Interestingly, RNA structural analysis showed that ss-Ino-RNA had increased formation of secondary structures. Our data further revealed that extracellular ss-Ino-RNA was taken up by scavenger receptor class-A (SR-A) which activated downstream MAP Kinase pathways through Toll-like receptor 3 (TLR3) and dsRNA-activated protein kinase (PKR). Our data suggests that ss-Ino-RNA is an as yet undescribed virus-associated innate immune stimulus.During viral infections, single- and double-stranded RNA (ssRNA and dsRNA) are recognized by the host and induce innate immune responses. The cellular enzyme ADAR-1 (adenosine deaminase acting on RNA-1) activation in virally infected cells leads to presence of inosine-containing RNA (Ino-RNA). Here we report that ss-Ino-RNA is a novel viral recognition element. We synthesized unmodified ssRNA and ssRNA that had 6% to16% inosine residues. The results showed that in primary human cells, or in mice, 10% ss-Ino-RNA rapidly and potently induced a significant increase in inflammatory cytokines, such as interferon (IFN)-β (35 fold), tumor necrosis factor (TNF)-α (9.7 fold), and interleukin (IL)-6 (11.3 fold) (p<0.01). Flow cytometry data revealed a corresponding 4-fold increase in influx of neutrophils into the lungs by ss-Ino-RNA treatment. In our  Inflammatory responses that are generated during viral infections are critical for antiviral immune responses. The exact viral recognition elements that activate cells to induce pro-inflammatory signals are not completely characterized. Viral recognition elements such as dsRNA and 5′-triphosphate single-stranded RNA are recognized by several cellular pathways including scavenger receptor class-A (SR-A), dsRNA-activated protein kinase (PKR), retinoic acid inducible gene-1 (RIG-I), Toll-like receptor 3 (TLR3), melanoma differentiation-associated antigen 5 (MDA5), NLR family, pyrin domain-containing 3 (NLRP3) and mitochondrial antiviral signaling proteins (MAVS) In addition to the innate immune inflammation, viral recognition elements trigger establishment of an antiviral state, under which each cell resists viral infection. The resistance to viral infection is in part through inhibition of viral replication by perturbation of RNA and protein synthesis. Determining the exact mechanisms by which immune and antiviral responses are activated is essential for understanding viral pathogenesis.Based on previous data published by our laboratory and others, we hypothesized that any ssRNA, of cellular or viral origin, modified by the presence of inosines (Ino-RNA) was an innate recognition element. RNA with high inosine content is not commonly found in normally growing eukaryotic cells but it is present during infections with DNA and RNA viruses such as polyomavirus, Rous-associated virus, vesicular stomatitis virus, measles virus, and respiratory syncytial virus Extracellular Ino-RNA is generated during viral infections. Cell lysis occurs frequently during viral infections, which results in the release of cell content, including intracellular generated Ino-RNA, into the extracellular space. Extracellular dsRNA has been shown to be able to stimulate antiviral responses in neighboring, uninfected cells Here, using RSV infection as a model, we report that the presence of inosines in ssRNA is a potent inducer of inflammatory cytokines and the antiviral state during virus infection. Our studies showed that in PHBE cells, primary human alveolar macrophages, or C57BL/6 mice, treatment with Ino-RNA significantly induced interferon (IFN-β), tumor necrosis factor (TNF-α), and interleukin (IL-6). Furthermore, treatment of epithelial cells with Ino-RNA led to a reduction in RSV replication. Structural analysis revealed that the presence of inosines increased formation of secondary structures. Extracellular Ino-RNA was internalized by SR-A-mediated endocytosis, which activated downstream MAP kinase pathways through the well-characterized dsRNA specific proteins, TLR3 and PKR. Based on our studies, we suggest that Ino-RNA, of viral or cellular origin, in the surrounding tissue after release from infected cells is a signal for the presence of virus infections.2 humidified chamber. BEAS-2B bronchial epithelial cell line was maintained in Dulbecco's Modification of Eagle's Medium/Ham's F-12 with 5% fetal calf serum. Cells, at 90% confluence, were treated with different RNA preparations and total cellular RNA or proteins were then extracted at indicated times.Primary human bronchial epithelial cell (PHBE) were grown in bronchial epithelial basal medium (BEBM) supplemented with growth factors  at 37°C in a 5% CO6 cells/ml.Isolation of primary human alveolar macrophages was approved by a written consent by the University of North Carolina School of Medicine Committee on the protection of the rights of human subjects. Healthy, non-smoking male volunteers, 18 to 40 year of age, underwent fiberoptic bronchoscopy with lavage to procure human alveolar macrophages. Samples were put on ice immediately after aspiration and centrifuged at 300 x g for 10 min at 4°C. Cells were washed twice with RPMI-1640, and re-suspended in RPMI-1640 at 1×10For virus infection, BEAS-2B or PHBE cells were seeded and allowed to reach 70% confluency. Cells were then treated for 10% Ino-RNA, N-RNA, or IFN-α. After 24 hr, cells were infected with respiratory syncytial virus (RSV-A2) at multiplicity of infection (MOI) of 0.1 plaque forming unit (PFU)/cell. Following infection for 24 hr, cells were harvested for total RNA extraction.Viral titers were measured by standard plaque assay using HEp-2 cells. Plaques were visualized by using goat anti-RSV antibody  and HRP-conjugated anti-goat secondary antibody .pGEM-Luc (p-Luc) vector , was linearized with HindIII and purified by Wizard DNA Cleanup kit . Run-off transcription of normal p-Luc RNA (transcript a) was performed using the Megascript T7 kit  according to the manufacturer's protocol with nucleotide triphosphates at 7.5 mM each. p-Luc RNA containing 6% Inosine (transcript b) was transcribed by ATP, GTP, CTP and UTP concentrations to 5.6 mM each, and including 9.4 mM ITP in the reaction. RNA containing 10–11% Inosine (transcript c) was transcribed by adjusting ATP, GTP, CTP and UTP concentrations to 2.8 mM each, and including 9.4 mM ITP in the reaction. RNA containing 16% Inosine (transcript d) was transcribed by further reducing GTP concentration to 1.0 mM, where ATP, UTP, and CTP were kept at 2.8 mM each, ITP concentration was kept at 9.4 mM. All transcription reactions were performed at 37°C for 2 hr, followed by digestion of the template DNA and purification using Megascript purification kit according to manufacturer's protocol . The transcripts were then quantitated by UV absorbance, and were analyzed by 1% agarose gel electrophoresis.2  for 2 hr at 37°C. Digested products were analyzed by high performance liquid chromotography (HPLC) using C-18 reverse phase column. Relative nucleoside composition was determined by peak integration using Breeze software , and is represented as a ratio of area under each nucleoside peak.Transcripts were characterized for inosine content by digestion of 3.5 mg of transcript with 2 units each of snake venom phosphodiesterase and calf Intestinal alkaline phosphatase in buffer containing 10 mM Tris pH 8.8, and 2 mM MgClTRIzol total RNA isolation reagent  was used for RNA extraction. First strand cDNA was synthesized using superscript reverse transcriptase . cDNA synthesis was performed using 0.5 µg total RNA in reverse transcription reaction. Following reverse transcription, 2 µl of cDNA was amplified by RT-PCR. Each experiment was performed in duplicate in 96 well plates using 1 X Sybr Green master mix  in a final volume of 25 µl. The statistical analysis was performed using parametric two-tailed unpaired t-test with Welch's correction. The sequences of primers were as follows:CAGCAGTTCCAGAAGGAGGAIFN-β forward, AGCCAGTGCTCGATGAATCTIFN-β reverse, GGAGAAGGGTGACCGACTCATNF-α forward, TGCCCAGACTCGGCAAAGTNF-α reverse, ATT CTG CGC ACG TTT AAG GAIL-6 forward, ATC TGA GGT GCC CAT GCT ACIL-6 reverse, IL-8 forward, tctggcaaccctagtctgctIL-8 reverse, gcttccacatgtcctcacaaTACCATGAAGGTCTCCGCRANTES forward, GACAAAGACGACTGCTGGRANTES reverse, CACAAACACAATGCCATTCARSV-NS1 forward, AGAGATGGGCAGCAATTCATRSV-NS1 reverse, GGACCTGACCTGCCGTCTAGGAPDH forward, TAGCCCAGGATGCCCTTGAGGAPDH reverse, in vitro kinase assays, cell extracts from BEAS-2B cells were prepared using a buffer containing nonidet-p40 detergent and were used for in vitro PKR activation as previously described with minor modification For total cellular protein isolation, PHBE cells were washed 2X in phosphate-buffered saline (PBS) and equal numbers of cells were lysed using 1X SDS-sample buffer with 2.5% β-mercaptoethanol. Prior to electrophoresis, proteins were denatured by heating the samples at 95°C for 5 min. To shear the chromosomal DNA, samples were passed through a 26G needle several times. The proteins were then resolved on a 10% SDS-PAGE and were electrotransferred onto nitrocellulose membranes. The immunoblotted proteins were visualized using the enhanced chemiluminescence (ECL) western blot detection system . For Signaling antibodis were used according to the manufacturer's instructions. Rabbit antibodies were used to detect p38 MAPK, phospho-p38 MAPK, JNK, phospho-JNK, phospho-eIF-2α, MDA5 and RIG-I , TLR3 , PKR and phospho-PKR .The sequences targeted by chemically synthesized small interfering RNAs  in transient knockdown experiments were as follows:GGTATAGCCAGCTAACTAGTLR3 gene, GCAGGGAGTAGTACTTAAATAPKR gene, GGAAGAGGTGCAGTATATTRIG-I gene, GGTGAAGGAGCAGATTCAGMDA5 gene, PHBE cells were transfected with TLR3-siRNA, RIG-I-siRNA, MDA5-siRNA, or control-siRNA (30 nM) using DharmaFECT transfection reagent  according to the manufacturer's instructions. 72 hr after the transfection, cells were stimulated with N-RNA or 10% Ino-RNA. Then, cytokine production was investigated at mRNA level and the phosphorylation state of MAPKs was determined by western blot analysis after.2. Loading buffer consisted of the running buffer with 40% sucrose. For Denaturing gel electrophoresis, we used 1% agarose gel with 2% formaldehyde and 10 mM MOPS. Prior to loading, samples were heated for 10 min at 65°C. For RNA structural analysis using fluorescence spectrum we used acridine orange RNA-binding dye. RNA at 5 µg/ml was dissolved in buffer containing 12 mM Hepes (PH 7.5), 120 mM KCl, 4 mM MgCl2. Spectral analysis was performed on a SpectraMax Gemini XS  with excitation at 460 nm and emission maximum at 530 nm.Non-denaturing gel electrophoresis of RNA was performed using 0.6% agarose gel in the presence or absence of 120 mM KCl, 4 mM MgClAll experiments were performed in accordance with the Animal Welfare Act and the U.S. Public Health Service Policy on Humane Care and Use of Laboratory Animals after review of the protocol  by the NIEHS animal care and use committee. 6–10 week old male C57BL/6 mice  were used in all experiments. Mice were housed and maintained in specific pathogen-free conditions. For treatments, anesthetized mice were made to aspirate intratracheally N-RNA, Ino-RNA at indicated concentration or vehicle control (PBS). After indicated times, bronchoalveolar lavage (BAL) was performed using phosphate buffered saline. ELISA was then used to measure the levels of cytokines in the BAL fluid. For PKR knockout experiments, 8–12 week old male mice genetically-deficient in PKR and wild-type control C57BL/6/SV129 were used. After 4 hr treatments, lung tissues were harvested for RT-PCR.For flow cytometry, BAL cells were first treated with rat anti-mouse CD16/CD32  and as immunoglobulin receptor blockers . Then, one microgram of each monoclonal antibody specific to CD11c (APC), Gr-1 (PE-Cy7) and major histocompatibility complex Class II (MHC II)- FITC , were added and incubated for 30 min on ice. Cell acquisition and analysis were performed on a BD LSR-II flow cytometer using FACSDiva software .10% Ino-RNA was labeled with Cy3 using Mirus RNA labeling kit . Labeled RNA was then purified with RNAeasy mini kit . Cells were grown in glass-bottom microwell dishes  and were treated with 10 µg/ml of Cy3-labeled 10% Ino-RNA alone or were first treated with 10 µg/ml of dextran sulfate or feutin . After 5 min, cells were washed 3 X in PBS, and mounted with mounting medium .Gene expression analysis was conducted using Agilent Whole human Genome 4x44 multiplex format oligo arrays  following the Agilent 1-color microarray-based gene expression analysis protocol. Starting with 500 ng of total RNA, Cy3 labeled cRNA was produced according to manufacturer's protocol. For each sample, 1.65 µg of Cy3 labeled cRNA was fragmented and hybridized for 17 hr in a rotating hybridization oven. Slides were washed and then scanned with an Agilent Scanner. Data was obtained using the Agilent Feature Extraction software (v9.5), using the 1-color defaults for all parameters. The Agilent Feature Extraction Software performed error modeling, adjusting for additive and multiplicative noise. The resulting data were processed using the Rosetta Resolver® system (version 7.2) .in vitro synthesis of normal RNA (N-RNA) and inosine-containing RNA (Ino-RNA)  . The inoIno-RNA) . QuantitTo assess the effect of Ino-RNA on innate inflammatory responses, we first treated PHBE cells with N-RNA and Ino-RNA (10 µg/ml) containing 6%, 10% and 16% inosine incorporation . RT-PCR Next, we examined whether the induction of inflammatory cytokines resulted in establishment of antiviral state during RSV infection. PHBE and BEAS-2B cells were treated with N-RNA or 10% Ino-RNA (10 µg/ml). After 24 hr, cells were infected with RSV at MOI of 0.1 pfu/cell. Cells were then collected after an additional 24 hr, for total RNA extraction, or 48 hr for plaque assays.Data in in vivo, we used C57BL/6 mice. Mice were treated with PBS, N-RNA or 10% Ino-RNA. After 24 hr, BAL fluid cells were harvested and used for ELISA and flow cytometry   . Similarin (PKR) , suggestTo identify the receptor that is involved in the recognition of Ino-RNA on epithelial cells, we tested SRs as possible candidates. Previous studies from our laboratory showed that SR-A is a cell surface receptor for dsRNA To characterize the downstream pathways, we used the endosomal acidification inhibitor bafilomycine A (BFA), which prevents adequate function of endosomal compartment Since our data showed that Ino-RNA contained more secondary structures, we next determined whether dsRNA-specific signaling pathways could be activated by Ino-RNA. PHBE cells were treated with Ino-RNA at 10 µg/ml and at various times, cell extracts were prepared for western blot analysis. The data showed that there was a rapid activation of dsRNA-specific protein kinase PKR, and the downstream signaling molecules such as eukaryotic initiation factor-2α (eIF-2α), p38 MAPK and JNK . TreatmeRecent reports have shown that TLR3, MDA5 and RIG-I -/- and wild type control mice were used. We examined the effect of PKR deficiency on cytokine expression. Total cellular RNA was extracted from lungs and was subjected to RT-PCR. As compared to the wild type, the lungs from PKR-/- mice exhibited significantly diminished expression of TNF-α, IFN-β and IL-6 . This is likely due to the fact that poly I∶C has been the most potent inducer of type-I interferons, and other ds- or ssRNA polynucleotides have not been as effective Our data, based on four different observations, demonstrated that incorporation of inosine in ssRNA increased secondary structures. First, our data showed that PKR, an intracellular dsRNA-specific kinase was activated by Ino-RNA more than with N-RNA  C and D.A report by Hartner et al. showed that, using ADAR1-deficient mice, ADAR1 was necessary for the maintenance of hematopoietic stem cells and suppression of interferon in vitro structural analysis data showing increased secondary structures after inosine incorporation are not consistent with previously published data Our in vitro transcription experiment, guanosines triphosphate are replaced by inosine triphosphate (It's known that, during virus infection, ADAR activation leads to adenosine to inosine conversion. In our hosphate , howeverRNA secondary structures are metastable, nonetheless they can activate innate immune responses. Interestingly, Nallagatla et al. reported that secondary RNA structures capable of activating PKR were also formed in 5′-triphosphate ssRNA Previous work showed that SR-A was a surface receptor for extracellular dsRNA in vivo data showing an increase in neutrophil infiltration into the lungs of Ino-RNA-treated mice (Our global transcriptomic analysis on RNA isolated after 2 hr of treatment showed that 132 genes were preferentially induced by Ino-RNA . Severalted mice .We therefore suggest that fof RNA with secondary structures through an increase in inosines and subsequent activation of intracellular signaling pathway is a critical step in cellular and immune activation.


Acanthaster planci are intense disturbances that can decimate coral reefs. These events consist of the emergence of large swarms of the predatory seastar that feed on reef-building corals, often leading to widespread devastation of coral populations. While cyclic occurrences of such outbreaks are reported from many tropical reefs throughout the Indo-Pacific, their causes are hotly debated, and the spatio-temporal dynamics of the outbreaks and impacts to reef communities remain unclear. Based on observations of a recent event around the island of Moorea, French Polynesia, we show that Acanthaster outbreaks are methodic, slow-paced, and diffusive biological disturbances. Acanthaster outbreaks on insular reef systems like Moorea's appear to originate from restricted areas confined to the ocean-exposed base of reefs. Elevated Acanthaster densities then progressively spread to adjacent and shallower locations by migrations of seastars in aggregative waves that eventually affect the entire reef system. The directional migration across reefs appears to be a search for prey as reef portions affected by dense seastar aggregations are rapidly depleted of living corals and subsequently left behind. Coral decline on impacted reefs occurs by the sequential consumption of species in the order of Acanthaster feeding preferences. Acanthaster outbreaks thus result in predictable alteration of the coral community structure. The outbreak we report here is among the most intense and devastating ever reported. Using a hierarchical, multi-scale approach, we also show how sessile benthic communities and resident coral-feeding fish assemblages were subsequently affected by the decline of corals. By elucidating the processes involved in an Acanthaster outbreak, our study contributes to comprehending this widespread disturbance and should thus benefit targeted management actions for coral reef ecosystems.Outbreaks of the coral-killing seastar  Acanthaster planci , a region where these disturbances occur with a periodicity of ∼20 years and, along with bleaching events and cyclones, are the major drivers of community dynamics on coral reefs Acanthaster aggregations and quantified their impacts on corals, other sessile communities, and resident coral-feeding fishes. In contrast to prior studies that were mostly restricted to reporting a posteriori observations of the consequences of Acanthaster on reefs, we quantitatively describe the processes leading to the community changes resulting from Acanthaster outbreaks.Here we describe an This study was approved and conducted as part of ongoing research of the Centre de Recherches Insulaires et Observatoire de l′Environnement .Acanthaster were progressively reported from the different high volcanic islands of the Society Archipelago  and then from the Australes (Rurutu) in French Polynesia. These islands are scattered over a broad geographical scale spreading 675 km north-south and 330 km east-west. Our study was conducted in Moorea  at each of three sites . The second sampling approach consisted of a yearly survey of seastar aggregations conducted at a large scale all around Moorea and its ∼100 km perimeter of reefs. The small-scale surveys were initiated between 2003 and 2005 depending on the different assemblages sampled (see Sampling section below), whereas the large-scale survey of seastars started in 2006, once Acanthaster aggregations were observed to spread to multiple sides of the island. Surveys were conducted until 2010, as long as remaining aggregations were observed. All the sampling was conducted using SCUBA on the outer reef slopes where, in Moorea as in other islands in French Polynesia, the highest coral biomass and the most diverse reef communities are concentrated Acanthaster aggregations were systematically first observed (refer to results of this study). Around Moorea, the outer reef habitat typically extends from the water surface at the crest of the barrier-reef where oceanic waves break, down to a depth of ∼35 m where sand plains begin. A peak in diversity and coral coverage is typically observed at the 10–20 m depth range Acanthaster outbreak, Moorean reefs underwent the additional impacts of the tropical cyclone Oli  whose immediate effects were partially captured by the present study.Starting in 2002, unusually elevated densities of Acanthaster outbreak (see above). The composition of the sessile communities  was sampled in n = 10 random quadrats by recording the type of substrate beneath 81 points defined by a grid of 10 cm-mesh within the 1 m2 areas. As major reef corallivores, populations of the outbreaking seastar Acanthaster and resident coral-feeding butterflyfishes were surveyed in n = 3 replicate belt-transects (50×4 m) placed randomly along constant depth contours. These fishes belong to the genus Chaetodon which encompasses several specialized coral-feeding species whose populations are tightly associated with corals C. ornatissimus, C. reticulatus, C. trifascialis, C. lunulatus, C. auriga, C. lunula, C. pelewensis, C. ulietensis, C. unimaculatus, C. quadrimaculatusAcanthaster aggregations at an island-scale around Moorea, we developed the SCUBA-tow technique, an adaptation of the manta-tow −1). In clear oceanic waters surrounding Moorea, this position of the observer allowed for a survey of the reef substrate in an ∼30 m-wide band in the 10–30 m depth range. Due to the cryptic character of AcanthasterAcanthaster feeding-scars remain clearly visible for ∼3 weeks before being covered by colonising algae and other sessile organisms  post-hoc test was used to compare data among groups. Linear Mixed Models (LMMs) were used to test for correlations between the dynamics of the different reef communities: Acanthaster, corals, other sessile communities, and coral-feeding butterflyfishes. LMMs have the advantage of taking into account correlated observations, and were also used to examine how populations of different coral genera were affected during the decline of coral communities facing Acanthaster. This was performed by drawing a linear regression between the coverage of each coral genus (dependant variable Genus cover) and the coverage of the overall coral community  as quantified at our nine reference reef locations on four different years during the process of the outbreak . For these regressions, autocorrelations were tested for the fixed effects of the covariable Coral cover, the random effects of the grouping factor Reef location (as the result of the interaction Site × Depth), and their interaction Coral cover × Reef location, and were taken into account in the calculation of parameters where significant Acanthaster and butterflyfish densities were log(x+1) transformed, and arcsin(√x) transformation was applied to percent-cover data. All statistics were computed in R version 2.12.0 (R Development Core Team 2008) complemented by the NLME package Variability in the coverage of benthic communities and in the density of butterflyfish assemblages was tested using three-way nested ANOVAs in which Acanthaster was observed at 18 m-depth at the outer reef site Tiahura in October 2003 (Acanthaster feeding-scars (>150 scars per 2 min-towing section) were mostly concentrated on the north-eastern corner of Moorea, with elevated densities also found near Tiahura and on the central west side of the island  and residence times of predators , which, combined with the sequential time of arrival of seastar swarms at the different reef locations, resulted in complex spatio-temporal variability in the rate of predation on corals. This generated asynchronicity in the decline of corals among sites and depths ), p<0.01; see Acanthaster had fallen to zero on all surveyed reef locations, and no additional individuals were observed during subsequent surveys.Expanding waves of ef-slope . This paAcanthaster predation resulted in a sharp collapse of coral populations and communities (Fisher's p<0.05 between consecutive samplings). This decline of corals progressively affected the different reef locations, as the seastars migrated through the reef system (p(a)<0.05). In contrast, no significant trend was observed in the cover of macro-algae, coralline algae, soft corals, or sponges (p(a)>0.05, see f system  and 5. TAcanthaster predation, which resulted in a sequential extirpation of taxa from local communities  = 0.38±0.03 SE). The collapse of Acropora populations was followed by those of sub-branching Pocillopora  = 0.11±0.02 SE). Populations of encrusting Montipora, massive Porites, and other hard-coral assemblages also declined, showing a synchronized collapse with the entire coral communities (refer to the intercepts of regressions in p(b)>0.05). The calcifying hydrozoan Millepora was rarely preyed upon by the seastars, and its populations did not vary with the decline in live coral. The selective predation of Acanthaster on corals, combined with the sequential time of arrival of seastars at the different reef locations and the variability in the observed peak densities, generated high spatio-temporal heterogeneity in the structure of benthic communities. Within the seven years of the Acanthaster outbreak on Moorea (2003–2010), a gradual shift of the outer-reef system was observed, from a coral-dominated one, to an ecosystem where space was mainly occupied by turf algae, coralline algae, rubble, and sand . Far from being unorganized and random events, outbreaks of the coral predator Acanthaster planci appear as ordered, relatively slow and diffusive biological disturbances. On the reefs surrounding the high volcanic islands of French Polynesia, these outbreaks were observed to originate from localized source areas situated at the base of outer-reef slopes, and to progressively spread to the entire reef systems by aggregative migrations of seastars. This pattern of propagation of Acanthaster from deeper parts of reefs toward unaffected locations also coincides with reports of previous outbreaks from other regions AcanthasterAcanthaster is observed. We advocate the importance of monitoring the ocean-orientated bases of reefs, particularly those where coral-rubble and coralline algae are abundant. These measures could help detect Acanthaster outbreaks at the earliest stages and, when appropriate, improve the efficiency of control efforts.Our observations of an Table S1Ratios of the density of feeding-scars to the number of predator seastar Acanthaster as observed in transect-counts on Moorean reefs during the outbreak.(PDF)Click here for additional data file.


Lethrinus harak, L. lentjan and Lutjanus fulviflamma) were collected in mangrove, seagrass and coral reef habitats and analyzed for stable isotope ratios in the juvenile and adult otolith zones. δ13C signatures were significantly depleted in the juvenile compared to the adult zones, indicative of different habitat use through ontogeny. Maximum likelihood analysis identified that 82% of adult reef L. harak had resided in either mangrove (29%) or seagrass (53%) or reef (18%) habitats as juveniles. Of adult L. fulviflamma caught from offshore reefs, 99% had passed through mangroves habitats as juveniles. In contrast, L. lentjan adults originated predominantly from coral reefs (65–72%) as opposed to inshore vegetated habitats (28–35%). This study presents conclusive evidence for a nursery role of Indo-Pacific mangrove habitats for reef fish populations. It shows that intertidal habitats that are only temporarily available can form an important juvenile habitat for some species, and that reef fish populations are often replenished by multiple coastal habitats. Maintaining connectivity between inshore vegetated habitats and coral reefs, and conserving habitat mosaics rather than single nursery habitats, is a major priority for the sustainability of various Indo Pacific fish populations.Mangroves and seagrass beds have long been perceived as important nurseries for many fish species. While there is growing evidence from the Western Atlantic that mangrove habitats are intricately connected to coral reefs through ontogenetic fish migrations, there is an ongoing debate of the value of these coastal ecosystems in the Indo-Pacific. The present study used natural tags, viz. otolith stable carbon and oxygen isotopes, to investigate for the first time the degree to which multiple tropical juvenile habitats subsidize coral reef fish populations in the Indo Pacific (Tanzania). Otoliths of three reef fish species ( Coastal habitats such as mangroves and seagrass beds are acknowledged as important nursery habitats for various species of reef fish, most of which are important to fisheries Until very recently, only indirect and circumstantial evidence existed in support of the paradigm that various species of coral reef fishes use mangroves or seagrass beds as essential juvenile habitat. Evidence was mostly based on higher juvenile densities and lower predation risk in these habitats as compared to the adult coral reef habitat  of the United Republic of Tanzania (URT) in 2009 Sonneratia alba along the sides of the Manyema creek. The creek receives substantial freshwater input only during heavy rainfall. The seagrass bed along the shoreline of the mainland lies at 0.5–5 m depth and the nearshore reefs along the island of Mbudya lie at 2–4 m depth, depending on the tides, while the deep offshore coral reefs are located at 15–20 m followed by mudflats at greater depths The Kunduchi area in Dar es Salaam Tanzania, where the study was conducted, has only one mangrove-lined creek (Manyema Creek), an extensive shoreline seagrass bed, two nearshore islands with fringing coral reefs which are separated from the mainland by a 15 m deep channel running almost parallel to the coastline, and several offshore submerged deep coral reefs . The manz. Lethrinus harak, L. lentjan, and Lutjanus fulviflamma, were selected for this study. Studies based on size-frequency data suggest that these species undergo ontogenetic mangrove/seagrass-to-reef habitat shifts L. lentjan and L. fulviflamma were caught from both the nearshore reef around Mbudya Island (about 3 km to mangrove) and two offshore reefs (∼9 km to mangrove), whereas L. harak could only be caught from the nearshore reef due to their very low abundances on the offshore reefs Three fish species, viL. harak (≥15 cm TL) from the seagrass beds, both the inner and outer otolith zones were analyzed to additionally determine the degree to which large juveniles from seagrass beds had spent their earlier juvenile stage in mangrove habitat and had moved to seagrass beds afterwards.After cleaning with deionized water, otoliths were mounted on glass plates and embedded in Araldite resin. The embedded otoliths were then cross-sectioned in the transverse plane through the core. For juvenile fishes from the mangroves and seagrass bed the outer otolith margin which reflects the current habitat was analyzed. For larger fishes from the reef, both the juvenile zone (which is the area directly adjacent to the core) which reflects earlier life in putative nurseries, and the outer otolith margin reflecting the adult reef habitat, were analyzed. The location for sampling the juvenile zone in adult otoliths was based on the mean otolith width of the mangrove/seagrass juvenile fish. For large 3 powder from a crater with a diameter of approximately 0.35 mm. Two craters were drilled per sample on opposite sides of the cross section to provide enough otolith powder for analysis. The powder (weight ≥10 µg) was collected with a scalpel and put into a glass tubes for further analysis. A few drops of pure (100%) orthophosphoric acid were added to the tube containing the powder at 80°C to dissolve all CaCO3. The isotope ratios of 12C/13C and 16O/18O of the released CO2 were measured using a Gas Bench mass spectrometer equipped with an automated carbonate extraction line (Kiel device). The NIST SRM 8544 (NBS 19) was used as a carbonate standard, which was routinely monitored during sample runs. The precision of analyses based on the measurements of this standard was within 0.05‰.The sectioned otoliths were drilled with a micromill that produced otolith CaCO13C) and oxygen (δ18O) signatures of the juvenile (inner) otolith sections from adult reef fish were compared to that of the outer otolith section from juvenile fish living in mangrove or seagrass habitats to determine whether adult fish had passed through either of these juvenile habitats. Fishes caught from the coral reef spanned a wide size range  across years for the 3 species, but never both stable isotope signatures together, meaning that habitats could always be distinguished throughout time based on at least one stable isotope signature. Notwithstanding some temporal variability, there was little overlap among habitat signatures . A Gabriel post-hoc test was used for comparison of means, while a Games-Howell post-hoc test was used when the requirement for homogeneity of variance was violated. Significance levels of p<0.05 were used in all tests. A quadratic discriminant function analysis (QDFA) using the jack-knife classification was done to examine classification success of assigning individuals to their known origin. SPSS 20 for Windows was used for all analyses Combined stable carbon  due to growth. We tested reef fish between 16 and 40 cm in length, comprising fish from a wide range in age (between ∼3 and 28 years). None of the regressions for either carbon or oxygen showed a significant relationship with size  for any species, except for δ13C in Lethrinus lentjan which showed a positive relationship (p = 0.01) but with a low R2 explaining only a small proportion of the overall variability . The observation that increase in body size has little to no effect on stable isotope signatures in our fish was further supported by lack of such a relationship in the fishes collected from the seagrass beds as well, which had a sufficiently large size range to test for this potential ontogenetic effect . These results show that potential ontogenetic effects are minor compared to habitat differences. Signatures from the inner (juvenile) part of adult reef fish otoliths were used in the MLA as the unknown mixed dataset to estimate the origin of these fishes. Finally, to determine in which habitat large juvenile L. harak, collected in seagrass habitat, had spent their earlier juvenile stage, juvenile seagrass and mangrove signatures were used as the baseline, and adult outer margins as unknown mixed sample. Maximum likelihood estimates and standard deviations were generated in HISEA by bootstrapping with 500 simulations.A maximum likelihood analysis  ‘HISEA’ developed by Millar 13C and/or δ18O signatures  and relatively high for L. fulviflamma (61%)  differed significantly based on otolith δgnatures , Fig. 2.ma (61%) . The claL. fulviflamma from the nearshore reef, the δ13C and/or δ18O signatures from juvenile margins of nearshore and offshore reef fish individuals were significantly different compared to the adult margin signatures (L. harak fish from the reef were only collected on nearshore reefs. Over 81% of these fish originated from either mangrove (29%) or seagrass (53%) habitats, while the remainder (18%) had grown up on the reef  compared to a much smaller amount of fish that had passed through seagrass and mangrove habitats (28%). Nearshore adult reef fish of this species also predominantly originated from coral reefs (65%). L. fulviflamma showed contrasting results: for adults collected on offshore reefs, mangroves were the dominant juvenile habitat (99%) and none of these fish had a juvenile signature that indicated a contribution from the coral reef. In contrast, nearshore L. fulviflamma adults mainly showed a coral reef signature (81%) in the juvenile zones of their otoliths, compared to that from seagrass (19%) or mangrove (0.03%) habitat. However, L. fulviflamma densities on nearshore reefs were at least an order of a magnitude smaller compared to those on offshore reefs , followed by L. lentjan (35%) and L. fulviflamma (19%), while for offshore reefs this was 28% and 100% for the latter two species, respectively. The large contribution of coral reefs to the nearshore as well as offshore adult populations of L. lentjan (65–72%) suggests that L. lentjan populations may be largely self-replenishing by the reef habitat. In contrast, the juvenile source habitats that were important for L. fulviflamma showed a large contrast for adults collected from nearshore vs. offshore reefs. However, considering that reef fish densities for this species were 13 times higher on offshore than nearshore reefs , most of the reef fish in this coastal area had likely originated from mangroves in terms of total population size. The observed variability in juvenile habitat contribution to adult populations indicates clear species-specific differences in nursery habitat dependency as well as presence of spatial differences in degree of population replenishment by nursery habitats This study is one of the first to present conclusive evidence that mangrove and seagrass habitats replenish reef fish populations in an Indo-Pacific locality, and is the first to identify the relative importance of multiple potential juvenile habitats in replenishing adult populations. The importance of these putative juvenile habitats differed among fish species, and among reefs located at different distances from these habitats. For adults collected on nearshore reefs, the combined contribution of mangrove and seagrass habitat was highest for L. harak were replenished to a greater degree by seagrass beds than by mangroves. This result corresponds to previous visual census data which also showed that mangroves are less important as a juvenile habitat for L. harak compared to seagrass L. lentjan showed a higher contribution from the coral reef to both nearshore and offshore adult populations than seagrass bed and mangrove combined, whereas visual census surveys suggest that this species primarily uses seagrass beds as juvenile habitats The results from our otolith study supported those from field surveys for some species, but not for others. Adult populations of Lutjanus fulvus  vs. Caribbean: 36% for Haemulon flavolineatumHaemulon flavolineatum and 99% for Lutjanus apodusBecause of the temporary nature of the Indo-Pacific mangroves' availability to fishes due to the tidal regime, and the relatively low number of species using them as juvenile habitats L. fulviflamma on offshore reefs), but act together in replenishing adult populations. Previous studies have shown high (88–99%) contributions from single juvenile habitats to adult reef fish populations, but none of these studies included multiple juvenile habitats, or the coral reef itself was not taken into account as a possible juvenile habitat Using three potential juvenile habitats  as a source, the current study suggests that no single habitat maintains reef fish populations of these species (except perhaps Lethrinus harak in seagrass beds had utilized mangroves during their earlier life stage. Such niche shifts are very common in tropical coastal fish species The usage of coastal habitat mosaics by juveniles of some reef fish could provide an insurance effect e.g.  against In conclusion, our results provide strong support for a nursery role of Indo-Pacific mangroves for certain species of reef fishes, but also indicate that seascape structure plays a vital role, and habitat mosaics rather than individual habitats should be conserved to maintain effective replenishment of offshore fish stocks.


Positive-sense RNA viruses are responsible for frequent and often devastating diseases in humans, animals, and plants. However, the development of effective vaccines and anti-viral therapies targeted towards these pathogens has been hindered by an incomplete understanding of the molecular mechanisms involved in viral replication. One common feature of all positive-sense RNA viruses is the manipulation of host intracellular membranes for the assembly of functional viral RNA replication complexes. This review will discuss the interplay between cellular membranes and positive-sense RNA virus replication, and will focus specifically on the potential structural and functional roles for cellular lipids in this process. One group of viruses responsible for a wide range of diseases in humans, animals, and plants are classified as positive-sense RNA viruses due to their genome structure, which consists of one or more single-stranded RNA molecules that in many respects resemble cellular mRNAs. Clinically important members of this group cause significant morbidity and mortality, include viruses from the  threats ,2. Curre threats ,4. An al threats –9. Howevin vitro [in vitro viral replicase activity [The small genomes of viruses relative to other organisms requires that they appropriate cellular machinery to complete their replication cycle, which for positive-sense RNA viruses is depicted schematically in in vitro ,28,40–45activity –45. And activity –63.et al. forming part of this special issue of Viruses.Although the observations outlined above provide substantial validation for the important role that cellular membranes play in positive-sense RNA virus replication, they provide only circumstantial evidence for the specific membrane components involved in the process and their precise molecular functions with respect to viral genome replication. Intracellular membranes contain diverse protein and lipid constituents and play a variety of roles during normal cellular physiology and metabolism, which include facilitating the spatial separation of cellular processes and the consequent differential concentrations of crucial cellular components, providing structural integrity to maintain organelle shape, and contributing functional co-factors for multiple processes such as signal transduction and biosynthesis. In this review, we will discuss three potential roles that lipid constituents of host cell membranes play in positive-sense RNA virus genome replication. These include: (i) providing a scaffold for targeting and assembly of RNA replication complexes; (ii) inducing alterations in membranes structure to potentially shield viral RNA replication intermediates from cellular innate antiviral pathways; and (iii) serving as functional co-factors for optimal enzymatic activity of viral replicase proteins. We will use examples of plant, insect, and mammalian viruses to highlight specific aspects of these potential roles, and will focus primarily on lipids rather than membranes in general. Several excellent reviews have recently been published –14 for r1.1.The interior of a cell is a highly organized environment, where membrane-bound organelles and specific organelle membranes represent one mechanism whereby cells enforce spatial constraints on particular metabolic processes. For example, the enzymes involved in electron transport and respiration are concentrated on the inner mitochondria membrane. Similarly, viruses may use intracellular membranes as simple molecular scaffolds on which to assemble their replication complexes, thereby increasing replication efficiency by concentrating essential viral and potential cellular components within a smaller microenvironment in the cell. Furthermore, membranes may provide a convenient platform to coordinate various steps in the replication cycle see , such as1.2.Positive-sense RNA viruses induce a number of distinctive membrane structures, which include the “membranous webs” of hepatitis C virus , the cluThe structural changes that must occur within lipid bilayers to produce the virus-induced alterations in intracellular membranes are substantial. For example, the outer mitochondrial membrane invaginations induced by Flock House virus  see als and the An alternative mechanism to induce membrane curvature is modification of lipid structure, either through changes in the polar headgroup or acyl chain composition. For example, lysophospholipids, which contain only one acyl chain per phospholipid molecule, and special lipids such as cholesterol or cardiolipin, which has four acyl chains attached to diphosphatidylglycerol, can have profound impacts on membrane curvature and plasticity . Acyl chRegardless of the mechanism whereby membrane curvature occurs, the function of these curious virus-induced structures remains enigmatic. One interesting possibility is that virus-induced membrane structures shield viral products that have the potential to activate cellular innate immune pathways. The replication of positive-sense RNA viruses involves the formation of dsRNA intermediates that serve as templates for genome amplification (see below, 1.3.de novo synthesis of a functional RNA replication complex in cell-free extracts has been accomplished for both poliovirus [One major goal of positive-sense RNA virus research is the complete isolation, characterization, and de novo synthesis of fully functional RNA replication complexes, a generic example of which is shown schematically in liovirus  and seveliovirus , and thiA substantial focus in the field of positive-sense RNA virus research has been the identification and characterization of the protein components necessary for replication complex function. The impact of lipids on this process has received far less attention in the past, in part due to some of the technical difficulties in lipid research noted above. However, there is both circumstantial and direct evidence that lipids can serve as functional co-factors for viral replicase proteins ,28,40–452.Despite the axiom that cellular membranes are essential host factors for positive-sense RNA virus replication, the specific role of individual membrane components, and in particular lipids, represents a vastly understudied area of virus biology and pathogenesis. We have discussed three potential structural or functional roles that cellular membrane-resident lipids may play in the assembly and function of positive-sense RNA virus replication complexes. These roles are not mutually exclusive, and it is possible that lipids contribute to several steps in the virus life cycle via multiple mechanisms, some of which we currently recognize, and others that remain to be discovered. The recent increase in targeted and genome-wide screens to identify host factors that impact positive-sense RNA virus replication –63, seve


In vitro cleavage assay using the recombinant human Dicer protein also showed that synthetic 7SL RNA was processed by Dicer into fragments of different lengths. Further functional analysis suggested that 7SL RNA-derived small RNAs do not function like miRNAs, neither do they regulate the expression of 7SL RNA. In conclusion, the current study demonstrated that Dicer can process 7SL RNA, however, the biological significance remains to be elucidated.It has been reported that decreased Dicer expression leads to Alu RNAs accumulation in human retinal pigmented epithelium cells, and Dicer may process the endogenous SINE/B1 RNAs  into small interfering RNAs (siRNAs). In this study, we aimed to address whether Dicer can process Alu RNAs and their common ancestor, 7SL RNA. Using Solexa sequencing technology, we showed that Alu-derived small RNAs accounted for 0.6% of the total cellular small RNAs in HepG2.2.15 cells, and the abundance decreased when Dicer was knocked down. However, Alu-derived small RNAs showed different characteristics from miRNAs and siRNAs, the classic Dicer-processed products. Interestingly, we found that small RNAs derived from 7SL RNA accounted for 3.1% of the total cellular small RNAs in the control cells, and the abundance dropped about 3.4 folds in Dicer knockdown cells. Dicer-dependent biogenesis of 7SL RNA-derived small RNAs was validated by northern blotting.  During the past 65 million years, Alu elements have evolved to more than one million copies in the primate genomes. This makes them the most successful transposable elements in terms of copy number in vitroAlu elements can be transcribed by two independent polymerases in two different ways. “Free Alu RNAs” are transcribed by Pol III from their own promoters, while “embedded Alu RNAs” are transcribed by RNA polymerase II (Pol II) as part of protein- or non-protein-coding RNAs 7SL RNA, a component of the signal recognition particle (SRP), is the common ancestor of Alu RNAs. Alu elements probably originate from duplication of the 7SL RNA gene In this study, we addressed whether there are endogenous Alu-derived siRNAs in HepG2.2.15 cells, and whether 7SL RNA is processed by Dicer. Using Solexa sequencing technology, we demonstrated that although the abundance of Alu-derived small RNAs was reduced in Dicer knockdown cells, their characteristics were totally different from those of classic siRNAs. Interestingly, we found that 7SL RNA can be processed by Dicer, and the small RNAs derived from 7SL RNA were characterized.To address whether Alu RNAs are processed by Dicer, we sequenced small RNAs extracted from Dicer knockdown and the control HepG2.2.15 cells using Solexa technology. A total of 14,289,326 genome-matching sequence reads were identified from the control cells, and 14,623,845 from Dicer knockdown cells. The majority (48%) of small RNAs in the control cells were derived from annotated miRNA loci, and as expected, the fraction of miRNA was diminished to 32% in Dicer knockdown cells .Alu-derived small RNAs were identified in our sequencing datasets using BLAST and SOAP2 software 7SL RNA, the evolutionary precursor of Alu, is a highly structured RNA containing partial double-stranded regions in vitro cleavage assay using the recombinant human Dicer protein. As shown in To validate Dicer-dependent biogenesis of 7SL RNA-derived small RNAs, we performed northern blotting analysis. As shown in Small RNAs derived from longer non-coding RNAs, such as tRNAs or snoRNAs, have been shown to function like miRNAs MiRNA is incorporated into the RNA-Induced Silencing Complex (RISC) with Argonaute proteins to regulate gene expression Growing evidence has suggested that small RNAs are implicated in epigenetic regulation of gene expression in vitro . Dicer knockdown was performed as described previously Human embryonic kidney cells (HEK293T from ATCC) and HepG2.2.15 cells . Membranes were incubated with primary antibody, followed by horseradish peroxidase-linked secondary antibody and detected with ECL plus reagents (Millipore). The primary antibodies include: anti-Dicer , anti-Ago2  and anti-β-actin .http://transpogene.tau.ac.il/) and the human 7SL RNA gene sequence (NR_002715.1), respectively.Small RNAs ranging from 16 to 30 nt were gel-purified, and those with 5′-phosphate and 3′-hydroxyl group were ligated with the 3′- and 5′-adaptors. The ligation products were then gel-purified, reversely transcribed, and PCR-amplified. The PCR fragments were then sequenced using Illumina GAII platform. Low quality sequencing reads were discarded, and the adaptor sequences were removed before analysis of small RNA sequences. The sequences were mapped to the reference human genome (hg19) using SOAP2 program with at most two mismatches. For annotation, small RNA sequences were aligned to miRBase15.0, Rfam 9.1 and GeneBank using BLAST and SOAP2 software. Alu or 7SL RNA-derived small RNAs were identified by mapping the small RNA sequences to the human Alu sequences extracted from the TranspoGene database , and small RNAs were extracted using the E.Z.N.A.® miRNA Isolation Kit .32P]ATP labeled oligonucleotides complementary to 7SL sRNA5cd. U6 was used as loading control.To detect the full-length 7SL RNA, 10 µg total RNAs were separated by electrophoresis in an 8% polyacrylamide gel and transferred to a nylon membrane. The membrane was hybridized with [γ-32P]ATP labeled oligonucleotides complementary to 7SL sRNA5cd. U6 was used as loading control.To detect small RNAs derived from 7SL RNA, 30 µg small RNAs were separated by electrophoresis in a 15% polyacrylamide gel and transferred to a nylon membrane. The membrane was hybridized with , immunoprecipitated with an anti-Ago2 antibody  and then bound to Protein-G Sepharose  beads. The beads were washed extensively with Ago2 IP washing buffer  before elution with 0.1 M glycine (pH2.3). Ago2-bound RNAs were then isolated using Trizol LS reagent (Invitrogen). To detect 7SL sRNA5cd, the eluted RNA was poly A tailed using a Poly (A) Polymerase Tailing Kit , reversely transcribed using RT primer (ATTCTAGAGGCCGAGGCGGCCGACATGTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTVN) and PCR amplified using the following primers: GAGTTCTGGGCTGTAGTGCGCTA and ATTCTAGAGGCCGAGGCGGCC, which only detected 7SL sRNA5cd but not the full-length RNA . MiR-17 was detected using the miR-17 RT-PCR kit from RiboBio (Guangzhou).Immunoprecipitation of Ago2 binding RNA was carried out as previously described Figure S1Quantification of small RNA northern blotting. 7SL sRNA5cd was represented by the 23 nt band.(TIF)Click here for additional data file.Figure S2Dicer cannot cleave the control RNA. 12% polyacrylamide gel electrophoresis of LacZ RNA digested by the recombinant human Dicer protein.(TIF)Click here for additional data file.Figure S3Knockdown of Ago2 in HepG2.2.15 and HEK293T cells. (A&C) representative western blot of Ago2, β-actin was used as loading control. (B&D) The relative levels of Ago2 mRNA determined by real-time RT-PCR. (A&B) Hep2.2.15 cells, (C&D) HEK293T cells.(TIF)Click here for additional data file.Figure S47SL sRNA5cd is not associated with Ago2 protein. (A) RT-PCR can specifically detect 7SL sRNA5cd but not full-length 7SL RNA. (B) RNA immnuoprecipitation experiment indicated that 7SL sRNA5cd was not associated with Ago2 protein, as a positive control, miR-17 was co-immunoprecipitated with Ago2 protein.(TIF)Click here for additional data file.Table S1Alu-derived small RNAs.(XLS)Click here for additional data file.Table S27SL RNA-derived small RNAs.(XLS)Click here for additional data file.


MicroRNAs’ dysregulation and profiling have been demonstrated to be clinically relevant in urothelial carcinoma (UC). Urine cytology is commonly used as the mainstay non-invasive test for secondary prevention and follow-up of UC patients. Ancillary tools are needed to support cytopathologists in the diagnosis of low-grade UC. The feasibility and reliability of microRNAs profiling by qRT-PCR analysis (miR-145 and miR-205) in archival routine urine cytology smears (affected by fixation/staining [Papanicolau] and room temperature storage) was tested in a series of 15 non-neoplastic and 10 UC urine specimens. Only samples with >5,000 urothelial cells and with <50% of inflammatory cells/red blood cells clusters were considered. Overall, a satisfactory amount of total RNA was obtained from all the considered samples . Twenty nanograms of total RNA have been calculated to be the minimal total RNA concentration for reliable and reproducible miRNAs expression profiling analysis of archival cytological smears . miR-145 and miR-205 were significantly downregulated in UC samples in comparison to non-tumor controls. These findings demonstrate that urine archival cytology smears are suitable for obtaining high-quality RNA to be used in microRNAs expression profiling. Further studies should investigate if miRNAs profiling can be successfully translated into clinical practice as diagnostic or prognostic markers. Our data provide further evidence on miRNAs as a promising class of diagnostic, prognostic, and predictive biomarkers.Written informed consent was obtained from all participants involved in the study, which was approved  by the institutional review board of the University Hospital of Padua .From the archives of the Surgical Pathology and Cytopathology Unit at Padua University (Cytopathology Unit) was retrieved a total of 10 consecutive low-grade UC and 15 consecutive non-neoplastic cytology samples reported in 2009 (>36 months old). Original routine cytological slides  were jointly re-assessed by two pathologists (RC and MF) accordingly to the current WHO criteria The presence of >5,000 well-preserved and well-visualized urothelial cells (as observed in 10 fields at 4x magnification) were appointed as minimum accepted cellularity. In neoplastic specimens, only slides with a proportion of tumor cells >25% were selected. Slides with >50% of urothelial cells obscured by either white or red blood cells were excluded .Overall, the male:female ratio was 8∶2 and mean age was 81.7±7.7 years  for UC patients, while male:female ratio was 8∶7 and mean age was 64.5±11.2 years  for the non-neoplastic patients.3 cells/well) were seeded in 150 µl of the complete medium in a 16-well microtiter plate specifically designed to measure cellular impedance  and placed into the xCELLigence system for data collection after a 30 min incubation at room temperature. The xCELLigence software was set to collect impendence data (reported as cell index) at least once every 15 min for 4 days. Overall, 106 cells at exponentially growth phase was yielded from each cell line for total RNA extraction, after routine spotting, fixation, and staining (as for the urine samples).As reference for the qRT-PCR analyses, Capan-1 and BxPc3 cell lines were purchased from the American Type Culture Collection  and maintained as recommended. Cell proliferation and counting was determined by monitoring each cell line with the xCELLigence system . xCELLigence cell index impedance measurements were performed according to the instructions of the supplier. Cells  for total RNA isolation according to the manufacturer’s instructions hsa-miR-145  and hsa-miR-205 , the NCodeTM miRNA qRT–PCR method (Invitrogen) was used on the LightCycler 480 Real-Time PCR System , as reported elsewhere  ACG CAA ATT CGT GAA GCG TT -3′; Invitrogen). All the reactions were run in triplicate, including no-template controls. A total of seven serial RNA dilutions for each non-neoplastic urine sample  and a total of eight RNA dilutions for cell lines  were tested for miR-205 and RNU6B in order to define the minimal adequate RNA quantity to use for miRNAs qRT-PCR analysis. To support the data, differences in miR-145 and miR-205 expression were tested in non-neoplastic and UC urine cytological specimens.RNA dilutions were retro-transcribed by using the VILO cDNA Synthesis kit (Invitrogen). To detect and quantify mature e of the qRT-PCR, according to the equation e = –10/slope. The ideal slope is −3.32, which corresponds to an amplification efficiency of 100% (e = 2). A slope comprised in the range of −3.6 to −3.1 is considered acceptable for qRT-PCR results α = 0.05. Differential expression between non-neoplastic and UC samples were tested for both miR-205 and miR-145 using the Welsh two-sided t-test. All statistical analyses were performed using the R software .Statistical analyses for testing the qRT-PCR efficiency were based on the results of experiments on routine cytology samples and two cell lines. The experiments were performed separately for RNU6B, miR-145and miR-205. The threshold cycle (Ct) was plotted against the log10 amount of cDNA input (ldose). The focused parameter was the slope of the relationship between ldose and Ct, since it was used to estimate the efficiency 260/280 ratio of 1.96±0.34 . Only one sample presented a lower RNA purity with a OD260/280 ratio of 2.80 (case #9), but was retained in the analysis. As expected, the purity and quantity of extracted RNA were higher in the two cell lines : CAPAN-1 RNA yield 10.8 µg and OD260/280 ratio of 1.85; BxPC3 RNA yield 15.2 µg and OD260/280 ratio of 1.92.As showed in e of the qRT-PCR obtained by cytology smear and to test the relationship between Ct and ldose, a covariance analysis (ANCOVA) model was applied to the two cell lines (Capan-1 and BxPC3). The best cut-off for RNA amount to obtain a proper qRT-PCR analysis was selected in terms of the estimated slope . When considering amount of total RNA ≥1 ng, the ANCOVA analysis (R2 = 0.99) showed that the slope of the relationship between ldose and Ct was equal to −3.561 , with efficiency e = 1.81. The estimated models are Ct = 27.65–3.55*ldose for BxPc3 and Ct = 24.97–3.55*ldose for Capan-1  showed that the slope of the relationship between ldose and Ct was −3.649 , with efficiency e = 1.75. The estimated model was Ct = 29.09–3.64*ldose both for BxPC3 and Capan-1  showed that the fixed effect slope of the relationship between ldose and Ct was equal to −2.966 , with efficiency e = 2.37. The estimated model was Ct = 33.23–2.966*ldose. For miR-205, when considering all dilutions, the LME analysis (R2 = 0.54) revealed that the fixed effect slope of the relationship between ldose and Ct was −1.73 , with efficiency e = 4.78. When considering RNA amount ≥20 ng, the LME analysis (R2 = 0.99) showed that the fixed effect slope of the relationship between ldose and Ct was −3.41 , with efficiency e = 1.94. These data pinpoint 20 ng of total RNA as the minimum cut-off to use in miRNA expression profiling in archival urine cytology specimens.To validate these results in long-stored urine archival smears, a total of 15 non-neoplastic urine smears were investigated, by applying a LME model including a random effect for the subject. The minimal RNA amount to perform an adequate qRT-PCR analysis was selected in terms of the fixed effect slope . All theBoth miR-145 and miR-205 were significantly down-regulated in UC specimens in comparison with non-neoplastic smears  . These fe.g. for urine in UC or effusions for carcinomas metastatic to serosal cavities.Formalin fixed and paraffin embedded (FFPE) tissues contain virtually all the information that confirm the central position of clinical pathology in diagnosis, prognosis, therapy recommendation, and disease mechanism insight. Well along in this pathway, cytopathology runs a parallel and successful track: many patients cannot undergo an open biopsy and cytological smears are the only tissue available for diagnosis. Moreover, disease and treatment modifications can be monitored via repeated sampling, as UC involves the urinary tract and represents the fourth most common cancer in men in the western world. UC is characterized by frequent recurrences comprised between 50% and 70%, and 10–15% of patients develops muscle invasion over a 5-yr period, requiring in many cases a longtime surveillance The gold standard for primary detection and follow-up of UC consists of cystoscopy and cytology As aforementioned, various molecular urinary biomarkers have been developed to improve UC detection; however, none of these showed a satisfying sensitivity or specificity for replacing cystoscopy, even coupled with cytology. A relatively new class of biomarkers (i.e. miRNAs) has been proposed for diagnostics, treatment monitoring and therapeutic approach in oncology.260/280 nm ratio between 1.7 and 2.3) of total RNA.In this paper, we first defined the minimal total RNA concentration for a reliable and reproducible miRNAs expression profiling analysis. Then, in order to demonstrate that archival cytology specimens are still informative for diagnostic purpose, we examined total RNA from 15 non-neoplastic and 10 UC cytology samples obtaining from each a good quantity (ranged between 0.06–4.44 µg) and good purity  miRNAs and provided evidence that miRNAs exist in a stable form in various body fluids, such as blood, urine, saliva, and peritoneal fluid Of interest, the method here presented, after a preliminary assessment of specimen cellularity and tumor cell ratio, could be virtually applied to whatever sample stored in cytopathology archives, from fine-needle aspirates to effusions and bronchoalveolar lavages. Further studies should evaluate this fascinating possibility.Our preliminary data opens a broad field of immediately available and low cost diagnostic samples to shed new light on urinary tract cancer. Larger studies should assess the wide spectrum of significant miRNAs involved in bladder cancer to be applied in clinical practice as diagnostic, prognostic, and predictive markers.


Lignins have a major impact on the agro-industrial uses of plants. Until now, most of the strategies considered for lignin reduction have targeted the monolignol pathway since the genes involved in these metabolic steps have been identified in many plants. Less is known about the other steps and in particular on lignin polymerization in the cell wall. While it is established that peroxidases are involved in the polymerization of lignin precursors, it is not yet clear whether laccases (EC 1.10.3.2) participate in constitutive lignification.AtLAC4 and AtLAC17) that are highly expressed in Arabidopsis stems were studied. AtLAC17 was specifically expressed in the interfascicular fibers while AtLAC4 was expressed in vascular bundles and interfascicular fibers. Arabidopsis T-DNA insertion mutants were selected and characterized. Two double mutants were obtained by crossing the AtLAC17 (lac17) mutant with two AtLAC4 mutants (lac4-1 and lac4-2). The single and double mutants displayed normal growth, except the lac4-2 lac17 mutant that sometimes had a semi-dwarf phenotype and collapsed vessels. While the single mutants had moderately reduced lignin levels, the stems of lac4-1 lac17 and lac4-2 lac17 had lignin content reduced by 20% and 40%, respectively. This lower lignin level improved their saccharification yield. Thioacidolysis revealed that disrupting AtLAC17 mainly affected the deposition of G lignin units in the interfascicular fibers and that complementation of lac17 with AtLAC17 restored the normal lignin profile. This study provides evidence that both AtLAC4 and AtLAC17 contribute to the constitutive lignification of Arabidopsis stems and that AtLAC17 is involved in the deposition of G lignin units in fibers, suggesting a role in early lignification .In order to address this issue, laccase genes  targeting several laccase genes was tested in different plants including Arabidopsis and poplar. These miRNAs were expressed constitutively under the control of the CaMV 35S promoter or of lignin-specific promoters such as Arabidopsis and preliminary results for poplar will be presented.Results obtained using of this miRNA strategy in


RNA interference (RNAi) is a powerful tool to study gene function in organisms that are not amenable to classical forward genetics. Hence, together with the ease of comprehensively identifying genes by new generation sequencing, RNAi is expanding the scope of animal species and questions that can be addressed in terms of gene function. In the case of genetic mutants, the genetic background of the strains used is known to influence the phenotype while this has not been described for RNAi experiments.Tribolium castaneum that RNAi against Tc-importin α1 leads to different phenotypes depending on the injected strain. We rule out off target effects and show that sequence divergence does not account for this difference. By quantitatively comparing phenotypes elicited by RNAi knockdown of four different genes we show that there is no general difference in RNAi sensitivity between these strains. Finally, we show that in case of Tc-importin α1 the difference depends on the maternal genotype.Here we show in the red flour beetle These results show that in RNAi experiments strain specific differences have to be considered and that a proper documentation of the injected strain is required. This is especially important for the increasing number of emerging model organisms that are being functionally investigated using RNAi. In addition, our work shows that RNAi is suitable to systematically identify the differences in the gene regulatory networks present in populations of the same species, which will allow novel insights into the evolution of animal diversity. Drosophila and mouse mutants can depend on the genetic background of different strains, e.g. [E.coli, rice and C.elegans [Drosophila melanogaster while a few non-saturating screens have been performed in other insects including the red flour beetle Tribolium castaneum [For a long time, the identification of gene functions has been based on classical forward genetic screens where mutants are randomly generated, e.g. by chemical or transposon mediated mutagenesis. The established mutant strains are then screened for phenotypes and subsequently the disrupted gene is identified and further analyzed -5. Imporns, e.g. -13. The .elegans -16. In y.elegans . The unb.elegans ,19. Withastaneum -22. The The discovery of RNA interference (RNAi) in animals  has openC. elegans and Tribolium the RNAi effect is transmitted even from injected parents to the offspring [The RNAi response of some organisms is systemic, i.e. dsRNA delivered into the body cavity is distributed throughout the organism and enters all cells. Hence, local injection leads to systemic gene silencing ,26,29,30ffspring ,29-32.Tribolium castaneum is robust, systemic, splice variant specific and feasible at all developmental stages [Tcdfd [Tcdistalless [Tckrüppel [Tcknirps . The strength of the RNAi response can be experimentally modulated by varying the concentration of injected dsRNA or by varying the time between injection of the mother and collection of the phenotypic offspring [RNAi in the red flour beetle l stages -35. Mores [Tcdfd , Tcdistastalless ,36Tckrüpckrüppel  and Tcknffspring ,34,38-40Tribolium , females of the black strain [pig19 males [karyopherin multi-gene family of nuclear import receptors [importin α genes exist: importin α1, importin α2, and importin α3 [importin α family function the same way and it has been shown that they act redundantly on many cargoes but there are also cargoes, which require a specific Importin α for their nuclear import [S. cerevisiae, estimated 57% of steady-state nuclear proteins use this import system [Tribolium.In an ongoing genome wide RNAi screen in k strain  were inj19 males . In the eceptors . In metaortin α3 . Importiortin α3 . The Importin α3 . The Importin α3 ,48. All r import ,45,49-54Tc-importin α1 in two Tribolium laboratory strains, black and San Bernadino (SB). Surprisingly, we find that RNAi knock-down leads to qualitatively different phenotypes depending on the strain. Further, we show that this is neither due to a general difference in RNAi sensitivity of these strains nor to nucleotide sequence divergence between them or differential embryonic expression. Instead, we find that the genotype of the injected female determines the RNAi phenotype of the offspring. These results show that the phenotypes generated in RNAi experiments can depend on the genotype of the used strain and we suggest that a proper documentation of the strain is an essential piece of information when publishing RNAi studies in any species.In this work, we quantitatively compare the RNAi phenotypes of TC000963 .The RNAi phenotype of the iB_00198 dsRNA fragment revealed by the iBeetle screen was marked by a highly penetrant and specific loss of the labrum . In order to follow up this phenotype, we first analyzed the phylogenetic relationship of the targeted gene. The iB_00198 sequence is part of the coding sequence of Tc-importin α1 open reading frame were generated . The resulting cuticles were scored for deletion or malformation of different parts of the body  resulted in a much stronger cuticle phenotype than observed in the screen. Most frequent were abdominal malformations , ranging from mild phenotypes in which the abdomen was constricted  to cuticles which showed an inversion of abdominal segments into the interior of the cuticle  were injected into black female pupae, which were mated with black males  into the Tc-importin α1 in the black strain using 0.3 μg/μl dsRNA resulted in a very similar phenotypic pattern as shown for 1 μg/μl  and mated them with black males. The phenotype of the offspring was intermediate between black and SB injected females  genetic background, the injected females became sterile not allowing judging the cuticle phenotype of the offspring  and Tcgiant (Tcgt), which elicit well quantifiable defects in the offspring of injected pupae [Tcdll was injected into both strains, 50 cuticles of the first egg collection (d 9 after injection) were analyzed regarding the number and state of the remaining leg segments. The cuticles were grouped into four different classes of phenotypic strength. In the strongest class only a coxa remained present  and calculated the average phenotype strength, which turned out to be similar .First, we checked ed pupae ,36. dsRNTc-gt dsRNA . The number of deleted trunk segments was used as measure for the phenotypic strength. This number varied from zero to six deleted trunk segments .The analogous experiment was performed with Tc-wingless (Tc-wg) and Tc-six3 in order to compare the RNAi response after larval RNAi (lRNAi). Knock-down of Tc-wg via injection of dsRNA (1 μg/μl) into late larval stages (L6) of SB and black lead to pupae with reduced genital lobes, an increased distance between the pupal wings and a reduced maxillary diameter .Further, we performed larval RNAi for r Figure C. The laof dsRNA  μg/μl inTc-six3 dsRNAi injection (0.5 μg/μl) in L6 larvae led to pupae with reduced eye size .ection 0. μg/μl inection 0. μg/μl inTc-importin α1 phenotype in the respective genetic backgrounds. This is similar to findings in other model organisms, where the phenotype of mutant alleles of some  genes is different depending on the genetic background of the strain.Taking into account the experimental variability inherent to RNAi experiments, these data suggest that our strains do not have a generally different RNAi response. Moreover, the nucleic acid sequence of the dsRNA fragments is almost identical in both strains (99.4%), making different RNAi efficiencies due to mismatches unlikely. Hence, the strain specific phenotypic difference we observed was likely due to different modulation of the black and SB strains were aligned with the sequenced strain (GA-2) and with Importin α orthologs of other species.Next, we asked whether differences of the amino acid composition of Tc-importin α1 protein could be the reason for the differences. We isolated and sequenced the coding sequences of both strains. The Tc-importin α1 amino acid sequences (526 amino acids) of the black and the GA-2 strains have identical sequences whereas the SB strain has different amino acids at two sites  whereas the SB strain shows an asparagine (N). In metazoa, this position is either occupied by a threonine (T)  or asparagine (e.g. C. elegans) (not shown). All three amino acids are similar regarding their polar but uncharged side chains.The SB strain encodes a leucine , whereas the two other Tribolium strains show a glutamine , which is also found in the two hymenopterans Nasonia vitripennis (N. vitripennis) and Apis mellifera (A. mellifera). M. musculus carries an alanine (A) at this position, which is hydrophobic and not polar, whereas D. melanogaster has a glutamic acid, which is acidic and negatively charged. None of the two sites is predicted to be the target of phosphorylation, N-glycosalation nor N-myristoylation by ExPASy and Prosite analysis. Taken together, the observed amino acid substitutions may lead to altered binding affinities, which might influence the phenotype. However, this is difficult to test because we do not know which of the many nuclear proteins likely to be imported by Importin αs actually elicit the observed phenotypes. An alternative explanation would be that the mutations lead to differential splicing of the gene in the different strains.The second different amino acid is located within the eighth ARM domain (position 376). Here, the Tc-importin α1 dsRNA (1 μg/μl) was injected in SB and black females and these were afterwards mated with males of the other strain, respectively. Therefore, zygotic expression of genes was based on heterozygous condition for black/SB in all offspring while the maternal contribution was either of the black or the SB type.Our data hinted at the genetic background as cause for the different phenotypes. However, it remained open whether this would be based on zygotic gene expression (i.e. expression of the embryonic genome) or whether different maternal inputs would be involved . In order to distinguish between these possibilities, SB females mated with black males showed a similar phenotype as described for the SB strain . This allowed detecting relative increase or decrease of the other importin αs. Both Tcimportin α2 and 3 levels were higher in all samples in the black strain compared to the SB strain but none of these differences was significant.By quantitative RT-PCR, we determined the amount of mRNA present in ovaries where maternal load of oocytes takes place, freshly laid eggs which reflect the final maternal load and embryos at 10–12 hours of development, where zygotic transcription has started. The qPCR data were normalized to the expression of the ribosomal protein  samples   was obtained from the iBeetle genome browser . The following primers were used to amplify the open reading frame froman embryonic cDNA pool (0-48 h) via standard PCR: 5’-ATGTCGGGCTCCGCTCACAA-3’ and 5’-TTAAAAATGGAATCCTCCCATCGGCACCG -3’. The Tc-importin α1 open reading frame was cloned into the pJET1.2 vector.Tcimportin α1a, 709 bp; see Additional file Tcimportin α1b, 872 bp; see Additional file Tcimportin α1a and Tcimportin α1b), 1 μg/μl (Tcgt and Tcdll) and for larval RNAi 1 μg/μl (Tcwg) and 0.5 μg/μl (Tcsix3). Pupal injections were performed as described [The sequences of the fragments used for RNAi are given in the Additional file1 B. The templates for the non-overlapping fragments were generated by PCR from a plasmid template using following primers: 5’-TAATACGACTCACTATAGGAGTCTGGAGGAGGGTTCTTGC-3’ and T7 Primer (5’-TAATACGACTCACTATAGG-3’) for the 5’ fragment . Deconvolution was performed with the “No Neighbour” method followed by a maximum projection using ImageJ (version 1.44 o). Pupae were analyzed and documented using a Leica M205 FA fluorescence stereomicroscope.Cuticles were documented using a Zeiss LSM 510 as described ,69. TcdlTc-six3 lRNAi in late pupal stages (fully sclerotized mandibles) was measured by freehand selection tool and the mean was calculated. The mean value provided the basis for the phenotype comparison. The phenotype level was chosen arbitrarily.lRNAi pupae were analyzed using ImageJ (version 1.44 o). The diameter of the second segment of the maxillary palpus . Phylogenetic analysis was conducted using MEGA version 5 [Importins of the different species were obtained using amino acid sequence of Tc-Importin α1 as query for a BLASTp search  at NCBI ersion 5 . The mulFor correspondence analysis , the lab® reagent . 1 μg/μl total RNA was converted to cDNA by using the MAXIMA® First Strand cDNA Synthesis Kit for RT-qPCR . Quantitative PCR was performed using HOT FIREPol® EvaGreen® qPCR Mix Plus (ROX)  and the CFX96™ Real-Time PCR System . For the qRT-PCR the following primer pairs were used: Tc-importin α1: 5’-CCGTATGCTGTGCTAATCGAG-3’ and 5’-CGTCCCGAAGAAGTGTTCAAT-3’, Tc-importin α2: 5’-AAAGTCTACGAACGGGCTTTG-3’ and 5’-GAACTGAATCTCCCCATTTGC-3’, Tc-importin α3: 5’-TGAGGAGTGCAATGGCTTAGA-3’ and 5’-TCATCCGCATCACCACTAAAG-3’ and Tc-rpS3: 5’-ACCTCGATACACCATAGCAAGC-3’ and 5’-ACCGTCGTATTCGTGAATTGAC-3’. All primers were designed to span an intronic sequence and were validated by gel analysis. To calculate primer efficiency (E=10^(−1/m)), a dilution series was performed. Data was normalized by the formula: rel. expression=Rtarget^Cqtarget/Rref.^Cqref. For better comparison of the results in the two strains, the expression levels were normalized such that the genes with the lowest relative expression were set to the same values in the SB and black strains. Specifically, the difference between the means of Tc-importin α1 expression levels in the SB and the black strains were calculated. Subsequently, all SB expression levels were reduced by this mean difference  and from eggs (0–2 h and 10–12 h) using TRIzole Figure . This noThe authors declare that they have no competing interests.JS identified the phenotype in the iBeetle screen, PK did the experiments, analyzed the data and wrote the manuscript, CSE contributed to the design of the study and the analysis of the data, GB conceived of the study, analyzed the data and finalized the manuscript. All authors read and approved the final manuscript.Genomic structure of Tc-importin α1, dsRNA fragments, alignments, tables with cuticle analyses, Tc-importin α1 RNAi phenotype in the GA-2 and pig19 strains, qPCR of importin paralogs in Tc-importin α1 RNAi.Click here for file


In actively dividing eukaryotic cells, chromosome ends (telomeres) are subject to progressive shortening, unless they are maintained by the action of telomerase, a dedicated enzyme that adds DNA sequence repeats to chromosomal 3′end. For its enzymatic function on telomeres, telomerase requires nuclear import of its protein component (hTERT in human cells) and assembly with the RNA component, TERC. We now confirm a major nuclear localization signal (NLS) in the N-terminal region of hTERT and describe a novel one in the C-terminal part. Using an siRNA approach to deplete several import receptors, we identify importin 7 as a soluble nuclear transport factor that is required for efficient import. At the level of the nuclear pore complex (NPC), Nup358, a nucleoporin that forms the cytoplasmic filaments of the NPC, plays an important role in nuclear import of hTERT. A structure-function analysis of Nup358 revealed that the zinc finger region of the nucleoporin is of particular importance for transport of hTERT. Together, our study sheds light on the nuclear import pathway of hTERT. The molecular mechanisms of DNA-polymerases result in incomplete replication of DNA at telomeres, the ends of linear chromosomes. As a consequence, DNA-ends were predicted to shorten with each round of replication, leading to a limited number of possible somatic cell divisions Nucleocytoplasmic transport in general occurs through the nuclear pore complexes (NPCs) that are embedded between the inner and the outer nuclear membrane. NPCs are composed of a set of ∼30 different nucleoporins (Nups), which occur in a copy number of eight or multiples of eight S. cerevisiae protein Mtr10, which is related to a transportin variant, was identified as an important transport factor for the RNA component of yeast telomerase With respect to telomerase, surprisingly little is known about its nuclear import pathway. Very recently, a bipartite NLS, as it is characteristic for the importin α/β-pathway, was described in hTERT In this study, we confirm the activity of the previously identified NLS of hTERT and describe a second, albeit less active, NLS in the C-terminal part of the protein. Furthermore, we identify the nuclear import receptor importin 7 as a major factor that mediates transport of hTERT into the nucleus. Efficient transport also requires the cytoplasmic nucleoporin Nup358, which was previously implicated in nuclear import of a subset of proteins HeLa-P4 cells Cells were transfected with small interfering RNAs (siRNAs) from Ambion, Eurofins or Santa Cruz  at a final concentration of 100 nM, using Oligofectamine (Invitrogen), according to the instructions of the manufacturer. 72 h after siRNA treatment, cells were plated on coverslips, transfected with reporter protein constructs of interest and analyzed after incubation for additional 20–24 h. For depletion of Nup358, cells were transfected twice with the siRNA, as described previously 2-M9 2-cNLS Xenopus laevis5′-AAAGACGGCGCTCTACAGATGA and 5′-TCATCTGTAGAGCGCCGTCTTT. 5′-AGTCTGCCGTTGCCCGCGGCGCCCAGGCGT and 5′-ACGCCTGGGCGCCGCGGGCAACGGCAGACT were used for the hTERT K236AR237A-mutation and 5′-TTCCGCAGAGAAGCGGCGGCCGAGCGT and 5′-ACGCTCGGCCGCCGCTTCTCTGCGGAA for the hTERT K649AR650A-mutation. All constructs were verified by sequencing .The plasmid coding for myc-EZI was kindly provided by Tohru Itoh GST-Nup358-fragment 2, 5% glycerol, 1% BSA, 1 mM DTT, 1 µg/ml each of aprotinin, leupeptin and pepstatin) for one h at 4°C. Some reactions also contained 2.5–10 µg RanQ69L, loaded with GTP. After the reaction, beads were washed three times with binding buffer without BSA. Bound proteins were analyzed by SDS-PAGE, followed by Coomassie-staining.5 µg of GST-tagged proteins were immobilized on 10 µl of glutathione-sepharose beads. After blocking of non-specific binding sites with 1% BSA, beads were incubated with 5 µg His-importin 7 in a final volume of 1 ml of binding buffer  and affinity-purified. The antibody against importin β was raised in rabbits against the full-length protein and affinity purified. Antibodies against Nup358 were described previously HEK 293T cells were transfected with plasmids coding for GFP or TERT-GFP. After 48 h, cells were trypsinized, washed with PBS and lysed in GFP-buffer . The lysate was cleared by centrifugation at 16,000 g for 10 min and diluted with GFP-buffer lacking NP-40 to yield a final concentration of the detergent of 0.2%. 8 µl of pre-equilibrated GFP-trap beads (Chromotek) were added. After 2.5 h at 4°C, beads were washed three times with GFP-buffer lacking NP-40 and BSA. Bound proteins were analyzed by SDS-PAGE, followed by immunoblotting.Immunofluorescence staining was essentially performed as described For quantification of subcellular localization of reporter proteins, cells were grouped into three categories: N>C (majority of the reporter protein in the nucleus); N = C ; C>N . Quantification was performed from at least three independent experiments, counting ∼100 cells with similar expression levels. Statistical significance of the data was analyzed by a two-tailed, heteroscedastic students t-test. p-values <0.05 were considered as biologically significant.2-independent medium (Gibco). FLIP-experiments were carried out at 37°C in a temperature controlled chamber attached to a confocal microscope  equipped with an argon laser and a 63x Plan-Neofluar 1.3NA water-corrected objective. For analysis of nuclear import during 146 seconds, a nuclear region was subjected to 180 bleach intervals with the argon laser set to 100%. After each interval, the fluorescence emission was collected during a period of 154 ms at 2% laser intensity with the pinhole set to 2.0 airy units. Signal intensities were analyzed from cytoplasmic regions of a bleached (Fbc) and a neighboring unbleached cell as a reference (Frc), and a background region (Fbg). Cytoplasmic fluorescence intensities for each time point were expressed as F(t) = (Fbc–Fbg)/(Frc–Fbg), normalizing the pre-bleach fluorescence to 1. 10–20 cells were analyzed per condition.24 h after transfection of hTERT-GFP constructs, HeLa cells grown on LabTec-chambers (Nunc) were transferred to COhttp://elm.eu.org), one region was identified that is enriched in basic amino acids, corresponding either to a bipartite or a monopartite NLS (amino acids 222–240 of the full-length sequence). Of particular interest was the short peptide PKRPRR at position 235–240, listed by the program as a “variant of the classical basically charged NLS”  experiments in living cells. GFP-tagged wild type and mutant hTERT were expressed in HeLa cells and the fluorescence in the nuclei of expressing cells was repeatedly bleached. The rate of loss in fluorescence intensity in the cytoplasm of bleached cells was taken as a measure for the efficiency of nuclear import of the fusion proteins. As shown in 2-cNLS, a reporter protein whose nuclear import depends on the importin α/β-dimer. It is mostly nuclear under steady-state conditions, although the nuclear export signal (NES) in the reporter leads to its constant shuttling between the nucleus and the cytoplasm. As a result, inhibition of nuclear import can be readily detected. Indeed, import of NES-GFP2-cNLS into the nucleus was clearly inhibited in importin β-depleted cells .Second, we performed live cell imaging of nuclear import by FLIP in importin 7-depleted cells, similar to the experiment described in Third, we used a biochemical approach and immunoprecipitated overexpressed hTERT-GFP and, as a control, GFP, using the GFP-trap Xenopus laevis, which shows 93% of sequence identity compared to the human protein, but has a base-substitution at the position that is targeted by our siRNA. Two additional substitutions were introduced by site-directed mutagenesis, rendering the resulting mRNA insensitive towards the siRNA. As shown in Fourth, (and most importantly), we designed an siRNA-insensitive expression construct for HA-tagged importin 7. The best control to exclude off-target effects in siRNA experiments is to rescue the phenotype by expression of the gene of interest from such siRNA-insensitive plasmids. For our experiments, we used importin 7 from Nucleoporins play very general roles in nuclear import and export of proteins, as they form the permeability barrier of the NPC. Besides this common function, certain nucleoporins have been shown to affect the localization of individual proteins in a transport receptor and/or cargo-specific manner In a previous study, we showed that different regions in Nup358 are involved in nuclear import of different substrates whose transport is strongly promoted by this nucleoporin We next addressed the question of how Nup358 and importin 7 operate together to promote nuclear import of hTERT-GFP. The nucleoporin could have a direct function, binding the import substrate or the receptor-substrate complex prior to translocation across the nuclear pore. Alternatively (or in addition), Nup358 could function during the recycling of importin 7 from the nucleus back to the cytoplasm, as suggested for importin β Finally, we analyzed the effects of co-depletion of importin 7 and Nup358 on nuclear import of wild-type hTERT-GFP and of the import-restricted mutant KR236AA. As shown in We also tried to rescue import defects in Nup358-depleted cells by overexpression of HA-tagged importin 7. In contrast to certain cargos whose cognate import receptors become rate-limiting for nuclear import in the absence of the nucleoporin Extra-nuclear functions of telomerase have been reported Being characterized as a bipartite NLS, the identified sequence (aa 222–240) implied the classical importin α/β -dimer as an import receptor for hTERT In the majority of nuclear import pathways, RanGTP plays a crucial role as it binds to all members of the importin β-family We have previously described Nup358 as a nucleoporin that affects nuclear accumulation of a subset of cellular proteins Together, our study provides important information about nuclear import of hTERT at the level of the cargo, the soluble import receptor and the nuclear pore complex. It will be interesting to analyze whether importin 7 and Nup358 play a role in maintaining high levels of nuclear TERT-activities and, thus, to prevent chromosomal damage. Interestingly, elevated levels of importin 7 transcripts were found in various tumor cells Figure S1Importin 7 functions as an import receptor for hTERT. HeLa cells expressing hTERT-GFP that had been treated with either control siRNAs or with siRNAs against importin 7 were analyzed for the dynamics of nuclear import of the reporter protein by FLIP. The graphs show the mean loss in fluorescence in three independent experiments, analyzing a total of 45 cells per condition. This is the same experiment as the one shown in (TIF)Click here for additional data file.Figure S2Alignment of the N-terminal region of five mammalian TERT-sequences (amino acids 1-349 for the human sequence). The NLS-region of the human sequence and the conserved basic residues are depicted in red. Asterisks, identical residues; colons, conservative changes.(TIF)Click here for additional data file.