysis of gene expression profiles. These techniques are highly dependent on the quality of total RNA -3 obtain2OH) and subsequently methylene bridges are formed between neighbouring bases that resulted to continue degradation over-time [RNA is a particularly labile bio-molecule and is much more susceptible to degradation by endogenous- and exogenous-nucleases and to non-specific degradation by divalent cations, heat, elevations in pH, and storage of tissue or cells over extended periods prior to RNA extractions, which result in falsely altered gene expression patterns . Particuver-time . Alterna260 nm/A280 nm by spectrophotometer [Obtaining high quality RNA is mandatory and depends upon the technique that has been employed . Generalotometer . Purity otometer , and hen2 ultracentrifugation). Moreover, GTC salt denatures the cellular proteins and inactivates RNases ensuring that isolated RNA is not degraded. Many commercial reagents  are based on this principle. In contrast, the principle of SGC technology (Qiagen RNeasy Mini column) is a combination of the selective binding properties of a silica-based membrane with the speed of microspin technology, which allows saving time, money, and efficient use of small and precious biological samples. The comparison between GTC and SGC techniques are summarized in Table Today, several other new techniques and kits are being offered for RNA isolation -17, whicIn the present study, we have tested a strategy to store lung tissues for the period of more than 2.5 yrs without any chemical treatment or processing, and investigated which RNA extraction technique (i.e. SGC and GTC) is preferable in terms of RNA recovery, purity, intactness, and amplification of various fragment sizes. A similar investigation was performed with fresh lung tissues.Lung tissues were harvested from 4 months old male C57BL mice. Lung samples were either immediately dipped into liquid nitrogen, cut down into small fine pieces on sterile plate on ice using sterile blade and frozen at -80°C for 2.5 years until processing and analysis (n = 3 mice). Fresh lung tissue was processed and analysed immediately (n = 3 mice).260) whereas purity was determined as per discussed above. The importance of RNA integrity and various fragment sizes were determined with qRT-PCR. For the assessment of RNA integrity, we used 28S and 18S subunits of ribosomal RNA, an internal control for RNA intactness. On the other hand, for the assessment of various fragment sizes of RNA, an amplification factor of Glyceraldehydes 3-phosphate dehydrogenase-3' un-translated region , Glyceraldehyde 3-phosphate dehydrogenase  and Porphobilinogen deaminase , the respective distance of which to the 3'ends of messenger RNA (mRNA) were 200 bp, 700 bp, and 1400 bp } was added, and with the help of a disposable syringe and a 20 G sterilized needle, tissue pieces were disrupted. The lysed-disrupted samples were kept at room temperature (RT) for 10 min, centrifuged for 15 min at 12,000 rpm to get rid off cellular debris, and supernatant was collected. In order to investigate the reproducibility of each method, collected supernatant was then divided into three equal volumes, i.e. 600 μl.Collected supernatant was directly applied on the RNeasy column  and total cellular RNA was isolated as per the steps given for the Qiagen RNA Miniprep kit. In contrast, using GTC technique collected supernatant was mixed with 200 μl of chloroform  and 80 μl of 2 M NaoAc. The samples were vortexed and centrifuged at 4°C for 15 min at 12,000 rpm. The upper phase was transferred carefully into fresh 1.5 ml eppendorf tubes. For RNA precipitation, 500 μl of cold isopropanol  with 70 μl of 2 M NaoAc was added to the supernatants and incubated for 2 hrs at -20°C. Samples were centrifuged at 4°C for 15 min at 12,000 rpm. The obtained pellets were washed with 500 μl of 70% chilled ethanol and centrifuged again at 4°C for 15 min at 12,000 rpm. The washed pellets were then air dried and finally dissolved in 40 μl of RNase free water.The total RNA quantification and purity was determined with an Ultraspec 2100-spectrophotometer .For total RNA quality determination, 1.2% of agarose gel was prepared by dissolving 1.2 g of agarose powder  in 100 ml of 1× Tris-Borate-EDTA buffer . The mix was cooked until get boiled, and then 5 μl of 1% ethidium bromide  was added, mixed, and poured in to gel electrophoresis unit. RNA samples (1.0 μg each) were mixed with 2 μl of gel loading solution  and were directly applied on a gel's wells. The integrity of total RNA was assessed on the basis of visualisation of 28S and 18S ribosomal RNA subunits under gel documentation system 2000  as a band at positions 4.8 kb and 1.8 kb, respectively.12–18 primer  and Superscript™ II Reverse Transcriptase kit . The reaction was incubated at 65°C for 5 min, at 37°C for 60 min, and at 72°C for 15 min in RoboCycler® Gradient 40 . The cDNA qualities for all the samples were evaluated by performing GAPDH qualitative PCR (data not shown). The qRT-PCR for determining the amplification factor of target genes i.e. 28S, 18S, GAPDH-3' UTR, GAPDH, and PBGD were performed in a 96-well format iCycler Detection System . The reactions (20 μl) were set up with the SYBR™ Green PCR mix  according to the manufacturer's protocol. The thermal cycle conditions used for all reactions were as follows: Step 1: 95°C 15 min; followed by 40 cycles of Step 2: 95°C 50 sec; sequence-specific oligonucleotide primer's annealing temperature -mean CT). Unless stated otherwise, mean values ± s.d. are given. Since the aim of this study was to determine the most appropriate technique to isolate RNA, out of two tested techniques (i.e. SGC and GTC) in frozen- and fresh- lung samples, respectively, t-test (and non-parametric tests) was performed to determine the level of significance of differences between SGC and GTC. If normality and equal variance were not given at p > 0.1, Mann-Whitney rank sum test was used using GraphPad Prism 4 software program . Values of p < 0.05 were considered to be significant.The total amount of target gene copy numbers were determined by applying the formula 2 raised to the power mean of cycle threshold } in comparison with RNA isolated by the SGC technique, which was true for both in frozen  and in fresh  lung tissues Figure .260/A280) of 1.8 with both, the GTC and the SGC technique, in frozen and fresh lung tissues, respectively . qRT-PCR revealed that the mean copy numbers of 28S sequence was significantly higher in RNA isolated with the SGC compared to the GTC technique Figure . This diLastly, to assess, which RNA extraction technique would reliably conserve a longer fragment from poly-A tail of mRNA, qRT-PCR was performed to determine the short , medium , and long  fragment sizes of RNA, respectively. The qRT-PCR for the GAPDH-3'UTR revealed that both techniques have the potential to amplify fragments of a size <200 bp in comparable manner Figure . RNA isoThe demand for high quality of RNA has been dramatically increased with the extensive application of DNA-microarray technology and real-time PCR-based expression profiling. A number of RNA extraction techniques are available to generate high quality RNA for use in gene expression profiling experiments . SeveralThe first step of evaluation after RNA extraction is the measurement of total recovery followed by the determination of purity and intactness of an isolated RNA. Our results demonstrate that irrespective of using RNA from frozen or fresh samples, the total recovery of RNA was higher with GTC- than SGC-based techniques Figure . SimilarNext, the most important parameter in the evaluation procedure is to assess the intactness of RNA, which can be determined by analyzing the 18S and 28S subunits of ribosomal RNA on either the ethidium bromide-stained agarose gel ,24 or miIn general, approximately 80 percent of the total RNA is ribosomal RNA and 15 percent is transfer RNA with protein-encoding messenger RNA constituting only a small portion (i.e. 5 percent) of the total RNA. To obtain an overall impression of RNA intactness, it is equally important to evaluate the quality of mRNA as well. By selecting three different sequences, which were classified according to their distance to the poly-A tail of mRNA, we asked to evaluate, which technique was able to conserve a longer fragment of mRNA from poly-A tail (3' to 5'). Our results demonstrated that any-one technique can be selected to isolate RNA either from frozen or fresh lung tissues for those sequences that are close to the poly-A tail of mRNA (<200 bp) and fragments lesser than 700 bp for downstream analysis Figure  &6B.Notably, the choice of the technique for RNA isolation had a significant impact on the potential to obtain appropriate copy numbers of bigger fragments as was demonstrated for the 1400 bp fragment of PBGD. In this respect, the SGC technique proved to be superior to the GTC technique both in RNA preparations from frozen and fresh lung tissues. The generation of high copy numbers of PBGD in RNA isolated from fresh as well as from frozen tissue suggests that the SGC-based technique is not only compatible with fresh but also with frozen tissues. We suggest that the reason for the superiority of the SGC technique again relates to the selective adsorption to silica-gel membranes and the avoidance of toxic chemicals and long-lasting protocol steps as discussed above.Comparing the SGC and GTC technique, we conclude that both techniques performed well in terms of total recovery and purity of RNA, isolated from 2.5 yrs old frozen and fresh lung tissues. With respect to RNA intactness, however, the SGC-based technique turned out to be superior to the GTC-based technique because of the conservation of an intact form of ribosomal RNA and conservation not only of short- to medium-sized but also of longer-sized RNA fragments. We may further conclude from our data that long-term storage of frozen lung tissues has only minor effects when compared to fresh lung tissues.The authors declare that they have no competing interests.JPM participated in the design of the study and performed qRT-PCR experiment, statistical analysis and drafted manuscript. VM carried out RNA isolation from frozen lung tissues, cDNA synthesis and qualitative PCR from frozen and fresh lung tissues. BPK carried out RNA isolation from fresh lung tissues. CS helped to analysis qRT-PCR data. HF conceived and supervised this work and helped to draft the manuscript.


BMC Biology reveals surprising tolerance to helix-disrupting changes in a region previously thought crucial for translocation, and suggests roles for two hinge-like segments of the bridge helix in coordinating modules that move during the nucleotide-addition cycle.The RNA polymerase 'bridge helix' is a metastable α-helix that spans the leading edge of the enzyme active-site cleft. A new study published in http://www.biomedcentral.com/1741-7007/8/134See Research article:  DNA-dependent, multisubunit RNA polymerases are conserved in core structure and are required for gene expression and regulation in all cellular organisms. To accomplish these roles, RNA polymerase has evolved into a complex molecular machine in which precisely orchestrated movements in a network of flexible modules mediate steps in a nucleotide-addition cycle of four basic steps: translocation of RNA and DNA chains through the polymerase; binding of the nucleotide triphosphate (NTP) substrate; catalysis; and pyrophosphate release. A key component of this network is the bridge helix, which occupies a critical position spanning the main channel of RNA polymerase just downstream of the active site Figure . After tMethanocaldococcus jannaschii) that can be reconstituted in vitro from individual subunits. The most recent results from this systematic dissection of the bridge helix, published in BMC Biology by Weinzierl [Testing the contributions to RNA polymerase function of these two proposed actions of the bridge helix  - or revealing other bridge-helix roles - is made difficult by the small movements involved relative to the size of the polymerase and by the inability of crystal structures to report molecular dynamics. Over the past few years, Weinzierl and colleagues have developed and exploited a novel approach that augments conventional structure-function studies by assaying RNA polymerase with systematically altered bridge-helix structures . This ameinzierl , suggestBacillus subtilis) or the newly described plant RNA polymerases IV and V . Thus, kinking focused at these two points of the bridge helix , on the basis of the effects of the proline substitutions that increase polymerase activity . Such a view is consistent with impairment of catalysis by substitutions that disrupt fork loop-HN interaction [N and HC to significant alterations such as the two-amino-acid deletions [In this view, bridge-helix conformation influences formation of the trigger helices  in response to DNA and RNA sequence or transcription regulators that interact with the RNA polymerase clamp, cap, or anchor and affect bridge-helix conformation through Heraction  and witheletions  and helieletions , as the tour de force mutagenesis of the bridge helix [in vitro assays specific for individual steps in the cycle is available. The nonspecific RNA-synthesis assay used in the robotic approach does not identify which step in the cycle is stimulated by HN and HC proline substitutions or inhibited by other alterations; even template engagement could be affected, as faster recycling of RNA polymerase could also increase RNA yield. Thus, much important biochemistry remains before we will fully understand RNA polymerases. Among the most important objectives should be to devise an assay that unambiguously and directly reports effects on translocation. A second key goal should be the determination of additional crystal structures of DNA-bound RNA polymerases that capture more extensive conformational changes, such as clamp opening, that might reveal the predicted changes in bridge-helix conformation.The results of Weinzierl's ge helix  yield sege helix  using bi


Sinorhizobium meliloti is a model soil bacterium known for its ability to establish a beneficial nitrogen-fixing intracellular symbiosis with alfalfa. Despite the predicted general involvement of Hfq in the establishment of successful bacteria-eukaryote interactions, its function in S. meliloti has remained unexplored.The bacterial Hfq protein is able to interact with diverse RNA molecules, including regulatory small non-coding RNAs (sRNAs), and thus it is recognized as a global post-transcriptional regulator of gene expression. Loss of Hfq has an extensive impact in bacterial physiology which in several animal pathogens influences virulence. S. meliloti mutants, 2011-3.4 and 1021Δhfq, were obtained by disruption and deletion of the hfq gene in the wild-type strains 2011 and 1021, respectively, both exhibiting similar growth defects as free-living bacteria. Transcriptomic profiling of 1021Δhfq revealed a general down-regulation of genes of sugar transporters and some enzymes of the central carbon metabolism, whereas transcripts specifying the uptake and metabolism of nitrogen sources (mainly amino acids) were more abundant than in the wild-type strain. Proteomic analysis of the 2011-3.4 mutant independently confirmed these observations. Symbiotic tests showed that lack of Hfq led to a delayed nodulation, severely compromised bacterial competitiveness on alfalfa roots and impaired normal plant growth. Furthermore, a large proportion of nodules (55%-64%) elicited by the 1021Δhfq mutant were non-fixing, with scarce content in bacteroids and signs of premature senescence of endosymbiotic bacteria. RT-PCR experiments on RNA from bacteria grown under aerobic and microoxic conditions revealed that Hfq contributes to regulation of nifA and fixK1/K2, the genes controlling nitrogen fixation, although the Hfq-mediated regulation of fixK is only aerobiosis dependent. Finally, we found that some of the recently identified S. meliloti sRNAs co-inmunoprecipitate with a FLAG-epitope tagged Hfq protein.Two independent S. meliloti RNA chaperone Hfq contributes to the control of central metabolic pathways in free-living bacteria and influences rhizospheric competence, survival of the microsymbiont within the nodule cells and nitrogen fixation during the symbiotic interaction with its legume host alfalfa. The identified S. meliloti Hfq-binding sRNAs are predicted to participate in the Hfq regulatory network.Our results support that the  Escherichia coli as a host factor essential for Qβ RNA bacteriophage replication  and exposed to Konica Minolta medical films.To verify the expression of the 3 × FLAG tagged Hfq protein, 0.05 OD whole cell protein fractions of the hfqFLAG were cultured in 200 ml of TY broth to OD600 nm 0.6. Total RNA was prepared from 25 ml of each culture as previously described [® resin (Sigma #A2220) were added to the cleared lysates followed by incubation overnight with shaking at 4°C. The suspensions were centrifuged; the beads were resuspended in 1 ml lysis buffer and transferred to spin columns, followed by five washes in 1 ml of the same buffer. Protein/RNA complexes were recovered from beads by incubation with 15 ng of 3 × FLAG Peptide® (Sigma #F4799) followed by elution in 100 μl of water. Phenol:chloroform extracted RNA was concentrated by ethanol precipitation and resuspended in 70 μl of water. Aliquots of 10 μg of total RNA and 10 μl of the co-inmunoprecipitated RNA were subjected to Northern analysis with the Smr sRNAs probes as described [For co-inmunoprecipitation, the 1021 wild-type strain and its derivative 1021escribed . The remescribed .hfq mutant and CoIP experiments; RIO, performed proteomics of the 2011-3.4 mutant and competition tests, and contributed to the design of the study; AP, performed RT-PCR experiments; EJ, contributed to the proteomic profiling; JL, contributed to the analysis of proteomic data and revised the manuscript; RR, contributed to the design of the study, analyzed data and critically revised the manuscript; NT, revised the manuscript; JIJZ, conceived and designed the study, obtained 1021Δhfq and 1021hfqFLAG strains and wrote the paper. All authors read and approved the final manuscript.OT-Q carried out transcriptomics, nodulation tests/microscopy of the 1021ΔS. meliloti 1021 and 1021Δhfq derivative strainDifferentially accumulated transcripts in . List of down- and up-regulated genes grouped by functional categories according to the S. meliloti genome database and KEGG.Click here for fileS. meliloti 2011 wild-type and 2011-3.4 insertion mutant derivativeDifferentially accumulated proteins in . List of down- and up-regulated proteins and their adscription to functional categories according to the S. meliloti genome database and KEGG.Click here for fileOligonucleotide sequences. Sequences of the oligonucleotides used in this study.Click here for file


Most of the cell biological aspects of retroviral genome dimerization remain unknown. Murine leukemia virus (MLV) constitutes a useful model to study when and where dimerization occurs within the cell. For instance, MLV produces a subgenomic RNA  that is co-packaged with the genomic RNA predominantly as FLSD' heterodimers. This SD' RNA is generated by splicing of the genomic RNA and also by direct transcription of a splice-associated retroelement of MLV (SDARE). We took advantage of these two SD' origins to study the effects of transcription and splicing events on RNA dimerization. Using genetic approaches coupled to capture of RNA heterodimer in virions, we determined heterodimerization frequencies in different cellular contexts. Several cell lines were stably established in which SD' RNA was produced by either splicing or transcription from SDARE. Moreover, SDARE was integrated into the host chromosome either concomitantly or sequentially with the genomic provirus. Our results showed that transcribed genomic and SD' RNAs preferentially formed heterodimers when their respective proviruses were integrated together. In contrast, heterodimerization was strongly affected when the two proviruses were integrated independently. Finally, dimerization was enhanced when the transcription sites were expected to be physically close. For the first time, we report that splicing and RNA dimerization appear to be coupled. Indeed, when the RNAs underwent splicing, the FLSD' dimerization reached a frequency similar to co-transcriptional heterodimerization. Altogether, our results indicate that randomness of heterodimerization increases when RNAs are co-expressed during either transcription or splicing. Our results strongly support the notion that dimerization occurs in the nucleus, at or near the transcription and splicing sites, at areas of high viral RNA concentration. Retroviridae, underlying the importance of RNA dimerization for virus replication. Packaging of two genome copies increases the probability of recombination events by template switching upon the reverse transcription, thus promoting genetic diversity [cis-element (Psi) and its interaction with the trans-acting Gag factor [The dimeric nature of the genome is strongly conserved among iversity . Dimeriziversity . RNA striversity -8. Dimerg factor ,9-18. Dig factor  and coulg factor -21, and Presumably, dimerization occurs in the cell prior to RNA packaging as supported by recent microscopy studies at single-RNA-detection sensitivity ,23. Moreenv mRNA, MLV produces an alternatively spliced 4.4-Kb RNA, called SD' RNA  or by direct transcription of SDARE (trSD').In addition to the A Figure . This alIn vitro, the two RNAs harbored similar dimerization abilities and formed Psi-dependent heterodimers (FLSD') [The FL and SD' RNAs harbor the same Psi sequence responsible for their co-packaging.  (FLSD') . Analysi (FLSD') . This pr (FLSD') ,37,38. HIt has been reported that co-packaging of two MLV RNAs was dependent on the distance between their transcription sites ,28. Thesin vitro and in vivo  RNAs were transcribed from pFL* and SDARE molecular clone (pSD'), respectively [trSD' and FL RNAs are equally transcribed in both contexts .  MSD1 in ,28,39-43ectively  . Cells were stably transfected with wild-type replication-competent MLV clone (here named pFL) and pcDNA-hygro plasmid   Figure . Neverthin vitro [Such a link between splicing and dimerization provides possible clues to the packaging process of spliced viral RNAs. Although the genomic RNA is preferentially packaged, the subgenomic RNAs are also specifically packaged into infectious HIV and MLV particles, although to a lower extent ,46-48. Sin vitro ,50. It iin vitro transcription than post-synthesis [in vitro [Altogether our results showed that MLV RNAs preferentially dimerize when they undergo splicing or co-transcription. In contrast, the distance between transcription sites could hinder RNA dimerization. At least two non-exclusive hypotheses could explain these results. Host factors could play a role in dimerization ,51. For ynthesis ,36,49. Ain vitro . Furtherin vitro .Our results suggest that viral RNAs dimerize in the nucleus and presumably traffic out of the nucleus as dimers. Importantly, the MLV packaging signal (Psi) which overlaps the DLS, also contributes to nuclear export of the FL RNA ,53. TherSM and MM conceived the study and analyzed the data. SM performed the laboratory work. MM wrote the manuscript. The authors read and approved the final manuscript.


Treatment with transcription inhibitors redistributes GANP from NPCs into foci throughout the nucleus. These results establish GANP as an integral component of the mammalian mRNA export machinery and suggest a model whereby GANP facilitates the transfer of NXF1-containing mRNPs to NPCs.Bulk nuclear export of messenger ribonucleoproteins (mRNPs) through nuclear pore complexes (NPCs) is mediated by NXF1. It binds mRNPs through adaptor proteins such as ALY   However, GANP residues 1–1259 have no counterpart in MCM3AP, and MCM3AP can be transcribed independently of GANP. We propose elsewhere that they should be referred to as independent but overlapping genes .Although the yeast mRNA export machinery is well characterized and is partly conserved in mammalian cells, there are important functional differences. First, the TREX (Drosophila Xmas-2 (43% identity) GANP contains regions of homology to two classes of protein involved in nuclear trafficking A. ResiduImmunoblotting with sheep antibodies raised against a unique region of GANP (residues 1050–1250) that is absent from MCM3AP recognized a 210 kDa band, which was abolished following small interfering RNA (siRNA) depletion D. Confoc+RNA export via RNA fluorescence in situ hybridization (FISH). Nuclear accumulation of poly(A)+RNA was observed with two independent siRNAs directed against the unique region of GANP, but not with control siRNA that differed by two bases from that used to deplete GANP ete GANP A and 2D.n nuclei A, as obsei +RNA did not accumulate at the nuclear envelope of GANP-depleted cells but instead accumulated in a distinct punctate focal pattern throughout the nucleus, excluding nucleoli  cellulose. Endogenous GANP specifically copurified with the poly(A) fraction, as did NXF1, a known component of nuclear mRNPs (To test whether GANP is associated with nuclear messenger ribonucleoproteins (mRNPs), we harvested nuclear poly(A)ar mRNPs A. Converar mRNPs A. These +RNA accumulation in a pattern identical to that of CFP-GANP(1-313) (NXF1 is a major metazoan mRNA export factor P(1-313) D. This eP(1-313) B. These P(1-313) .+RNA from the nucleus suggest a role for GANP in mammalian mRNA nuclear export whereby GANP recruits NXF1-containing mRNPs in the nuclear interior and delivers them to the NPC. One prediction of this hypothesis is that GANP depletion should reduce the association of NXF1 with NPCs. The observations that GANP interacts directly with NXF1, that it is present in both the nuclear envelope and the nuclear interior, and that it is required for efficient export of poly(A)The proportion of GANP located at NPCs was higher than that seen for NXF1 A, consisNuclear mRNP export is a dynamic process, and most actively transcribed genes localize at focal concentrations of RNA polymerase II (RNA Pol II) deep in the nucleus called transcription factories GAL1 to NPCs Our results are consistent with a model for GANP function in mammalian mRNA nuclear export whereby GANP is recruited to mature mRNPs after NXF1 is attached, enhancing the delivery of mRNPs to the NPC C. In conOur data establish a role for GANP distinct from those proposed previously


Information technology (IT) may improve the quality, safety and efficiency of medicine, and is especially useful in intensive Care Units (ICUs) as these are extremely data-rich environments with round-the-clock changing parameters. However, data regarding the implementation rates of IT in ICUs are scarce, and restricted to non-European countries. The current paper aims to provide relevant information regarding implementation of IT in Flemish ICU's .The current study is based on two separate but complementary surveys conducted in the region of Flanders (Belgium): a written questionnaire in 2005 followed by a telephone survey in October 2008. We have evaluated the actual health IT adoption rate, as well as its evolution over a 3-year time frame. In addition, we documented the main benefits and obstacles for taking the decision to implement an Intensive Care Information System (ICIS).Currently, the computerized display of laboratory and radiology results is almost omnipresent in Flemish ICUs, , but the computerized physician order entry (CPOE) of these examinations is rarely used. Sixty-five % of Flemish ICUs use an electronic patient record, 41.3% use CPOE for medication prescriptions, and 27% use computerized medication administration recording. The implementation rate of a dedicated ICIS has doubled over the last 3 years from 9.3% to 19%, and another 31.7% have plans to implement an ICIS within the next 3 years. Half of the tertiary non-academic hospitals and all university hospitals have implemented an ICIS, general hospitals are lagging behind with 8% implementation, however. The main reasons for postponing ICIS implementation are: (i) the substantial initial investment costs, (ii) integration problems with the hospital information system, (iii) concerns about user-friendly interfaces, (iv) the need for dedicated personnel and (v) the questionable cost-benefit ratio.Most ICUs in Flanders use hospital IT systems such as computerized laboratory and radiology displays. The adoption rate of ICISs has doubled over the last 3 years but is still surprisingly low, especially in general hospitals. The major reason for not implementing an ICIS is the substantial financial cost, together with the lack of arguments to ensure the cost/benefit. Over the past decades there have been substantial changes in medicine, with more effective but also increasingly complex therapies. This results in an increased life expectancy on the one hand, but also in an increased number of medical errors on the other hand. In 2003, the Institute of Medicine published the groundbreaking report "To err is human, building a safer health system". This report estimated that at least 44,000 people die in US hospitals each year as a result of medical errors that could have been prevented . FurtherThe intensive care unit (ICU) has several typical characteristics which make it favorable for computerization, because caring for the critically ill is even more complex, resulting in substantially higher numbers of medical errors and costs ,4. DonchFor the above reasons it is advisable to study the current level of intensive care computerization, both for general and dedicated specialized IT applications used in the ICU. General IT applications are the electronic patient record, the computer laboratory system ), the computer radiology system (i.e. Picture Archiving and Communication System (PACS)), and the Computerized Physician Order Entry (CPOE) applications. The term CPOE can be confusing however, because some authors restrict its use to prescribing medication, and add the term "order communication system" for laboratory and radiology requests. In this paper, we will use CPOE in the broader sense, and we will specify reference to medication CPOE, laboratory CPOE or radiology CPOE. The dedicated IT solution for the ICU is often described as an ICU Patient Data Management System (PDMS), but we prefer the term "Intensive Care Information System" (ICIS), which describes the broader functionalities of more advanced IT programs better, i.e. doing more than mere data storage and representation. These systems are developed in order to meet the specific requirements to optimize data processing and workflow support in critical care medicine. In our survey, an ICIS has to fulfil all of the following conditions: (i) automated collection of physiological and monitoring variables from monitors and ventilators, (ii) incorporation of CPOE for medication prescription and (iii) one bedside personal computer for every ICU bed.In this paper, the present IT adoption rate in ICUs in Flanders (Belgium) is evaluated, as well as its evolution within a 3-year time frame. This includes both the use of general hospital information system components such as the electronic patient record, CPOE for medication, radiology and laboratory requests and the computerized display of these results, as well as ICU-specific IT software such as ICIS. Furthermore, we have explored the main benefits and obstacles for taking the decision to implement an ICIS as perceived by ICU directors.This study is based on two separate but complementary surveys conducted in the region of Flanders.The first survey was performed in January 2005 and consists of a written questionnaire which was sent to the medical directors of all Flemish ICUs. Six weeks later the non-respondents were sent a reminder. After another month the remaining non-respondent ICU directors were contacted by phone, and a second reminder was sent when necessary.The second survey was carried out in October 2008 (i.e. 3.5 years after the first survey). This telephone survey was carried out by K.C., who interviewed each ICU director, or the ICU head nurse if the medical director suggested that the head nurse was the more competent person regarding IT use in the ICU.The Local Ethical Committee of Ghent University Hospital approved the study, and informed consent was waived. All answers were kept confidential.http://www.health.fgov.be). They are located in three different types of hospitals: general hospitals (52/63 or 82.6%), tertiary non-academic referral hospitals (8/63 or 12.7%), and university hospitals (3/63 or 4.7%). General hospitals have approximately 250 to 700 beds, tertiary non-academic referral hospitals 500 to 1,100 beds and university hospitals 700 to 1,600 beds. The number of ICU beds consists of 5 to 7% of the total number of hospital beds . Especially the larger hospitals have 24/7 junior or senior critical care physicians available, whereas smaller ICUs usually have anesthesiologist-intensivists  who combine their anesthesia care with intensive care.Flanders has 6,117,440 inhabitants and represents the largest region of the federal state of Belgium. The federal state contains 10,580,000 inhabitants. The Region of Flanders had a total of 54 ICUs in 2005, and a total of 63 ICUs in 2008. Differences between these two numbers are mainly due to changing alliances between hospitals, or new approved ICUs  which have been approved by the government. All these ICUs provide mechanical ventilation and are approved by the national government  were general hospitals, 3 (9.7%) were tertiary non-academic referral hospitals and 2 (6.4%) were university hospitals. The number of ICU beds per ICU varied between 6 and 56.For the 2008 telephone survey, a 100% response rate was obtained (63/63). It must be stressed that both the 2008 telephone survey and the 2005 questionnaire probed into the use of hospital information system components and the availability of ICIS. For this reason, the results concerning the IT adoption rate, and its evolution over time, are highly accurate. The results regarding the benefits of and obstacles for implementing an ICIS have mainly been derived from the 2005 written questionnaire.• Electronic patient recordIn 2008, 41 out of 63 ICUs (63.1%) use the hospital electronic patient record within the ICU. This is a limited increase compared to 2005 (16/31 ICUs or 51.6%). Of these 31 hospitals, an additional 7 ICUs shifted to an electronic patient record during the 3-year time frame.• Medication CPOE and the computerized recording of medication administrationA larger proportion of ICUs report using medication CPOE in 2008 compared to 2005: 41.3% vs. 19.3%. Currently, 11 of these CPOE programs are part of an ICIS, 7 have been bought commercially for use in the entire hospital and another 8 CPOE programs are hospital-specific and have been developed in-house. Many of the latter less sophisticated programs needed extensive adaptations, however, especially for continuous infusion pump recording. Another 5 ICUs are using Microsoft Office documenting (i.e. Excel), but as this software has not been developed specifically for medication CPOE we did not include it as such. One ICU that implemented an ICIS more than a decade ago still uses paper-based medication prescriptions, and chose to discard the available CPOE functionality due to integration problems with the pharmacy department. It is important to note that only 6 of the CPOE programs that are not part of a dedicated ICIS also provide facilities for computerized medication administration recording by the nursing staff. In the other ICUs, print-outs of the medication CPOE are taken and used as part of the paper charts.• The computer laboratory system.At present, every Flemish ICU uses the computerized display of laboratory results, whereas this was only 70.9% in 2005 ).A larger proportion of ICUs report using the computerized display of radiology results and the CPOE module for radiology requests in 2008 compared to 2005   had implemented an ICIS and another 20 (31.7%) ICUs were planning to implement a system within the next 3 years (i.e. before 2012), of which 5 were scheduled to go live in 2009.In 2005, 5 out of 31 (16.1%) of responding hospitals had an ICIS in place. Another 7 hospitals (22.5%) had the intention of implementing an ICIS within the next 3 years (i.e. before 2009), while the remaining 19 hospitals (61.3%) had no explicit intention to implement an ICIS. None of the seven ICUs from the 2005 survey which intended to adopt an ICIS within the following 3 years, actually did so. Finding adequate financing, together with anticipated integration problems with the hospital information system, were the main reasons for postponing the implementation. However, 3 hospitals which showed no interest in adopting an ICIS in 2005, did implement an ICIS before the end of 2008  Figure . ICUs wiAll ICISs are commercial systems, and the market is shared between a variety of software vendors  coupling, has been carried out in 83% of ICISs, and most systems are integrated with the electronic patient record and laboratory system as well. Direct integration with the radiology system and the pharmacy department on the one hand, and ICIS on the other hand is technically more demanding, resulting in an integration rate of only 25%.The regular use of data extraction tools for management or scientific purposes is still limited (4 out of 12), and is only accomplished in university or tertiary non-academic referral hospitals.Data are obtained from the written 2005 questionnaire. The main anticipated benefits of an ICIS according to ICU directors are listed in order of importance in Table Almost 40% of ICU directors are convinced that investing in an ICIS should be a top priority for their ICU. However, around 80% of ICU directors doubt whether there is enough evidence to ensure the cost/benefit of an ICIS.The major reason for not implementing an ICIS is the substantial financial cost. One hospital even invested in a new paper medical record recently. The extra cost per ICU bed for installing an ICIS ranged between 20,000 and 25,000 Euros, which is a substantial investment. Twenty-nine out of 31 respondents hold the opinion that the government should finance at least 40% of total costs, and 1 out of 3 hospitals even feel that governmental financial assistance should cover over 70% of the implementation cost. Some ICU directors in our study even suggest full reimbursement by the government.To our knowledge, this is the first study evaluating the actual use of IT applications in the critical care environment in a European country, together with its evolution over time. We have evaluated both the adoption rate of hospital-wide IT-applications, which are part of the hospital information system, as well as the use of dedicated ICU IT applications (i.e. ICIS).In our study, we found that laboratory results and radiology images are digitally available and are used in respectively 100% and 93.5% of the Flemish ICUs. But the use of IT for computerized requests of laboratory analyses and radiology investigations is still rare. Although few surveys have been conducted in this respect, we have noted substantial national variability regarding this issue -18. In lIn Flanders, the ICIS adoption rate is currently 19% and this low rate correlates well with the rate mentioned in the Canadian report by Lapinsky et al.. HoweverIn line with the survey by Jha et al. the Flemish larger and teaching hospitals are the leading ICUs investing in an ICIS (see Figure In contrast to the USA or the UK, there are no Flemish governmental financial incentives for the computerization of ICUs ,28. Yet In our survey, it became clear that ICISs are not always used to their full capacity. In fact, two Flemish centres which implemented an ICIS experienced major difficulties in linking their systems to the hospital information system. These problems created failure for automated charting in one hospital, and failure of the pharmacy linking in the other. Furthermore, at least 5 other hospitals admit that the implementation schedule of an ICIS has been significantly delayed due to integration problems with the hospital information system and/or pharmacy department.There are several limitations to our study. First, the response rate of the 2005 survey was only 57.5%. Nevertheless, this is in accordance with other response rates of similar written questionnaires ,17. We sSecond, we did not use a validated questionnaire. This implies limited applicability for parallel follow-up study. Finally, the responses indicate self-reported IT implementation rather than direct observation.Nearly all ICUs in Flanders use hospital-wide available IT applications such as computerized laboratory and radiology displays, although the computerized request for laboratory and radiology is still an exception. Furthermore, the adoption level of medication CPOE and ICIS remains relatively low. The implementation rate of ICISs has nearly doubled over the last three years, and has the potential to increase to 50% by the end of 2011. Major obstacles to implement specialized IT solutions are the high initial costs and maintenance costs, the complexity of integrating ICISs with existing hospital-information systems, and the unclear return on investment.IT: Information Technology; ICU: Intensive Care Unit; PACS: Picture Archiving and Communication System; CPOE: Computerized Physician Order Entry; ICIS: Intensive Care Information System.The authors declare that they have no competing interests.KC has made substantial contributions to the original concept, coordination and design, acquisition of the data, analysis and interpretation of the data, and drafting of the manuscript. SV participated in the design and the coordination of the study, the acquisition and analysis of the data, and drafting the manuscript. CD participated in the design and coordination of the study. DB participated in the design and helped to draft the manuscript. KS, SVH and FDT participated in the design of the study, and have been involved in revising it thoroughly. JD made substantial contributions to the concept and design, analysis and interpretation of the data, and has revised the manuscript thoroughly. All authors have reviewed and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1472-6947/10/62/prepub


An affiliation for the fourth author was not indicated. Affiliation #1 for Luonan Chen should be: Key Laboratory of Systems Biology, SIBS-Novo Nordisk Translational Research Centre for PreDiabetes, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China. Affiliations #3 and #4 are his second and third affiliation, respectively.


APOBEC3G is a cytidine deaminase with potent antiviral activity. The protein deaminates single-stranded DNA but is known to bind cellular and viral RNAs. There is increasing evidence that RNA binding of APOBEC3G is important for packaging into viral particles. However, there is no consensus yet on the type of RNA involved. APOBEC3G is a cytidine deaminase that was recently identified as the prime substrate of the HIV-1 viral infectivity factor Vif ). While The study by Bach et al.  attemptsThere clearly is a trend in the field to accept the importance of RNA in the packaging of APOBEC3G into HIV-1 virions. But which RNA is it? There is no simple answer to this conundrum. Bach et al. point out that they and Wang et al. use a highly quantitative assay for determining encapsidation of 7SL RNA. So part of the difference may be attributed to differences in sensitivity. Some of the confusion may also come from the use of different experimental model systems, i.e. virus-like particles (VLP) versus whole virus. Also, it often is not appreciated that packaging of APOBEC3G into HIV virions or VLPs is as much a qualitative as it is a quantitative problem. For instance, the cytidine deaminase APOBEC3A is packaged into HIV virions but does not associate with viral cores and has no antiviral activity . HoweverIn conclusion, the final verdict on what RNA guides the packaging of APOBEC3G into HIV-1 virions is still out and awaits further investigation. In particular, it remains to be determined whether APOBEC3G encapsidation entails a specific  and/or a non-specific component (e.g. cellular RNA). However, it is our (not completely unbiased) opinion that viral genomic RNA is excellently placed to make the finals.The authors declare that they have no competing interests.KS wrote the first draft and MAK made critical suggestions for improvement. All authors read and approved the final manuscript.


In recent years, many efforts have been made to computationally predict C-to-U RNA editing sites in the mitochondria of seed plants, but there is still no algorithm available for C-to-U RNA editing site prediction in the chloroplasts of seed plants.RNA editing is a type of post-transcriptional modification of RNA and belongs to the class of mechanisms that contribute to the complexity of transcriptomes. C-to-U RNA editing is commonly observed in plant mitochondria and chloroplasts. The .In this paper, we extend our algorithm CURE, which can accurately predict the C-to-U RNA editing sites in mitochondria, to predict C-to-U RNA editing sites in the chloroplasts of seed plants. The algorithm achieves over 80% sensitivity and over 99% specificity. We implement the algorithm as an online service called CURE-Chloroplast CURE-Chloroplast is an online service for predicting the C-to-U RNA editing sites in the chloroplasts of seed plants. The online service allows the processing of entire chloroplast genome sequences. Since CURE-Chloroplast performs very well, it could be a helpful tool in the study of C-to-U RNA editing in the chloroplasts of seed plants. RNA editing is a kind of RNA processing  that can alter the genetic information of RNA via insertion, deletion or substitution of single or multiple nucleotides. In plant mitochondrial and chloroplast transcripts, several cytidines can be converted to uridines by a deamination process -3. Generin vivo site recognition mechanism of C-to-U RNA editing in plant organelles is still not fully understood [Takakia lepidozioides [Biologically, the derstood . While sderstood -22, littderstood -26. It sderstood ,28. Withderstood , REGAL iderstood ,31 and Pderstood  and our derstood  considerozioides . HoweverAlthough the chloroplast C-to-U RNA editing sites of rice , maize , tobaccoHowever, in the public sequence database, there are many chloroplast transcripts with undetermined RNA editing status. An accurate computational prediction method would be helpful in filling the gap between the sequence data and the RNA editing annotations. It would also reduce the need for experimental determinations, which are costly and time consuming. In this paper, we will extend our algorithm CURE, which can accurately predict C-to-U RNA editing sites in mitochondria, to predict C-to-U RNA editing sites in the chloroplasts of seed plants. We will also present the online prediction service, CURE-Chloroplast.There are three main RNA editing databases: dbRES , REDIdb This dataset has significant lineage bias. Most of the editing sites are from angiosperms. Only one gymnosperm plant is included. Since several editing sites in the chloroplasts of the gymnosperm are not conserved in angiosperms , we needThe basic algorithm for CURE-Chloroplast is the same as the CURE algorithm, which we have already successfully developed to predict mitochondria C-to-U RNA editing sites . The CURThe alignments for training can be prepared by two different methods. One is to create alignments from the CDS sequences of each gene with the ClustalW program; the other is to create alignments directly from the whole genome sequences using the TBA program . Since mAs we have mentioned in the dataset section, several editing sites in gymnosperms are not conserved to the angiosperms. Thus, we need to modify the CURE algorithm to make it applicable to gymnosperms. An extended definition of an EPES was proposed. If a column in the multiple sequence alignments contains only T, and this column is at the middle position of a codon, this column is also defined as an EPES. This extended EPES definition is based on the fact that the protein translated from the edited version of the transcript is usually more conserved across species than the sequence predicted from the genomic DNA. Thus, if a cytidine is mapped by this extended EPES, it is likely to be edited to restore the conservation at the protein level.Six summary statistics were used to describe the performance of CURE-Chloroplast: sensitivity, specificity, accuracy, positive predictive value (PPV), balanced accuracy (BA) and the Matthew's correlation coefficient (MCC) (Eq. 1~Eq. 6).TP, TN, FP and FN are the number of true positives, true negatives, false positives and false negatives, respectively.CURE-Chloroplast was implemented on a Linux server with two Xeon dual-core CPUs and 4 GB of memory. Users can either paste a single sequence or upload a FASTA file containing multiple sequences via the web-based user interface. The server-side PHP scripts process the sequences and carry out the prediction. The results are presented in a web-based result browser. The plain text-based result file can be downloaded within 24 hours after the prediction is made.CURE-Chloroplast users are allowed to adjust several parameters after the advanced mode is enabled. The "Up Bound" and "Low Bound" parameters define the working region of a K-NN classifier, which is called a "micro-analyzer" in the CURE-Chloroplast system. When the submitted sequence is mapped by an EPES with an RNA editing conservative ratio between the Low Bound and Up Bound, the K-NN classifier will be used to decide whether or not the EPES mapping cytidine should be predicted as an editing site. If the users enlarge the working region bounded by these two parameters, the editing statuses of more cytidines are determined by the K-NN classifier rather than the RNA editing conservative ratio. According to our experience, the default parameters are suitable for most organisms.The CURE-Chloroplast service has two more options than the CURE service. One is the "Cons-T EPES" option. If this option is turned on, the extended EPES definition will be used. The other is the "Positive strand only" option. If this option is turned on, CURE-Chloroplast will only scan the positive strand of the input sequence.We used leave-one-species-out cross-validation to estimate the performance of CURE-Chloroplast. When we were testing the performance on one organism, all the data relating to that organism, including sequences and editing sites, were removed from the training set. The algorithm was retrained on the remaining data. The details of CURE-Chloroplast performance can be found in Table Pinus thunbergii was more than twice that of the original definition, the PPV only decreased by about 20%. The extended EPES definition successfully improved the performance on gymnosperms. All the following tests on gymnosperms were carried out using the extended EPES definition.The extended EPES definition was applied for gymnosperm plants. With the extended EPES definition, the sensitivity on The pioneer research on predicting the C-to-U RNA editing sites in mitochondria was carried out on the balanced dataset . We alsoTo further eliminate the concerns of over-fitting algorithm, we carried out an independent data test. We randomly selected 10%, 20% and 30% of the dataset as the test samples. The remaining data were used as the training set. The performance estimated with this independent test was similar to the leave-one-species-out cross-validation performance Table , indicatCuscuta reflexa and Cuscuta gronovii, were recently determined [Cuscuta reflexa and three of the four known editing sites with only three false positives in Cuscuta gronovii. The overall performance in this full-blind validation achieved 94.7% sensitivity, 99.8% specificity, 78.3% PPV and 99.7% accuracy.It should be noted that the RNA editing sites of the chloroplast genes of two parasitic flowering plants, termined . These dArabidopsis thaliana with different parameters and illustrated an ROC-like curve  are the only organism with comprehensive C-to-U RNA editing information in the corresponding lineage, the low performance is expected. When the editing sites of more organisms in these lineages are determined, the performance of CURE-Chloroplast is expected to improve. However, we have to emphasize that CURE-Chloroplast is currently only developed for seed plants.Another more telling explanation for the low performance of CURE-Chloroplast with regard to these two organisms is the phylogenetically skewed knowledge of C-to-U RNA editing in chloroplasts. The performance of a lineage is associated with the abundance of data, and especially the number of organisms in that lineage with systematically determined editing sites Figure . The perCURE-Chloroplast predicts C-to-U RNA editing sites in the chloroplasts of seed plants with quite well performance. The predictive result is expected to improve as more data becomes available. The online service allows the processing of the entire chloroplast genome sequence. Although the predictive ability of CURE-Chloroplast is currently restricted within the seed plant lineage, CURE-Chloroplast is still a useful tool for studying C-to-U RNA editing in chloroplasts.Project name: CURE-ChloroplastProject home page: Operating system(s): Online service, platform independentProgramming languages: Java, PHP, JavaScriptOther requirements: The web browser must support JavaScriptLicense: FreeRestrictions for non-academic use: Please contact the authors before non-academic applicationPD designed the algorithm, carried out the programming, implemented the online service, analyzed the results and partially wrote the manuscript. LJ collected the data, prepared the dataset, carried out the evaluation and optimization of the system, analyzed the results and partially wrote the manuscript. YL directed the entire study, analyzed the results and partially wrote the manuscript.RNAE performance on seed plants. The prediction performance comparison between CURE-Chloroplast and RNAE.Click here for fileCURE algorithm document. The description of CURE algorithm in details.Click here for fileArabidopsis thalianaROC-like curve on . The analysis of CURE-Chloroplast prediction performance under different algorithm parameters.Click here for file


Chemotherapy with the cytotoxic drug procarbazine (PCB) causes permanent infertility in most male patients. Since many patients treated with this cytotoxic drug are of reproductive age, it is important to develop a method to protect spermatogenesis and fertility. It has been hypothesised that 'spermatogenic arrest' by pharmacological intervention may render the testes less susceptible to the effects of chemotherapy. The present study investigated whether recovery of fertility in a male rat model could be achieved by suppression of spermatogenesis with high doses of clomiphene citrate (CC) prior to PCB administration. It was demonstrated that young male rats treated with a combination of CC and PCB partially recovered spermatogenesis and achieved almost normal fertility. In contrast, animals treated with PCB alone exhibited abnormal spermatogenesis and remained infertile.


Target recognition in RNA silencing is governed by the “seed sequence” of a guide RNA strand associated with the PIWI/MID domain of an Argonaute protein in RISC. Using a reconstituted in vitro target recognition system, we show that a model PIWI/MID domain protein confers position-dependent tightening and loosening of guide-strand-target interactions. Over the seed sequence, the interaction affinity is enhanced up to ˜300-fold. Enhancement is achieved through a reduced entropy penalty for the interaction. In contrast, interactions 3′ of the seed are inhibited. We quantified mismatched target recognition inside and outside the seed, revealing amplified discrimination at the third position in the seed mediated by the PIWI/MID domain. Thus, association of the guide strand with the PIWI/MID domain generates an enhanced affinity anchor site over the seed that can promote fidelity in target recognition and stabilize and guide the assembly of the active silencing complex. RNA silencing refers to a group of related gene silencing mechanisms mediated by short (18–30 nucleotide) single-stranded RNA molecules. These mechanisms include RNA interference (RNAi) mediated by small interfering RNAs (siRNAs), posttranscriptional gene regulation by microRNAs (miRNAs), and the suppression of parasitic mobile genetic elements by repeat-associated small interfering RNAs (rasiRNAs) and (some) Piwi-interacting RNAs (piRNAs) . The mecTarget selection in RNA silencing is governed by the “seed sequence” at the 5′ end of the guide strand  and acceCaenorhabditis elegans lin4-lin14 interactions  greatly enhances the affinity of the seed-target interaction. Enhancement is achieved through a reduced entropy penalty for the interaction, probably as a result of immobilization or preordering of the guide. We examined the position specificity of binding modulation by AfPiwi, showing that enhancement is strictly localized to the seed region. Guide-target interactions more 3′ along the guide, including around the scissile phosphate region of the duplex, are inhibited by AfPiwi. We also assess the capacity of the complex for mismatched target recognition, showing that mismatches can have a major influence on the affinity of seed-target recognition, with the impact of mismatches at position 3 amplified through association with the protein. We discuss potential molecular explanations for our observations and explore their implications with respect to the mechanisms of RNA silencing.Seed-target recognition does not occur in isolation but in the context of large multicomponent effector complexes, such as RISC or RITS. Crystal structures show that the seed region of the guide strand is anchored via its phosphodiester backbone to the PIWI/MID domain of Argonaute and that this complex provides the platform for target mRNA recognition . It is bSome of our experiments were performed using DNA mimics of the guide and target strands, so we first determined the structure of AfPiwi in complex with DNA mimics of guide and target strands, and compared the structure to a similar complex with RNA . AfPiwi The structure superimposes closely with the RNA complex  Figure A, with tProkaryotic Argonaute proteins display greater slicing activity using DNA guide strands , consistd), enthalpy (ΔH), and entropy (ΔS) of an interaction by measuring the heat released or absorbed when a ligand is titrated into a solution of a macromolecule  (Thermodynamic parameters for the interactions are displayed in  strand)  shows thThe RNA/RNA and DNA/RNA enhancement experiments presented in this section were conducted using target strands lacking a T1 nucleotide to maintain the same number of base pairs in isolation and in the presence of AfPiwi (the G1-T1 base pair is unwound in the presence of AfPiwi). Measurement of the enhancement of a similar RNA-RNA interaction with a flush 5′ end results in an apparent reduced enhancement  and a series of step-translated 8-mer DNA target strands (Tscan1 to Tscan5) . Because a DNA-DNA interaction covering nucleotides 2–8 is enhanced 21-fold , 4–11 (Tscan4), and 5–12 (Tscan5) are all inhibited, to a small extent (between 2.2-fold and 7.6-fold), by AfPiwi. Thus, toward the 3′ end of the guide, beginning at around position 9, AfPiwi weakens the guide-target interaction.We wanted to assess whether there was any variation in the influence of AfPiwi at different positions along the guide strand. We therefore performed a series of “scanning” experiments using a 12-mer DNA guide (G Tscan5) . The G1211) with G12, covering nucleotides 1–11 of the guide  to that of Watson-Crick base pairs . Some reG:U wobble mismatches at positions 4 and 5 have a relatively mild impact on seed-target recognition in the presence of AfPiwi  strand -target duplex. Consistent with these predictions, the recent crystal structure of ) strand  shows thendo conformation found in A-form duplexes. The enhanced binding affinity is in part attributed to the influence of preorganization, thereby modulating the entropy of the interactions. LNA-containing duplexes also display substantially increased melting temperatures. In general, the mobility of single stranded nucleic acids and the increase in order concomitant with their interaction may render them particularly susceptible to binding enhancement by this mechanism. It seems likely that nucleic acid interactions in other macromolecular complexes will be subject to similar effects.These observations are reminiscent of the properties of Locked Nucleic Acids (LNAs), which also confer enhanced affinity for complementary nucleic acid strands . LNA nucThe Argonaute-guide-strand complex forms the core of the RNA silencing effector complexes RISC and RITS . It is kAssociation of the seed region of the guide strand with the PIWI/MID domain protein generates a high-affinity recognition site for the target, with an affinity far exceeding that for the corresponding interaction in isolation. Furthermore, this extends to rescue of otherwise “melted” seed-target interactions, whose interaction would otherwise be completely abolished as the ambient temperature exceeds the melting temperature of the duplex. The short length of the seed region (six or seven nucleotides) renders this region particularly susceptible to this effect, which would be even more pronounced in the higher temperature environment of mammalian cells.In addition, the strong seed anchor site may stabilize and facilitate the subsequent steps in the formation of the active slicing complex, where it appears the guide-target duplex must be propagated through Argonaute accompanied by extensive conformational change . BecausePerhaps unexpectedly, guide-target interactions 3′ of the seed region are weakened by AfPiwi. This asymmetry in activity (enhancement over the seed and inhibition at more 3′ positions) is consistent with previous observations for the RISC complex as a whole, showing preferential target interaction at the 5′ end of the guide . The inhtrans-binding proteins  and facilitate complex recycling—are tolerated as part of a secondary recognition region, dependent on the establishment of a strong recognition anchor over the seed.AfPiwi was purified as described , with thhttp://www.ambion.com/techlib/misc/oligo_calculator.html.Oligonucleotide extinction coefficients were determined using the Ambion Extinction Coefficient Calculator, available at −1cm−1) was determined using the Expasy ProtParam tool, available at http://us.expasy.org/tools/protparam.html.The AfPiwi extinction coefficient  was the same as the sequence of RNA used in a previously described structure   were dialyzed using Spectra/Por Biotech Cellulose Ester (CE) dialysis membrane (MWCO 500 Da) (Spectrum Laboratories Inc). The RNA guide strand used in Most samples were dialyzed overnight at 4°C prior to ITC analysis. The following buffer was used: 50 mM Tris (pH 7.5), 150 mM KCl, 5% (v/v) glycerol, 10 mM MgClKa because the transition is very sharp and too few titration points are collected near equivalence [Samples concentrations were chosen to optimize ITC C values ([macromolecule] / dissociation constant) but were limited at the lower end by the magnitudes of the heats and at the upper end by the solubility of AfPiwi. Most runs were conducted with C values in the range of 1 to 300; for an accurate determination of the binding constant, a C value between 1 and 1000 is required.  TypicaAnalysis was performed on a VP-ITC instrument  at 20°C. Two schedules were used: (1) dummy injection followed by 16 × 17 μl injections (2 s/μl), and (2) dummy injection followed by 19 × 15 μl injections (2 s/μl). Injection spacing was usually 360 s (to obtain flat baseline between injections), stirring was at 300 rpm, and the reference power was either 5 or 10 μcal/s.a (see also Data analysis was performed using Origin versions 5.0 and 7.0. These programs model experimental isotherms according to equations defined by see also . Baselin


Pol) and RNA replication templates at these sites to establish active replication complexes. During replication, 1a provides RNA capping, NTPase and possibly RNA helicase functions. Here we identify in BMV 1a an amphipathic α-helix, helix A, and use NMR analysis to define its structure and propensity to insert in hydrophobic membrane-mimicking micelles. We show that helix A is essential for efficient 1a–ER membrane association and normal perinuclear ER localization, and that deletion or mutation of helix A abolishes RNA replication. Strikingly, mutations in helix A give rise to two dramatically opposite 1a function phenotypes, implying that helix A acts as a molecular switch regulating the intricate balance between separable 1a functions. One class of helix A deletions and amino acid substitutions markedly inhibits 1a–membrane association and abolishes ER membrane invagination, viral RNA template recruitment, and replication, but doubles the 1a-mediated increase in 2aPol accumulation. The second class of helix A mutations not only maintains efficient 1a–membrane association but also amplifies the number of 1a-induced membrane invaginations 5- to 8-fold and enhances viral RNA template recruitment, while failing to stimulate 2aPol accumulation. The results provide new insights into the pathways of RNA replication complex assembly and show that helix A is critical for assembly and function of the viral RNA replication complex, including its central role in targeting replication components and controlling modes of 1a action.Brome mosaic virus (BMV) protein 1a has multiple key roles in viral RNA replication. 1a localizes to perinuclear endoplasmic reticulum (ER) membranes as a peripheral membrane protein, induces ER membrane invaginations in which RNA replication complexes form, and recruits and stabilizes BMV 2a polymerase  transfer their genetic material between host cells as RNA of mRNA polarity, which are translated into proteins immediately upon entry. One immediate function of these proteins is to establish RNA replication compartments on intracellular membranes to copy the incoming viral RNA. Although much is known about the viral protein and RNA components in such replication complexes, little is understood about how the multiple protein–membrane–RNA interactions required for replication complex assembly are regulated. To study this, we used a well-established model virus that encodes only two replication proteins: an RNA polymerase enzyme that copies the viral RNA and an assembly-coordinating protein that guides the rearrangement of intracellular membranes to form replication compartments and recruits the viral RNA template and polymerase to these sites. We identified a small helix in this guiding replication protein that is essential for efficient association with and rearrangement of the correct intracellular membrane type and for regulating a switch between at least two different functional states of the replication guide protein. Mutations in this small helix interfere with separable guide protein functions, revealing new insights into the sequential steps in positive-strand RNA virus RNA replication complex formation. Positive-strand RNA viruses comprise over one-third of all virus genera and cause numerous diseases of humans, animals and plants A universal feature of positive-strand RNA virus RNA replication is its close association with intracellular membranes. One or more viral nonstructural proteins target the viral replication complex to its preferred membrane type and often, if not always, induce membrane rearrangements. The responsible viral proteins can be true integral membrane proteins such as the flock house virus protein A that builds replication complexes on outer mitochondrial membranes Pol), respectively, which are required for RNA replication. RNA3 and RNA4 encode the 3a movement protein and the coat protein, respectively, required for systemic spread in plants Saccharomyces cerevisiae by expressing the viral RNA replication and/or capsid proteins together with one or more genomic RNAs Brome mosaic virus (BMV), a member of the alphavirus-like superfamily of human, animal, and plant viruses, is among the best-studied positive-strand RNA viruses for RNA replication. BMV has three genomic RNAs, RNA1, RNA2 and RNA3, and a subgenomic mRNA, RNA4. RNA1 and RNA2 encode nonstructural replicase proteins 1a and 2a polymerase  and resides fully on the cytoplasmic side of the ER membrane, but that amino acids 368–478 contain sequences important for ER membrane binding Previously, using membrane affinity and protease sensitivity assays, we showed that BMV 1a strongly localizes to the cytoplasmic face of the ER membrane despite lacking any detectable trans-membrane domain To test the functionality of helix A for membrane association, the 105, 35 and 18 aa regions described above were fused to the N-terminus of GFP to produce E-GFP, 35H-GFP, and 18H-GFP, respectively . Lysates13C chemical shifts, NMR showed that aa 397–406 in the peptide had a >80% probability to be in a helical structure . For each 1a mutant class, similar results were obtained with all members, and representative results are shown in Pol fusion protein to allow direct fluorescence microscopy detection rather than immunofluorescence, which is often compromised by low 2aPol detection sensitivity The localization of wt 1a and selected representatives of the Class I and II 1a derivatives in cells co-expressing 2aPol fluorescence in the absence of 1a was mostly faint and diffusely cytoplasmic with a few punctate dots. When co-expressed with wt 1a, GFP-2aPol co-localized with 1a in typical partial to almost complete ring-like perinuclear ER structures  and centrifuged 5 min at 4°C at 2000×g to yield pellet and supernatant fractions. For RNase treatment, 1 U of micrococcal nuclease was added to 100 µl of supernatant and pellet fractions, incubated at 30°C for 15 minutes and inactivated by addition of 2 µl of 0.5 M EGTA (pH 8.0). RNA was isolated and prepared by the hot phenol method in vitro transcribed and capped  from EcoRI-linearized plasmid pB1TP3 or its derivatives, pB2TP5 and pB3TP8, respectively BMV RNA1 or its mutants, RNA2, and RNA3 were 15N, (U)-13C labeled amino acids were purchased from Cambridge Isotope Laboratories. Labeled Fmoc-phenylalanine was purchased from Isotech. Wang resin was loaded with Fmoc-15N, (U)-13C-alanine using N, N-Diisopropylcarbodiimide and 4-diththylaminopyridine. The synthesis was carried out at a scale of 12.5 micromoles with a 3-fold excess of each amino acid. Coupling times for the first three and final 5 couplings were fixed at one hour each. The remaining 6 couplings were programmed as extended couplings. The cleaved and deprotected peptide was purified by HPLC using a C18 Vydac column (250×10 mm). Mass confirmation was done using a Bruker Biflex III MALDI-TOF.The helix A peptide, representing amino acids 392–409 of BMV 1a (GenBank accession number ABF83485), was synthesized on an Applied Biosystems 432A synthesizer using standard Fmoc chemistry with HBTU/HoBT coupling 2H2O using a Varian VNMRS 600 MHz spectrometer equipped with a 5 mm cryogenic triple resonance probe. DSA  and deuterated SDS were purchased from Aldrich. The 3D data were collected using HIFI, a rapid methodology for collection of multidimensional NMR spectra 1H{15N}HSQC, HNCO, CBCA(CO)NH, HNCACB, HNCA, HN(CO)CA, HN(CA)CO, HN(CA)CB. All experiments were standard Varian Biopack pulse sequences modified for the HIFI method 15N-edited 1H-1H NOESY and 3D 13C-edited 1H-1H NOESY spectra were used as input for the ATNOS/CANDID/CYANA suite of programs http://www.pymol.org). The NMR and structural data described have been deposited in BioMagResBank (http://www.bmrb.wisc.edu) under BMRB accession number 20027.NMR spectra were collected from a solution of 400 mM peptide in 100 mM SDS and 5% Pol were fixed with 5% formaldehyde, spheroplasted with lyticase, and permeabilized with 0.1% Triton X-100. Spheroplasts were then stained by using rabbit anti-1a serum, followed by Texas red-conjugated donkey anti-rabbit antibodies. Fluorescent images were acquired with a Bio-Rad 1042 double-channel confocal microscope system.Confocal microscopy was as described 4 and 1% uranyl acetate. Cells then were dehydrated via a series of step-wise increasing ethanol concentrations ranging from 50% to 100%, and infiltrated and embedded with Spurrs resin. Samples were sectioned and placed on nickel grids, washed, incubated in 15 min in 2% glutaraldehyde, poststained with 8% uranyl acetate and Reynold's lead citrate, and viewed with a Philips CM120 microscope.Samples were prepared for electron microscopy as described


Different phylogenetic methods have been developed to predict secondary structures in RNA viruses, for high-resolution thermodynamic scanning and for detecting SSSV. These approaches have been successfully in predicting cis-acting signals in different members of the family Picornaviridae and Caliciviridae. In order to gain insight into the identification of cis-acting signals in viruses whose mechanisms of replication are currently unknown, we performed a phylogenetic analysis of complete genome sequences from 49 Human Norovirus (NoV) strains.Discrete RNA structures such as cre motif at positions 240 through 291 of the RNA dependant RNA polymerase (RdRp) sequence (relative to strain [EMBL:EU794713]). This sequence region has a high probability to conform a stem-loop.The complete coding sequences of NoV ORF1 were obtained from the DDBJ database and aligned. Shannon entropy calculations and RNAalifold consensus RNA structure prediction identified a discrete, conserved, invariant sequence region with a characteristic AAACG Hepatovirus cre stem-loop.A new predicted stem-loop has been identified near the 5' end of the RdRp of Human NoV genome. This is the same location recently reported for  The results of these studies are shown in Fig. cre sequence motif is embedded in a sequence region with a very high probability to conform a stem-loop structure cre sequeRNA structure predictions are consistent with previous analyses based on the thermodynamic folding of individual sequences ,17,18. ACaliciviridae, and they are non-enveloped viruses with positive, single-stranded RNA genomes. They also share other important features with picornaviruses, like having a VPg protein covalently linked to the 5' of the genomic RNA [NoV belong to the family omic RNA . Neverthomic RNA . NoV areomic RNA .cis-acting signals in the 5' and 3' regions as well as at the start of the sg RNA transcript of NoV [Despite the importance of these outbreaks, our understanding of the RNA structures or sequences required for NoV replication has been limited. Previous reports have identified the poly-pyrimidine tract-binding protein (PTB), poly-A binding protein (PAB) and La autoantigen to interact with the 3' untranslated region of the Norwalk virus genome . Very ret of NoV .Caliciviridae, NoV are thought to replicate in a manner typical of positive-stranded RNA viruses, through the synthesis of a full-length anti-genomic strand (reverse complement copy) using the viral RdRp translated initially from the RNA genome entering the cell [As a member of the family the cell . The minthe cell .in vitro due to the lack of a standard cell culture to grow these viruses, the probability that this predicted structure will acts as a functional element may open new avenues to our understanding of molecular mechanisms of NoV replication.Although the presence of this new putative cis-acting signal predicted in this study was not yet investigated Picornaviridae, like Poliovirus (PV) and Human Rhinovirus 14 (HRV-14), revealed a critical conserved AAACA/G cre sequence motif in the 5' half of the loop sequence that is essential for its function [cre elements of other picornaviruses and are important for RNA replication [Extensive mutagenesis studies performed in members of the family function . Similarlication ,24,25.cre act as the template for VPg uridylylation through a "slide-back" mechanism catalyzed by the 3Dpol (RdRp) [Paul and colleagues (2003) have shown that the PV l (RdRp) ,26,27. Tl (RdRp) .pol (RdRp) generates VPg-poly(U) and that this uridylylated VPg can prime the replication of polyadenylated RNA [de novo [de novo initiation of replication of antigenomic RNA [Interestingly, recent studies have shown that incubation of VPg with NoV 3Dated RNA . In cont[de novo . These fomic RNA . Besidesomic RNA .cre-like sequence motif with members of the family Picornaviridae [It is possible that the predicted stem-loop identified near the 5' end of the NoV RdRp coding region, which share a aviridae , will beaviridae .The authors declare that they have no competing interests.JC conceived the study. MV and JC designed, performed and revised the phylogenetic analysis. MV, RC, MPM and JPL contributed to the interpretation and discussion of the results found. JC wrote the manuscript. All authors read and approved the final manuscript.Origins of Norovirus strains. The table provides the strain names and accession numbers of their respective sequences of ORF1 of Norovirus strains included in this study.Click here for file


High-throughput functional genomic procedures depend on the quality of the RNA used. Copurifying molecules can negatively impact the functionality of some plant RNA preparations employed in these procedures. We present a simplified, rapid, and scalable SDS/phenol-based method that provides the high-quantity and -quality RNA required by the newly emerging biotechnology applications. The method is applied to isolating RNA from tissues of two biotechnologically important crop plants, sugarcane and citrus, which provide a challenge due to the presence of fiber, polysaccharides, or secondary metabolites. The RNA isolated by this method is suitable for several downstream applications including northern blot hybridization, microarray analysis, and quantitative RT-PCR. This method has been used in a diverse range of projects ranging from screening plant lines overexpressing mammalian genes to analyzing plant responses to viral infection and defense signaling molecules. Preparation of high-quality RNA is critical for functional genomic studies. Isolating high-quality RNA from biotechnologically important crops such as sugarcane and citrus is complicated by the presence of high concentrations of intrinsic polysaccharides, polyphenols, and other secondary metabolites. Levels of these compounds increase in plants under biotic and abiotic stresses, such as pathogen infection or drought , 2. ThesSeveral methods exist for isolating RNA from tissues of species with a high content of polysaccharides or polyphenols. These methods mainly use denaturing agents such as guanidine- and phenol-based extraction buffers in combination with isopropyl alcohol precipitation –6, deterWe have developed a simple, rapid, and scalable procedure for isolation of high-quality RNA from sugarcane and citrus to facilitate the application of functional genomic studies in these crops. The procedure is a simplified SDS/phenol extraction method with sequential steps of purification from polysaccharides and polyphenols using 2-mercaptoethanol/PVP-binding, chloroform partitioning, and sodium acetate/ethanol- and LiCl–mediated precipitations. It relies on two extraction steps using automated homogenization from small amounts of tissue and extraction buffer and on two rounds of precipitation. In citrus, only one extraction step is needed. High yields and quality of RNA are consistently obtained from multiple samples. Low and high molecular weight-RNA as well as low- and high-abundant RNA isoforms can be recovered. The simplified RNA isolation method was compared with other RNA extraction methods used for functional genomic studies in sugarcane and citrus, namely, those based on guanidine thiocyanate –21, TRIZBamHI-digested vector-pZero  and placed under the control of the maize ubiquitin 1 (Ubi1) promoter and the 35S cauliflower mosaic virus terminator, yielding pZeroUbi1:mammalian gene. The construct for citrus transformation was generated by cloning the mammalian gene into the binary vector pBIN34S [Constructs carrying a synthetic mammalian gene (0.455 kilobase [kb]), codon optimized for expression in either monocots or dicots, were generated. The sugarcane construct consisted of the mammalian gene cloned into the  pBIN34S  to produSaccharum spp. hybrid, cultivar CP72-1210) and transformed with pZeroUbi1:mammalian gene together with the Ubi1:BAR-pUC8 (pAHC20) plasmid α-dATP by random priming using Klenow Exo− DNA polymerase . Hybridization and washes were conducted in accordance with the method of Church and Gilbert  and 5′-ttgccacgatgtccatgatg-3′ [R]), DIR1 (5′-cattcggcaaaacaacagaca-3′ [F] and 5′-gcgtccaaagaaacagatga-3′ [R]), DIR11 (5′-atcaatcaagcacaatataa-3′ [F] and 5′-agatcgtgaaaagatacatt-3′ [R]), DIR12 (5′-gcttgatcgactagcgc-3′ [F] and 5′-gcacaagaagcagctg-3′ [R]), DIR16 (5′-cctgggcgcttctaccaac-3′ [F] and 5′-acacttgtcgatcaagcgtcg-3′ [R]), and ubiquitin (5′-ccaaaccccgacgatcc-3′ [F] and 5′-tctcgtacttgtgccggtcc-3′ [R]).First-strand cDNAs were synthesized from DNase I-treated aRNA  for extraction and two rounds of precipitation to yield high-quantity and -quality RNA from 100 mg to 60 g of tissue in an efficient manner. The method is simplified in terms of requiring less manipulation, with only two extraction steps and two rounds of precipitation. It is also considered as a rapid isolation method due to the reduced time needed for automated tissue homogenization (30 seconds) and RNA precipitation (one precipitation for one hour and another for 16 hours). Optimization of the TENS-PCI micro-scale isolation method was achieved by reducing the leaf tissue weight from 1.0 g usually required for grasses  to 0.1 gμL). Higher-throughput preparations could be easily achieved with this method by adapting the use of a higher-capacity homogenizer. Automated tissue homogenization allowed the extraction of 24 samples in a very short period of time (30 seconds) in small tubes (2 mL), using a reduced extraction volume (600 An important component of the TENS-PCI extraction buffer was the presence of high levels of NaCl (0.1 M) used to increase the solubility of polysaccharides, reducing their coprecipitation with RNA in subsequent steps . Higher Two precipitation steps were included in the TENS-PCI method as opposed to the SDS-phenol method of Carson and Botha , 28 that260 : A230 that are close to 2.0  into the TENS-PCI extraction buffer as well as the adoption of only two rounds of selective precipitation proved to be efficient in removing polysaccharides and phenols. The TENS-PCI method yielded RNA with low levels of polysaccharides/phenols as indicated by the spectrophotometric ratios of Ae to 2.0 . Ratio vctively, , suggestctively, . Sorghum mosaic virus (SrMV) pathogen or treatment with the stress-regulated hormones, salicylic acid (SA), and methyl jasmonate (MeJA). For example, average A260 : A230 ratios of 2.37 ± 0.12 and 1.98 ± 0.06 were obtained with RNA extracted with TENS-PCI from plants treated with MeJA for 24- and 48-hours, respectively, compared to those of RNA extracted from the control plants at 24- and 48-hour treatment with water/ethanol, which were 2.21 ± 0.11 and 2.25 ± 0.14, respectively. Previous reports showed that stress treatments such as pathogen infection or water and nutrient deficiencies can enhance the levels of accumulation of polysaccharides and secondary metabolites, particularly phenols, in plants , 2. Our 260 : A280 ratio values around 2.0  by 4.7-fold in leaf as compared to the two tested kits, the RNeasy (0.11 μg/mg tissue) and FastRNA Pro Green (0.11 μg/mg tissue)  , althoug tissue) . We further assessed the performance of the TENS-PCI method across tissues and stress-related treatments, by checking the integrity of the RNA isolated from different sugarcane tissues such as leaf, stem and root, as well as from plants treated with the SrMV pathogen or the stress-regulators, SA and MeJA, using chip-based microcapillary electrophoresis. We similarly tested RNA obtained with the two kits, FastRNA Pro Green and RNAeasy Mini Plant (guanidine isothiocyanate). RNA degradation was evaluated using the ratios of the area under the curve of the peaks corresponding to the signal intensity of the large subunit to that of the small subunit (LS : SS) rRNA . Values of LS : SS rRNA ratios of about 2.0 are considered to be indicators of good RNA integrity, depending on the tissue analyzed and the biological system used , 48. Elaborate measures designed to improve RNA purity can increase the risk of RNA that is degraded in the process. The simplified TENS-PCI method produced a high-purity intact RNA from leaves, stems, and roots, showing a non-significant variation among these tissues, as reflected by the ratios of the LS : SS rRNA peak areas of the RNA profiles of the tested samples . RNA of Low-integrity RNA was obtained with the FastRNA Pro Green kit as shown by the low LS : SS ratios for the three tested tissues, leaf, root, and stem . The RNeOver 350 transgenic sugarcane and citrus plants were generated that constitutively expressed a mammalian gene. These were screened by Southern blot hybridization for presence and copy number of the gene (data not shown). To demonstrate the suitability of TENS-PCI for detecting specific transcripts, we tested the expression of the mammalian gene in leaves of transgenic sugarcane and citrus lines using northern blot hybridization. RNA isolated from the same lines using the RNeasy Plant Mini and the FastRNA Pro Green kits was also tested. Binding of the probe was the strongest and the most specific with RNA extracted by the TENS-PCI method, resulting in an intense band corresponding to the expected mammalian transcript size, of about 1.20 kb Figures , indicatThe inconsistency in RNA quality provided by the tested kits can impede the quantitation of the signal strength across multiple samples. Using the TENS-PCI micro-scale method, we were able to measure the transcriptional levels of multiple transgenic lines in a short time to identify high expressers of a mammalian gene. Our results were corroborated by Western analysis of protein levels and measurement of enzyme activity (data not shown). Thus, the TENS-PCI method represents a good option when limited amounts of tissue samples are available, since it consistently produces high-quantity and -quality RNA and does not interfere with the detection of specific transcripts.Important sources of variability in genomic data include variations in tissue processing, RNA preparation and assay protocols, as well as inherent tissue heterogeneity , 50. Of Feldman et al.  have desr] = 0.89–0.96; t-test P ≤.05) . Lower correlations (r = 0.65–0.70) were obtained from comparisons between replicate experiments that used RNA extracted with the RNeasy-guanidine isothiocyanate method . The detection of differential gene expression as well as the small variation between replicate experiments shows that the TENS-PCI RNA is of high quality to generate biologically significant and reproducible microarray data.Microarray analysis performed with aRNA derived from the TENS-PCI method revealed significant differences in relative gene expression ratios, as reflected by the two-fold up- or down-regulation of the identified genes (Sorghum mosaic virus (SrMV). Plant stress responses are largely mediated by phytohormones that trigger conserved defense mechanisms, with intricate signaling pathways leading to protection. Salicylic acid (SA) and the jasmonates (including methyl jasmonate (MeJA)) are major signaling molecules that regulate such protective responses via synergistic and antagonistic actions, referred to as signaling cross-talk –55. To fdirigent (DIR) family [O-methyltransferase (OMT) [chitinase gene [DIR genes in sugarcane maturing stems. MeJA has previously been shown to induce a number of DIR genes in sugarcane root [OMT has been reported to be expressed in mature sugarcane stems [OMT to be induced in barley by MeJA [The micorarray analysis identified three major transcript profiling groups  that con) family , 56 and se (OMT)  genes asase gene   as well as two rounds of precipitation (sodium acetate/ethanol and lithium chloride). This method represents a good option since it combines the advantages of high RNA recovery , high RNA integrity, reproducibility among biological and experimental replicates, and applicability to a wide range of tissues. We have demonstrated that this method accelerates the screening of transgenic plants with tissues rich in polysaccharides and secondary metabolites, using northern blot analysis. We have further shown that the high-quality RNA obtained by the TENS-PCI method can be easily amplified to generate reproducible and biologically significant gene expression data. We provide evidence of the utility of the RNA extracted by the TENS-PCI method in sensitive assays by showing that several defense- and stress-responsive marker genes are differentially regulated during the transcript profiling of part of the sugarcane transcriptome in response to pathogenic and hormonal stresses. These data corroborate with previously reported findings on the signaling pathways governing the plant stress response. We anticipate that the application of the TENS-PCI method in novel high-throughput functional genomic technologies such as next generation DNA sequencing will shed more light into the cross-talk signaling in biotechnologically important crops with complex genomes, such as sugarcane.Supplementary FIGURE 1S: Reproducibility of the data obtained from the microarray analysis of gene expression profiling of the sugarcane stem, using amplified RNA (aRNA).Click here for additional data file.