Escherichia coli sRNA-mRNA pair, MicA-ompA, specifically demonstrating that the addition of the poly-A-tail to either RNA does not affect its subsequent binding interactions with partner molecules. We then apply this method to investigate the novel interactions of a Vibrio cholerae Qrr sRNA with partner mRNAs, hapR and vca0939; RNA-RNA pairings that are important in mediating pathogenic virulence. The calculated binding parameters allow insights to be drawn regarding sRNA-mRNA interaction mechanisms.Characterisation of RNA and its intermolecular interactions is increasing in importance as the inventory of known RNA functions continues to expand. RNA-RNA interactions are central to post-transcriptional gene regulation mechanisms in bacteria, and the interactions of bacterial small non-coding RNAs (sRNAs) with their mRNA targets are the subject of much current research. The technology of surface plasmon resonance (SPR) is an attractive approach to studying these interactions since it is highly sensitive, and allows interaction measurements to be recorded in real-time. Whilst a number of approaches exist to label RNAs for surface-immobilisation, the method documented here is simple, quick, efficient, and utilises the high-affinity streptavidin-biotin interaction. Specifically, we ligate a biotinylated nucleotide to the 3′ end of RNA using T4 RNA ligase. Although this is a previously recognised approach, we have optimised the method by our discovery that the incorporation of four or more adenine nucleotides at the 3′ end of the RNA (a poly-A-tail) is required in order to achieve high ligation efficiencies. We use this method within the context of investigating small non-coding RNA (sRNA) - mRNA interactions through the application of surface technologies, including quantitative SPR assays. We first focus on validating the method using the recently characterised  RNA is a multifaceted molecule with an ever-expanding repertoire of intra-, inter-molecular and ligand-binding functions. These include acting as the messenger for gene expression (mRNA), regulating gene expression , catalysing biological processes  and providing mechanisms for viral infection V. cholerae quorum regulatory RNAs (Qrr sRNAs). Specifically, V. cholerae expresses four Qrr sRNAs that are thought to be functionally redundant, and that are involved in regulating mRNA expression of specific transcripts, thereby controlling biofilm formation and regulating pathogenic virulence vca0939 mRNA by preventing the formation of a translation-inhibiting stem-loop structure within the 5′ UTR vca0939 gene encodes a GGDEF domain-containing protein that synthesises the intracellular signalling molecule cyclic di-GMP which is involved in controlling biofilm formation hapR expression, by pairing to the 5′ UTR, thereby blocking the ribosome binding site Vibrio species have been shown to contain a conserved 21-nucleotide sequence predicted to imperfectly base-pair with the mRNA targets hapR and vca0939The interactions of bacterial trans-acting small non-coding RNAs (sRNAs) with their mRNA targets are the subject of much current research. sRNAs, which range from 50–200 nucleotides, have become increasingly recognised as a novel and ubiquitous class of gene expression regulator Sensor surface-based technologies such as surface plasmon resonance (SPR) and biolayer/dual polarisation interferometry are becoming increasingly popular for studying intermolecular interactions since both techniques offer sensitive detection in real-time. However, oriented immobilisation of one of the interacting partners in a manner that generates a homogenous surface, and allows observation of physiologically-relevant interactions, remains a challenge for most molecules, including RNA The main classes of immobilisation that are currently available are physical adsorption, covalent attachment and bioaffinity immobilisation in vitro transcription from a DNA template. For example, including a 5′-biotin-modified guanosine analogue in place of the usual GTP within the transcriptional mix allows T7 RNA polymerase to synthesise a 5′-biotin tagged RNA A number of approaches currently exist to incorporate bioaffinity tags, such as biotin, into RNA molecules. For example, RNA can be chemically synthesised to include the biotin label, but this approach is realistically limited to RNAs less than approximately 40 nucleotides in length due to the exponential decay in yield with increasing oligonucleotide length E. coli in response to cellular stress. MicA’s interaction with its target transcript, ompA, has been well characterised with a low nanomolar equilibrium dissociation constant (KD) previously determined by electrophoretic mobility shift assay (EMSA) analysis ompA interaction when monitoring binding of either MicA or ompA to its immobilised biotin-tagged partner RNA by SPR. With the methodology validated, we have demonstrated the use of this approach to explore the interaction of a V. cholerae Qrr sRNA with its mRNA targets.With the aim of devising an SPR study to explore sRNA-mRNA interactions, we undertook biotin-tagging of RNA molecules for surface immobilisation using the approach of ligating a biotinylated nucleotide to the 3′ end of the RNA using T4 RNA ligase. Crucially, we discovered that the presence of multiple adenine nucleotides at the 3′ end of the RNA was essential for high-efficiency ligation. This requirement was demonstrated for RNAs of a range of sizes, including substrates up to several hundred nucleotides in length. Validation that our optimised method did not impact RNA function was achieved using MicA, an sRNA produced in rpoS constructs, (in vitro transcription using a MegaScript T7 kit (Life Technologies) from PCR templates generated by gene synthesis from overlapping primers 5′-TAATACGACTCACTATA) and up to 3 guanines at the 5′ end to enhance the yield from transcription. For rpoS constructs (rpoS-Blunt II TOPO (encoding -576 to +10 of rpoS with a 5′ T7 promoter sequence) was used as template DNA. For RNAs incorporating 3′ poly-A-tails, between four and eight adenines were appended to the native sequence with, in each case, the extension length being limited by the need to maintain the native structure of the RNA as determined by MFold analysis RNAs, excluding structs, , with bonstructs , the pla2, 5 mM DTT and 1 mM ATP. Reactions were incubated at 37°C for 60 min. To achieve high ligation efficiencies, the RNA had a poly-A-tail of four to eight adenine nucleotides appended to the 3′ end (as described above in ‘RNA Synthesis’). Subsequent separation of the RNA and RNA-biotin conjugate from the other reaction components was achieved by two cycles through a Micro Bio-Spin 6 size exclusion column (Bio-Rad). Chromatographic clean up was confirmed using an analytical gel filtration column  on a liquid chromatography system (Dionex).Reaction mixtures contained 5 µM RNA, 10% (v/v) DMSO, 5 µM U-biotin phosphate with biotin linked through the 3′ phosphate via an extended organic linker; Ligation reactions were analysed on 20% (w/v) polyacrylamide (19∶1 acrylamide:bis-acrylamide) denaturing-gels containing 7 M urea. Gels were stained with SYBR-Gold and visualised using a UV transilluminator. RNA bands were quantified using Gene Tools software (SynGene). These data were used to determine the yield of biotin-labelled RNA, calculated as a percentage of the total RNA present. To confirm that U-biotin had been ligated to the RNA, the RNA was transferred to a Nylon+ (Bio-Rad) membrane by electro-blotting in 0.5×TBE, and cross-linked to the membrane using UV light. The membrane was probed with streptavidin-horseradish peroxidise conjugate, and detected with enhanced chemiluminescence.Aliquots (10 µL) of 1 mg/mL streptavidin in phosphate-buffered saline pH 7.4 (PBS) were pipetted onto NHS-activated slides , incubated for 30 min at 37°C in a humidified chamber, washed three times with PBS containing 0.05% (v/v) Tween (PBS-T), twice with water, and then air-dried. The slide was incubated in 50 mM ethanolamine at room temperature for 30 min prior to washing, as described above. Once air-dried, 10 µL of 400 nM test sample (biotin-tagged RNA) and control samples  were pipetted onto the streptavidin, and incubated for 30 min at 37°C in a humidified chamber, washed three times with PBS and then air-dried. The slide was blocked with 200 nM bulk mRNA (Sigma) for 30 min at 37°C in a humidified chamber prior to washing as above. The slide was then incubated overnight at room temperature with 400 nM Cy-labelled probe RNA in PBS, washed three times with PBS and air-dried. The slide was imaged at 550 nm excitation using a slide scanner .2 or, depending on the size of the RNA, ∼1–10 fmol/mm2, calculated using the standard manufacturer’s conversion of 1 RU = 1 pg/mm2) of sample were immobilised. The same procedure was used to immobilise biotin-tagged control molecules on the control flow cell. The blank flow cell was left untreated. To monitor interactions with binding partner RNA molecules, single-cycle kinetic experiments were conducted. This involved consecutive 2 min injections of 5 different concentrations of binding partner molecule (in either the 0–0.25 µM or 0–10 µM range), each separated by a 1 min dissociation phase and a final dissociation of ∼8 min. The experiment was run at 60–90 µL/min in HBS buffer using a T100 Biacore instrument  and the single cycle kinetic method within the Biacore control software. Data were analysed using T100 BiaEvaluation software  with curve fitting to the data achieved using a 1∶1 binding model.On-chip RNA immobilisation on the test flow cell was achieved by injecting 10 nM biotin-tagged RNA in HEPES-buffered saline pH 7.4  over a streptavidin-coated sensor chip  at 10 µL/min until ∼200 RU  onto a poly-A-tailed RNA was also tested, and the results of two typical ligation reactions are shown in 5-biotin compared to 63% on average for U-biotin; The efficiency of ligating a longer biotinylated oligoribonucleotide , it could be equally applicable to shorter chemically synthesised RNA molecules incorporating a poly-A-tail. However, if the method is to be applied to such chemically synthesised RNAs, then the 5′ end should not be a monophosphate as that would allow undesired circularisation and/or concatenation of the RNA in the presence of RNA ligase.We have demonstrated that our strategy can be used to efficiently label eleven different RNA molecules that range in length from 61 to 592 nucleotides, suggesting that the approach could prove to be applicable for any RNA. The method is straightforward, fast (∼90 minutes when starting from poly-A-tailed RNA) and requires only commercially-available reagents and equipment  in addition to the RNA to be biotin-tagged. The poly-A-tailed RNA can be readily prepared by including the adenines in the E. coli sRNA, MicA, to its mRNA target, the 5′ UTR of ompA. This interaction has been well characterised, with the affinity of MicA-ompA identified to be in the low nanomolar range ompA , known to interact with the 5′ UTR of its mRNA target, hapRhapR, only specific binding to the biotin-tagged Qrr sRNA was seen (The ability to simply and efficiently biotin-label large RNAs (≥60 nucleotides in length) has allowed us to apply our method to the study of biologically relevant RNA-RNA interactions using surface technologies. Here, we examine the binding of the ompA was biotin-labelled (D for the interaction (ompA (without a poly-A-tail) was tested as the binding partner Click here for additional data file.


NLS1,1), truncation at 238 (BRMS1ΔNLS2), or switching the location of NLS1 and NLS2  did not affect nuclear localization; but, replacement of NLS1 with NLS2  or truncation at 197 (BRMS1ΔNLS which removes both NLS) promoted cytoplasmic localization. MDA-MB-231 human metastatic breast cancer cells transduced with BRMS1NLS1,1, BRMS1NLS2,2 or BRMS1NLS2,1 were evaluated for metastasis suppression in an experimental xenograft mouse model. Interestingly, while NLS2 was not necessary for nuclear localization, it was found to be important for metastasis suppression since BRMS1NLS2,2 suppressed metastasis by 85%. In contrast, BRMS1NLS2,1 and BRMS1NLS1,1 did not significantly suppress metastasis. Both BRMS1 and BRMS1NLS2,2 co-immunoprecipitated with SIN3A in the nucleus and cytoplasm; however, BRMS1NLS1,1 and BRMS1NLS2,1 were associated with SIN3A in the nucleus only. Moreover, BRMS1 and BRMS1NLS2,2, but not BRMS1NLS1,1 and BRMS1NLS2,1, down-regulated the pro-metastatic microRNA, miR-10b. Together, these data demonstrate an important role for NLS2 in the cytoplasm that is critical for metastasis suppression and is distinct from nuclear localization.Breast cancer metastasis suppressor 1 (BRMS1) is a predominantly nuclear protein that suppresses metastasis in multiple human and murine carcinoma cell lines. BRMS1 interacts with several nuclear proteins including SIN3:HDAC chromatin remodeling complexes that are involved in repressing transcription. However, recent reports suggest BRMS1 may function in the cytoplasm. BRMS1 has two predicted nuclear localization sequences (NLS) that are located near the C-terminus . We hypothesized that nuclear localization sequences of BRMS1 were essential for BRMS1 mediated metastasis suppression. Replacement of NLS2 with NLS1 (BRMS1 Molecules regulating gene transcription either directly or indirectly have the potential to dramatically impact the metastatic process. Since the discovery of the metastasis suppressor BRMS1 in 2000 As a transcriptional regulatory molecule, it is not surprising that BRMS1 has been involved with modulation of multiple molecular pathways associated with metastasis. In fact, it has been suggested that BRMS1 robustly blocks the overall process of metastasis through small, albeit significant, inhibition of each step in the metastatic cascade To begin exploring possible cytoplasmic roles, we generated mutations at the two nuclear localization (NLS) regions. We were surprised to find that, although NLS2 was not important for active transport into the nucleus, it was critical for metastasis suppression. We identified potential cytoplasmic functions of BRMS1 through interaction with SIN3A that correlates with the ability of BRMS1 to suppress metastasis. This study adds to our understanding of the BRMS1 metastasis suppressor protein that will expand our knowledge of metastatic disease.All animal studies were carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the University of Alabama at Birmingham Institutional Animal Care and Use Committee (protocol #071106666). Animals were sacrificed by cervical dislocation following anesthesia (ketamine/xylazine) and all efforts were made to minimize suffering.2+/Mg2+ free Dulbecco's phosphate buffered saline (CMF-DPBS). Cells were maintained on 100 mm tissue culture dishes  at 37°C with 5% CO2 in a humidified atmosphere. Neither antibiotics nor antimycotics were used and all cell lines were found to be negative for Mycoplasma spp. contamination using a PCR-based method  or PlasmoTest kit .The metastatic human breast carcinoma cell line, MDA-MB-231, and those constitutively expressing BRMS1 or BRMS1 mutants was described previously GFP-GST fused BRMS1 or mutants were generated by amplifying GST-BRMS1 by PCR from pGEX-2TK/BRMS1 vector. The product was cloned into Gateway system entry vector pENTR/SD/D-TOPO according to the user manual (Invitrogen). Recombination between pENTR/SD/D-TOPO/BRMS1 and destination vector pcDNA-DEST53 was done to generate pcDNA-DEST53/GST-BRMS1 expression vector. BRMS1 mutants were created by QuickChange II site-directed mutagenesis  as described previously The metastatic human breast carcinoma cell line, MDA-MB-231, was transduced with a HIV type 1-based, lentiviral vector system to constitutively express BRMS1 or BRMS1 mutants as described previously The monoclonal antibody directed against BRMS1 (3a1.21) was described previously The transfected COS7 cells with GFP-GST-BRMS1 or -mutants were permeabilized with 1% Triton X-100 for 10 min. at room temperature. Blocking was accomplished with 10% BSA in PBS for 30 min. after 3×5 min. washes with PBS. Texas Red conjugated anti-phalloidin antibody  1∶2,000 dilution in 2% BSA in PBS was incubated for 1 hr. at room temperature. After 3×5 min. washes with PBS, the coverslips were inverted on DAPI-containing mount media , sealed with nail polish, and observed under a Nikon eclipse TE 2000-U fluorescent microscope.Interaction of endogenous BRMS1 and SIN3A in MCF10A and MCF10CAa.1 cell lines were analyzed by PLA according to the manufacturer's protocol . Briefly, cells were seeded on 8-well chamber slides for 24 hours, fixed with 4% paraformaldehyde, permeabilized with 0.5% triton X-100, and blocked with Duolink blocking solution. The antibodies for SIN3A and BRMS1 were incubated at 1∶100 dilution and the anti-mouse minus and anti-rabbit plus probes were used at 1∶5 dilution. Images were taken as described for fluorescence microscopy.miRNA expression was determined as described previously ad libitum.Experimental metastasis assays (tail vein injection) with athymic mice (10 per group) were performed as previously described The number of lung metastases was compared for BRMS1- and BRMS1 mutant-transduced cell lines to the parental MDA-MB-231 line. A Kruskal-Wallis ANOVA of ranks procedure was used with Dunn's post hoc test. All calculations were performed using SigmaStat (SPSS). Statistical significance was defined as a probability p≤0.05.et al. The functional properties of BRMS1 protein are not currently well understood. BRMS1 has a glutamate-rich region, two coiled-coil regions important for protein-protein interactions, and two nuclear localization sequences (NLS) . An addihttp://expasy.org) for cellular localization prediction. The two mutant proteins that are lacking NLS1  had negative NLS scores using PSORT II and had significant cytoplasmic prediction with WoLF PSORT . Although there were many changes in gene expression between the groups, hierarchical clustering analyses did not reveal correlations to the metastasis data (data not shown) indicating that other modifiers of gene expression may be involved. These arrays include>60,000 transcripts; however, small RNA including miRNA are not included. We therefore proceeded with analyzing selected miRNA associated with metastasis (metastamir) that are known to be regulated by BRMS1 including miR-146a, -146b, and -10b We hypothesized that nuclear localization of BRMS1 would be required for metastasis suppressive function. Several mutations were generated in order to test this hypothesis; and, surprisingly, we found the second so-called NLS, which does not play a role in active transport into the nucleus, was critical for metastasis suppression. This was contradictory to our original hypothesis but suggested important functional requirements for BRMS1. Somewhat unexpectedly, BRMS1 interacted with SIN3A in the cytoplasm. Concomitantly, the pro-metastatic gene, miR-10b, was down-regulated by both wild-type and the NLS2,2 BRMS1 mutant protein. Taken together, these data demonstrate critical functional requirements of NLS2 in the BRMS1 protein. Moreover, the implication is that BRMS1 protein interactions in the cytoplasm may be, at least partially, responsible for metastasis suppression.The interpretation regarding cytoplasmic roles for BRMS1 is supported by recent clinical reports et al. It is clear from data published by Rivera One clone expressing the NLS1,1 mutation did not metastasize as well as the MDA-MB-231 parental cells. The simplest explanation relates to heterogeneity within the MDA-MB-231 population . The poorly metastasizing clone with NLS1,1 was most likely not metastatic to begin with. Although heterogeneity is the likely explanation, we conservatively interpret the findings as NLS1,1 does not consistently suppress metastasis. This is an important consideration and the reason why it is essential to assess multiple cell clones when analyzing tumors, especially for their capacity to metastasize Composition of SIN3 complexes dictates function Metastasis is clearly a relevant therapeutic target for patients with cancer. BRMS1 and SIN3 complexes regulate specific coding and non-coding metastasis associated genes depending on the specific composition of the complex. It follows that SIN3A:BRMS1 complexes are likely regulated by the environments surrounding cancer cells. This study has added an additional level of regulation for such complexes: subcellular localization. Although we have not identified mechanisms of trafficking between the nucleus and cytoplasm, we have identified important requirements for the metastasis suppressive function of BRMS1. These findings may help explain clinical studies of BRMS1 to further our understanding of the most deadly aspect of cancer.


We show that the binding of AtRH2 and AtRH5 helicases to the TBSV (−)RNA could unwind the dsRNA structure within the RIII(−) REN. This unique characteristic allows the eIF4AIII-like helicases to perform novel pro-viral functions involving the RIII(−) REN in stimulation of plus-strand (+)RNA synthesis. We also show that AtRH2 and AtRH5 helicases are components of the tombusvirus VRCs based on co-purification experiments. We propose that eIF4AIII-like helicases destabilize dsRNA replication intermediate within the RIII(−) REN that promotes bringing the 5′ and 3′ terminal (−)RNA sequences in close vicinity via long-range RNA-RNA base pairing. This newly formed RNA structure promoted by eIF4AIII helicase together with AtRH20 helicase might facilitate the recycling of the viral replicases for multiple rounds of (+)-strand synthesis, thus resulting in asymmetrical viral replication.Replication of plus-strand RNA viruses depends on recruited host factors that aid several critical steps during replication. Several of the co-opted host factors bind to the viral RNA, which plays multiple roles, including mRNA function, as an assembly platform for the viral replicase (VRC), template for RNA synthesis, and encapsidation during infection. It is likely that remodeling of the viral RNAs and RNA-protein complexes during the switch from one step to another requires RNA helicases. In this paper, we have discovered a second group of cellular RNA helicases, including the eIF4AIII-like yeast Fal1p and the DDX5-like Dbp3p and the orthologous plant AtRH2 and AtRH5 DEAD box helicases, which are co-opted by tombusviruses. Unlike the previously characterized DDX3-like AtRH20/Ded1p helicases that bind to the 3′ terminal promoter region in the viral minus-strand (−)RNA, the other class of eIF4AIII-like RNA helicases bind to a different  Tomato bushy stunt virus (TBSV) to aid viral replication. The authors find that eIF4AIII-like helicases bind to the replication enhancer element (REN) in the viral (−)RNA and they promote (+)-strand TBSV RNA synthesis in vitro. Data show that eIF4AIII-like helicases are present in the viral replicase complex and they bind to the replication proteins. In addition, the authors show synergistic effect between eIF4AIII-like helicases and the previously identified DDX3-like Ded1p/AtRH20 DEAD box helicases, which bind to a different cis-acting region in the viral (−)RNA, on stimulation of plus-strand synthesis. In summary, the authors find that two different groups of cellular helicases promote TBSV replication via selectively enhancing (+)-strand synthesis through different mechanisms.Genome-wide screens for host factors affecting tombusvirus replication in yeast indicated that subverted cellular RNA helicases likely play major roles in virus replication. Tombusviruses do not code for their own helicases and they might recruit host RNA helicases to aid their replication in infected cells. Accordingly, in this paper, the authors show that the yeast eIF4AIII-like Fal1p and Dbp3p and the orthologous plant AtRH2 and AtRH5 DEAD-box helicases are co-opted by  Host factors co-opted for replication of plus-stranded (+)RNA viruses are critical in each step of the well-orchestrated infection process. After translation of the viral mRNA-sense genomic RNA(s), the viral (+)RNA and the viral replication proteins together with host RNA-binding proteins (RBPs) are recruited to the site of viral replication in membranous cellular compartments. Ultimately, the process leads to the assembly of the membrane-bound viral replicase complexes (VRCs), followed by the activation of the polymerase function of the viral RNA-dependent RNA polymerase (RdRp), and initiation of complementary RNA synthesis on the viral (+)RNA template Although the roles of host factors in facilitating the replication process of (+)RNA viruses have been extensively characterized in recent years Tomato bushy stunt virus (TBSV) is a plant RNA virus with a single ∼4,800 nt genomic RNA and has two essential replication proteins, p33 and p92pol, required for TBSV replicon (rep)RNA replication in yeast (Saccharomyces cerevisiae) model host pol, and the tombusviral (+)repRNA, which serves both as a template and as a platform during VRC assembly and activation TDH2 and TDH3 in yeast) In spite of our growing understanding of TBSV replication and TBSV-host interaction, many questions remain. Indeed, multiple genome-wide screens and global proteomics approaches with TBSV using yeast as host identified ∼500 host factors, which interact with viral replication proteins or affect TBSV replication pol for initiation of (+)-strand RNA synthesis. Additional characterization of Ded1p and the similar yeast Dbp2p (similar to human p68) DEAD-box helicases revealed that these helicases play major and overlapping roles in (+)-strand synthesis The many cellular RNA helicases identified in TBSV screens are intriguing because, tombusviruses and other small RNA viruses do not code for their own helicases Arabidopsis RH2 and RH5 helicases bind to a critical replication enhancer element (REN) present in a 5′ proximal region of the TBSV minus-strand RNA. We show that these cellular helicases can locally unwind the double-stranded (ds) structure within the REN of the replication intermediate in vitro. These activities by the host helicases enhance in vitro replication and plus-strand RNA synthesis, and the accumulation of TBSV RNA in yeast and plants. We also demonstrate that AtRH2 and AtRH5 helicases work synergistically with the DDX3-like AtRH20 helicase (that binds to the 3′ promoter sequence) to facilitate plus-strand synthesis in an asymmetric manner. Altogether, these co-opted host DEAD box helicases greatly enhance TBSV replication by interacting with the viral (−)RNA and the replication proteins within the VRCs.In this paper, we characterized the novel pro-viral functions of two yeast RNA helicases, which were among those identified in previous screens, and their plant orthologs in TBSV replication. We found that the yeast Dbp3p  and Fal1p (eukaryotic translation initiation factor 4AIII-like) DEAD box helicases, which are involved in ribosome biogenesis in yeast To characterize the functions of host RNA helicases in TBSV replication, first we overexpressed 5 yeast RNA helicases in yeast and tested their effects on TBSV replicon (rep)RNA accumulation via Northern blotting . These hArabidopsis AtRH2  and AtRH5  helicases Cucumber necrosis virus, CNV, which is very closely related to TBSV) was also robust in Nicotiana benthamiana host plants when AtRH2 and AtRH5 RNA helicases were overexpressed  together with the orthologous ensified  and the ensified . Therefoin vitro RNA binding experiments with affinity-purified recombinant helicase proteins. Using four different cis-acting regions present in the TBSV (−)repRNA , which carries a well-defined RNA replication enhancer (REN) element  and Dbp2p (human p68-like) and the ortologous AtRH20 DEAD-box helicases −)repRNA , we founin vitro .cis-acting elements , . Purification of His6-tagged AtRH2 and His6-AtRH5 (using plasmids pET30-RH2 or pET30-RH5) was carried out using ProBond (Invitrogen) resin , following otherwise the same protocol as for the MBP-tagged proteins. Purified proteins were aliquoted and stored at −80°C. Proteins used for the replication assays were at least 95% pure, as determined by SDS-PAGE (not shown).Recombinant MBP-tagged helicase proteins, the MBP-tagged TBSV p33 and p92 replication proteins and several truncated MBP-tagged p33C derivatives (described earlier) were expressed in 6-tagged helicase proteins (200 µg) were loaded onto MBP columns, containing bound MBP-tagged p33C derivatives and incubated with mixing for 25 min at 4°C 6-tagged proteins by SDS-PAGE, followed by Coomassie blue staining or Western blotting with an anti-His antibody.For in vitro pull-down assay, purified His32P-labeled or unlabeled four separate regions R134 or (−)R124 were annealed to unlabeled DI-72(+) RNA in STE buffer  by slowly cooling down the samples (in 20 µl) from 94°C to 25°C in 30 min.Partial dsRNA duplexes [(−)R124/(+)DI-72 and ((−)R134/(+)DI-72] for in vitro. Purity of dsRNAs was tested with agarose gel-electrophoresis.Complete DI-72 dsRNA duplexes were prepared using Replicator RNAi kit (Finnzymes). Briefly, DI-72 (+)-strand RNA, which was synthesized with T7 transcription, was used as a template for synthesis of DI-72 dsRNA by Phi6 RNA polymerase 2, 1 mM DTT, 1 mM EDTA 5% glycerol, 6 U of RNasin and 0.1 mg tRNA in a 10 µl reaction volume. Approximately 0.1 pmol of 32P-labeled RNA probes, 0.6 µg of purified recombinant proteins and 0.15 or 0.3 µg of unlabeled RNA were used in template competition assay. For the assay, we used 0.02 µg MBP-p33C, 0.1 pmol of 32P-labeled SLR2 RNA (the stem-loop sequence from RII) EMSA was performed as described previously 32P-labeled RIII(−) or RI/II/III(−) were annealed to unlabeled DI-72 (+)RNA in STE buffer by slowly cooling down the samples (in 20 µl) from 94°C to 25°C in 30 min. 0.6 µg of purified recombinant helicase proteins (in MBP elution buffer) or MBP as a negative controls were added separately to the partial dsRNA duplex in the RdRp buffer. 2 mM of ATP was added to the reaction. Reaction mixtures were incubated for 15 min at room temperature and loaded onto 5% nondenaturing polyacrylamide gel as described previously Strand separation assay was performed as published 2), followed by loading onto 5% nondenaturing polyacrylamide gel.Some samples were treated with proteinase K after the assay. The incubation with proteinase K lasted for 10 min at 37°C using 0.5 µl of proteinase K from stock of 20 mg/ml  or “+bridge” and “Δbridge” constructs  were used as competitors in 0.1 to 0.3 µg amounts in the competition assay. After the formation of RNA–protein complexes during incubation of the reaction mixtures at room temperature for ∼15 min, we transferred the reaction mixtures to a 96-well plate on ice. To cross-link the RNA and protein, we irradiated the reaction mixtures on ice at 254 nm wave-length for 10 min using an UV Stratalinker 1800 (Stratagene). Then, we digested the unprotected RNAs by 1 mg/ml RNase A for 15 min at 37°C. Samples were mixed and boiled for 10 min in 1× SDS loading dye. Analysis was performed using SDS-PAGE and phosphorimaging The UV-cross-linking assay was performed as described − plates and then pre-grown overnight at 29°C in selective media containing 2% glucose − media containing 2% galactose and 50 µM CuSO4, yeast were grown for 36 hours in SC-ULH− media containing 2% galactose at 23°C. Pelleted yeasts (about 200 mg) were used for affinity-purification of FLAG-p33 and FLAG-p92 with anti-FLAG M2 agarose as published previously 6-AtRH2 or His6-AtRH5 proteins were detected with anti-His antibody from mouse  and AP-conjugated anti-mouse  followed by NBT-BCIP detection Yeast strain Δdbp3 was transformed with plasmids pGBK-HIS-Cup-Flag33/Gal-DI-72, pGAD-Cup-Flagp92 and pYC-Gal-6×HisRH2 or pYC-Gal-6×HisRH5 to co-express the cellular helicases with the viral replication proteins in yeast cells actively replicating the TBSV repRNA. The transformed yeast strains were selected on SC-ULHSSA1 as a positive control −/Leu2− synthetic minimal medium plates. After transformed colonies were picked with a loop and re-suspended in water, we streaked them onto TLH− (Trp2−/Leu2−/His2−) plates to test for helicase protein-p33 interactions as described The split-ubiquitin assay was based on the Dualmembrane kit3 . pGAD-BT2-N-His33, expressing the CNV p33 replication protein (bait construct), has been published earlier S. cerevisiae strain BY4741 with three plasmids: pGBK-HIS-Cup-Flag33/Gal-DI-72, pGAD-Cup-Flag92 and one of the following plasmids: pYC-His-RH2, pYC-His-RH4, pYC-His-RH5, pYC-His-RH7, pYC-His-Dbp3, pYC-His-Dbp5, pYC-His-Dbp7, pYC-His-Fal1, pYC-His-Tif1 (empty pYC plasmid was used as a control). After the selection of transformed yeast cells on SC-ULH− plates, they were pre-grown in SC-ULH− media containing 2% glucose for 24 h at 29°C. Then cells were centrifuged at 2,000 rpm for 3 min, washed with SC-ULH− media containing 2% galactose and resuspended in SC-ULH− media containing 2% galactose and 50 µM CuSO4. After growing yeast cells for 14 h at 23°C, they were used for total RNA extraction and Northern blotting and Western blotting as previously published To study the effect of over-expression of yeast and plant helicase proteins on DI-72 repRNA replication in yeast, we transformed − media containing 2% glucose at 29°C. Then yeast cells were centrifuged at 2,000 rpm for 3 min, washed with SC-ULH− media containing 2% galactose and resuspended in SC-ULH− media containing 2% galactose and 50 µM CuSO4 in the presence or absence of 1 mg/ml Doxycycline. After growing for 24 h at 23°C, and yeasts were pelleted and the replicase was purified according to a previously published procedure 2, 10 mM KCl, 0.5 M NaCl, and 1% [V/V] yeast protease inhibitor cocktail (Ypic)] and homogenized in FastPrep Homogenizer  by glass beads. After the membrane fraction was solubilized with 1 ml TG buffer containing 1% Triton X-100, 1% [V/V] Ypic, Flag-p33 and Flag-p92 were affinity purified on anti-FLAG M2-agarose affinity resin (Sigma). Replicase complex was eluted with 200 ml elution buffer [50 mM Tris–HCl [pH 7.5], 10% glycerol, 15 mM MgCl2, 10 mM KCl, 50 mM NaCl, 0.5% Triton X-100, and 0.15 mg/ml Flag peptide (Sigma)].After yeast strain TET:Fal1 was transformed with plasmids pGBK-HIS-Cup-Flag33/Gal-DI-72 and pGAD-Cup-Flag92, it was pre-grown in SC-ULH32P-labeled RNA was performed at 37°C for 30 min in a 1× RNase ONE buffer containing 0.1 µl of RNase ONE (Promega) In vitro RdRp activity assays with the purified tombusvirus replicase preparations were performed by using DI-72 (−)RNA, RI/II (−)RNA or partial dsRNA [such as (−)RI/II/IV/(+)DI-72 or [(−)RI/III/IV/(+)DI-72] or complete dsRNA templates. RNase ONE digestion to remove single-stranded pol and 200 ng purified MBP-tagged helicase proteins. The assays were performed as published 32P-labeled dsRNAs generated in the CFE assays, we prepared the RNA samples in 1× RNA loading dye (containing 25% formamide), followed by dividing the samples into two equal fractions; one half was loaded on the gel without heat-treatment, while the other half was heat-treated for RNA denaturation at 85°C for 5 min and analyzed by PAGE We prepared cell-free extract (CFE) from BY4741 or Δdbp3/ts-Fal1 yeast strains as described earlier Figure S16-tagged Dbp3p (DDX5-like), Dbp5p, Dbp7p, Fal1p (eIF4AIII-like) and Tif1p (eIF4A-like) DEAD-box helicases from plasmids. These yeast helicases have been identified in previous high throughput screens with TBSV and yeast host. The TBSV repRNA levels were normalized based on rRNA loading. Bottom panel: Northern blot analysis shows the level of ribosomal RNA loading. (B) Top panel: Detection of the overproduced His6-tagged Dbp3p, Dbp5p, Dbp7p, Fal1p and Tif1p DEAD-box helicases by Western blotting using anti-His antibody in yeast. Bottom panel: Detection of Flag-tagged p33 and p92pol by Western blotting using anti-Flag antibody. The total protein level in each sample was analyzed by SDS-PAGE and Coommassie-blue staining. Note that all the helicases expressed in yeast are His6-tagged at the N-terminus.Over-expression of selected yeast RNA helicases enhanced TBSV repRNA accumulation in yeast. (A) The wt yeast strain was used for the overexpression experiments. Top panel: Northern blot analysis of TBSV repRNA accumulation in yeast overproducing the His(PDF)Click here for additional data file.Figure S2In vitro binding assay with 0.6 µg of purified AtRH5, the yeast Dbp3p and Fal1p helicases. The assay contained the 32P-labeled DI-72 (−)repRNA (∼0.1 pmol) plus increasing amount of unlabeled competitor RNAs, including RI(−), RII(−), RIII(−) or RIV(−). The free or helicase-bound ssRNA was separated on nondenaturing 5% acrylamide gels, followed by quantification of the bound RNA by a Phosphorimager. See further details in AtRH5, and the yeast Dbp3p and Fal1p helicases bind to the RIII(−) replication enhancer element in the TBSV (−)RNA. (A–C) (PDF)Click here for additional data file.Figure S3in vitro. (A) Schematic representation of the 621 bp DI-72 RNA/RNA duplex used in the tombusvirus replication assay. (B) Representative denaturing gel of 32P-labeled RNA products synthesized in vitro using DI-72 RNA/RNA duplex template by the purified tombusvirus replicase obtained from yeast with depleted Fal1p in the presence of 0.4 µg of purified recombinant cellular helicases (except 1.0 µg in case of AtRH20) is shown. Note that lanes 1–6 show samples from the in vitro replication assays with the combination of two cellular helicases  in the presence of purified AtRH5 or AtRH2. The 32P-labeled RNA template was RII(+)-SL (∼0.1 pmol), which is the p33RE [part of RII(+)], and binds selectively to p33. The assay contained 0.02 µg of purified recombinant MBP-p33C, plus 0.02, 0.06, 0.2 or 0.6 µg of purified recombinant AtRH5 or AtRH2, as shown. The samples in lanes 6 and 13 contained 0.6 µg of purified recombinant AtRH5 or AtRH2 in the absence of p33C.AtRH2 and AtRH5 do not inhibit the binding of p33 replication protein to the TBSV (+)RNA. (A) Scheme of the in vitro TBSV (+)RNA binding assay. (B) (PDF)Click here for additional data file.


When KM-Luc/GFP malignant fibrous histiocytoma-like cells stably expressing the firefly luciferase gene were injected into the SiLN, metastasis could be detected in the proper-ALN within 3 to 9 days, using in vivo bioluminescence imaging. The metastasis route was found to be via the efferent lymphatic vessels of the SiLN, and metastasis incidence depended on the number of cells injected, the injection duration and the SiLN volume. Three-dimensional contrast-enhanced high-frequency ultrasound imaging showed that the blood vessel volume and density in the metastasized proper-ALN significantly increased at 14 days after tumor cell inoculation into the SiLN. The present metastasis model, with lymph nodes similar in size to those of humans, has potential use in the development of ultrasound imaging with high-precision and high-sensitivity as well as other imaging modalities for the detection of blood vessels in lymph nodes during the progression of metastasis.Animal studies of lymph node metastasis are constrained by limitations in the techniques available for noninvasive monitoring of the progression of lymph node metastasis, as well as difficulties in the establishment of appropriate animal models. To overcome these challenges, this study has developed a mouse model of inter-lymph-node metastasis via afferent lymphatic vessels for use in the development of imaging modalities. We used 14- to 18-week-old MRL/MpJ−/ Human lymph nodes are round or kidney-shaped organs of the immune system, 2 to 20 mm in diameter, that are distributed throughout the body The two major transplantation models of metastasis currently used are referred to as the spontaneous and the experimental metastasis models However, the spontaneous metastasis model is limited by its poor performance for the dynamic quantitative assessment of the progression of lymph node metastasis using non-invasive approaches. This is maybe because both the latency period and the initial site of the metastasis vary considerably, and also because conventional imaging modalities are unable to identify changes in the internal structure of lymph nodes, of conventional mice, with a diameter of 1–2 mm in vivo bioluminescence imaging and three-dimensional contrast-enhanced high frequency ultrasound imaging.This study describes the development of a mouse model of lymph node metastasis via the afferent lymphatic vessels, which is suitable for use in the development of imaging modalities for dynamic quantitative assessment of metastatic progression. We used MRL/lpr mice developing systemic lymphadenopathy within 3 to 4 months of birth in vivo studies were carried out in strict accordance with the recommendations in the Guide for Proper Conduct of Animal Experiment and Related Activities in Academic Research and Technology, 2006. The protocol was approved by the Institutional Animal Care and Use Committee of Tohoku University . All surgery was performed under sodium pentobarbital anesthesia, and all efforts were made to minimize animal suffering.All gld/gld malignant fibrous histiocytoma-like (MRL/N-1) cells l-glutamine-penicillin-streptomycin  and 1% Geneticin G418 . Before conducting the metastasis experiments, the absence of mycoplasma contamination in the cell cultures was ensured by testing with a mycoplasma detection kit .KM-Luc/GFP cells, which stably express a fusion of the luciferase (Luc) and enhanced-green fluorescent protein (EGFP) genes, were prepared by transfection of MRL/MpTn-MRL/lpr mice, which develop systemic lymphadenopathy 4 cells injected in 1 min (n = 4); Group 2: 5×104 cells injected in 1 min (n = 4); Group 3: 1×105 cells injected in 1 min (n = 4); Group 4: 1×105 cells injected in 2 min (n = 6); and Group 5: 1×105 cells injected in 3 min (n = 21). PBS (30 µL) was injected into the SiLN as a negative control . Metastasis to proper-ALNs was analyzed using an in vivo bioluminescence imaging system  The metastasis model was developed by injecting KM-Luc/GFP cells, suspended in 30 µL PBS, into the SiLN as follows: Group 1: 1×10On day 14 post-injection of KM-Luc/GFP cells, luciferin was intraperitoneally injected into each mouse, and 10 min later, the mice were sacrificed by ether inhalation. The ALNs (proper and accessory) and SiLN were surgically removed, and immediately placed in 6-well plates. Bioluminescence was then quantified for 30 s using the IVIS.ex vivo observation of bioluminescence, harvested lymph nodes were fixed overnight in 18.5% formaldehyde in phosphate-buffered solution (PBS), dehydrated, and embedded in paraffin. The embedded specimens were cut into 4-µm-thick serial sections and either stained with hematoxylin and eosin (H&E), or immunostained for luciferase after antigen retrieval. For the latter procedure, sections were incubated for 10 min at 37°C with 0.1% trypsin in Tris buffer, and blocked with 10% rabbit serum  for 10 min at room temperature (RT) to reduce non-specific protein binding. To detect luciferase protein, sections were incubated overnight at 4°C with a goat polyclonal anti-luciferase antibody . Immunoreactivity was detected using biotinylated polyclonal rabbit anti-goat immunoglobulin  for 30 min at RT, peroxidase-conjugated streptavidin (Nichirei Bioscience Inc.) for 30 min at RT, and diaminobenzidine (DAB) as a chromogen in accordance with the manufacturer’s protocol.Following n = 4) or PBS . Tissues were weighed, frozen in liquid nitrogen, homogenized with reporter lysis buffer (Promega Co.), and centrifuged at 12,000×g for 2 min at 4°C. Supernatant samples (10 µL each) were examined for luciferase activity using 50 µL luciferase assay reagent containing D-Lucifer (Promega Co.). Bioluminescence was measured at RT for 10 s using a luminometer . Results are presented in arbitrary units (AUs).To determine luciferase activity in the organs post-inoculation, the inoculated SiLN, the ipsilateral proper- and accessory-ALNs, ipsilateral mandibular lymph node, ipsilateral lumbar aortic lymph node, caudal mesenteric lymph node, contralateral proper-ALN, contralateral SiLN, liver and lungs were surgically removed on day 14 post-inoculation of KM-Luc/GFP cells ; an infrared photodynamic camera ; or a high-speed fluorescence video camera system  comprising a high-power xenon light source  and a charge coupled device (CCD) camera  with a lens . The IVIS emitted and detected light at wavelengths of 710 to 760 nm and 810 to 875 nm, respectively, whereas the PDE emitted light at 760 nm and detected light at wavelengths greater than 820 nm. After injection of indocyanine green  n = 1) or 0.5 to 3.0 mL/h , using an infusion pump . After the PDE had been used to investigate the effect of injection speed into the SiLN on the incidence of metastasis to the proper-ALN , the HS-FVCS was used to determine whether metastasis from the SiLN to the proper-ALN had occurred via the veins or the afferent lymphatic vessels (n = 2). Under anesthesia, an arc-shaped incision was made in the abdominal skin from the subiliac to the proper axillary region, and 30 µL of 1.8 µg/µL fluorescein isothiocyanate-labeled bovine serum albumin  was then injected into the SiLN at 1.0 mL/h by means of an infusion pump. A high-power xenon light source with an appropriate filter  was used to deliver excitation light at a wavelength of 490±2 nm, and a CCD camera with an appropriate filter  was employed to detect light at a wavelength of 510±2 nm.After anesthetization via intraperitoneal injection of 5% sodium pentobarbital , visualization of the metastatic route was performed using at least 1 of 3 imaging systems: an n = 1), 30 µL Indian ink was injected into the SiLN, and 10 min later, the skin from the subiliac to the proper axillary region, including the veins and afferent lymphatic vessels, was surgically removed and dissected, and the tissue stained with H&E to observe the cross-sectional surface.To confirm the route of metastasis histologically (12 bubbles/mL Acoustic liposomes (ALs) were used as ultrasound contrast agents, and were prepared as described previously 5 KM-Luc/GFP cells in 30 µL PBS  or PBS only , metastasis to the proper-ALN was confirmed by bioluminescence imaging on days 0, 9 and 14. On the same days, a 3D vascular image of the proper-ALN was reconstructed using ALs and a high-frequency ultrasound imaging system  with a 35-MHz center-frequency transducer  at 100% transmittance power. Mice anesthetized with 2% isoflurane were placed on a stage maintained at 38°C  during the scanning period. An ultrasound transmission gel  was placed on the proper-ALN, the focal length of the transducer adjusted using a stage control system , and the working distance of the scan determined by moving the transducer over the proper-ALN using the stage control system.To reveal changes in the angiogenic vessel density within the metastasized proper-ALNs, contrast-enhanced high frequency ultrasound (CE-HFUS) with ALs was used. Subsequent to injection of the SiLN, over a 1 min period, with either 1×10n = 4) in vivo AL half-life of 284±13 s . Multiple 2D images of the proper-ALNs were then captured over a period of 156±12 s , shorter than that of the in vivo half-life of the ALs, and blending algorithms were used to convert these 2D slices into 3D volumetric images. Blood vessel structures were mapped and reconstructed by tracking the flow of ALs through the vessels, using the software provided with the VEVO 770 system. Regions of interest were drawn manually on every 2D image along the lymph node margins. The blood vessel volumes and densities within the proper-ALNs were calculated using VEVO770 contrast-mode software. Extracted 2D slices were obtained for analysis at 61±5 s .Generating a 3D dataset requires assessment of multiple 2D ultrasound images that are then reconstructed to achieve the desired 3D image. In our previous experiments in vivo and ex vivo bioluminescence results were determined using Spearman’s rank correlation coefficient test. Comparisons of metastasis incidence between the various inoculation conditions were performed using Fisher’s exact probability test. A P value less than 0.05 was considered to be an indication of statistical significance. Statistical analyses were performed using Excel 2007  with Statcel2 software.All measurements are presented as either the mean ± SD or mean ± SEM. Differences between groups were determined by two-way ANOVA followed by the Tukey-Kramer test. Correlations between l, by the duration of time that had elapsed post-injection , and the flow was observed with the IVIS  and PDE in vivo bioluminescence signals were detected in the ALNs within 3 to 9 days, but were not evident in other organs until day 14 , the luciferase activity was found to be at background levels.Using IVIS, l day 14 a. Ex vivper-ALNs b, but nooper-ALN . To furtN n = 4; . The res4 to 1×105 cells), with all 4 mice examined exhibiting metastasis (an incidence of 100%) upon injection of 1×105 cells (5) and injection duration (3 min) were held constant. Specifically, the incidence of metastasis was found to be 100% at SiLN volumes less than 100 mm3, but significantly reduced at volumes greater than 150 mm3  was found to be not statistically significant, there appeared to be a tendency toward greater metastasis with injections of shorter duration b. To con n = 39; .n = 7) a time period shorted than the in vivo half-life of the ALs  . The period of time over which the images were captured may therefore be sufficiently short to avoid detection of leakage of ALs from vasculature of the proper-ALNs P<0.05); blood vessel density increased from 1.00 (day 0) to 1.01±0.07 (day 9) and 1.24±0.03 (day 14) (P<0.05). On day 14, both blood vessel volume (P<0.05) and blood vessel density (P<0.01) were significantly higher than in the negative control group.CE-HFUS enables real-time reconstruction of co-registered sections into a 3D image that reflects tumor vascularity /lpr mice lpr) mutant gene have defects in the Fas gene gld  gene, a missense mutation in the Fas ligand gene The present study has developed a novel model of lymph-node metastasis, based on the injection of KM-Luc/GFP cells into the SiLNs of MRL−3 min−1 values greater than 4.72×102  metastasis achieved using cells mm4.72×102 . These fThe American Joint Committee on Cancer (AJCC) classifies lymph node metastases on the basis of histological results; isolated tumor cells (cell clusters with diameters ≤0.2 mm), micrometastases (0.2 mm<tumor cell clusters ≤2 mm) and macrometastases (tumor cell clusters >2 mm) Currently, a lymph node size greater than 10 mm in the short-axis diameter, determined by CT and MRI, is considered to be the most important radiologic criterion for lymph node metastasis 3 in volume, it cannot rely on the diffusion of metabolites to meet its needs and therefore must recruit and develop new vessels through the well-known process of angiogenesis As angiogenesis has been found to be an important marker of cancer progression The intravital imaging systems currently used in preclinical cancer research employ magnetic resonance imaging (MRI) at a resolution of 10 to 100 µm, X-ray computed tomography imaging (CT) at a resolution of 50 µm, ultrasound imaging at a resolution of 50 µm, and positron emission tomography at a resolution of 1 to 2 mm In conclusion, we have established a mouse model of inter-lymph node metastasis via afferent lymphatic vessels, using mice exhibiting remarkable systemic lymphadenopathy, with the proper-ALNs and SiLNs 6 to 12 mm in diameter (similar in size to human lymph nodes). This model may be used for the development of highly functionalized, high-resolution ultrasound as well as other imaging modalities for the detection of blood vessels in lymph nodes during metastatic progression.Figure S1Malignant fibrous histiocytoma (MFH)-like cells.(TIF)Click here for additional data file.Video S1Visualization of the route of lymph node metastasis.(AVI)Click here for additional data file.


We aimed to better discriminate metastasized (lymphogen/occult/both combined) from non-metastasized seminoma based on post-transcriptional changes examined in the peripheral blood.Total RNAs including small RNAs were isolated from the peripheral blood of patients suffering from metastasized testicular tumours  and non-metastasized patients . Small RNA next generation sequencing  was employed to examine post-transcriptional changes. We searched for small RNAs showing at least 50 reads and a significant ≥ 2-fold difference using peripheral blood small RNAs of non-metastasized tumours as the reference group. Candidate small RNAs were examined in univariate logistic regression analysis and combinations of two small RNAs were further examined using support vector machines.7, 1.2x107 and 1.2x107 small RNA reads were detectable in non-metastasized, lymphogen and occult metastasized seminoma, respectively of which 73-76% remained after trimming. From these between 80-82% represented annotated reads and 7.2-7.8% (1.6-1.7x104) were annotated small RNA tags. Of them 137 small RNAs showed > 50 reads and a ≥ two-fold difference to the reference. In univariate analysis we detected 33-35 different small RNAs which significantly discriminated lymphogen/occult/combined metastasized from non-metastasized seminoma and among these different comparisons it were the same small RNAs in 44-79%. Many combinations of two of these small RNAs completely discriminated metastasized from non-metastasized seminoma irrespective of the metastasis subtype.On average 1.3x10Metastasized (either lymphogen or occult) seminoma can be completely discriminated from non-metastasized seminoma with a combination of two small RNAs measured in the peripheral blood. Testicular tumor, as the most common tumor in young men, is associated with a 5 year survival rate close to 100% in early stages. Pure seminoma are the most frequent histological subtype (55%) nowadays and more than 70% of patients present without visible metastasis at primary staging ,3. UntilRecently, other authors started to examine whether a certain set of micro RNAs (miRNA) might be suitable for discriminating between seminoma bearing patients and healthy persons -8. ExpreThe average age at diagnosis was 39.1 (+/- 7.2) years for non-metastasized, higher  for lymphogen metastasized and lower  for occult metastasized seminoma. Primary tumor size was comparable between lymphogen and occult metastasized seminoma , but smaller (23.8 mm) in non-metastasized seminoma (Table Per 2.5 ml whole blood we isolated about the same amount (mean) of total RNA from seminoma patients without metastasis , lymphogen metastasis  and occult metastasis  representing annotated small RNAs. Of them 137 small RNAs showed > 50 reads and a two-fold difference to the reference. In univariate analysis we identified 33/34 and 35 small RNA species which significantly discriminated lymphogen/occult metastasized and both metastasis subtypes combined from non-metastasized seminoma, respectively  from non-metastasized seminoma based on small RNA changes examined in the peripheral blood. A whole genome screening on small RNA species was performed employing NGS. We demonstrated each metastasis subtype and its combination to be completely discriminated from non-metastasized seminoma using two small RNAs combined.MicroRNAs are previously described to be involved in different distant  and local (e.g. changes in tumor microenvironment) processes in metastatic cascade. Interestingly, the microRNAs identified in our study are involved in local tumour microenvironmental changes only. For instance, members of the Let-7 family are involved in inflammatory processes as a part of the metastatic cascade. The Let-7 family targets oncogenes such as HMGA2 and KRAS and seem to be a key component in a so called epigenetic switch -15. CancRecently other authors demonstrated that microRNA 371-73 cluster and microRNA 302 discriminated seminoma bearing patients from healthy persons -8. We diThe complete separation of metastasis from non-metastasized seminoma using a combination of two small RNA species points to the significant diagnostic potential of these biological marker which is in line with previous examinations on the transcriptional level showing the superiority of molecular marker over epidemiological or clinical-histological parameter ,19. AfteInteresting, the discrimination in our study occurred irrespective of the metastasis subtype and with the same microRNAs in up to 79%. This was expected, since we recently demonstrated lymphogen and occult metastasized seminoma to be indistinguishable on the transcriptional and post-transcriptional level (revised version submitted).Noteworthy, these changes were examined in the peripheral blood of seminoma patients taken at the time of the primary tumour’s diagnosis. It can be hypothesized that our measurements are linked to seminoma tumour cells circulating in the peripheral blood.Our study has certain weaknesses such as the low number of cases examined . However, the molecular biological methodology applied provides a deepness, which under financial considerations does not allow large scale studies. Hence, this study provides hints towards certain small RNA species comprising a significant diagnostic potential for prediction of seminoma metastasis based on examinations in the peripheral blood, but certainly these candidate small RNA species have to be examined on a larger independent group for validation purposes. The validation should also include haematogenous metastasized seminoma , not included in this study, to prove the usability in this tumor stage as well. Furthermore, we did not adjust the p-values for multiple comparisons, because of the explorative character of this study and the low sample size. Based on experiences with previous work we expect about 10-20% of our candidates being false positives/negatives [Metastasized (either lymphogen or occult) seminoma can be completely discriminated from non-metastasized seminoma with a combination of two small RNAs measured in the peripheral blood.Non-metastasized seminoma (n = 5) received no adjuvant treatment and were free of relapse/progress for at least two years of follow up. Occult metastasized patients (n = 5) presented without visible metastasis at primary staging, received no adjuvant treatment, and developed retroperitoneal tumour progress during the follow up of 12 month. For patients with detectable metastasis at primary staging (n = 5) we focused on clinical stage IIb and IIc, to include lymphogenic metastatic spread only and to provide a high level of diagnostic accuracy (avoiding doubtful lymph nodes). Lymphogen metastasis and non-metastasized patients were matched with occult metastasized patients considering demographic and histological parameters where applicable as well as quality criteria for isolated RNA (Table All testicular tumours n = 15) were examined by an experienced pathologist for histological and TNM classification (Table 5 were exWhole blood samples (2.5 ml) were taken intraoperative from cubital vein directly into the PAXgene Blood RNA system . The tube was gently inverted (10 times), settled at room temperature overnight and stored at -20° until use. The Ethical Review Committee of the Medical Association Hamburg approved the study and all human samples were obtained with written informed consent.After thawing the PAXGene tubes, and washing and centrifugation of the samples the cells became lysed (Proteinase K) followed by adding Lysis/Binding Solution taken from the mirVana Kit . With the mirVana kit total RNA including small RNA species was isolated by combining a Phenol-Chloroform RNA precipitation with further processing using a silica membrane. After several washing procedures DNA residuals became digested on the membrane , washed, RNA was eluted in a collection tube and frozen at -80°C. Quality and quantity of isolated total RNA were measured spectrophotometrically . RNA integrity was assessed by the 2100 Agilent Bioanalyser  and DNA contamination was controlled by conventional PCR using actin primer.For analysis, we used only RNA specimens with a ratio of A260/A280 ≥ 2.0 (Nanodrop) and RNA integrity number (RIN) between 6.0-9.3 for small RNA Next Generation Sequencing . For RNA samples with RIN below 7.0 or questionable 28/18S bands (indications of RNA degradation) we performed additional checks to exclude the presence of RNAses and true RNA-degradation.http://www.mirbase.org/) and known non-coding RNAs . Small RNAs showing a significant and ≥ 2-fold differences in gene expression among groups and at least 50 reads were further analysed on their ability to separate both groups using univariate logistic regression analysis [We performed a genome wide small RNA sequencing using the SOLiD5500xl Next Generation Sequencing Technology . In brief, total RNA was purified (PureLink microRNA isolation Kit), enriched small RNAs were ligated to SOLiD adaptors, reverse transcribed (SOLiD RT primer and ArrayScript RT), cDNA was purified , a cut-off size of 60-80 nt was selected , cDNA was in-gel amplified and samples were barcoded using SOLiD 3′Barcode primer at the same time. Amplified cDNA was purified  and used in emulsion PCR (SOLiD EZ Bead). Thereafter, the emulsion was broken to recover enriched beads and the so-called di-base probes were used by the SOLiD system in the sequencing-by-ligation procedure. Beside the SOLiD5500xl inherent software (LifeScope) used for image and signal processing, software CLC Genomics Workbench 5.1 (CLC bio) was used for clustering, counting, and annotation of all generated 75 bp reads. After discarding reads without adaptor sequence and being shorter than 15 bp (trimming) reads were assigned to known microRNAs (Sanger miRBase release 18, analysis  and combbp: Base pair; cDNA: Copy deoxyribonucleic acids; cS: clincal stage; CT scan: Computed tomography; DNA: Deoxyribonucleic acids; miRNA: micro ribonucleic acid; NGS: Next generation sequencing; qRT-PCR: Quantitative real time polymerase chain reaction; RNA: Ribonucleic acid; RIN: RNA integrity number; TNM-Classicfication: Tumor nodus metastasis classification.The German Ministry of Defence supported this work. All authors declare that they have no competing interests.CR has developed the study design, acquisition and interpretation of data and drafted the manuscript, DD has substantially contributed in the acquisition of data, MP and H-US have substantially contributed in the study design, interpretation of data and in revising the manuscript, WW and CM have made substantial contributions to acquisition of data and in revising the manuscript, BM-M has performed the statisitcal analysis of data and revised the manuscript, VM has made substantial contributions to technical procedures, data analysis and revised the manuscript, MA has developed the study design, supervised all technical/laboratory procedures and quality checks, analysis and interpretation of data and drafted the manuscript. All authors read and approved the final manuscript.


Basidiomycota, especially at the RNA level, suggesting that these important and potentially beneficial fungi are not affected by the plant cultivars nor by GM traits (Bt toxin production). Our results suggest that even though DNA- and RNA-derived soil fungal communities can be very different at a given time, RNA composition may have a predictive power of fungal community development through time.We assessed soil fungal diversity and community structure at two sampling times (t1 = 47 days and t2 = 104 days of plant age) in pots associated with four maize cultivars, including two genetically modified (GM) cultivars by high-throughput pyrosequencing of the 18S rRNA gene using DNA and RNA templates. We detected no significant differences in soil fungal diversity and community structure associated with different plant cultivars. However, DNA-based analyses yielded lower fungal OTU richness as compared to RNA-based analyses. Clear differences in fungal community structure were also observed in relation to sampling time and the nucleic acid pool targeted (DNA versus RNA). The most abundant soil fungi, as recovered by DNA-based methods, did not necessary represent the most “active” fungi (as recovered via RNA). Interestingly, RNA-derived community compositions at t1 were highly similar to DNA-derived communities at t2, based on presence/absence measures of OTUs. We recovered large proportions of fungal sequences belonging to arbuscular mycorrhizal fungi and  Numerous soil processes are primarily, and some even exclusively, carried out by soil fungi. Two of the most important processes are the facilitation of nutrient uptake and degradation of crop remains Recently, methodological advances have allowed enhanced screening of soil microbial communities, revealing high diversity and temporal turnover based on sequencing of 18S rRNA gene variants Bacillus thuringiensis toxin coding gene introduced in maize) crops on arbuscular mycorrhizal fungi (AMF). Some of these studies have reported no consistently significant impacts on AMF In this study, we examined the potential impact of four maize plant cultivars, consisting of two genetically modified cultivars (GM) and two near-isogenic non-GM cultivars, on soil-borne fungal communities in a pot-based experiment. GM plants were incorporated into the study to address concerns that some transgenic crops may adversely affect soil processes, including important groups of beneficial soil fungi. To date, most studies on this topic have been focused on the effects of Bt  by 454 pyrosequencing, and the relative impacts of plant cultivar, plant age and nucleic acid pool were assessed. A previous study using the same system Zea mays L.) cultivars used in this study consisted of “Monumental MON810” (GM event MON810) and “DK 3421YG” (GM event MON810) and two near isogenic non-GM cultivars of these lines . For brevity, different cultivars are abbreviated by letters, Monumental = M, DKC = D, and GM cultivars are indicated with “GM” . Both GM cultivars had been transformed to express the CryIAb gene .The four different maize (Trifolium pratense L. and Lolium perenne L.), which had been sown after maize in the autumn of 2007 and mown twice a year. Soil chemical properties were pH (CaCl2-extractable) = 5.8, P (CaCl2-extractable) = 5.1 mg kg−1, N = 1.36 g kg−1, OM = 1%. In each pot , one of four different maize (Zea mays L.) cultivars was grown.Intact pot-size soil cores from an organically managed agricultural field were transferred to pots in order to maintain natural stratification and integrity of fungi inhabiting the soil. No specific permits were required for this field sampling. This field is not a protected area and does not involve endangered or protected species. Soil cores were collected randomly from within a homogeneous 10×10 m plot in September 2009. The standing crop was a grass-clover mixture . One seedling was kept per pot after two weeks. Hoagland solution  was applied twice during the first month of plant growth. On November 24th, after 47 days (t1) of plant growth, soil samples were taken in the following way: one core (diameter 1 cm) per pot was taken and the part originating from 5–11 cm depth was immediately transferred to dry ice and subsequently stored at −80°C. Cores were taken 5–6 cm from the edge, which was approximately halfway between the edge of the pot and the stem of the plant. On January 20th 2010, after 104 days (t2) of growth, samples were again taken as above, but the position of cores was shifted 45° in relationship to the first core to minimize potential disturbances from the first sampling event. At the end of the experiment , total above- and belowground plant biomass was harvested.The experimental design was 2 GMs×2 non-GMs×3 replicate pots×2 sampling times×2 types of nucleic acids. Two seeds were sown into each pot on October 1RNA and DNA were simultaneous extracted from two grams of fresh bulk soil (soil without plant roots) per sample using the RNA PowerSoil kit and the DNA Elution accessory kit . The DNA from total RNA samples was digested by DNase I (RNase-free DNase set Qiagen 79254) according to manufacturer recommendations. The total RNA was measured with a ND-1000 spectrophotometer , and the quality of the total RNA was checked with Experion (BioRad). The cDNA was synthesised from the total RNA using random hexamer primers and the superscript double-stranded cDNA synthesis kit  exactly as described in Verbruggen et al. 2012.Glomeromycota . Thermocycling conditions were: denaturing at 94°C for 30 s , and every two cycles the annealing temperature was lowered by 2°C until 47°C was reached, which was the annealing temperature used for the final twenty cycles . Extension conditions were 68°C for 2 min. for all cycles. Reactions contained about 25 ng of DNA or cDNA template added to a standard PCR mix. Four PCR reactions of 25ul each per biological replicate of a sample were carried out. Primers contained 12 different multiplex tags for sample identification were sequenced in 1/8 lane plate of Roche 454 automated sequencer and GS FLX system using titanium chemistry . Sequence analysis was performed using QIIME 1.2.1 scripts DNA and cDNA templates derived from each of the soil pots at t1 = 47 days and t2 = 104 days of plant age were utilized for nucleic acid amplification and subsequent 454 pyrosequencing. In these procedures, a fungal-specific primer set was used, consisting of the FR1 primer After denoising, chimera detection and removal of non-fungal reads , a total of 60,845 reads were used for analyses. The average length of reads of fungal 18S rRNA was 375 bp. The numbers of reads of fungal DNA-based ranged from 1,025–6,970 while from RNA-based ranged from 2,448–9,000. Although we sought to mix PCR products in equal amounts prior to sequencing, product amounts were quantified using a spectrophotometer. Such measures may not be entirely accurate, and may have caused undesired variations in template amounts and subsequently sequence numbers.In order to compare samples, we discarded singletons and rarefied the OTU table to the lowest numbers of reads obtained for any single sample. Doing this, we ended up with 906 reads per sample, yielding between 76 and 97 OTUs per sample, and OTU coverage ranged from 92 to 97% as determined by Good’s coverage estimator . OTU numFungal diversity (Shannon), evenness and richness (Chao-1) indices per plant cultivar were not different . SimilarDifferences were detected in soil fungal community composition associated with the different plant genetic backgrounds examined in the experiment. There is a significant effect of plant cultivar in DNA-based analysis for sampling time t2 and no significant effect of GM for both DNA and RNA-based analysis at the same sampling time t2 . There iWhen OTU occurrence between sampling times was compared using a presence/absence measure of similarity (Jaccard), a very clear separation of treatments was observed. All nucleic acid type/time combinations formed distinct clusters, with the exception of the RNA-based OTUs at sampling time t1 and the DNA-derived OTUs at sampling time t2, which grouped very close together and were overlapping . MoreoveAscomycota was the most abundant phylum independent of the sampling time, while in the RNA-based fungal community, Basidiomycota was the most abundant phylum for both time points. The Glomeromycota were more abundant in the RNA-derived datasets, and increased in relative abundance with plant age (t2)  .Chytridiomycota, Ascomycota  and Glomeromycota  were significantly higher in the DNA-derived dataset, while the relative abundance of Basidiomycota  was higher in the RNA dataset , environmental  dataset .Glomeromycota  significantly increased with plant age, while members of the Ascomycota  decreased (Ascomycota (Dothideomycetes) and Glomeromycota  significantly increased with plant age, while Ascomycota (Sordariomycetes) and Basidiomycota  significantly decreased , Basidiomycota  and Glomeromycota  were significantly different between DNA and RNA-based analysis  and Glomeromycota  were significantly different between DNA and RNA-fungal community based analysis (At the same time point of sampling (t1 or t2), the relative abundances of fungal groups based on DNA and RNA analyses were not the same. At sampling time t1, members of the analysis . Also, aanalysis .Despite having to restrict our analysis to relatively small numbers of sequence reads per sample, we still recovered 92 to 97% of the total OTU-level fungal diversity in the soil samples studies as determined by Good’s coverage estimator. The numbers of OTUs obtained in this study were rather low compared to other soils i.e. forest soils Under the greenhouse conditions used in this study, no differences in soil fungal diversity (Shannon), evenness and richness (Chao-1) indices could be detected with respect to the GM nature of the plant. However, there was a difference in fungal richness depending on the type of nucleic acid (DNA or RNA) and the time point of soil sampling. The fungal community assessed by DNA-based analysis was richer than fungi community assessed by RNA-based analysis.Glomus-like fungi, as determined by PCR-DGGE community profiling. Although these authors did observe differences between GM plants and their nearly isogenic parental lines, these differences were not greater that those observed between different non-GM cultivars. These previous studies have zoomed in specifically on AMF, yet relatively few studies have examined complete fungal community responses to GM plants i.e. the t1 RNA-based community appears to foreshadow the t2 DNA-based community). It should be noted, however, that we did not examine any samples in the intervening time between the two sampling points, and additional observations and time points would be necessary to confirm the time-scale and generality of this relationship between RNA versus DNA template pools.The fungal community structure was also not affected by non-GM and GM plants. This result is in agreement with previous studies that have examined the colonization and community structure of mycorrhizal fungi in response to GM lines of maize i.e. 18S rRNA content) may be differentially correlated with activity for different species Although DNA and RNA clearly give rise to potentially complementary windows of observation, it should be kept in mind that ribosome number (In conclusion, soil fungal community proved to be dynamic with changes over time across nucleic acid pools, but not revealing any detectable impact of maize cultivar or the GM nature of the plant.Nucleotide sequences were deposited in GenBank-SRA under the Study Accession No. ERP002065 for agriculture soil samples.


Apis mellifera (Hymenoptera: Apidae), as an example, the electrophoretic rRNA profile of insects is explained. This profile differs significantly from the standard benchmark since the 28S rRNA of most insects contains an endogenous “hidden break.” Upon denaturation, the masking hydrogen bonds are disrupted, releasing two similar sized fragments that both migrate closely with 18S rRNA. The resulting rRNA profile thus reflects the endogenous composition of insect rRNA and should not be misinterpreted as degradation.The integrity of extracted ribonucleic acid (RNA) is commonly assessed by gel electrophoresis and subsequent analysis of the ribosomal RNA (rRNA) bands. Using the honey bee,  Gene expression studies employing methods such as RT-PCR, quantitative RT-PCR, and microarrays are now routinely conducted in many areas of molecular biology. An essential prerequisite for these studies is successful extraction of the ribonucleic acids (RNA). Extracting RNA is generally complicated by its high lability caused by presence of ribonucleases (RNases) within the sample as well as in the environment. For this reason, RNA degradation can be a significant issue, and thus the integrity of the extracted RNA is usually determined before its application in down-stream procedures.Gel electrophoresis and inspection of the prominent bands of the highly abundant ribosomal RNAs (rRNAs) is the most common method for assessing the integrity of extracted RNA. Analyzed in particular are the large rRNAs, designated 16S and 23S in prokaryotes and 18S and 28S in eukaryotes. An intact sample is generally defined as displaying clear and distinct bands for both these large rRNA species. This is based on the assumption that rRNA integrity reflects the integrity of the other fractions of RNA, for example the mRNA fraction in the case of expression studies. While rRNA integrity may not necessarily be an accurate measure of mRNA quality, it is certainly useful as a readily available indicator of the general state of the purified RNA.Apis mellifera (Hymenoptera: Apidae), as obtained via electrophoresis with the Agilent 2100 Bioanalyzer, is reported here. Using these results as an example, the electrophoretic rRNA profile of insects in general is detailed. A reminder and a short review explaining whythis profile differs significantly from the standard rRNA integrity benchmark is provided.The rRNA profile of the honey bee, A. mellifera RNA was obtained from brains of forager bees collected into liquid nitrogen. The brains were dissected frozen on a precooled aluminium block (-80° C) surrounded by dry ice. Single brains were then homogenized in 320 µl RLT lysis buffer  using a rotor-stator homogenizer. Subsequently, total RNA was extracted via silica-matrix spin columns (QIAGEN RNeasy Mini Kit) including an on-column DNase I treatment , both according to the manufacturer's instructions. For quality and integrity analysis, A. mellifera brain RNA (70 ng) was electrophoretically separated with an Agilent 2100 Bioanalyzer using an RNA 6000 Nano Chip Kit. When RNA was heat-denatured prior to separation (as recommended), RNA was incubated at 70° C for 2 min.A. mellifera, extracted RNA was analysed for integrity via gel electrophoresis. To this end, an Agilent 2100 Bioanalyzer was used. This instrument is one of the recent microfluidic capillary electrophoresis systems that have become the current gold standard for RNA quality assessment. Separation is based on a non-denaturing gel matrix; however, samples are usually heat-denatured prior to separation  in the entation . Analyseentation .A. mellifera (Hymenoptera)   display noptera) . Furthernoptera) . SurprisCtenomys .A. mellifera , disruption of these hydrogen bonds occurs, and the two fragments co-migrate with the 18S rRNA. The 5.8S rRNA is also base-paired to this 28S complex and is likewise released in denaturing conditions. Therefore, the typically observed insect rRNA profile reflects endogenously present components of the insect rRNA rather than degradation during the extraction process. A lack of awareness of the rRNA composition in insects has led to poor interpretation of insect rRNA profiles both in daily experiments as well as in publications.


Alu elements , as well as the transcriptional levels of the ADAR1 enzymes. This analysis was performed on five fetal and adult human tissues: brain, heart, liver, kidney, and spleen, as well as on human embryonic stem cells (hESCs), which represent the blastocyst stage in early human development. Our results show substantially greater editing activity for most adult tissue samples relative to fetal ones, in six of the eight genes tested. To test the effect of reduced A-to-I RNA editing activity in early human development we used human embryonic stem cells (hESCs) as a model and tried to generate hESC clones that overexpress the ADAR1–p110 isoform. We were unable to achieve overexpression of ADAR1–p110 by either transfection or lentiviral infection, though we easily generated hESC clones that expressed the GFP transgene and overexpressed ADAR1-p110 in 293T cells and in primary human foreskin fibroblast (HFF) cells. Moreover, in contrast to the expected overexpression of ADAR1-p110 protein following its introduction into hESCs, the expression levels of this protein decreased dramatically 24–48 hr post infection. Similar results were obtained when we tried to overexpress ADAR1-p110 in pluripotent embryonal carcinoma cells. This suggests that ADAR1 protein is substantially regulated in undifferentiated pluripotent hESCs. Overall, our data suggest that A-to-I RNA editing plays a critical role during early human development.Post-transcriptional events play an important role in human development. The question arises as to whether Adenosine to Inosine RNA editing, catalyzed by the ADAR (Adenosine Deaminase acting on RNA) enzymes, differs in human embryogenesis and in adulthood. We tested the editing of various target genes in coding  and non-coding sequences at their  RNA editing is a site-specific modification of an RNA sequence that yields a different product than that encoded by the DNA template. The most prominent RNA editing event in human cells is the substitution of adenosine to inosine (A-to-I), catalyzed by members of the double-stranded RNA-specific Adenosine Deaminase Acting on the RNA (ADAR) family of enzymes. Since inosines (I) in mRNA are recognized as guanosines (G) by the ribosome during the course of translation, RNA editing can lead to the formation of an altered protein if editing results in a codon exchange. Thus, RNA editing is an essential post-transcriptional mechanism for expanding the proteomic repertoire Alu repetitive elements Alu repeats in those genomes Only a handful of known editing sites within coding sequence have been well characterized Literature describing RNA editing in human embryogenesis is limited. Low availability of human fetal samples and the complexity of measuring global RNA editing in various tissue samples are among the obstacles to such studies. In addition, the study of RNA editing role in stem cell biology is in a very early stage. Two recent publications have reported the involvement of ADAR enzymes and A-to-I editing in the regulation of adult stem cells, such as human neural progenitor cells Alu sequences were recently observed during spontaneous differentiation of hESCs; and ADAR1 knockdown was shown to result in increased expression of genes involved in differentiation Edited RNA was recently shown to escape nuclear retention in undifferentiated hESCs, suggesting a specified role for non-coding edited RNA in hESCs Alu elements of five genes: BRAC1, CARD11, RBBP9, MDM4, and FANCC. We compared RNA editing in samples derived from human fetal tissue and adult tissue, and assessed mRNA expression levels of ADAR enzymes. Simultaneously, we analyzed the editing efficiency of the same sites in hESCs. Finally, to further study the function of ADAR1 in early human embryogenesis we attempted to generate overexpression of ADAR1 in hESCs.In the current study we analyzed the RNA editing levels of single sites at three coding genes: BLCAP, FLNA, and CYFIP2 Ten human embryos aborted at 10–20 weeks gestation due to social reasons were obtained from women who signed consent forms that were approved by the Helsinki Committee for Genetic Experiments on Human Subjects at Rambam Health Care Campus, Haifa, Israel. Tissue samples were dissected within one hour post mortem and placed immediately in liquid nitrogen for subsequent storage at −80°C until RNA isolation. Adult tissue RNA was obtained from different sources. Brain, spleen, and liver samples were obtained from commercial sources: Clontech , Ichilov hospital , and Ambion  FirstChoice® Human Total RNA Survey Panel (Ambion RNA samples are comprised of pooled RNA from 3 donors). Adult heart and kidney RNA samples were obtained from commercial sources and isolated from biopsies. For adult tissue biopsies, written informed consent was obtained from each patient prior to surgery and approved by an institutional review board in accordance with guidelines for experiments on human subjects. Adult heart RNA was obtained from Clontech , Ambion (pooled RNA from 3 donors), and isolated RNA from a biopsy obtained during elective coronary artery bypass surgery . Adult kidney RNA was obtained from Clontech , Ambion (pooled RNA from 3 donors), and isolated RNA from a biopsy of normal renal tissue obtained during elective surgery.http://www.atcc.org, and the 2102 Ep cell line was a gift from Prof. P. Andrews, University of Sheffield, UK).Human embryonic stem cell (hESC) lines H9.2 I6 and I3.2 were grown as described Frozen tissue samples (∼1 mg) were homogenized in 1 ml TRizol reagent , using Diax-100 Heidolph homogenizer . Total RNA was isolated from tissue samples and from hESC samples according to manufacturers' instructions. One µg RNA was subjected to SuperScript™ II Reverse Transcription (RT) reaction (Invitrogen) to generate cDNA. The cDNA samples were subjected to Quantitative Real-Time PCR analysis . Relative quantification (RQ) was performed in triplicate and normalized by the internal endogenous GAPDH expression. The reaction was performed using SybrGreen (ABI) with the following primers:ADAR1common Fw: ACAGCCAAAGACACTCCCTCTC, Re: GGCTCAGCATGGCTATCTGG. ADAR1-p110 Fw: GGCAGCCTCCGGGTG, Re: CTGTCTGTGCTCATAGCCTTGA. ADAR1-p150 Fw: CGGGCAATGCCTCGC Re: AATGGATGGGTGTAGTATCCGC. ADAR2 Fw: CCGCAGGTTTTAGCTGACG Re: CGGTCAGGTCACCAAACTTACC. ADAR3 Fw: TTGGAAGGAGGCACCGACA, Re: CTTATTGGTTTCTCTGGGGCTG GAPDH: Fw: AGCCACATCATCGCTCAGACA, Re: GTACTCAGCGGCCAGCAT.Editing quantification by primer extension combined with Sequenom analysis: RNA editing quantification was carried out using MALDI-TOF mass spectrometer  as described Plasmid construct: pCDNA3.1/HisC-ADAR1-p110  was used for the transfection experiments. For lentivirus infection, the ADAR1-p110 cDNA was excised from pCDNA3.1, using BamH1 and XhoI, and subcloned into the lentiviral construct PTK . Plasmid Transfection: 3 µg pCDNA3.1-p110 plasmid was transfected using Lipofectamine 2000 transfection agent (Invitrogen) into I3.2 and H9.2 hESCs and 293T cells. Following several days of G418 selection, 40 stable clones were mechanically selected, re-plated, and allowed to expand.6 in a 10 cm plate, and supplemented with 25 µM chloroquine. The medium was switched 24 hr post transfection, and the supernatant containing the viruses collected 48–72 hr post transfection for either direct infection or freezing until infection.Virus construction and infections: for infection of hESCs, constructs harboring transgen, packaging (Gag-Pol), and VSVG (envelope) DNA  were transfected using CaCl2/HBS  into 293 T cells, which were plated 24 hr before transfection, at a density of 2.5×10-associated protein kinase (ROCK) Inhibitor Y-27632  was added to the hESC culture medium for 1 hr before collagenase treatment. After detachment from the MEF feeder layer using collagenase, the hESCs were treated with 0.5% trypsin and re-plated as single cells on fibronectin-covered 6-well dishes, at a density of ∼2.5×105 cells per well, in MEF-conditioned medium (CM) supplemented with Rock inhibitor. Two days post transfection, supernatant from 293T cells was collected, filtered through a 0.45 μ filter and concentrated using Amicon  at 5000 g for 20 min. Concentrated viral supernatant was added to the hESCs CM and supplemented with ROCK inhibitor and 8 µg/ml Polybrene . Twenty-four hours later, a second round of infection was conducted. Forty-eight hours post infection, hESCs were detached from fibronectin and either re-plated on MEF for further expansion or harvested for subsequent analysis.One day prior to the infection, 10 µM RhoProtein was extracted from cells using RIPA lysis buffer . Extracted proteins were separated by migration on SDS Polyacrylamide Gel Electrophoresis (SDS PAGE), followed by transferring for 1 h in 300 mA onto a nitrocellulose membrane. The membrane was then blocked with 5% milk powder in TBST , and incubated with anti ADAR1-p-110 primary antibody , overnight at 4°C. In the morning, the antibody residues were washed using TBST, and the membrane incubated with secondary goat anti–rabbit conjugated to horseradish peroxidase (HRP) antibody, diluted 1∶5000 in blocking solution for 1 hr. After washes with TBST, the reaction was performed using the ECL kit. Protein loading was verified using anti-actin Mab and secondary rabbit α-mouse antibody.GCATCCTCTCTCGCTTCTTG; and Genomic OCT4 segment: Fw: CCTTCCCTCCTTTACCCTACTCC Re: CCACCCCTGCTGCCTCTATT.Genomic DNA was extracted by the Wizard Genomic DNA purification kit  according to the manufacturer's instructions. PCR was performed using DreamTaq green master mix  with the following primers: primers corresponding to the Rev Response Element (RRE) of the lentivirus construct: Fw-ACGGTACAGGCCAGACAATTA Re-GGTGCAAATGAGTTTTCCAGA; primers corresponding to a segment including part of the CMV promoter (left primer) and part of the ADAR1-p110 sequence (right primer):Fw: TACATCAATGGGCGTGGATA Re: To analyze editing levels of different fetal tissues we collected 10 human embryos of 10–20 weeks gestation, which were aborted due to social reasons. Fetal tissues were dissected and RNA was isolated from five tissues: brain, heart, kidney, liver, and spleen. Editing levels were measured using MALDI-TOF mass spectrometer, and compared to commercial RNA samples from respective human adult tissues.Alu elements in non-coding regions Alu repeat sequences containing transcripts were selected for investigation due to their relevance to cancer and development, and their capability to regulate cell growth, proliferation, and normal differentiation. The panel included retinoblastoma binding protein 9 (RBBP9), Caspase recruitment domain family 11 (CARD11), Fanconi anemia complementation group c (FANCC), double minute 4 (MDM4), and breast cancer 1 (BRCA1).In light of the large number of A-to-I editing sites in the human genome, and the lack of an affordable and validated high throughput method to analyze them, a panel of eight functional genes for which editing activity had been validated and calibrated The highest level of RNA editing was detected at the BRCA1 gene. The tumor suppressor nuclear phosphor protein, BRCA1, is known as a breast and ovarian cancer susceptibility gene, presenting in 21%-40% of women with these types of cancer. The BRCA1 mutation can function as a predictive marker of response to chemotherapy CARD11 is a protein that participates in apoptosis signaling through protein-protein interactions The RBBP9 gene encodes a protein that is involved in the complex that binds to retinoblastoma (RB) tumor suppressor and regulates its activity as a monitor of apoptosis. RBBP9 is involved in malignant transformation of cells MDM4 expression is elevated or overexpressed in 10–20% of over 800 diverse tumor types FANCC is essential for protection against chromosome breakage, and is involved in protein complexes that functionally interact and inhibit the pro-apoptotic protein kinase PKR. Mutations in these genes cause increased binding of PKR to FANCC and increased PKR activation, leading to growth inhibition of hematopoietic progenitors and bone marrow failure in Fanconi anemia (FA) disease , a ubiquitously expressed cell membrane anchoring protein, is involved in cell cytoskeleton element remodeling. RNA editing of FLNA results in Q to R substitution at amino acid 2341 of the human protein FLNABLCAP is expressed predominantly in the brain and B lymphocytes CYFIP2 is an evolutionarily conserved gene that is expressed predominantly in brain tissues, kidney, and white blood cells. CYFIP2 transcripts undergo A-to-I editing at amino acid 320, resulting in K to E substitution Alu elements in four of the five non-coding regions tested  Q/R editing site, a well studied ADAR2 editing substrate The ADAR3 expression pattern was tissue- type dependent and not developmentally dependent. The ADAR3 transcript expression level in the brain was lower in most embryos than in adult samples , with onAlu elements in hESCs, as well as promiscuous editing of SINEs in mouse, were shown to be highly dependent on ADAR1 To study the mechanisms regulating reduced RNA editing and ADAR1 expression in fetal tissues and hESCs, we attempted to generate ADAR1 gain of function in hESCs. We chose the ADAR1-p110 isoform since this isoform was found to be reduced in most fetal tissue samples compared with respective adult tissue samples , and sinWith the intention of generating hESC ADAR1-p110 overexpressing clones, we transfected H9.2 hESCs with neomycine selectable plasmid, containing human ADAR1-p110 gene conjugated to N-terminal 6XHis-tag and Xpress epitope under the CMV promoter Of the ADAR1-p110 OE hESC clones generated, none overexpressed ADAR1-p110. In representative data for six selected hESC clones, none of the neomycin resistant clones exhibited increased mRNA  or proteIn contrast, we were easily able to generate extensive OE of ADAR1-p110 following transfection of 293T cells with the same vector. Transfected 293T cells showed ADAR1-p110 OE both by RNA expression  and by pTo eliminate the possibility that the ADAR1-p110 protein level was reduced in hESC neomycine resistant clones due to extended passages, we generated stable ADAR1-p110 OE under CMV promoter using lentivirus infection, and analyzed the protein level immediately after the infection. Lentivirus infection is a robust procedure for introducing exogenous genes into hESCs The 293T cell line, and the primary cell human foreskin fibroblast (HFF), were infected simultaneously as control cells. Infection efficiency was estimated using a similar lentivirus construct harboring GFP under the CMV promoter. The infection rate was ∼50% for H9.2 cells and ∼90% for 293T and HFF cells . InfecteEmbryonal carcinoma (EC) cells are derived from teratocarcinoma benign tumors. EC cells are considered to be pluripotent cells since they exhibit both the signature expression of pluripotent markers and the ability to differentiate in-vivo into derivatives of the three germ layers Using RNA samples derived from multiple tissues, obtained from 10 pregnancies aborted at ages 10–20 weeks of gestation, as well as from hESCs, we were able to test RNA editing in early human embryogenesis. We compared the editing level of five embryonic tissues to respective adult tissues in five editing sites of non-coding sequence and three editing sites in coding regions. We found A-to-I RNA editing to be significantly higher in adult than fetal tissue of the same origin in two of three known coding regions, and in four of five non-coding regions  and 2.Alu element sites in BRCA1 gene were increased, and those of the coding sequences in CYFIP2 and FLNA genes were reduced in glioblastoma multiforme brain tissue Studies comparing normal and malignant human tissue samples, showed levels of RNA editing to be elevated or reduced in a gene specific manner. Editing levels of the In the current study, the editing of CYFIP2 was ∼70% in the adult brain samples and ∼10% in the fetal brain samples . Other tTwo of the genes we tested did not show statistical difference in RNA editing between adult and fetal tissues. BLCAP editing was found to be significantly reduced in only one fetal tissue. This result concurs with previous results, which found no difference in the BLCAP editing level of lung and oral cavity cancer versus normal tissue 2CR editing levels, were markedly increased during rat development Knockout experiments in mice have demonstrated that RNA editing is crucial for normal development One study that examined the involvement of RNA editing in human brain development reported Q/R GluR-5 and GluR-6 editing sites to be developmentally up regulated, while GluR-B editing levels remained constant Alu sequences decreases during spontaneous differentiation of hESCs and that ADAR1 knockdown results in elevated expression of differentiation related genes Originally isolated from the Inner Cell Mass (ICM) of human blastocysts, hESCs represent a very early stage of human development. These cells have virtually unlimited self-renewal capacity and developmental potential To further study ADAR1 function in early development we attempted to generate ADAR1 gain of function in hESCs. We hypothesized that since editing and ADAR1 mRNA expression levels are reduced in early human development, and knockdown of ADAR1 affects the undifferentiated state of hESCs, ADAR1 could play an important role in controlling differentiation, self renewal, and pluripotency in hESCs. However, we were unable to achieve significant overexpression of ADAR1 mRNA or protein in the 40 antibiotic resistant clones we obtained from transfecting hESCs, even though these clones integrated into the plasmid containing the ADAR1-p110 isoform .To eliminate the possibility of ADAR1 silencing during the passaging of hESC resistant clones, we used the lentivirus infection system to overexpress ADAR1-p110. As shown in Overall, the differences observed in RNA editing between human embryonic and adult tissues, together with the unsuccessful ADAR1 overexpression in hESCs, indicate significant involvement of ADAR1 regulation during early human development.Table S1Oligonucleotide list for the RNA editing analysis by SEQUENOME MassArray technology. List of primer sequence and localization.(DOCX)Click here for additional data file.Table S2Mann-Whitney test P values for differences in RNA editing levels between adult and fetal tissues. Fetal and adult editing level were compared for statistical difference using Mann-Whitney Statistical analysis. Significant differences (p<0.05) were found in RNA editing between adult and fetal samples for 23 of 26 comparisons (shaded) of six genes . FANCC exhibited no significant difference between adult and fetal samples in RNA editing, and adult BLCAP editing was significantly higher only in the spleen.(DOCX)Click here for additional data file.Table S3Editing % of the GluR-B Q/R site. RNA editing levels were determined for samples of fetal tissues and adult tissues using the Sequenom Mass ARRAY compact analyzer. In fetal tissues GluR-B expression was observed in brain and most kidney and heart samples. In adult, GluR-B was not expressed in heart samples. For all tissues tested, GluR-B Q/R editing was essentially completely edited .(DOCX)Click here for additional data file.