Post-transcriptional regulatory networks are dependent on the interplay of many RNA-binding proteins having a major role in mRNA processing events in mammals. We have been interested in the concerted action of the two RNA-binding proteins hnRNP A1 and HuR, both stable components of immunoselected hnRNP complexes and having a major nuclear localization. Specifically, we present here the application of the RNA-immunoprecipitation (RIP)-Chip technology to identify a population of nuclear transcripts associated with hnRNP A1-RNPs as isolated from the nuclear extract of either HuR WT or HuR-depleted (KO) mouse embryonic fibroblast (MEF) cells. The outcome of this analysis was a list of target genes regulated via HuR for their association (either increased or reduced) with the nuclear hnRNP A1-RNP complexes. Real time PCR analysis was applied to validate a selected number of nuclear mRNA transcripts, as well as to identify pre-spliced transcripts (in addition to their mature mRNA counterpart) within the isolated nuclear hnRNP A1-RNPs. The differentially enriched mRNAs were found to belong to GO categories relevant to biological processes anticipated for hnRNP A1 and HuR  indicating their concerted function in mRNA metabolism. This is brought about by the extensive interplay amongst discrete sets of RBPs and their associated (pre)- and mRNA-target molecules in the form of ribonucleoprotein (RNP) complexes .Heterogeneous nuclear ribonucleoproteins (hnRNPs) are a major group of mostly nuclear RBPs in dynamic association with nascent hnRNA (pre-mRNA) and processed mRNA in the form of hnRNP complexes ,4. LikewThe hnRNP proteins comprise a group of over 20 polypeptides in the range of 32 to 110 kDa and designated in order of increasing molecular weight as hnRNP A-U ,9. They An additional RNA-binding protein of importance to mRNA maturation processes is HuR, a ubiquitously expressed and best characterized member of the small family of embryonic lethal abnormal vision (ELAV) Hu proteins  . Like moDespite the early recognition that under normal cellular growth the bulk (over 90%) of HuR is in the nucleus ,20, it wA breakthrough in the study of the assemblies of cellular mRNAs with their RNA-binding proteins (referred to as the Ribonome) was the application of the Ribonomics platform by Keene’ lab . This prThe basis of the RIP-Chip application in the present study stems from our recent reports for the existence of a broad range of interactions between HuR and hnRNP proteins existing within endogenous hnRNP/mRNP, as well as HuR assemblies, as they are obtained from extracts of mammalian (human/mouse) cell origin ,32. Spec2.2.1.The general strategy applied in the present study was based on our previous identification of hnRNP A1 (as well as hnRNP C1/C2) as associating with HuR ,32 and w2.1.1.MEF nuclear extracts were first incubated with a mouse IgG2 fraction (same isotype of the 4B10 hnRNP A1 antibody) that served as control IP (IgG-co) and, then, with the equivalent amount of the 4B10 antibody. Triplicate experiments were performed on an equal number of HuR WT and HuR KO cells. The immunoselected nuclear hnRNP A1-RNPs and their respective IgG-control assays from either HuR WT or HuR KO cells, together with an aliquot of the nuclear extract taken prior to IP, were the basis of obtaining the RNA population for subsequent microarray analysis.To test the specificity and efficiency of the anti-hnRNP A1 IP reactions, in direct comparison to the IgG-control assays, and to verify the presence or absence of co-precipitated HuR we applied western blotting analysis on an aliquot of the immunoselected nuclear RNP complexes. A representative picture referring to one of the triplicate IPs on HuR WT and HuR KO nuclear extracts is shown . In HuR 260) of the isolated RNA from the triplicate experiments, the percentage (about 8%) of RNA isolated from the hnRNP A1-RNP pellets relative to their respective total RNA was similar between HuR WT and HuR KO cells. Contrary, a much lower amount of RNA (much below 0.2%) was obtained from the IgG-control nuclear pellets, indicative of a highly specific IP reaction with practically undetected RNA background levels. This too low amount of isolated nuclear IgG-control RNA  could not meet the requirement of microarray analysis and was, thereafter, excluded from any further analysis. Instead, we relied on the total nuclear RNA expression levels in HuR WT and HuR KO cells in order to estimate the relative enrichment of the RNA transcripts within the immunoselected hnRNP A1-RNP complexes.The RNA-extraction protocol was applied to all immune pellets (hnRNP A1-RNP and IgG-control assays) of HuR WT and HuR KO cells to obtain any tightly associated RNA transcripts, as well as to the nuclear extracts for the total nuclear RNA. Based on the estimated amount  and after setting a baseline of two-fold change in their expression level in KO vs. WT cells, we obtained a number of 529 differentially expressed transcripts, which are presented in the Table S1. This list of transcripts contained 261 up-regulated and 268 down-regulated RNAs. In We first compared the expression level of the detected total nuclear RNA transcripts between HuR KO and HuR WT cells by statistical analysis coupled to fold change filtering. In particular, The differentially expressed RNA transcripts in the total nuclear RNA population in HuR WT and KO cells were subjected to Gene Ontology (GO) analysis on the basis of Biological Processes (BPs); a restricted number of BPs were likely to be affected by the HuR’s absence. More specifically, in the case of the up-regulated transcripts the prominent GOs referred to regulation of transcription, cell cycle and oxidation-reduction while discrete GO categories were ascribed for the down-regulated transcripts, affecting BPs like development, transport and apoptosis.t-test: p-value < 0.05 and FDR < 0.05). To assign any of these transcripts as true target genes of the immunopurified complexes, we considered that their enrichment, calculated as the relative abundance in the IP reaction compared to the corresponding expression level in total nuclear RNA, should be above 2. In this way, we have directly related the association of the hnRNP A1-RNP transcripts to their expression level in the total nuclear RNA population. As anticipated, Gapdh mRNA, serving as an abundant mRNA and a reported non-target of HuR and of hnRNP A1-RNP complexes [We then proceeded to the identification of RNA transcripts associated with the hnRNP A1-RNP complexes in nuclear extracts of either HuR KO or HuR WT cells, aiming to identify enriched transcripts in the IP reactions, as compared to the input RNA , we obtained 601 nuclear transcripts stably associated with HuR WT hnRNP A1-RNPs and 731 transcripts in the HuR KO complexes. These numbers corresponded to 2.1% and 2.4% of the total genes on the IlluminaBeadChip platform for HuR WT and HuR KO cell types, respectively. The targets with the highest enrichment scores depicted an up to 5-fold increase in the IP reaction as compared to total RNA. Moreover, as seen in Following the application of a high stringency enrichment score  and HuR-lacking (KO) nuclear RNP complexes. This allowed us to proceed to estimates of their differential association in the two cell types. We stress again that as target genes we considered those with a relative abundance in IP pellets, as compared to total RNA, greater than 2-fold. As a representative case of this normalization effect we refer here to the Gbp2 mRNA; its expression level was significantly affected in the HuR KO compared to HuR WT cells while it had the same degree of enrichment in the IP pellets of the two cell types (see Table S2 and sub-section 2.3.). If we had not normalized for its nuclear expression level, Gbp2 mRNA would have appeared, instead, with a reduced association in the HuR KO IP pellets as a result of its reduced expression.This first analysis of target genes for HuR WT and HuR KO cells is shown in the Venn diagram  that indIn order to address the RNA targets related to HuR, we performed hierarchical clustering on the union of the identified hnRNP A1-RNP-associated transcripts in HuR WT and HuR KO cells . This diThe identity of all associated RNAs in HuR KO against HuR WT cells, based on the degree of their enrichment (in the range between 4 to 0.1 fold changes), is presented in Table S2. For the sum of 973 target genes in both cell types, an estimated number of 670 (over 50%) were in the range of 1.40 to 0.70 and were considered as RNA transcripts practically non-affected by the absence of HuR. Furthermore, 336 and 160 transcripts had a fold change in their enrichment over 1.40 or below 0.70 and were taken to represent target genes with increased or reduced association, respectively. This was again in line with the data on the hierarchical clustering presented in vs. HuR WT cells, represented by the separate heat map in In an effort to further investigate the influence of HuR’s presence on the regulation of the target genes, we compared the list of differentially expressed transcripts in total RNA presented in Table S1 to the one of hnRNP A1-RNP-associated targets presented in Table S2. Only 29 common genes were found. Even when relaxing the statistical criteria while retaining the expression fold change cut off at the level of 2-fold, the number of common genes remains small, as only 88 genes characterized as hnRNP A1-RNP targets displayed a significant alteration at the level of gene expression (Table S3). For a more detailed bioinformatic analysis, we provide for the entire list of the identified targets their expression fold change in the total nuclear RNA population of HuR KO For the purpose of gaining further insight concerning the biological functionalities of RNA transcripts in association with the immunopurified hnRNP A1-RNP complexes in HuR WT and HuR KO cells, the list of identified targets were subjected to GO analysis focused on the GO category of “Biological Processes” (BPs). The GOs ascribed to the nuclear hnRNP A1-RNP-associated target genes separately for the HuR WT and HuR KO cells are given in 2.3.The validity of the microarray data in defining target genes associated with HuR-containing and HuR-lacking hnRNP A1-RNP complexes was tested by RT-qPCR. We selected for validation 8 target genes with likely relevance to hnRNP A1 and/or HuR biological function shown in Ccnh, Tceal5, Ccnc) or reduced  association with the HuR-lacking hnRNP A1-RNP complexes, while two  had minor changes in their association state in the two cell types. In addition, an indication of the changing ratio in the expression level of the validated mRNAs in total nuclear RNA in HuR KO vs. HuR WT cell is presented  based on the microarray data, suggesting that it could be characterized as absent. However, Tceal5 mRNA was clearly identified in HuR KO cells and, moreover, had a high affinity for the respective hnRNP A1-RNP complexes , we point to a disproportional increase in Ccnh mRNA affinity for the HuR-lacking RNP complexes  was designed to span an exon-exon junction and to yield the spliced product, alone. The RNA obtained from the triplicate experiments on HuR WT and HuR KO cells was used to estimate the actual mRNA levels of the selected target genes both in the total nuclear RNA population and in the RNA fraction associated with the hnRNP A1-RNP complexes (IPs). The level of Gapdh mRNA was used as an internal non-target normalization index in the qPCR reaction. On the basis of the estimated mRNA enrichment in IP reactions by RT-qPCR , as wellresented . As seenresented . A firstomplexes . AnotherThe early loading of the hnRNP proteins on the nascent RNA pol II transcripts of pre-spliced mRNAs has been well documented ,2. This Tceal5 gene organization consisting of three exons (E1–E3). Exons E1–E2 and the two intervening sequences (I1 and I2) are located at the 5′ end, while exon E3 contains the open reading frame (ORF) and the 3′UTR of the gene. In the spliced mRNA, the joined E1–E2 exons form the major part of a 5′UTR region. The pair of primers pE1/pI1 that corresponded to the first exon (E1) and intron (I1) was initially used to detect the Tceal5 pre-mRNA product shown in Tceal5 gene, at the level of pre-mRNA splicing.An unexpected finding of the above assay was the detection of the Tceal5 pre-mRNA in both HuR-lacking and HuR-containing cells, in clear opposition to the absence of its mature mRNA in HuR WT cells compare  and 4a. 3.Our present study is the first report on the application of a holistic approach to identify the HuR-dependent pattern of hnRNP A1-RNP-associated RNA transcripts, using Ribonomics on HuR-containing and HuR-lacking MEF cells. By taking into account that the bulk of cellular HuR, as for most of hnRNPs, resides in the nucleus ,20 and tet al., [et al. [Several reports already exist on the application of the RIP-Chip platform on immunoselected either hnRNP-  or HuR- et al.,  that proet al.,  the RNA et al.,  and our  [et al.   effects cannot be excluded. We also inferred from As our main effort was to define the RNA components of hnRNP A1-RNP complexes from HuR WT and KO cells, we have not looked into any possible alterations in the protein composition of the isolated complexes. Nonetheless, we have commented in the Results on the apparent absence of major changes in the nuclear level of hnRNP A1 in the HuR-containing vs. HuR WT nuclear extracts, special reference is made to the genes up-regulated in HuR KO with a role in transcription, proteasome assembly, cell signaling and myogenesis. In the down-regulated RNA transcripts we note the high ranking of matrix metallopeptidases and ubiquitin specific peptidases functioning in the degradation of extracellular matrix and progression of inflammatory responses, as well as the members of the cytochrome P450 family having a role in detoxification. After normalizing for the expression level of RNA transcripts in the nuclear pool of HuR WT and HuR KO cells, the nature of the mRNA targets and their degree of enrichment in the hnRNP A1-RNPs of either cell type have been clearly linked to their affinity for the isolated RNP complexes. As seen in the priority list of the top HuR WT and HuR KO RNA targets  on non-overlapping sites of the target RNA ,50. AlonThe absence of HuR had an overall greater impact on the hnRNP A1-RNP-bound RNA compared to its presence as supported by all findings  involvement of HuR in nuclear mRNA processing was that of the Tceal5 mRNA transcript, a member of the Tceal family functioning as RNA polII elongation factors. As also outlined in the Results, Tceal5 pre-mRNA, although associated with hnRNP A1-RNP complexes in both HuR WT and HuR KO cells, did not process to mature mRNA in the presence of HuR. Most likely, removal of HuR from the RNP complexes released this blockage, which suggests a post-transcriptional regulatory mechanism of HuR via competition with hnRNP proteins, possibly at the level of splicing. Similar to Tceal5, additional cases of HuR’s participation in the regulation of gene expression at the post-transcriptional level might be unraveled in future studies.Finally, in our present study we have chosen to work on two essential multifunctional RBP proteins; HuR a known major modulator in inflammation as well as cancer and hnRNP A1 a prototype hnRNP protein involved in splicing  that also exhibits altered expression in carcinogenesis. We believe that expanding the current study on MEF cells to include other cell types, such as macrophages and epithelial cell cultures, could possibly provide for new biological markers for disease diagnosis and prognosis, as well as novel molecular targets for therapeutic intervention.4.4.1.The initial construction of the HuR knockout (KO) mouse embryos and the isolation of Mouse Embryonic Fibroblast (MEF) cells from the E12.5 stage of either HuR WT or HuR KO embryos has been described before, as is their maintenance in cell culture . HuR WT 4.2.et al. [7 cells was resuspended in 750 μL buffer A  in the presence of 1 mM PMSF and 0.5% Triton X-100. The cell suspension was allowed to stay on ice for 5 min with gentle shaking and cell lysis followed by homogenization in a glass homogenizer with 10 strokes, on ice. The homogenate was centrifuged at 4000 rpm, 10 min at 4 °C in an Eppendorf centrifuge and the supernatant (representing the cytoplasmic extract) was removed. The nuclear pellet was, thereafter, resuspended in half the volume (350 μL) of buffer A without Triton X-100 and with the addition of 2 μg/mL protease inhibitor mix  . After sonication (2 × 5 s) on ice, the resulted lysate was layered on top of two volumes of a 30% sucrose cushion in the same buffer used for nuclei suspension and centrifuged at 5000 rpm, 15 min at 4 °C. The new supernatant (nuclear extract) was carefully collected from the top of the sucrose cushion and immediately frozen in liquid nitrogen. A similar extraction protocol was performed on triplicate experiments for HuR WT and HuR KO cells, alike. Protein estimates with the BioRad reagent were performed on a small aliquot of the nuclear extract. The total nuclear protein corresponding to the 3 × 107 cell pellet of either HuR WT or HuR KO cultures was about 2.5–3.0 mg.Nuclear extracts were obtained from MEF cells using the classical protocol of Dreyfuss et al.  that is 4.3.4.3.1.7 cells) were subjected to immunoprecipitation (IP) assays applying the basic procedure used before [et al. [et al. [2, 0.05% NP-40) with 1 mM PMSF, 5 times with a 5 min interval in between.Fractionated nuclear extracts from the triplicate experiments  and stored nuclear aliquots were subjected to RNA extraction. First, the washed immune pellets were resuspended in 100 μL NT2 buffer and 1 mM PMSF and an aliquot (1/20th) removed and stored for protein resolution and western blotting. Similarly, an aliquot corresponding to 1/50th of the total extract applied per IP reaction was also stored (see immunoblotting). The remaining main portion of immune pellets and nuclear extracts were subjected to proteinase K digestion, quickly spun to remove the empty beads, and processed through the RNA extraction protocol using the RNeasy Mini Kit column  following the manufacturer’s instructions. On-column DNA digestion  was used to ensure the absence of DNA from the samples. The quantification and quality analysis of RNA was performed on a Bioanalyzer 2100 .4.3.3.p-value > 0.01 in all tested conditions were not considered. Significantly enriched probe sets in IP samples as compared to total nuclear RNA in HuR KO and HuR WT cells were identified by t-test and characterized as hnRNP A1-RNP targets by applying the following thresholds: p-value < 0.05, False Discovery Rate (FDR) < 0.05 and a fold change >2. Similarly significantly differentiated genes in total nuclear RNA of HuR KO as compared to HuR WT cells were identified by t-test with the aforementioned thresholds. Probe sets not annotated with a RNA RefSeq were not considered for further analysis. The mouse platform contained 45281 probes corresponding to 30,855 different RNAs, mostly mRNAs but also a low percentage of non-coding RNAs. This is expected to provide whole-genome transcript coverage. GO-based analysis was performed with the web application StRAnGER [p-value cutoff of the hypergeometric test was set at 0.01.Synthesis of cDNA and biotinylated cRNA was performed with the Illumina TotalPrep RNA Amplification Kit  using 500 ng of total or immunoprecipitated RNA. Hybridization was performed onto Illumina MouseWG-6_V2_Expression BeadChips  according to manufacturer’s instructions. Three biological replicates were used for each condition. The raw data were analyzed using GeneArmada software . BrieflyStRAnGER  on the b4.4.Protein analysis was performed on an aliquot of nuclear extracts and immune pellets. Samples were directly mixed with SDS sample buffer and subjected to SDS-PAGE (10% polyacrylamide gel). Following transfer to nitrocellulose, the immunoblotting protocol used to detect the antigen-antibody reactions on the membrane was applied as before .4.5.For the real time PCR assays on selected mRNA transcripts, 250 ng RNA obtained from the immunopurified hnRNP A1-RNP complexes or total nuclear RNA was reverse-transcribed using Superscript III  reverse transcriptase and an oligo-dT primer according to the manufacturer’s instructions. In the case of pre-mRNA targets the reverse transcription step was performed with random hexamer primers. Real-time PCR reaction products were synthesized with the application of appropriate primer pairs and quantified by the incorporation of SYBR Green I  on an iQ5 Real-Time PCR Detection System  using Gapdh mRNA as an internal control for normalization. All assays were performed in triplicate in a 25 μL reaction. Specificity of the amplified PCR product was assessed through a melting curve analysis and agarose gel electrophoresis of a small aliquot of the reaction and staining with ethidium bromide. For each qPCR reaction set a standard curve was obtained using serial dilutions of total nuclear RNA and calculations were performed according to the Relative Standard Curve Method as in Applied Biosystems technical notes .The list of synthesized primers that were applied in qPCR reactions are provided in File S1. For mRNAs one primer per pair was designed to span an exon-exon junction, while for pre-mRNAs one primer was designed to anneal to an exon and its pair to an intron.5.Our present study aimed at investigating the nuclear role of HuR in the context of its association with endogenous hnRNP complexes. To this extent, we report here on the novel application of the Ribonomics (RIP-Chip) platform on hnRNP A1-RNP complexes immunoselected from the nuclear extracts of HuR-lacking and HuR-containing mouse embryonic fibroblast (MEF) cells. This led to the identification of HuR-dependent RNA transcripts in stable association with nuclear hnRNP A1-RNP complexes as a specific sub-fraction of the total HuR-bound nuclear targets. Amongst the identified RNA transcripts were some known hnRNP A1 as well as HuR mRNA targets, proving the validity of our experimental approach. The nature and degree of enrichment of the detected RNA targets in the immunopurified hnRNP A1-RNPs have been linked to their affinity for the isolated RNP complexes by normalizing for their expression level in the nuclear pool of either HuR WT or HuR KO cells. All findings, in particular those that were based on the presence of specific GO categories and on the RT-qPCR validated mRNA targets (including some unspliced pre-mRNA forms), led to the recognition of a significant change in the HuR-lacking (KO) RNA transcript population, compared to HuR-containing (WT) cells. This reflected upon a dynamic composition of hnRNP A1-RNPs in relation to HuR’s presence that is likely to affect, to a greater or lesser degree, the many anticipated nuclear functions regulated by HuR.


The isothiocyanate sulforaphane (SFN) possesses interesting anticancer activities. However, recent studies reported that SFN promotes the formation of reactive oxygen species (ROS) as well as DNA breakage.2O2). We observed that SFN was devoid of either RNA damaging and RNA protective activity in human leukemic cells. It was able to potentiate the RNA damage by doxorubicin and spermine. In the first case, the effect was attributable to its ability of modulating the bioreductive activation of doxorubicin. For spermine, the effects were mainly due to its modulation of ROS levels produced by spermine metabolism. As to the cytotoxic relevance of the RNA damage, we found that the treatment of cells with a mixture of spermine or doxorubicin plus SFN increased their proapoptotic potential. Thus it is conceivable that the presence of RNA damage might concur to the overall toxic response induced by a chemical agent in targeted cells.We investigated whether SFN is able to damage RNA, whose loss of integrity was demonstrated in different chronic diseases. Considering the ability of SFN to protect from genotoxicity, we also examined whether SFN is able to protect from RNA damage induced by different chemicals (doxorubicin, spermine, S-nitroso-N-acetylpenicillamine, HSince RNA is emerging as a potential target for anticancer drugs, its ability to enhance spermine- and doxorubicin-induced RNA damage and cytotoxicity could represent an additional mechanism for the potentiating effects of SFN associated with anticancer drugs. Broccoli and broccoli sprouts contain wide amounts of glucosinolates Genomic DNA breaks represent an important trigger of apoptosis In the present study, we performed our investigation to see whether SFN is able to target and damage RNA. We used nutritional and supranutritional concentrations of SFN.RNA may be more susceptible to damaging agents than DNA for different reasons. RNA is indeed mostly single-stranded and its bases are neither protected by hydrogen bonding nor located inside the double helix Despite its potential to affect cell physiology, potential triggers of RNA damage as well as its pathophysiological implications remain largely unknown. A significant loss of RNA integrity has been demonstrated in advanced human atherosclerotic plaques Taking into account the demonstrated ability of SFN to protect cells from genotoxic insult Blood donors provided written, informed consent for the study use of the samples at the time of donation. The described study was approved by the Comitato Etico dell'Azienda Unità Sanitaria Locale di Bologna.2 (7 mg/mL in RNase free water). The mixture was acidified with 50 µL of hydrochloric acid (19% v/v) per 1 mL of SNAP solution and incubated for at least 30 min at 4°C before use. The stock solution was prepared immediately before administration.Reagent grade chemicals were purchased from Sigma . SFN (Sigma) was dissolved in DMSO. The stock solution (50 mM) of S-nitroso-N-acetylpenicillamine (SNAP) was prepared by combining equal volumes of N-acetyl-D-penicillamine (19 mg/mL in 100% ethanol) and NaNO5 cells/mL were added with 10 µg/mL of phytohemagglutinin (Sigma).Human peripheral blood (60 mL) was obtained from normal healthy volunteers of AVIS . Human mononuclear cells were isolated by density gradient centrifugation using Histopaque-1077 (Sigma). Lymphocytes at a concentration of 4×105 cells/mL.Human leukemia Jurkat (acute T lymphoblastic leukemia) and HL-60 (acute promyelocytic leukemia) cell lines were purchased from Istituto Zooprofilattico Sperimentale Lombardia ed Emilia Romagna, Brescia, Italy). KU812F (chronic myeloblastic leukemia) cell line was purchased from LGC Standards . Jurkat, HL-60, KU812F cell lines and non-transformed human T lymphocytes were grown in suspension and propagated in RPMI 1640 supplemented with 10%  or 20% (HL-60) heat-inactivated bovine serum, 1% antibiotics . To maintain exponential growth, the cultures were divided every third day by dilution to a concentration of 1×102O2 . Cells were treated with different concentrations (0.0–0.5 mM) of SNAP, spermine NONOate, or doxorubicin for 24 h at 37°C. An additional time point (6 h) was included for spermine. For H2O2, the protocol was slightly modified. The cultures were treated with different concentrations of H2O2 (0.0–0.5 mM) in PBS for 6 h at 37°C. The range of concentrations of the potential RNA-damaging agents were selected considering the quantity of total RNA extracted per cell, as recently suggested Cells were treated with different concentrations of SFN (0–30 µM) for 6 or 24 h at 37°C. The RNA-damaging compounds used in this study were SNAP, spermine NONOate, doxorubicin, and HFor assessing the potential protective activity of SFN, two different treatment protocols were used:2O2).Pre-treatment protocol: the cells were incubated for 24 h with SFN, then were washed and treated with the RNA toxic compounds for 24 h (6 h for H2O2).Co-treatment protocol: the cultures were incubated for 24 h with SFN and the RNA toxic compounds , according to the manufacturer's recommendations. Briefly, cells were mixed with an adequate volume of Guava ViaCount Reagent (Millipore) and allowed to stain for at least 5 min at room temperature. The Guava ViaCount Reagent provides absolute cell count and viability data based on the differential permeability of DNA-binding dyes and the analysis of forward scatter. The fluorescence of each dye is resolved operationally to allow the quantitative assessment of both viable and non-viable cells present in a suspension.After cell treatment, RNA was isolated with an Agilent Total RNA isolation Mini Kit , according to the manufacturer's recommendations. Considering the high susceptibility of RNA to RNase, all the steps were performed by using nuclease-free water, RNase-free final collection tubes and RNase inhibitors. Briefly, 350–400 µL of lysis solution were added to cell pellet and the cell homogenate was centrifuged through a mini-prefiltration column. The flow-through was mixed with an equal volume of 70% ethanol, incubated for 5 min at room temperature and centrifuged through a mini-isolation column. The flow-through was discarded and the RNA-loaded column was transferred into an RNase-free final collection tube. Then, the purified RNA was eluted by addition of 10 µL of nuclease-free water.RNA analysis was performed by microfluidic capillary electrophoresis with the Agilent 2100 bioanalyzer. Tiny amounts of RNA samples are separated in the channels of the microfabricated chips according to their molecular weight and subsequently detected via laser-induced fluorescence detection. The result is visualized as an electropherogram where the amount of measured fluorescence correlates with the amount of RNA of a given size. A software algorithm then allows the calculation of an RNA Integrity Number (RIN). The RIN algorithm is based on a selection of informative features from the electropherograms. For this purpose, each electropherogram is divided into the following nine adjacent segments covering the entire electropherogram: a pre-region, a marker-region, a 5S-region, a fast-region, an 18S-region, an inter-region, a 28S-region, a precursor-region, and a post-region. In addition, several global features are extracted, i.e. features that span several segments. Among these, the average and maximum height, areas and their ratios, total RNA ratio and the 28S area ratio are the most important. The gradual degradation of RNA is reflected by a continuous shift towards shorter fragment sizes. For classification of RNA integrity, ten categories are defined from 0  to 10 (fully intact RNA) 6 cells were pre-treated with SFN 10 µM for 3 or 24 h. Then, samples were washed, treated with spermine 0.5 mM and additionally incubated for 3 or 24 h. At the end of the treatment, the samples were washed and 0.5×106 cells were stained with DCHF-DA 5 µM. After 20 min of incubation at 37°C, fluorescence intensity was monitored at 510 nm using VICTOR 3 V Multilabel Counter . Data were expressed as percentage of the control (untreated cells).2′,7′-dichlorofluorescein-diacetate (DCFH-DA) was used for ROS detection. DCFH-DA is cleaved intracellularly by nonspecific esterases to form 2′,7′-dichlorodihydrofluorescein (DCFH), which is further oxidized by ROS to form the highly fluorescent compound 2′,7′-dichlorodihydrofluorescein (DCF) 6 cells were treated with SFN 10 µM for 3 h or 24 h. In order to determine the DTNB reduction due only to the thioredoxin reductase activity present in the samples, two assays were performed. The first assay measured the total DTNB reduction by the sample and the second one measured the DTNB reduction by the sample in the presence of the thioredoxin reductase inhibitor solution (Sigma). The difference between the two analyses represents the DTNB reduction due to thioredoxin reductase activity alone. The results were expressed in U/mL and normalized to protein concentration determined by Bradford reagent (Sigma). Thioredoxin reductase (Sigma) was used as positive control.The thioredoxin reductase activity was determined by the colorimetric assay based on the reduction of 5,5′-dithiobis(2-nitrobenzoic) acid (DTNB) with NADPH to 5-thio-2-nitrobenzoic acid (TNB), which produces a strong yellow color that is measured at 412 nm by VICTOR 3 V Multilabel Counter (PerkinElmer). Briefly, 1.5×10i.e. glutathione (GSH) and glutathione disulfide (GSSG)] was measured by the Gluthatione Assay Kit (Sigma), in which catalytic amounts (nmoles) of GSH caused a continuous reduction of DTNB to TNB, spectrophotometrically measured at 412 nm by VICTOR 3 V Multilabel Counter (PerkinElmer). The GSSG formed is recycled by glutathione reductase and NADPH. The rate of TNB production is proportional to the concentration of glutathione within the sample. Briefly, 1.5×106 cells were pre-treated with SFN 10 µM for 3 h or 24 h. Then, samples were washed and treated with spermine 0.5 mM for additionally 3 h. At the end of the treatment, the samples were first deproteinized with a 5% 5-sulfosalicylic acid solution (Sigma), centrifuged to remove the precipitated protein and then assayed for total glutathione. The results were expressed as glutathione concentration (µM).The level of total glutathione [4 cells were stained with 100 µL of Guava Nexin Reagent  and incubated for 20 min at room temperature in the dark. Samples were then analyzed by flow cytometry.Flow cytometric procedures were performed with an EasyCyte 5HT flow cytometer. Approximately 5000 events (cells) were evaluated for each sample. In all cytofluorimetric determinations, cell debris and clumps were excluded from the analysis by gating. Experiments were conducted by using duplicate samples for each treatment, and each experiment was repeated at least three times. Cells were treated with 1) doxorubicin or SFN 10 µM plus doxorubicin for 24 h, or 2) SFN 10 µM for 24 h, washed and treated with spermine for 24 h. After 24 h, cells were washed and resuspended in drug-free culture medium for further 24 h. Aliquots of 2.0×10All results are expressed as the mean ± S.E. of at least three independent experiments. Differences among treatments were evaluated by ANOVA, followed by Bonferroni t-test, using GraphPad InStat version 3.00 for Windows 95 . P<0.05 was considered significant.To exclude that RNA fragmentation was an artifact associated with cell death, we first analyzed the effect of the treatment protocols on the cell viability. A general approach in performing genotoxicity test is to avoid the testing of doses that decrease viability, compared to the concurrent control cultures, by more than 60% vs 9.9, respectively)  . SimilarInterestingly, when SFN was tested in a cell-free system, it induced a significant RNA damage . The val2O2.In the second part of the study, we analyzed the ability of SFN to protect cells against the RNA-damaging activity of spermine, SNAP, doxorubicin, and H2O2 in Jurkat cells demonstrated that they did not induce a decrease in cell viability by more than 60% compared to the untreated cultures up to the highest concentration tested (0.5 mM) (data not shown). The viability of doxorubicin-treated cells was significantly decreased. The only concentration exhibiting a toxicity lower than 60% was 0.01 mM (data not shown). Concentrations of 0.5 mM for spermine, SNAP and H2O2, and 0.01 mM for doxorubicin were then used in the experiments aimed at evaluating the protective ability of SFN against RNA-damaging activity of the above reported compounds.The previously performed analysis of the cytotoxic potential of spermine, SNAP, doxorubicin, and H2O2, and doxorubicin induced a pronounced RNA damage in Jurkat cells , thus demonstrating a lack of cell-type specificity for these effects.To study and compare the cell-line specificity of the effects of SFN on doxorubicin- and spermine-induced RNA damage, the same experiments were performed using different cell lines, namely HL-60 and KU812F. Results shown in To test the possibility that the ability of SFN to enhance the RNA-damaging properties of doxorubicin and spermine was an artifact associated with a cytotoxic effect, we immediately analyzed the effect of the treatment protocols on Jurkat cell viability. No reduction of cell viability was observed after treatment with SFN plus doxorubicin or SFN plus spermine (data not shown).On the whole, our results indicated a general inability of SFN to protect RNA from the insult caused by the four chemicals. Because of SFN's capacity to potentiate the RNA-damaging properties of doxorubicin and spermine, we performed a series of experiments in order to elucidate its mechanism of action in Jurkat cells.We first analyzed the ability of SFN to induce thioredoxin reductase activity. Treatment of cells with SFN 10 µM for 24 h significantly up-regulated thioredoxin reductase specific activity .To know whether the effect of SFN on increasing the spermine-induced RNA damage was dependent on the increased formation of ROS, we determined the levels of ROS in cultures treated with SFN, spermine or SFN plus spermine (pre-treatment protocol). The formation of DCF was determined and quantified by means of a multilabel microplate reader. Results depicted in GSH depletion affects the antioxidant capacity of the coupled glutathione peroxidase–glutathione S-transferases system sensitizing target cells to oxidative stress. As shown in Finally, we analyzed the impact of RNA damage induction in the context of cell survival. In particular, the last part of our study was addressed to the study of the consequences and cellular handling of the RNA damage induced by doxorubicin and spermine alone and in combination with SFN. Flow cytometric analysis at 24 h post-spermine or -doxorubicin treatment showed that a significant proportion of cells underwent apoptosis, suggesting that this latter might be a relevant type of cell death in this exposure paradigm . To invein vitro experiments reported that total RNA is susceptible to free radical attack owing to its single-stranded structure and the absence of protective histones ad hoc studies. However, Martinet et al. indicated that if the quantity of total RNA per cell is in the order of 10 pg, the intracellular free radical production needed to destroy the intracellular RNA content will be approximately 50 nM vs. cytosolic or extracellular) might lie at the cross between the tendency of ROS to preferentially damage DNA, RNA or both. Such a hypothesis  is indirectly strengthened by the finding that spermine and H2O2, which generate ROS at the extracellular and cytoplasmic level, do promote RNA damage (see below).In the present study, we clearly demonstrated that SFN did not alter RNA integrity in different cell systems. This is quite surprisingly because (1) our study also indicated that 3 h-treatment of cells with SFN was able to elicit intracellular ROS and different In vitro experiments advocate the possibility of interaction between isothiocyanates and proteins having active thiol groups, such as thioredoxin and JNK phosphatase It is of worth to note that the treatment of RNA extracted from Jurkat cells with SFN 30 µM (a cell-free system) induced a significant RNA damage. These results cast light on the apparently unexpected inability of SFN to induce RNA damage in a cell system. Isothiocyanates compounds are normally unstable towards nucleophiles. Thus, it is conceivable that SFN interact with multiple biological nucleophiles such as cysteine in proteins and the tripeptide glutathione in a cell system The levels of ROS induced by SFN in Jurkat cells strongly decreased after 24 h of treatment with SFN. The dual effect of SFN on ROS levels can be associated with the dual effect of SFN on GSH levels. Over the last decade, a number of studies have shown that SFN is taken into cells almost entirely by conjugation with cellular GSH and is exported from the cell as a GSH conjugate 2O2, SNAP, doxorubicin or spermine for 24 h induced RNA damage in all the cell lines tested and in normal human T lymphocytes. Under the selected exposure conditions, they did not alter cell viability by more than 60% compared to the concurrent control cultures. Since indirect mechanisms related to cytotoxicity may lead to enhanced RNA fragmentation, this finding implies that RNA damage induced by the above reported compounds is not the result of aspecific death-related events, but rather is likely to depend on their direct action on RNA.Treatment of Jurkat cells with HIn this context, it is important to note that changes of RNA size/level distribution observed in the RNA electropherograms do not necessarily indicate potential damages by a xenobiotic. Indeed – although the inclusion of RNase-free reagents and RNase inhibitors prevent RNA from damage by endogenous and exogenous RNAase – some processes such as RNA synthesis mechanisms and stabilizing/repairing mechanisms, may affect the RNA electropherograms. However, these are common problems with one of the most used genotoxicity assay, the Comet test. In the Comet assay, increased DNA migration is associated with increased levels of DNA damage such as strand breaks and alkali-labile sites. Furthermore, increased DNA migration may also derive from the presence of single-strand breaks associated with incomplete excision repair sites. On the other hand, a decrease in DNA migration can result from the ability of crosslinks to stabilize DNA molecule 2O2. This latter finding is not in keeping with our previous data, which reported that SFN was capable to afford protection against DNA damage induced by H2O22O2 concentration (0.5 mM vs. 0.1 mM H2O2 in Ref. 19) was used throughout the present study. Such a higher dose of H2O2, selected to induce a significant RNA damage, is likely to yield a level of ROS that stoichiometrically overwhelms the scavenging potential of SFN. Also in support of our hypothesis is the inability of SFN at all concentrations studied (up to 100 µM) to scavenge H2O2, used at a very high concentration (7.5 mM), reported in a recent study SFN did not affect the RNA damage induced by HUnder our experimental conditions, the RNA damage induced by SNAP was not affected by SFN. SNAP exerts its toxic activity through the production of reactive nitrogen species Pre-treatment and co-treatment with SFN markedly enhanced the RNA-damaging activities of doxorubicin, one of the most effective anticancer drugs ever developed. Doxorubicin undergoes bioreductive activation by redox-cycling reactions. One-electron addition to the quinone moiety of doxorubicin is associated with the formation of a semiquinone. The latter quickly regenerates its parent quinone by reducing ground state oxygen to ROS, such as superoxide anion and its dismutation product, hydrogen peroxide. Toxicity of doxorubicin rests with the DNA intercalation of the semiquinone radical. The superoxide generated in this redox-cycling process induces additional, qualitatively different, DNA lesions E. coli thioredoxin system enhanced the redox-cycling of anthracyclines and increased the generation of superoxide anion The biological role of thioredoxin reductase is to transfer reducing equivalents from NADPH to various oxidized substrates. Thioredoxin reductase, together with its substrate thioredoxin, forms a redox system, which plays multiple roles and acts on different substrates such as lipoic acid, lipid hydroperoxides, vitamin K3 and ascorbyl free radicals In keeping with a previous study 2O2, and ammonia. Aminodialdehyde produced during the oxidation of spermine subsequently undergoes spontaneous β-elimination to form acrolein The polyamine spermine has an important role in the viability and propagation of most cells. However, many studies reported that spermine is also the source of cytotoxic metabolites Amine oxidase in fetal calf serum catalyses the oxidative deamination of spermine and produces aminodialdehyde, HIn our experimental settings, spermine induced a significant RNA damage, which was clearly potentiated by pre-treatment with SFN, while it remained unchanged in the co-treatment protocol.Interestingly, the RNA-damaging effects observed for spermine might directly depend on the formation of ROS promoted by spermine metabolism. Therefore, we performed a set of experiments aimed at determining whether and how much spermine gives rise to ROS under the treatment conditions adopted for evaluating RNA damage. Here the formation of ROS was directly monitored in experiments involving the sensitive probe DCFH, which – upon oxidation – is converted to its fluorescent derivative, DCF. Surprisingly, we did not observe an increase in the levels of ROS after 24 h-treatment with spermine or SFN plus spermine. However, when we analyzed the levels of ROS after 3 h of treatment, we recorded a significant increase in the ROS levels both in cells treated with spermine and in cells treated with the association. The highest levels of ROS observed after treatment with spermine easily explain their ability to attack RNA and initiate RNA damage. Despite the lack of a ROS increase after 24 h of treatment with spermine, we recorded RNA damage after 24 h of treatment with spermine. In this context, it is important to note that Sharmin et al. As regards the effect of SFN on the RNA damage induced by spermine, pre-treatment with SFN markedly enhanced the RNA-damaging potential of spermine. In an attempt to delineate the mechanism involved in this effect, we decided to investigate the levels of ROS induced by SFN plus spermine at 3 h of treatment. We recorded a statistically significant increase in the ROS level after the combined treatment . The increase in ROS production caused by SFN plus spermine was additive. This is not unexpected because other papers have shown that SFN causes a transient increase in ROS production As to the co-treatment conditions, SFN did not affect the RNA-damaging activity of spermine. Recent studies reported that spermine 2O2 lacked cell-type specificity, since it could be observed in HL-60, KU812F and normal T cells.Notably, the effects of SFN on the RNA damaging activity of doxorubicin, spermine, SNAP and HAs to the cytotoxic relevance of the events described in this study, we found that the treatment of cultures with spermine or doxorubicin, both alone and more strongly in combination with SFN, induced apoptosis. However, it is hard to distinguish between the relative importances of RNA damage as compared to other possible toxic effects promoted by the above reported chemicals. A lot of studies reported proapoptotic activity of SFN in many experimental models In conclusion, we demonstrated that RNA did not represent a target for the toxic action of SFN. Furthermore, SFN was unable to protect cells from RNA insult induced by different toxic agents.The induction of RNA damage still represents a maturing approach and its true potential remains to be defined and studied. As an example, an open question emerged from the present study which would deserve specific studies deals with the problem of RNA differential susceptibility to ROS arising from selected subcellular compartments. However, it is worth noting that RNA is being investigated as a potential target for new anticancer agents: the induction of damage to RNA is clearly an interesting and potentially useful therapeutic approach, as demonstrated by successful pre-clinical and clinical trials


Transcription and replication of the influenza A virus RNA genome are mediated by the viral RNA polymerase from a promoter consisting of the partially base-paired 3′ and 5′ termini of viral genome segments. Here we show that transcription and replication can be uncoupled by mutation of an unpaired adenosine in the 5′ strand of the promoter. This residue is important for transcription but not replication by being essential for the cap-binding activity of the RNA polymerase. As leotides  and F.To address the question of whether the unpaired nature of 5′10A or the chemical properties of the residue are important for supporting viral transcription, we generated (i) a 3′ uridine insertion opposite 5′ A10 to enable its base pairing (3′iU10), (ii) a 5′dA10 and 3′i10U double mutation as a control for 3′iU10, and (iii) a 5′A10U substitution to control for the chemical properties of position 10 . As showin vivo, we transfected 293T cells with plasmids expressing PB1, PB2, tandem affinity purification (TAP)-tagged PA , RNAs consisting of about 22 to 27 nucleotides (nt) of the 5′ end of vRNA, as cofactors (In the absence of an influenza A virus RdRp structure, we can currently only speculate about the exact mode of action by which 5′A10 controls capped RNA binding by the RdRp without interfering with the ability of the RdRp to replicate RNAs. Deletion of 5′10A may result in an altered vRNA promoter structure, in which, for instance, base pair formation between 5′9C and 3′9G can efficiently compete with the formation of the 5′ and 3′ intrastrand base pairs, as proposed by the corkscrew model. Indeed, the formation of these 5′ intrastrand base pairs was previously shown to be critical for endonuclease activity of the RdRp , 13, sugtructure . In addiofactors . Our dat


Trypanosoma brucei. RNA interference studies have determined that the proteins are essential and are involved in ribosome biogenesis. Here, we show that these proteins interact in vitro with the 5S rRNA with nearly identical binding characteristics in the absence of other cellular factors. The T. brucei 5S rRNA has a complex secondary structure and presents four accessible loops (A to D) for interactions with RNA-binding proteins. In other eukaryotes, loop C is bound by the L5 ribosomal protein and loop A mainly by TFIIIA. The binding of P34 and P37 to T. brucei 5S rRNA involves the LoopA region of the RNA, but these proteins also protect the L5 binding site located on LoopC.P34 and P37 are two previously identified RNA binding proteins in the flagellate protozoan  Trypanosomes are unicellular organisms that diverged early in the history of eukaryotic life The assembly of ribosomes is a highly conserved and coordinated process that involves over a hundred accessory proteins Xenopus laevis oocytes Other factors have been described that bind 5S rRNA. Under special circumstances, cells transcribe 5S rRNA in excess and accumulate it in the cytoplasm in storage particles for subsequent mobilization to the nucleolus. This event has been studied in detail in Trypanosoma bruceiRNA-binding proteins play essential roles in the many aspects of ribosome biogenesis, including RNA stabilization, processing and transport. Our laboratory has identified two novel, abundant and closely-related RNA-binding proteins in Specific silencing of the expression of P34 and P37 through RNA interference has shown that procyclic cells lacking these proteins exhibit defects in ribosome biogenesis and ultimately lose viability T. brucei 5S rDNA (GenBank M14817.1) was amplified using primers 5ST3Fwd (5′ ATTAACCCTCACTAAAGGGTACGACCATACTTGGCC 3′) and 5SRev (5′ AGAGTACAACACCCCGGGT 3′) to generate a template that was used for T3 polymerase directed in vitro transcription  in the presence of [α-32P]UTP.Assays involving radiolabeled RNA were performed as previously described Full length, radiolabeled 5S rRNA at a concentration of 1 nM was incubated for 20 minutes at room temperature with complementary individual deoxyoligonucleotides targeted against specific secondary structure domains of 5S rRNA  and addeE. coli strain M15. Expression was induced with 1 mM IPTG for 5 hours at 37°C. Cell pellets were frozen and thawed, resuspended in lysis buffer  and incubated with lysozyme (1 mg/mL) for 30 minutes. Cells were lysed by sonication (six 10 second bursts at 75 W with a 10 second cooling period between bursts) and DNase I was added to a final concentration of 5 µg/mL if the lysate was viscous. After centrifugation at 10,000×g to remove cellular debris, the cell lysate was incubated with a 50% Ni-NTA agarose slurry (QIAGEN) at a 1∶4 slurry∶lysate ratio for 1 hour at 4°C with gentle rotation. The mixture was applied to a column, and washed four times with wash buffer (same as lysis buffer with 20 mM imidazole). The recombinant protein was eluted with elution buffer (containing 250 mM imidazole) in four fractions of 500 µL each. Fractions containing recombinant protein  were pooled and desalted in a PD-10 column (GE) with storage buffer  and flash-frozen in 100 µL aliquots.Both P34 (GenBank AF020695) and P37 (GenBank AF020696) were cloned into plasmid pQE-1 (QIAGEN) and expressed as histidine-fusion proteins in A constant concentration of 5S rRNA  was used in all the reactions and increasing concentrations of recombinant protein were added in a total volume of 100 µL in binding buffer . After incubation for 20 minutes at room temperature, the reactions were loaded onto pre-wetted nitrocellulose filters. The filters were washed twice with buffer, once with ethanol and then dried. A nylon filter underneath the nitrocellulose filters was used to capture unbound RNA Radiolabeled RNA at a constant concentration (approximately 1 nM) was incubated with different concentrations of recombinant protein in binding buffer  for 20 minutes at room temperature. After incubation, the reactions were loaded onto a 6% native polyacrylamide gel  and electrophoresed at 100 V for 1.5 hours. Following electrophoresis, gels were dried and exposed to film and phosphorimager screens for signal quantification in a Bio-Rad GS-700 analyzer. The experiments were performed in triplicate and dissociation constants were calculated using Graphpad Prism 5.5′ GCGGGGUGCCAUACUUACAGGCCUCGC 3′ (LoopA/StemV) and 5′GGCCGAAUGCACCAUAUCCCGUCCGAUUUGUGAAGUUAAGCGGCC 3′ (β arm) (IDT). The oligonucleotides were dissolved in 50 mM Tris, pH 7.4, 300 mM KCl, 10 mM MgCl2 and heated at 55°C for 5 minutes and allowed to slowly reach room temperature before use. The binding buffer used and the conditions for incubation with protein in these competition assays were identical to those described above.For competition assays, the labeled RNA was preincubated with varying amounts of non-labeled RNA and then incubated with protein at a final concentration of 100 nM. Total yeast RNA (Applied Biosystems) was used as a nonspecific competitor. Ribo-oligonucleotides designed to mimic secondary structure elements were as follows: Full length, radiolabeled 5S rRNA at a concentration of 1 nM was incubated in the presence or absence of protein (100 nM) in binding buffer  for 20 minutes at room temperature. Complementary individual deoxyoligonucleotides targeted against specific secondary structure domains of 5S rRNA  were addXenopus L5 protein E. coli. 5S rRNA (ΔU) was generated by in vitro transcription as described above for use in filter binding assays.A point mutant in Loop C was constructed by site-directed mutagenesis  at one of the positions (U43) that interferes with binding of the In addition, we utilized two synthetic β arm oligonucleotides incorporating two relevant mutations in Loop C: the ΔU mutation previously described, and a mutation at a universally conserved G residue at position 41 to a U residue (ΔG). These oligonucleotides were used in unlabeled form in competition EMSAs.The three dimensional structure of eukaryotic 5S rRNA provides different domains for protein interaction. The 120 nucleotide molecule is typically folded in 5 stems  and 5 loops (named A–E) T. brucei database and although binding of T. brucei L5 to 5S rRNA has been identified It has been shown that TFIIIA binds eukaryotic 5S rRNA via a high affinity interaction with the loop A region, although binding also protects a larger area that extends towards loop E T. brucei 5S rRNA, we first analyzed the native folding of the RNA in silico. Mfold modeling of the T. brucei 5S yielded two structures . The structures differ only in the organization of the β arm. The main structure is depicted in Panel A, and it is typical of other 5S rRNA molecules previously described. There are several G∶U wobble pairs . In addition, Loop D is a canonical tetraloop , and Loop E has the typical eukaryotic signature 5′UUAGUA∶GAACC3′. A G residue is external and unpaired in this Loop (green circle) and the other components of the Loop are usually involved in noncanonical pairings (indicated with arrows in the figure). In contrast, Loops B and C differ considerably between the predicted structures . However, if the RNA has been preincubated with P34 (left panel) or P37 (right panel), this region is protected from annealing and cleavage (lane 9 vs lane 5). LoopA, however, is inaccessible to this particular oligonucleotide even in the naked RNA reaction (lanes 3 and 7), presumably because of its short size. Longer oligonucleotides directed against LoopA show that it is available in the naked RNA , lane 6.Taken together, these results suggest that the P34 and P37 establish the high-affinity interaction with 5S rRNA via the LoopA domain on the RNA, but upon binding protect a region that extends to LoopC. Alternatively, the binding of P34 and P37 could cause a conformation change on the RNA that would make the LoopC region inaccessible to the nuclease.Xenopus L5 T. brucei 5S rRNA and wild type 5S rRNA in filter binding assays with P34 and the results show , the significance of this observation is unclear at the moment. It could be that P34 relies on additional contacts elsewhere on the RNA.P37 seems to display more affinity for the Loop A region than P34 . As indicated above, the binding sites of P34 and P37 and L5 are not the same, although P34 and P37 binding does protect part of the L5 binding site.The intriguing presence of the trypanosome-specific factors in an association with the highly conserved 5S rRNA raises questions concerning the role of these 5S rRNA binding proteins relative to the other well characterized 5S rRNA binding proteins, TFIIIA and L5. No TFIIIA homologue has been described or identified by data mining to date. It is unclear whether P34 and P37 may perform some of the functions of TFIIIA, but given the major structural differences between these proteins  we consider this possibility unlikely. Furthermore, the protection of Loop C by P34 and P37 does not suggest a TFIIIA-like mode of binding to the rRNA. The T. brucei cells lacking P34 and P37 exhibit a marked reduction in the stability of 5S rRNA Saccharomyces cerevisiae when L5 is knocked out T. brucei. As previously indicated, L5 has been detected in association with 5S rRNA. If this association also involves P34 and P37, our data would suggest that the addition of L5 to the P34/P37:5S rRNA complex might take place through protein-protein interactions, rather than protein∶RNA interactions (given the large protection footprint of P34 and P37 on 5S rRNA). A trimolecular complex could be formed by a protein∶RNA interaction and a subsequent conformational change to allow for the binding of the second protein partner in the complex, in a manner analogous to the formation of the 42S complex in Xenopus. The Xenopus storage particle component p43 binds 5S rRNA primarily through interactions in the Loop D region Previous data from our laboratory using RNA interference demonstrated that


The variant cleaved the tat RNA sequence in vitro about 20 times more efficiently than the wild type ribozyme. Our results provide the first direct evidence that combined mutations at nucleotide 83 and 340 of RNase P catalytic RNA from Escherichia coli (G83 -> U83 and G340 -> A340) increase the overall efficiency of the ribozyme in cleaving an HIV RNA sequence. Moreover, the variant is more effective in reducing HIV-1 p24 expression and intracellular viral RNA level in cells than the wild type ribozyme. A reduction of about 90% in viral RNA level and a reduction of 150 fold in viral growth were observed in cells that expressed the variant, while a reduction of less than 10% was observed in cells that either did not express the ribozyme or produced a catalytically inactive ribozyme mutant. Thus, engineered ribozyme variants are effective in inhibiting HIV infection. These results also demonstrate the potential of engineering RNase P ribozymes for anti-HIV application.Using an in vitro selection procedure, we have previously isolated RNase P ribozyme variants that efficiently cleave an mRNA sequence in vitro. In this study, a variant was used to target the HIV RNA sequence in the  Human Immunodeficiency Virus (HIV) is the etiological agent of Acquired Immunodeficiency Syndrome (AIDS) Nucleic acid-based gene interference technologies, including conventional antisense molecules, aptamers, ribozymes, and small interfering RNAs (siRNAs), represent promising gene-targeting strategies for specific inhibition of mRNA sequences of choice. RNA enzymes are also being developed as promising gene-targeting reagents Escherichia coli) Ribonuclease P (RNase P) is a ribonucleoprotein complex responsible for the maturation of the 5′ termini of tRNAs in vivo. However, it has not been demonstrated whether RNase P ribozymes are also effective in inhibiting HIV-1 gene expression and replication. Meanwhile, increasing M1GS RNA catalytic efficiency in vitro and its efficacy in vivo is required in order to develop this ribozyme for practical use both as a research tool and as a therapeutic agent for gene-targeting applications. Using an in vitro selection procedure, we have previously isolated M1GS ribozyme variants that are more efficient in cleaving a specific mRNA sequence than that derived from the wild type M1 RNA tat gene, and investigated its activity in cleaving the target RNA sequence in vitro and its efficacy in inhibiting HIV-1 gene expression and growth in cultured cells. The tat gene encodes an essential viral transcription factor that is required for HIV-1 gene expression tat region in the HIV-1 genome by the ribozyme is expected to cleave the tat mRNA and in addition, the viral genomic RNA, leading to inhibition of viral replication. Our results indicate that the constructed ribozyme variant, when transiently or stably expressed in human cells, is more effective in inhibiting HIV-1 replication than the ribozyme derived from the wild type M1 RNA sequence. A reduction of more than 150 fold in the level of cell-free supernatant HIV-1 p24 was observed in cells that expressed the ribozyme variant. These results provide the first direct evidence that RNase P ribozyme can be effective in inhibiting HIV-1 gene expression and growth and furthermore, demonstrate the feasibility of developing highly effective RNase P ribozyme variants for anti-HIV-1 application by using in vitro selection procedures.Compared to hammerhead and hairpin ribozymes, RNase P ribozymes may possess several unique features as a gene targeting tool. For example, RNase P ribozymes (e.g. M1 RNA or M1GS RNA) can interact with specific cellular factors, which include the protein subunits of RNase P 83 -> U83 and G340 -> A340) 347C348 -> C347U348, C353C354C355G356 -> G353G354A355U356) at the catalytic domain (P4 helix) 5′-CCCGCTCGAGAAAAAATGGTGTCAGACTCATCAAGCTTCGAGCTATGACCATG-3′) as 5′ and 3′ primers, respectively. The DNA sequence that encodes substrate tat37 was constructed by PCR using pGEM3zf(+) as a template and oligonucleotides AF25 (5′-GGAATTCTAATACGACTCACTATAG-3′) and sTAT (5′-TTGATAGAGAAGCTTGATGAGTCTGACTGTTCGATCCTATAGTGAGTCGTATTA-3′) as 5′ and 3′ primers, respectively.Plasmid pV38, pFL117, and pC102 contain the DNA sequence coding for V38, M1 RNA, and mutant C102 driven by the T7 RNA polymerase promoter tat mRNA substrate (i.e. tat37) were synthesized in vitro by T7 RNA polymerase  following the manufacturer’s recommendations and further purified on 8% polyacrylamide gels containing 8 M urea. Subsequently, the M1GS RNAs were mixed with the [32P]-labeled RNA substrate. The cleavage reactions were carried out at 37°C in a volume of 10 µl for 30 min in buffer A  M1GS RNAs and the obs) and the values of the cleavage efficiency [(kcat/Km)s] were performed under single turnover conditions as described previously obs) at a fixed excess ribozyme concentration and the reaction followed pseudo-first-order kinetics. We assayed pseudo-first-order rate constants of cleavage (kobs) at each ribozyme concentration by determining the slope of a plot −ln[(Ft−Fe)/(1−Fe)] versus time using Kaleidagraph . Ft and Fe represent the fraction of the substrate at time t and at the end point of the experiments, respectively. We calculated the values of the overall cleavage rate [(kcat/Km)s] by determining the slope of a least-squares linear regression  of a plot of the values of kobs versus the concentrations of the ribozymes. These values were the averages of five independent experiments.Assays to determine the observed reaction rate (kd) of the M1GS-tat37 complexes were modified from Pyle et al d was then extrapolated from a graph plotting percentage of product bound versus ribozyme concentration. The values were the averages of five independent experiments.The procedures to measure the equilibrium dissociation constants  using Lipofectamine . Forty-eight hours post transfection, both the transfected cells and culture media were harvested. Cell-free supernatants were isolated from the culture media and viral production was quantified by assaying the levels of HIV-1 p24 in the supernatants with a p24 ELISA kit . RNA and protein samples were isolated from the transfected cells and the levels of total (unspliced and spliced) intracellular HIV RNA and viral p24 were determined by a real-time PCR assay and Western analysis, respectively.The DNA sequences coding for V38-TAT, M1-TAT, and C-TAT were cloned into retroviral vector LXSN and placed under the control of the small nuclear U6 RNA promoter, which has previously been shown to express M1GS RNA and other RNAs steadily The protocols for the construction of cells expressing different ribozymes were modified from Miller and Rosman NL4-3 at a multiplicity of infection (MOI) of 0.02–0.1. At different time points of infection, the infected cells and culture media were harvested. The infected cells were used to determine the levels of total HIV RNA and p24, while the culture media were used to determine the levels of cell-free-supernatant HIV p24.To study the antiviral activity of the ribozymes, H9 cells and the constructed ribozyme-expressing cells were infected with strain HIV32P]-radiolabeled DNA probes that contained the DNA sequence coding for M1 RNA and H1 RNA, respectively, and finally analyzed with a STORM840 phosphorimager. The radiolabeled DNA probe used to detect M1GS RNAs and H1 RNA was synthesized from plasmid pFL117 and pH1, by using a random primed labeling kit .For Northern analyses of the expression of the ribozymes, RNA fractions from M1GS-expression cells were isolated as described previously 5′-TTCCTGCCATAGGAGATGC-3′). The resulting cDNA (2 µl) was added to 48 µl of PCR mix containing 1×titanium Taq PCR buffer, 1 mM dNTPs, SYBR Green , 10 nM fluorescein, 1×titanium Taq DNA polymerase (Clontech), and 20 pmol each of 5′ primer HIV5 (5′-CATCCAGGAAGTCAGCCT-3′) and 3′ primer HIV3 (5′-TTCCTGCCATAGGAGATGC-3′). To normalize the RNA level, the level of actin mRNA was assayed by real-time PCR using the same PCR mix except for the primers which were Actin5 (5′-TGACGGGGTCACCCACACTGTGCCCATCTA-3′) and Actin3 (5′-CTAGAAGCATTGCGGTGGCAGATGGAGGG-3′), respectively For real-time PCR analyses of total intracellular HIV RNA, total cellular RNA samples were isolated from the cells that transiently or stably expressed the ribozymes, using Trizol reagent , and treated with RQ1 DNase . The treated RNA was reverse-transcribed in the presence of Powerscript reverse transcriptase  using RT primer . The real-time PCR results were derived from three independent experiments.Real-time PCR was carried out in an iCycler  and the PCR consisted of 35 cycles with denaturation at 94°C for 40 seconds, followed by primer annealing at 50°C for 40 seconds and extension at 72°C for 40 seconds. We also generated a standard (dilution) curve by amplifying different dilutions of the RNA transcript of the N, N’-methylenebisacylamide, transferred electrically to nitrocellulose membranes. We stained the membranes using the antibodies against HIV p24 and human actin in the presence of a Western chemiluminescent substrate kit  with an alkaline phosphatase-conjugated antibody, and analyzed the stained membranes with a STORM840 phosphorimager. The anti-p24 and anti-actin monoclonal antibodies were obtained from the NIH AIDS Research and Reference Program or purchased from Sigma , respectively. Quantification was performed in the linear range of protein detection.Western analyses were performed to determine the intracellular levels of HIV p24 protein. The polypeptides from cell lysates were separated on either 7.5% or 9% [vol/vol] SDS-polyacrylamide gels cross-linked with To determine the p24 level in the cell-free supernatants, the supernatants were isolated from the culture media collected from cultures of cells that transiently or stably expressed the ribozymes and were infected with HIV-1. The levels of HIV-1 p24 was assayed by a p24 ELISA kit .in vivo mapping approach tat RNA in HIV-1-infected cells. A position, 30 nucleotides upstream from the 3′ terminus of the exon 1 of the tat mRNA Most mRNA species inside cells are believed to be associated with proteins and are present in a highly organized and folded conformation. Thus, it is important to choose a targeting region that is accessible to binding of ribozymes in order to achieve efficient cleavage. Using dimethyl sulphate (DMS), we employed an in vitro selection procedure, we have isolated M1GS ribozyme variants that are more efficient in cleaving the mRNA sequence (TK mRNA) encoding the HSV-1 thymidine kinase (TK) than the ribozyme derived from the wild type M1 RNA sequence tat RNA sequence  Using an tat RNA sequence. Two other M1GS ribozymes, M1-TAT and C-TAT, were also constructed in a similar way and were included in the study. M1-TAT was derived from the wild type M1 sequence. C-TAT was derived from C102 RNA, a M1 mutant that contained several point mutations at the catalytic P4 domain and was at least 104-fold less active than M1 RNA in cleaving a pre-tRNA tat RNA region. These DNA sequences were under the control of the promoter for T7 RNA polymerase and M1GS RNAs were synthesized in vitro from these DNA sequences by T7 RNA polymerase. A substrate, tat37, which contained the targeted tat RNA sequence of 37 nucleotides, was used s] for these ribozymes in cleaving the tat RNA sequence substrate tat37. These results indicate that V38-TAT is about 20 fold more active than M1-TAT in cleaving tat37 , is similar to those of M1-TAT and V38-TAT  and a monoclonal antibody against human actin (anti-actin) . The latIn the second series of experiments, we investigated the effect of transient expression of various M1GS RNAs on the levels of total (spliced and unspliced) HIV-1 intracellular RNA. RNA samples were isolated from cells at 48 hours post-transfection. The levels of total HIV-1 intracellular RNA were determined using a real-time PCR assay with the expression level of the actin mRNA as the internal control. The results of three independent experiments indicated that a reduction of 91%±7%, 57±6% and 4±3% in the level of total HIV-1 intracellular RNA was observed in cells that expressed V38-TAT, M1-TAT, and C-TAT, respectively . The lowTo construct cell lines that express M1GS ribozymes, amphotropic packaging cells (PA317) The level of M1GS RNA in each cell clone was determined by Northern analysis with a DNA probe that is complementary to M1 RNA, using the expression of human H1 RNA as the internal loading control. Expression of the M1GS RNAs was detected in these cells . We onlyTo study the effect of stable expression of ribozymes on blocking HIV-1 production, cells were infected with HIV-1 at a multiplicity of infection (MOI) of 0.02–0.1 and three sets of experiments were carried out. First, supernatants were collected from cell cultures at 3 days postinfection, and the levels of p24 in the supernatants were determined using an anti-p24 ELISA assay. A reduction of about 91%±5%, 57±5%, 7±4% (average of three experiments) in the level of p24 was observed in cells that expressed V38-TAT, M1-TAT, and C-TAT, respectively . Since CSecond, we determined whether stable expression of M1GS ribozymes effectively reduces the level of total (spliced and unspliced) HIV-1 intracellular RNA. RNA samples were isolated from cells at 48–72 hours postinfection. Using the expression level of the actin mRNA as the internal control, we carried out a real-time PCR assay to determine the levels of total HIV-1 intracellular RNA. The results of three independent experiments indicated that a reduction of 92%±6%, 55±6% and 8±3% in the level of total HIV-1 intracellular RNA was observed in cells that expressed V38-TAT, M1-TAT, and C-TAT, respectively . The lowtat RNA sequence in cells expressing V38-TAT and M1-TAT results in the observed reduction of the levels of HIV intracellular RNA and p24, and viral production.The third set of experiments was carried out to study the magnitude and durability of the anti-HIV-1 effects of the stably-expressed ribozymes. The ribozyme-expressing cells were infected with HIV-1 at an MOI of 0.02–0.1. Cell-free supernatants containing newly produced viral particles were collected from the infected cultures at 3 day intervals through 15 days postinfection. The number of viral particles was determined by assaying the level of p24 antigen in the supernatants. The results of the experiments are summarized in Using ribozymes to inactivate mRNA of choice represents an appealing approach for gene therapy, since ribozyme cleavage of an mRNA target is highly specific and is irreversible in vitro in cleaving the HIV-1 RNA genomic sequence than the ribozyme (i.e. M1-TAT) derived from the wild type M1 RNA sequence. Moreover, V38-TAT reduced the level of HIV total intracellular RNA in cultured cells by more than 90% and was more effective in cultured cells than M1-TAT, which decreased the viral RNA level by about 55–60%. A reduction of about 150 fold in HIV-1 production was observed in the V38-TAT-expressing cells while a reduction of about 30 fold was observed in M1-TAT-expressing cells. In contrast, a reduction of less than 10% in the levels of p24 expression and viral total intracellular RNA was observed in cells that expressed C-TAT. C-TAT exhibited similar binding affinity to tat37 as V38-TAT and M1-TAT but was catalytically inactive due to the presence of the mutations at the catalytic domain (cat/Km)s] appeared to be more effective in cell culture. These results strongly suggest that increasing the catalytic efficiency of RNase P ribozymes may lead to an improved efficacy in inhibiting HIV-1 gene expression and growth in cultured cells.Understanding the mechanism of ribozyme cleavage and improving its efficacy is essential to develop M1GS ribozyme for practical gene-targeting applications including anti-HIV therapy. However, little is currently known about the rate-limiting step of M1GS RNA cleavage reaction in cells. Equally unclear is whether the efficacy of the ribozymes can be improved, and if so, how it can be improved. In the present study, we showed that an RNase P variant, V38-TAT, exhibited at least 20 times higher rate of cleavage c domain , Table 1in vivo applications. To our knowledge, this study represents the first to use RNase P ribozymes for inhibition of HIV gene expression and replication.It has previously been shown that custom-designed EGS molecules could induce human RNase P to cleave HIV RNA sequence in vitro and could inhibit HIV infection in cultured cells In vitro selection has been widely used to generate either new nucleic acid-based catalysts or more efficient variants from known ribozyme molecules in vitro also exhibits improved efficacy in inhibiting HIV-1 gene expression and growth in cultured cells. Thus, our study provides a direction for the engineering of highly active and effective RNase P ribozyme variants by carrying out selection procedures in order to improve the efficacy of the M1GS-based technology. Further characterization of the cleavage reactions of this as well as other RNase P variants both in vitro and in cultured cells should provide insights into the mechanism of how an RNase P ribozyme efficiently cleaves an mRNA substrate and develop guidelines for construction of effective gene-targeting ribozymes.tat sequence can be generated and become resistant to the ribozymes. In order to achieve effective anti-HIV therapy, multiple ribozymes should be designed to target different regions of the HIV genome that are highly conserved and may encode viral proteins that are essential for HIV replication HIV-1, a member of human retrovirus family, can easily introduce mutations in its genome during replication because of the low fidelity of reverse transcriptase for genome replication


Isolation of RNA from skin biopsies presents a challenge, due to the tough nature of skin tissue and a high presence of RNases. As we lacked the dedicated equipment, i.e. homogenizer or bead-beater, needed for the available RNA from skin isolation methods, we adapted and tested our zebrafish single-embryo RNA-isolation protocol for RNA isolation from skin punch biopsies.We tested our new RNA-isolation protocol in two experiments: a large-scale study with 97 human skin samples, and a small study with 16 mouse skin samples. Human skin was sampled with 4.0 mm biopsy punches and for the mouse skin different punch diameter sizes were tested; 1.0, 1.5, 2.0, and 2.5 mm. The average RNA yield in human samples was 1.5 μg with an average RNA quality RIN value of 8.1. For the mouse biopsies, the average RNA yield was 2.4 μg with an average RIN value of 7.5. For 96% of the human biopsies and 100% of the mouse biopsies we obtained enough high-quality RNA. The RNA samples were successfully tested in a transcriptomics analysis using the Affymetrix and Roche NimbleGen platforms.Using our new RNA-isolation protocol, we were able to consistently isolate high-quality RNA, which is apt for further transcriptomics analysis. Furthermore, this method is already useable on biopsy material obtained with a punch diameter as small as 1.5 mm. However, for -omics experiments such as transcriptomics, it is often difficult to obtain sufficient RNA from biopsies. This is primarily caused by the tough nature of skin material, which makes it hard to homogenize a skin sample, in addition to the high number of RNases in skin tissue that quickly degrade the biopsy RNA [The use of skin biopsies is an important method for studying the opsy RNA -5. Becauopsy RNA -8. More The limited amount of biopsy material from an experiment of 97 human skin biopsies, i.e. half of a 4.0 mm diameter biopsy, initiated the development of an optimal skin RNA-isolation protocol. At the same time, we investigated whether such a protocol would also allow for an experimental set-up with multiple skin biopsies per mouse, as this would imply the use of small biopsy punches 2.5 mm - 1.0 mm). Because, neither bead-beater nor homogenizer, which are commonly used for this type of RNA extraction [.0 mm. BeAfter testing of several adaptations on our zebrafish single-embryo protocol, it showed optimal performance by the addition of a ten minutes shake step ; and 5) average probe-signal intensity level after microarray analysis. We performed a human and a mouse study to assess the performance of our new RNA-isolation protocol.To evaluate our skin-biopsy RNA-isolation protocol, we defined five key criteria: 1) Weight of the biopsy; 2) RNA yield; 3) RNA Integrity Number (RIN ) value fFor the experiment with 97 human patients, skin biopsies were taken with a 4.0 mm biopsy punch. Tissue material from these biopsies was divided and one half was used for RNA extraction, while the other half was used for pathologic examination. The results of the five protocol-evaluation criteria are summarized in Table To investigate the relationship between the protocol-evaluation criteria we plotted the results of the individual biopsies against each other Figure , 3. As eAfter successful extraction of RNA from small human skin biopsies, we tested the performance of our new protocol in a limited experiment with mouse skin biopsies from test animals (un-)treated with UV light radiation  and pelleted for 30 s at 12,000 × g. Single biopsies were pulverized to a fine powder with a standard liquid nitrogen pre-chilled mortar and pestle. This powder was transferred to a 1.5 ml tube with 300 μl Qiazol (Qiagen). The homogenate was vortexed vigorously for 15 s and subsequently shaken for 10 min on a REAX 2000 (Heidolph). After a quick spin-down 60 μl chloroform was added to the homogenate, vortexed for 15 s and kept at room temperature for 3 min. The partly separated mixture was transferred as a whole to a pre-prepared phase-lock gel heavy containing tube and centrifuged for 15 min at 12,000 × g. The aqueous phase was transferred to a new 1.5 ml tube. RNA was purified by column precipitation according to the RNeasy MinElute Cleanup Handbook (version 2007) - Appendix D: RNA Cleanup after Lysis and Homogenization with Qiazol Lysis Reagent (Qiagen). At the end of the procedure, the RNA was eluted in 14 μl nuclease-free water. A PDF file with the complete protocol is available in a convenient format in the Additional file Gene expression was analyzed with Human Exon 1.0 ST Arrays in biopsies from all 97 patients. Sense-strand cDNA was generated from total RNA using Ambion WT Expression Kit (Applied Biosystems) conform the manufacturer's instructions. Further steps were performed using the manufacturer's protocols for the GeneChip platform (Affymetrix). Those included purification of double-stranded cDNA, synthesis of aRNA by in vitro transcription, recovery and quantification of biotin-labeled aRNA, fragmentation of this aRNA and subsequent hybridization to the microarray, post-hybridization washings and detection of the hybridized aRNA using a streptavidin-coupled fluorescent dye. Hybridized Affymetrix GeneChips were scanned using a GeneChip Scanner 3000-7G (Affymetrix). Image generation and feature extraction were performed using Affymetrix GCOS Software v1.4.0.036.Mus musculus microarray  containing 44,170 genes with 3 probes per target gene. Per RNA sample, 200 ng total RNA was amplified according to the Agilent LRILAK kit manual (Agilent technologies). Amino-allyl modified nucleotides were incorporated during the aRNA synthesis , 0.75 mM rCTP , 0.75 mM AA-rCTP (TriLink Biotechnologies). Synthesized aRNA was purified with the E.Z.N.A. MicroElute RNA Clean Up Kit (Omega Bio-Tek). Test aRNA samples were labeled with Cy3 and a Reference sample (made by pooling equimolar amounts of RNA from Test samples) was labeled with Cy5. 5 μg of aRNA was dried down and dissolved in 50 mM carbonate buffer pH 8.5. Individual vials of Cy3/Cy5 from the mono-reactive dye packs  were dissolved in 200 μl DMSO. To each sample, 10 μl of the appropriate CyDye dissolved in DMSO was added and the mixture was incubated for 1 h. Reactions were quenched with the addition of 5 μl 4 M hydroxylamine (Sigma-Aldrich). The labeled aRNA was purified with the E.Z.N.A. MicroElute RNA Clean Up Kit. The yields of aRNA and CyDye incorporation were measured on the NanoDrop ND-1000.Gene expression of mouse samples was analyzed with a 12 × 135 k http://www.ncbi.nlm.nih.gov/geo/.Each hybridization mixture was made up from 1.1 μg Test (Cy3) and 1.1 μg Reference (Cy5) sample. Samples were dried and 1.98 μl of water was added. The hybridization cocktail was made according to the manufacturer's instructions . 5.22 μl from this mix was added to each sample. The samples were incubated for 5 min at 65°C and 5 min at 42°C prior to loading. Hybridization samples were loaded onto the microarrays, and hybridized for 18 hours at 42°C with the Roche NimbleGen Hybridization System 4. Afterwards, the slides were washed according to the Roche NimbleGen Arrays User's Guide - Gene Expression Arrays Version 5.0 and scanned in an ozone-free room with a DNA microarray scanner G2565CA (Agilent Technologies). Feature extraction was performed with NimbleScan v2.5 (Roche NimbleGen). The array data have been deposited in NCBI's Gene Expression Omnibus and is accessible through GEO Series accession number GSE28463 http://www.R-project.org and Bioconductor http://www.bioconductor.org/ software.To generate the average log2 probe signal for the Affymetrix GeneChips, raw probe intensities without control probes were used. For the Nimblegen microarrays, raw sample channel data without control probes was used. Data handling, scatterplot generation and PCA analysis were performed using R-2.11.1 The authors declare that they have no competing interests.OB performed the data analysis and drafted the manuscript. WR and AdV provided the mouse biopsies. TR and AHZ provided the human biopsies. TMB helped to draft the manuscript. MdJ conceived of the study, and participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.RNA isolation protocol. Step-by-step protocol describing the isolation of RNA from skin biopsies.Click here for fileHuman biopsies results. Table showing the quality measure results for individual RNA samples isolated from human skin biopsies.Click here for fileScatterplots Human biopsies. Same figure as Figures Click here for fileMouse biopsies results. Table showing the quality measure results for individual RNA samples isolated from mouse skin biopsies.Click here for fileMouse biopsy sampling. Overview of resulting holes on mouse skin after sampling with different punch diameters.Click here for fileComparison to the gold standard methods. Table showing the quality measure results of this study compared to the gold standard methods for RNA isolation from skin biopsies.Click here for file


Hypoxia is central to both ischaemic and neoplastic diseases. However, the non-coding transcriptional response to hypoxia is largely uncharacterized. We undertook integrated genomic analyses of both non-coding and coding transcripts using massively parallel sequencing and interfaced this data with pan-genomic analyses of hypoxia-inducible factor (HIF) and RNApol2 binding in hypoxic cells. These analyses revealed that all classes of RNA are profoundly regulated by hypoxia and implicated HIF as a major direct regulator of both the non-coding and coding transcriptome, acting predominantly through release of pre-bound promoter-paused RNApol2. These findings indicate that the transcriptional response to hypoxia is substantially more extensive than previously considered. Cells respond to physiological insults by altering their transcriptional output. Hypoxia (low oxygen level) is an important environmental stress with a central role in many physiological responses including adaptation to altitude, exercise, growth and development, as well as major pathophysiological processes such as ischaemic vascular disease, inflammation, wound healing and cancer To date, pan-genomic analyses of the transcriptional response to hypoxia using microarrays have focussed on protein-coding RNAs (mRNAs) and some microRNAs (miRNAs) Here, we provide the first pan-genomic analysis of both non-coding and coding transcriptional responses to hypoxia, including lncRNAs, miRNAs, piwiRNAs, snRNAs, and tRNAs. The analysis reveals marked bidirectional hypoxia-inducible changes in transcript abundance across all classes of RNA and demonstrates the importance of HIF in regulating non-coding as well as coding transcriptional responses to hypoxia. Correlation with changes in histone marks and RNA polymerase 2 (RNApol2) positioning, indicates that activation of gene expression by HIF occurs commonly through long-range interactions that cause the release of pre-bound, promoter-paused RNApol2.To gain a global overview of the transcriptional response to hypoxia, we integrated expression profile and chromatin maps of human breast cancer MCF-7 cells incubated in either 21% ambient oxygen (normoxia) or 1% ambient oxygen (hypoxia) Fig. IndepenTo determine transcript abundance across all classes of RNA, the ribosome depleted, directional RNA-seq dataset was mapped sequentially to databases of different RNA classes Fig. 42989 net alSeveral microarray-based pan-genomic analyses have described heterogeneous responses in the miRNA response to hypoxia depending on cell type, hypoxic stimulus and profiling platform – reviewed in McCormick As few lncRNAs have been included in previous pan-genomic analyses our data identifies lncRNAs as a new class of transcript that are commonly regulated by hypoxia. The majority of these transcripts remain functionally uncharacterized. However, two lncRNAs (NEAT1 and MALAT1) included in a previous microarray-based analysis A proportion of reads did not map to an annotated transcript of any class. To define whether these represent previously non-annotated transcripts, they were first examined for contiguity, using the Cufflinks de novo transcript assembler. This identified 91 novel RNA transcripts with a median length of 9988 bp (range 269–38 2549 bp) many of which were upregulated in hypoxia  and S4 abona fide non-annotated transcripts, reference to UCSC data (http://genome.ucsc.edu/cgi-bin/hgTrackUi?hgsid=336849981&g=cpgIslandExt) revealed that 50 (55%) have a CpG Island within 1000 bp of the putative promoter. All 91 transcripts were also detected in the independently derived polyA+ RNA-seq dataset. Of the 91 non-annotated transcripts, 37 did not overlap with any annotated RefSeq gene, and were therefore classified as non-annotated intergenic transcripts  To pursue this further, we undertook polyA+ RNA-seq analysis of mRNA and lncRNA in hypoxic MCF-7 cells transfected with siRNAs targeting HIF-1α, HIF-2α or both isoforms simultaneously. Approximately 25% of all lncRNA transcripts detected in the ribosomal-depleted RNA-seq were detected in this analysis. Figbona fide genes  of the previously non-annotated transcripts consistent with assignment as de genes  1617.Consideration of changes in RNApol2 binding in hypoxia revealed that for the majority of hypoxia-inducible genes, RNApol2 was already bound at the promoter in normoxic cells. Although there were exceptions, at the majority of promoter sites hypoxia did not increase promoter-bound RNApol2 substantially. This is illustrated in FigOn close inspection the maximum RNApol2 signal was observed in the promoter-proximal region, 40–45 bp downstream of the transcriptional start. This suggests that promoter-proximal pausing is an important rate-limiting step for transcription of hypoxia-inducible genes and that reversal of pausing is a key step through which hypoxia regulates transcription de novo in hypoxia  in an atmosphere of either normoxia (21% oxygen) or hypoxia (1% oxygen). Expression of HIF-1α and/or HIF-2α subunits was suppressed as previously described Human MCF-7 breast carcinoma cells from the American Type Culture Collection (ATCC) were incubated for 24 h in an Total RNA was prepared using the mirVana miRNA Isolation Kit  and treated with DNaseI . Ribosomal RNAs were depleted using RiboMinus™ Locked Nucleic Acid probes . The resulting RNA was fragmented and end-repaired using the Illumina directional protocol. The final cDNA library was purified using Agencourt AMPure beads  optimised to retain small RNAs.Directional PolyA+ RNA libraries were prepared using the ScriptSeq™ v2 RNA-Seq kit  and non-directional libraries using the TruSeqRNA Sample Prep Kit .Chromatin immunoprecipitation was performed as previously described All libraries were sequenced using the GAII or HiSeq platforms (Illumina) according to the manufacturers protocol. Raw and mapped data for RNA-seq and ChIP-seq is available at EMBL-EBI Array Express .http://hannonlab.cshl.edu/fastx_toolkit/), RNA-seq reads were sequentially mapped to RNA databases and expression analysis performed using the edgeR  package. Remaining unmapped reads were examined for non-annotated reads using the Cufflinks de novo transcript assembler (http://cufflinks.cbcb.umd.edu). ChIP-seq databases were analysed as previously described After trimming adapter sequences using the FASTX-tool kit  and R (http://www.R-project.org).All assays were performed in duplicate or triplicate. Statistical analyses using two-tailed