Triaenodon obesus, grey reef sharks Carcharhinus amblyrhynchos and silvertip sharks Carcharhinus albimarginatus. Most individuals showed year round residency at Osprey Reef, although five of the 49 individuals tagged moved to the neighbouring Shark Reef (∼14 km away) and one grey reef shark completed a round trip of ∼250 km to the Great Barrier Reef. Additionally, individuals of white tip and grey reef sharks showed strong site fidelity to the areas they were tagged, and there was low spatial overlap between groups of sharks tagged at different locations. Spatial use at Osprey Reef by adult sharks is generally restricted to the north-west corner. The high residency and limited spatial use of Osprey Reef suggests that reef sharks would be highly vulnerable to targeted fishing pressure and that MPAs incorporating no-take of sharks would be effective in protecting reef shark populations at Osprey and Shark Reef.Although marine protected areas (MPAs) are a common conservation strategy, these areas are often designed with little prior knowledge of the spatial behaviour of the species they are designed to protect. Currently, the Coral Sea area and its seamounts  are under review to determine if MPAs are warranted. The protection of sharks at these seamounts should be an integral component of conservation plans. Therefore, knowledge on the spatial ecology of sharks at the Coral Sea seamounts is essential for the appropriate implementation of management and conservation plans. Acoustic telemetry was used to determine residency, site fidelity and spatial use of three shark species at Osprey Reef: whitetip reef sharks  Many marine apex predators are under threat from direct exploitation, mortality as bycatch, competition with fisheries and from other anthropogenic impacts such as habitat alteration or degradation Orcinus orca and African penguins Spheniscus demersus used behavioural information to prioritise habitats primarily used for the activity in which they are most receptive to anthropogenic disturbance In general, total protection only occurs once a species is significantly reduced Shark stocks are experiencing huge declines in numerous locations 2, extends east of the Great Barrier Reef Marine Park (GBRMP) to the edge of Australia’s Exclusive Economic Zone. In May 2009, the entire Coral Sea region was declared a Conservation Zone to provide interim protection while the area is being assessed for potential inclusion in the Commonwealth Marine Reserves. Activities taking place in the Coral Sea region, including tourism and commercial and recreational fishing, were allowed to continue. Commercial fishing activities are currently licensed and managed through the Australian Fisheries Management Authority (AFMA) and include long lining, deep water trapping, sea cucumber collecting and aquarium fish collecting. These activities have an estimated value of ∼ $AUS1 million per annum http://www.environment.gov.au/coasts/mbp/coralsea/publications/pubs/coralsea-reserve-proposal.pdf). Conservation groups and some scientists have proposed a total no-take model. To date, this no-take proposal has polarized the debate amongst the community and stakeholders. If MPAs are approved, the protection of sharks at Coral Sea seamounts should be an integral component of this planning. Therefore, knowledge on the spatial ecology of sharks at the Coral Sea seamounts is essential for the appropriate implementation of management and conservation plans.The Coral Sea region, an area of approximately 972 000 kmCarcharhinus amblyrhynchos, whitetip reef sharks Triaenodon obesus and silvertip sharks Carcharhinus albimarginatus are the most common species observed In the initial proposed multi-use model, our study location, Osprey Reef (a seamount) is listed as a Habitat Protection Zone that would allow limited commercial fishing  and recreational fishing. As with other isolated atolls and seamounts in the Indo-Pacific region, the shark assemblage at Osprey Reef is dominated by a few species The objective of this study was to examine the patterns of spatial use of reef shark species to determine if an MPA would be an appropriate management strategy for protecting shark populations at Osprey Reef and the neighbouring Shark Reef. The specific aims were 1) determine if reef sharks are permanent residents around Osprey and Shark Reef, 2) investigate the spatial use of sharks at Osprey Reef and 3) examine inter- and intra-specific spatial overlap of sharks at Osprey and Shark Reefs.All research methods were approved and conducted under Australian Fisheries Management Authority Scientific Permit #901193.2 , approximately 220 km east off the north-east coast of Australia, and 125 km from the edge of the Great Barrier Reef . Osprey 2 . The eas2) ∼14 km south of Osprey Reef  was deployed from March 2008 to June 2009. Receivers were attached to and suspended above the reef with stainless steel chains, shackles and buoys. The array was configured to be non-overlapping, with 25 receivers forming a ring around the perimeter of Osprey Reef, and 5 receivers within the lagoon . Howeverrey Reef . This isSharks were tagged at four locations: North Horn, Admiralty and False Entrance, which are located on the outer edge of the reef and are popular dive sites . The fouth March and the 13th April 2008, after all receivers had been deployed. In total, 18 adult whitetip reef sharks (112–150 cm TL), 27 grey reef sharks  and 4 adult silvertip sharks (157–230 cm TL) were tagged. With the exception of three grey reefs and one whitetip, all sharks were females.Animals were fitted with acoustic-coded V16 4H transmitters  . Sharks were tagged between 25Due to differences in behaviour and size, the three species were captured using different techniques. For whitetip reef sharks, animals were attracted to a closed crate containing fish frames (heads and skeletons) and secured to a barren reef outcrop. Sharks were then caught by a SCUBA diver gently grabbing the tip of the tail and quickly transferring the noose of a rope around the tail Grey reef sharks were caught by handline using circle hooks or using a hook-less method. In the hook-less technique, sharks were attracted using a large tuna head threaded onto a stainless steel chain attached to a rope. The tuna head was thrown from a 6×2 m hydraulic platform on the back of the research vessel into the water and quickly pulled back in again. When a grey reef shark pursued the bait onto the platform, this was quickly raised thereby stranding the shark. Acoustic tags were implanted as described for whitetip reef sharks.For silvertip sharks, two individuals were caught by hook and line, and internally tagged on the hydraulic platform of the boat as described for whitetip reef sharks. The other two individuals were tagged underwater. The transmitters were fitted with stainless steel tag heads and fitted to a modified spear gun. The sharks were attracted using the baitbox and a scuba diver shot the transmitter into the dorsal region of the shark.The number of days that each individual was detected at Osprey and Shark Reef was plotted on a timeline to determine if individuals are permanent residents at this seamount. Other data analyses were based on the number of hours each shark was present at each area (i.e. each receiver), and this was used as an indication of how often an individual and a species used that area of the reef. If a shark was detected by a receiver more than once in any particular hour, it was considered as having been present during that hour.O), permutated 1000 times. The degree of overlap is presented in a 0–1 scale, where 0 equals no overlap and 1 equals complete overlap. To complement the spatial overlap results, the proportion of hours each species was recorded at each receiver is graphically presented. Further spatial overlap analyses were also performed within species. For whitetip reef sharks, spatial overlap was tested between sharks tagged at three locations , and for grey reef sharks spatial overlap was run to compare adults tagged at two locations  and between life stages (adults from both locations vs. juveniles).The spatial overlap of the three species  was compared using niche overlap analysis conducted in EcoSim 700 For whitetip and grey reef sharks, circular statistics (Oriana 3 software) were also used to study the diurnal pattern of area use around receivers that had the highest number of hits (hereafter termed as key locations): N, W1, W6, W7 and, for Grey Reef sharks, also W2 . For eacn = 15, grey reef sharks: n = 16, silvertip sharks: n = 2). Input data were the number of detections per hour blocks. A FFT separates time-series data into frequencies and identifies any sinusoid patterns, or periodicity, in the dataset. A power spectrum is then constructed and the dominant frequencies are represented by peaks in the spectrum A Fast Fourier Transform (FFT) was also computed for each species to identify any temporal periodicity in shark activity around receivers N and W6, as these had the highest number of detections for all species. Only adult individuals that were regularly detected at either receiver throughout the course of the study were considered in this analysis , in particular between grey reef and silvertip sharks (O = 0.98). All three species spent the majority of time at N, W6 and W7 locations  was detected by receiver SR on the 16th of September 2008, 11.5 h after being within range of receiver S. It stayed in Shark Reef for at least one day, eight days later it was detected back at Admiralty, and remained in the Osprey area for the rest of the study. A third grey reef (7900) was detected at Shark Reef on the 27th of March 2009, after a last detection at North Horn 41 days earlier. This individual was not recorded after this last detection at Shark Reef. The fourth grey reef (7870) moved from receiver S to Shark Reef in one day, on the 15th January 2009, and remained in Shark Reef for the rest of the study. The silvertip shark (7864) was detected at Shark Reef on the 7th January 2009, after a last detection in Admiralty on the 28th December 2008. It was then within range of receiver SR until the 15th February 2009, after which there were no more detections.Five individuals, four grey reef and one silvertip sharks were detected at the single receiver at Shark Reef. Surprisingly, three of the grey reef sharks were juveniles (80–107 cm TL). One grey reef (7892) detected at W6  on the 2O = 0.91; O = 0.03) and False Entrance (O = 0.01)  (O = 0.27), and adult and juveniles were even more spatially separated  to their tagging location for the duration of the study . A high  = 0.11) . Adult gp<0.01). Whitetips tagged in Admiralty and False Entrance spent most of their time at receivers W6 and W7 (p<0.01) bimodal pattern in the use of W6, with more individuals being detected in the first hours of the night and in the first hours of the day (p<0.01) (A circadian (24 h) periodicity in the use of receivers N and W6 was present for all adults of the three species that were frequently detected at these sites . For whi6 and W7 . These a the day . At W7, (p<0.01) .p<0.01) (p<0.01) (p<0.01)  . The patp<0.01) . Grey re(p<0.01) , and the(p<0.01) .th June 2008 nd of October 2008. Finally, the two silvertip sharks for which detections stopped during the study were those that were externally tagged, so this could be a result of the tags being removed or falling off the sharks. Although the expulsion of internally implanted tags has been recorded in teleosts Results suggest that reef sharks at Osprey Reef are permanent residents of the area (Osprey and neighbouring Shark Reef), with very little emigration away from these areas. It is however important to note that not all animals were detected for the entire study period, and this needs to be considered when evaluating emigration and possible immigration of sharks to and from the Osprey population. There are a number of possible reasons why some animals (6 adult and 4 juvenile sharks) were not detected for the entire study: animals could still be present but were in areas of low receiver coverage, they could have emigrated from Osprey Reef, could have died during the study, or the transmitters failed. All four reasons are plausible, and it could well be a combination of them. As the results of whitetip reef shark movements suggest this species has small centres of activity, the three individuals that were not detected for the duration of the study could have moved to an area with no receiver coverage. For example, two of these individuals were tagged at False Entrance (W7) and they could have moved further south into the area where the two receivers were lost (W10 and W11). Also, there was only one receiver at Shark Reef, so animals could have moved there without being detected. Indeed, individuals moving to and from Shark Reef went days without being detected (11.5 hours to 41 days). For the four juvenile grey reef sharks, mortality could be responsible for the cessation of detections, as juvenile sharks are vulnerable to natural mortality by predation and starvation An interesting aspect of the population structure of whitetip and grey reef sharks at Osprey Reef is the sex ratio, which appears to be strongly biased towards females . This was reflected in the low number of males tagged in the current study (one whitetip and three grey reefs). So, do the few males mate with all females in the area, or is there a temporary emigration by females (as seen by shark 7908) or immigration of males from other areas? In Hawaii, female whitetip reef sharks show higher philopatry than males, which may suggest that males move more than females For whitetip reef sharks, the small area use reported at Osprey Reef resulted in very low overlap or mixing between sharks residing in different areas separated by only ∼10 km. This agrees with previous estimates that their movements are limited to 3–5 km Grey reef sharks at North Horn showed diel patterns of activity similar to whitetip reef sharks, but over a greater area. For example, during the day their centre of activity was around North Horn and W1, but at night they moved further down the west wall to the area around receiver W2 see . The lowSphyrna lewini in the Galapagos Islands, where sharks mainly use a few key areas around the Wolf Island hotspot The predictable use of particular sites by reef sharks suggests that determining the ecological significance of these sites is crucial for conservation planning. For example, whitetip and grey reef sharks showed peaks of activity in the use of the W7 area around the dusk period. False Entrance (W7) is an area where the reef wall is broken up, with many gullies and crevices (pers. obs). This may make it a good area for foraging at dusk, when reef fish are in the transitional stage from diurnal to nocturnal behaviour. Crepuscular peaks in activity have been observed for both captive and wild whitetip reef sharks (Whitney et al., 2007; The low spatial overlap between grey reef shark adults tagged 10 km apart was somewhat surprising, given that an individual moved to the GBR and returned within 4 months, and grey reef sharks display little site fidelity to reefs on the GBR The dissimilarity in movement patterns between grey reef sharks on the GBR and at Osprey Reef highlights the dissimilar behaviour that can be observed for the same species in different locations. It also shows the uniqueness of different systems when considering protection plans. The conservation plan currently implemented in the GBR is considered to have limited utility for grey reef shark protection Due to their isolation, oceanic seamounts can still constitute relatively pristine functioning systems that support a diverse array of species Overall, there is a lack of information regarding the spatial use of large mobile species, and this makes it difficult to provide technical advice on the design and spacing of MPAs Few tropical marine systems have remained unaffected by human exploitation


Our laboratory is blending new RNA chemistries, largely directed at the ribose 2'-hydroxyl group, with quantitative interpretation to create new kinds of chemical microscopes for accurate RNA structure analysis. This seminar will focus on two examples from retroviral biology. All retroviral RNAs contain a packaging element that directs the genome into nascent virus particles through a mechanism that has been unclear. Using SHAPE, we probed the structure of murine leukemia virus RNA inside virus particles and identified the binding site for the nucleocapsid domain of Gag. The RNA structural context is critical: High-affinity binding requires two specific UCUG-UR-UCUG motifs flanked by base-paired elements. Mutation of four guanosine residues in these two motifs - only 4 nucleotides per genomic RNA - reduced packaging to non-specific levels. These motifs only form in genomic RNA dimers; thus analysis of authentic full-length RNA was critical. This work illustrates how local context and RNA structure can create information-rich recognition signals from simple single-stranded sequence elements. In the second example, we discovered strong correlations between shRNA-mediated inhibition of HIV-1 replication in a cell-based assay and both the energetic cost of breaking secondary structures and the overall annealing energy between guide strand and folded target RNA. These correlations were only apparent when the energetic cost of disrupting pre-existing structure in the HIV-1 RNA was calculated using a secondary structure model derived from SHAPE probing information. We verified these correlations by design and evaluation of additional shRNAs and could achieve consistent inhibition of HIV-1 production by 90%. This approach emphasizes the profound, but ultimately predictable, role of RNA structure in tuning RNAi function and is likely to prove broadly useful in understanding large-scale biological recognition involving RNA.


Retinoic acid inducible gene-I (RIG-I) is a key regulator of antiviral immunity. RIG-I is generally thought to be activated by ssRNA species containing a 5′-triphosphate (PPP) group or by unphosphorylated dsRNA up to ∼300 bp in length. However, it is not yet clear how changes in the length, nucleotide sequence, secondary structure, and 5′ end modification affect the abilities of these ligands to bind and activate RIG-I. To further investigate these parameters in the context of naturally occurring ligands, we examined RNA sequences derived from the 5′ and 3′ untranslated regions (UTR) of the influenza virus NS1 gene segment. As expected, RIG-I-dependent interferon-β (IFN-β) induction by sequences from the 5′ UTR of the influenza cRNA or its complement (26 nt in length) required the presence of a 5′PPP group. In contrast, activation of RIG-I by the 3′ UTR cRNA sequence or its complement (172 nt) exhibited only a partial 5′PPP-dependence, as capping the 5′ end or treatment with CIP showed a modest reduction in RIG-I activation. Furthermore, induction of IFN-β by a smaller, U/A-rich region within the 3′ UTR was completely 5′PPP-independent. Our findings demonstrated that RNA sequence, length, and secondary structure all contributed to whether or not the 5′PPP moiety is needed for interferon induction by RIG-I. Our findings indicated that RNA sequences found within the NS1 segment of the influenza viral genome were capable of inducing IFN-β in vitro based on their specific sequences and structures. In addition, U/A-rich elements within the genome had the ability to induce IFN-β in a 5′PPP-independent manner. Our findings have demonstrated that RIG-I has evolved to interact with multiple U/A-rich RNA motifs commonly found in the UTRs of many diverse RNA viruses, thus facilitating its role as a key pathogen sensor against a broad range of viruses.The innate immune system has evolved to recognize pathogen-associated molecular signatures leading to activation of innate immune receptors The ends of the eight influenza A virus genomic RNA segments are highly conserved but only the shorter segments appear to provide optimal substrates for recognition by RIG-I After transfection into A549 cells, two sequences from the 5′ end of the cRNA  efficiently induced IFN-β message  at levelSince previous reports suggest that U/A-rich sequences are important for RIG-I recognition of RNA In contrast to all of the shorter 5′UTR RNAs, sequences derived from the larger 3′ UTR cRNA of NS1 (172 nt) and its complementary sequence (5′ UTR vRNA) resulted in only a partial decrease in IFN-β mRNA levels in the absence of the 5′PPP, exhibiting levels that were still much greater than the positive control IVT 9.2 RNA with a triphosphate . InteresThis surprising result led us to investigate the nature of our RIG-I ligands in more detail. Recent reports have indicated that IVT can result in the addition of non-templated complementary bases at the 3′ ends of transcripts Based on the sequence dependence seen for the 5′UTR cRNA, we performed mutational analysis on the U/A-rich cRNA and vRNA sequences and evaluated the consequences for IFN-β mRNA production. We substituted either all U nucleotides, or just a stretch of 6 conserved U residues, with C residues. The substitutions and their effect on the predicted RNA secondary structure is shown in To ensure that IFN-β gene induction of these RNAs was mediated through RIG-I, we used two different approaches. First, we used a conformational dependent antibody that only recognized the RNA-bound form of RIG-I . 24 hours after transfection of the appropriate RNAs, cells were fixed and stained. Although all cells expressing constructs containing a triphosphorylated 5′ end contained RNA-bound RIG-I . After tOne possibility to explain the ability of the 5′PPP-independent ligands to continue to signal after removal of the triphosphate group is that these ligands have a higher affinity for RIG-I than 5′PPP-dependent ligands. Since previous reports have shown that removal of the 5′PPP group from many RNA species decreases binding affinity for RIG-I , it is also possible that the 5′PPP-independent ligands do not show a similar affinity loss upon capping or dephosphorylation. To test these possibilities, we first determined the binding affinity of purified full-length RIG-I and a well-characterized fluorescein-labeled RNA molecule (FL-rU15). The dissociation constant was calculated from changes in the emission of polarized light that result from RIG-I/ligand binding obtained using a Beacon 2000 system (Invitrogen). RIG-I/FL-rU15 was then mixed with varying amounts of the unlabeled RNAs of interest and binding affinities determined from the competition with the labeled ligand .Although the binding affinities of the 5′PPP-independent vRNAs (U/A-rich and 5′UTR) are higher than the affinity of the 5′PPP-dependent 3′UTR vRNA  were grown in DMEM (Life Technologies) supplemented with 10% FBS, 100 units/mL penicillin, and 100 µg/mL streptomycin.In vitro transcribed RNA was prepared using the Ambion MEGAscript T7 High Yield Transcription kit according to the manufacturer's instructions. Templates were prepared by annealing complementary DNA oligos containing a T7 promoter followed by the desired target sequence. Transcription reactions proceeded for 4–16 hrs with no difference in biological activity. Following the reaction, the DNA template was digested with DNase I  and the RNA purified and isolated using TRIzol , followed by ethanol precipitation. Capped RNA was produced either by replacing the GTP in the transcription reaction with a 12∶1 ratio of m7G(5′)PPP(5′)G cap analog:GTP or by using the ScriptCap m7G Capping System  according to the manufacturer's instructions. CIP-ssRNA was made by removing the functional 5′PPP end with calf intestinal alkaline phosphatase  treatment.For transfections of A549 cells, 3 µg of RNA was used to transfect each well of a 6 well tissue culture plate using Lipofectamine 2000 (Invitrogen) as the transfection reagent. At designated time points, protein and RNA were harvested from duplicate wells for Western and qRT-PCR analyses. Total protein was separated on a 4–15% SDS-PAGE gel and the separated proteins were transferred to a nitrocellulose membrane. Western blots were performed using commercial antibodies purchased from Sigma (actin), Santa Cruz Biotechnology , and Cell Signaling (PKR). The relative amount of intracellular RNA for each gene of interest was quantified by qRT-PCR on a Stratagene Mx3000P , using the Superscript III Platinum SYBR Green One-Step qRT-PCR kit (Invitrogen) according to the manufacture's protocol, and expressed as a fold change. Primers used are available upon request.Secondary structures were predicted using the RNA secondary structure predicting program, mfold v3.2 4) cells were plated on Nunc LabTek II  chambered slide flasks in DMEM with 10%FBS, penicillin-streptomycin, and L-Glutamine. A549 cells were transfected with one µg of each RNA species using Lipofectamine 2000 . Cells were fixed 24 hours post-transfection using 4% paraformaldehyde and permeabilized using a 0.2% saponin/0.1% BSA/PBS buffer. Cells were blocked using CAS block (Invitrogen) overnight and probed with a conformational dependent RIG-I polyclonal antibody generated by Dr. Fujita, AlexaFluor goat anti-rabbit 549, and Hoechst 33342 (Invitrogen). Cells were visualized using a Zeiss fluorescent microscope with an axiocam HRM apotome attachment using AxioVision software .A549 (1×102 and 0.5 mM Tris[2-carboxyethyl] phosphine) and incubated for 30 s at 25°C. Experiments were performed in reduced light and data were plotted using KaleidaGraph (Synergy Software).Binding of RIG-I to labeled RNA was measured by a change in polarization (ΔmP) on a Beacon 2000 fluorescence polarization system (Invitrogen). RIG-I (0–1000 nM) and 0.1 nM 3′-fluorescein-labeled rU15 (FL-rU15) were mixed in binding reaction buffer , and then 10–1000 nM of various competitor RNAs was added. The solution was incubated at 25°C for 30 s before each measurement.In RNA competition assays, 0.1 nM FL-rU15 was pre-incubated briefly with 100 nM RIG-I in 50 mM HEPES, pH 7.5, 100 mM NaCl, 5 mM MgClIn vitro transcribed RNA samples (30 µg) were resolved on a 5% acrylamide gel at 90 mA for 2 hr in TAE buffer in the presence of RNase inhibitor  (10 U/ml). A 41-nt long chemically synthesized ssRNA (complementary strands) and its annealed 41 bp dsRNA product were used as controls. RNA was transferred to nylon membrane using electrophoretic transfer apparatus. The nucleic acids were not denatured prior to or after the transfer. After the transfer was complete, RNAs on the membrane was cross-linked by Stratagene cross-linker. Membrane was blocked using 5% BSA in TBS in the presence of RNase inhibitor for 3 hr and incubated with anti-dsRNA antibodies  (2 µg/ml) overnight at 4 C. Antibody signals were detected by chemiluminescence using secondary antibodies conjugated to horseradish peroxidase and an ECL detection kit .MALDI-TOF-MS of IVT RNA was performed using an Ultraflex III mass spectrometer  operated in positive linear mode. The matrix was made up of 1∶1 (v/v) mixture of 15 mg/ml of ammonium citrate in 0.1% trifluoroacetic acid/30% acetonitrile/water and 80 mg/ml of 2′,4′,6′-trihydroxyacetophenone monohydrate in ethanol. The sample was mixed 1∶8 with the matrix and analyzed with external calibration using synthetic oligodeoxyribonucleotides.Figure S1Induction of IFN-β message by IVT RNAs is sequence dependent. (A) The secondary structures of the 5′ UTR cRNA RNAs after base substitutions within the A rich regions were predicted using mfold (v3.2). (B) and (C) A549 cells were transfected with the RNA constructs shown and RNA was isolated 24 hr post-transfection. IFN-β mRNA levels were quantified using qRT-PCR. Error bars represent the standard deviation of triplicate qRT-PCR runs using RNAs from one of three representative experiments.(TIF)Click here for additional data file.Figure S25′PPP-independent induction of IFN-β message is sequence dependent. (A) The secondary structures of the U/A-rich regions or mutant RNAs after base substitutions as predicted by the program mfold (v3.2) are shown. (B) and (C) A549 cells were transfected with the indicated IVT RNAs and RNA was isolated 24 hr post-transfection to quantitate IFN-β message by qRT-PCR. Error bars represent the standard deviation of triplicate qRT-PCR runs using RNAs from one of three representative experiments.(TIF)Click here for additional data file.Figure S3Induction of IFN and IFN-stimulated genes is inhibited by reduction of RIG-I but not PKR using IVT capped RNAs. (A) A549 cells were transfected with the siRNAs shown or mock transfected. 24 hr later, cells were transfected again with the indicated IVT RNAs and the cells were processed 24 hr post-secondary transfection. (B) Protein lystaes were used to determine the levels of RIG-I and PKR by western blot analysis. (C) RNA isolated from A549 cells was used to measure IFN-β mRNA levels by qRT-PCR. Error bars represent the standard deviation of triplicate qRT-PCR runs using RNAs from one of three representative experiments.(TIF)Click here for additional data file.


Tick-borne encephalitis virus (TBEV) is a causative agent of acute central nervous system disease in humans. It has three subtypes, far eastern (FE), Siberian (Sib) and European (Eu) subtypes, which are distributed over a wide area of Europe and Asia. The objective of this study was to develop a simple and rapid assay for the detection of TBEV RNA by using reverse-transcriptase loop-mediated isothermal amplification (RT-LAMP) method that can differentiate the three subtypes of TBEV and can be used for clinical diagnosis and epidemiological study.In vitro transcribed RNA of Oshima strain that belongs to FE subtype was serially diluted and used to examine the sensitivity of the assay. Cross-reactivity of subtype-specific RT-LAMP assay was tested by using the RNA of Oshima and Sofjin (FE), IR-99 (Sib) and Hochosterwitz (Eu) strains. RNA extracted from the mixtures of TBEV and ticks, and of TBEV and human blood, and the mouse tissues infected with TBEV, were evaluated in the assay. Positive amplification was observed by real-time monitoring of turbidity and by visual detection of color change.Primers for TBEV-specific and subtype-specific RT-LAMP assay were designed to target the consensus sequence in NS1 of all subtypes and the consensus sequence in the E gene of each subtype, respectiveluy. 2 copies of target RNA per reaction volume. FE-specific RT-LAMP assay amplified viral genes of Oshima and Sofjin strains but not of IR-99 and Hochosterwitz strains, and of Japanese encephalitis virus. RT-LAMP assay for Sib and for Eu specifically amplified viral genes of IR-99 and Hochosterwitz strains, respectively. We also showed that tick or human blood extract did not inhibit the amplification of viral gene during the assay. Furthermore, we confirmed that the TBEV RT-LAMP could detect virus RNA from peripheral and central nervous system tissues of laboratory mice infected with TBEV.The sensitivity of TBEV-specific RT-LAMP assay was 10TBEV RT-LAMP assay offers a sensitive, specific, rapid and easy-to-handle method for the detection of TBEV RNA in tick samples and this may be applied in the clinical samples collected from TBE-suspected patients. Flavivirus in the family Flaviviridae, is a causative agent of acute central nervous system (CNS) disease in humans and animals  × 6.022 × 1023, where the RNA length is 4966 base. The RNA concentration was measured with a NanoDrop ND-1000 apparatus (Thermo Scientific). The target RNA copy number was calculated and serial dilutions ranging from 10-1 to 104 RNA copies were used to determine the range of quantification.To obtain a quantitative RNA standard with which to examine the detection limit of RT-LAMP assay, a plasmid containing full length sequence of the Oshima strain of TBEV was used for RNA transcription . The pla-1 to 106 pfu) of the Oshima strain of TBEV were prepared. Each diluted sample was added to 10 mg of pooled ticks  and homogenized in 1 ml of Isogen (Nippon Gene). A 200 μl volume of healthy human blood was mixed with 50 μl of the Oshima strain of TBEV prepared at different concentrations (10-1 to 106 pfu) and with 750 μl of Isogen-LS. RNA was extracted according to the manufacturer’s protocol. Total RNAs of mouse blood and plasma samples were extracted using Isogen-LS (Nippon Gene). Total RNAs of mouse livers, spleens and brains were extracted using RNeasy Lipid Tissue Mini Kit (Qiagen) according to the manufacturer’s protocol. RNA was dissolved in DPEC-treated water and stored at −80°C until use. The experiment using human blood was performed with the approval of ethics committee of the Institute of Tropical Medicine, Nagasaki University .A 250 μl volume of culture supernatant from infected BHK cells was mixed with 750 μl of Isogen-LS (Nippon Gene) and genomic viral RNA was extracted according to the manufacturer’s protocol. Ten-fold serial dilutions  with 5 pmol of the primers F3 and B3, 20 pmol of the primers LF and LB, and 40 pmol of the primers FIP and BIP. One microliter of the extracted RNA was used as template per reaction. The RT-LAMP reaction mixtures were incubated at 62.5°C for 60 min and inactivated at 80°C for 5 min.A real-time turbidity caused by the accumulation of magnesium polyphosphate was monitored spectrophotometrically at 650 nm with LA-320C Loopamp real-time turbidimeter . The results were analyzed by the LA-320C software package (Teramecs). The product was electrophoresed on an agarose gel in Tris-acetate-EDTA (TAE) buffer followed by staining with ethidium bromide. Amplified products were sequenced to show that they matched the expected nucleotide sequences. Positive amplification was shown by the specific ladder-like pattern on a UV transilluminator at 320 nm. For visualization of the positive reaction, fluorescent detection reagent  was added to the reaction mixture and a change of color was recognized directly (transparent to cloudy yellow color) or under UV irradiation (fluorescent green).One step rRT-PCR was done with One Step PrimeScript® RT-PCR Kit (TAKARA BIO INC.) according to the manufacturer’s instructions. TBEV-specific primers and probe that detect all TBEV subtypes described in the previous paper  were synThe authors declared they have no competing interests.DH designed the experiment, performed live virus infection, extracted RNA, did RT-LAMP assay, and wrote the manuscript. KA designed the primers for RT-LAMP assay, extracted RNA, and carried out RT-LAMP assay and rRT-PCR. KM participated in discussion of the results and helped in drafting the manuscript. All authors read and approved the final manuscript.


Hepatitis C virus (HCV) infection develops into chronicity in 80% of all patients, characterized by persistent low-level replication. To understand how the virus establishes its tightly controlled intracellular RNA replication cycle, we developed the first detailed mathematical model of the initial dynamic phase of the intracellular HCV RNA replication. We therefore quantitatively measured viral RNA and protein translation upon synchronous delivery of viral genomes to host cells, and thoroughly validated the model using additional, independent experiments. Model analysis was used to predict the efficacy of different classes of inhibitors and identified sensitive substeps of replication that could be targeted by current and future therapeutics. A protective replication compartment proved to be essential for sustained RNA replication, balancing translation versus replication and thus effectively limiting RNA amplification. The model predicts that host factors involved in the formation of this compartment determine cellular permissiveness to HCV replication. In gene expression profiling, we identified several key processes potentially determining cellular HCV replication efficiency. Hepatitis C is a severe disease and a prime cause for liver transplantation. Up to 3% of the world's population are chronically infected with its causative agent, the Hepatitis C virus (HCV). This capacity to establish long (decades) lasting persistent infection sets HCV apart from other plus-strand RNA viruses typically causing acute, self-limiting infections. A prerequisite for its capacity to persist is HCV's complex and tightly regulated intracellular replication strategy. In this study, we therefore wanted to develop a comprehensive understanding of the molecular processes governing HCV RNA replication in order to pinpoint the most vulnerable substeps in the viral life cycle. For that purpose, we used a combination of biological experiments and mathematical modeling. Using the model to study HCV's replication strategy, we recognized diverse but crucial roles for the membraneous replication compartment of HCV in regulating RNA amplification. We further predict the existence of an essential limiting host factor (or function) required for establishing active RNA replication and thereby determining cellular permissiveness for HCV. Our model also proved valuable to understand and predict the effects of pharmacological inhibitors of HCV and might be a solid basis for the development of similar models for other plus-strand RNA viruses. Upon entry into the host cell, its 9.6 kb genome is translated by a cap-independent, internal ribosomal entry site (IRES) mediated mechanism into a single large polyprotein. Viral and cellular proteases co- and post-translationally cleave this precursor into ten mature viral proteins, comprising three structural proteins , the ion channel p7 as well as the six non-structural (NS) proteins NS2, 3, 4A, 4B, 5A and 5B HCV is an enveloped plus-strand RNA virus and belongs to the It is interesting to note that although HCV establishes a persistent infection, it does not have a latent phase; throughout the course of the infection, which can be decades long in many patients, there is constant production of viral RNA, proteins and infectious particles. In most viral infections, presence of non-self structures, such as dsRNA or viral proteins, is readily detected by sensors of the immune system, leading to the production of type I interferon (IFN) and activation of the adaptive immune response Therefore, in our present study we quantitatively followed the onset of intracellular RNA replication within the first couple of hours upon introduction of HCV genomes into the host cells. Based on these data we developed a comprehensive mathematical model capable of precisely describing both, the dynamic and the steady state phases of viral replication. We then used this model to study aspects of the viral replication cycle that cannot directly be accessed experimentally.in vitro transcribed replicon RNA into the cells. As host cellular factors play a critical role in determining the efficiency of viral replication To assess the dynamics of HCV RNA replication, we performed quantitative, time resolved measurements of strand specific viral RNA and polyprotein concentrations over 72 h after viral RNA transfection into Huh7 cells. To achieve sufficiently strong replication that can be measured reliably, we used subgenomic reporter replicons carrying the firefly luciferase gene in front of the viral proteins Upon transfection of replicon RNA into Huh7-Lunet cells, the RNA was instantly translated into protein and at the same time was rapidly degraded . ConsequPcytR) can be translated by ribosomes, but not HCV RNA within the replication compartment (PR). Model equations (eq.) of our final model are given in the χ2 = 8.69, compare supplementary In order to comprehensively understand the observed HCV replication dynamics and its underlying molecular processes, we set up a mathematical model of the intracellular HCV RNA replication. Dahari and colleagues developed a similar model previously, upon which we could build here ab initio replication in our setting (in contrast to pre-formed steady-state replication), we introduced one additional RNA species punpR, representing the transfected “naked” replicon RNA and an according processing step (rate k0), subsuming any re-folding, association with RNA-binding proteins and other processes that might take place and be required before in vitro transcribed RNA assumes a translation-competent state (pUnpμ) as compared to “processed” or cell-derived RNA (pcytμ). The second step that we updated to reflect the current biological understanding of the molecular processes was the initiation of minus strand RNA synthesis . It has been described for HCV, but also for other viruses cis. This means that for reasons not yet fully elucidated, NS5B cannot initiate RNA synthesis from a free, cytosolic RNA genome, but only from the very RNA that it has been translated from. This implies a tight spatio-temporal coupling of (poly)protein production and initiation of RNA replication, i.e. initiation can only occur immediately after translation/polyprotein processing and therefore in close proximity to the translation complex (CT). As our model does not account for spatial effects (such as diffusion), we approximated this cis-process by requiring an active translation complex instead of free, non-translating RNA (PcytR) for the initiation of minus strand RNA synthesis  into replication vesicles. This is a major change to Dahari's original model, in which cytosolic RNA (pcytR) and NS proteins (cytE) could freely and independently enter the compartment. This model, comprising Dahari's original model with the described extensions, we then considered our base model.Based on biological reasoning, we extended and modified Dahari's original model at two steps of the replication cycle. For one, to account for nt state  and 2. Tsis RIP, . This ciχ2 = 8.69, base model: χ2 = 2.12) and was capable of adequately describing both, the highly dynamic initial phase as well as the ensuing steady state of viral RNA replication , which additionally takes into account the number of unconstrained parameters – assuming a difference in the formation of the RC and initiation of RNA synthesis– led to a slightly larger χ2 (5.84 vs. 5.60) but a significantly lower AIC (−21.31 vs. −0.68). Moreover, hypothesis (E) reached a steady-state within 80 h, while (F) did not. This comparison therefore identified the initiation of minus strand RNA synthesis (i.e. the formation of the RC) as the step in the model, at which alteration of a single reaction rate suffices to optimally transform replication dynamics from high permissive cells into the dynamics found in low permissive cells.Having established a base model for HCV replication, we next wanted to assess which factors could explain differences observed between high and low permissive cell lines. In our experimental measurements for two differently permissive cell lines, Huh7-Lunet (high permissive) and Huh-7 lp (low permissive), replication reached a steady-state within the period of observation (72 h), however, plateau levels of viral protein, plus-strand RNA and minus-strand RNA differed by approximately one order of magnitude; further, the onset of the net increase of plus-strand RNA was delayed significantly in the low permissive cells and also the minimum concentration of plus-strand RNA reached during net degradation in the first hours after transfection were significantly lower in low permissive cells compare . As bothrameters . While pHF  and with actively translated HCV RNA (CT) to create replication vesicles and to allow for initiation of minus-strand RNA synthesis , the initial concentration of the host factor (HF), as well as the total number of ribosomes available for viral RNA translation (ibototR). The full system of differential equations and detail on the modeling procedure can be found in the Materials & Methods section; more detail on parameter optimization and analysis are given in supplementary With this additional modification to the mathematical description of the formation of replication compartments, and calibration of the model to the experimental data from both cell lines , excellent agreement between the model and experimental data was achieved, both, for high and low permissive cells with an overall f −112.31. We therInterestingly, analysis of the fitted parameters showed that the concentration of the host factor was more than 10 fold higher in highly permissive Huh7-Lunet cells than in low permissive Huh-7 lp cells. This difference led to slower formation of the replication compartment in Huh-7 lp cells, which in turn resulted in the observed delay in early viral replication and in decreased steady state levels in these cells. Based on our model and computational analysis, we therefore propose that a host process is critically involved in the formation of replication vesicles and/or the initiation of minus-strand RNA synthesis, turning this into the rate-limiting step for HCV RNA replication in low permissive cells.a priori using the model, and retrospectively compared the results with the experimental data in order to assess the validity of model predictions. This approach of introducing targeted mutations into the HCV genome interfering with distinct functions in the viral RNA replication cycle allows validation of individual steps in the model, thus step-wise reconfirming model assumptions and parameters.While the model could be very well fitted to the original replication data, we then wanted to corroborate its applicability for predicting replication dynamics also under distinct conditions that were not part of the calibration process. For this purpose, we performed additional, independent experiments using mutant HCV replicons with defects at defined stages of the replication cycle. We predicted the impact of such defects on viral replication Pink of the plus strand replication initiation complex IpR to zero  of the NS5B polymerase. The measured RNA and protein data thus reflect only the effect of translation and degradation in the cytoplasm, with no RNA replication occurring. We predicted the impact of this intervention with our model by setting the formation rate  to zero , 5 and 7 to zero . The facpink and 4mk for the 3′-NTR exchange  2k), the export rate of RNA into the cytoplasm (rate Poutk) and the efficiency of plus- (rate 4pk) and minus- (rate 4mk) strand RNA synthesis inside the replication compartment, respectively , since this determines at what time and to what extent RNA is available for translation, as well as the RNA degradation rate RCμ inside the replication compartment. The availability of viral RNA for rapid genome replication and the replication process inside the membranous web itself are therefore key determinants of the initial replication dynamics and thus the efficiency of infection, and consequently constitute a very attractive target for anti-viral drugs. Interestingly, the rate of polyprotein translation (ck) only very mildly impacts replication dynamics.For the establishing phase of replication, this analysis showed that the most influential processes are the polyprotein translation  as compared to RNA degradation in the cytoplasm  and calculated the resulting levels of plus strand RNA over time (RCμ) was approximately equal to the degradation rate in the cytoplasm (pcytμ). Our model therefore predicts that the viral RNA must be protected from active degradation during replication in order for HCV to maintain robust persistent replication. While it is virtually impossible to reproduce the above findings in a biological experiment (i.e. increasing RNA degradation inside the replicative compartment), previous in vitro data actually showed that viral RNA in the cell, particularly the minus-strand, is highly resistant to nuclease treatment RCμ = 0.08 h−1), whereas RNA transfected into the cytoplasm decayed with a half-life of approximately two hours in the experiments using a replication-defective replicon . Although model estimations for both, pcytμ and RCμ, exhibit a rather broad confidence interval, simultaneous modification of both parameters shows that RCμ needs to be substantially lower than pcytμ in order to explain the observed kinetics . In fact, when analyzing the calibrated model, we found that the rate constant for this reaction (3k in −4 h−1*molecules−1) to fit the experimental data, and the concentration of “active” polymerase (E) was severely limiting the rate of RNA synthesis during the initial dynamic phase. This resulted in an extremely low efficiency of internal re-initiation, implying that most or all of the newly synthesized viral plus-strand RNA is exported to the cytoplasm, from where it must be re-imported for further rounds of RNA replication to occur. Hence, our model supports the notion that negative-strand initiation is very different from plus-strand initiation in that it most likely depends on actively translated RNA with the required NS proteins, mainly NS5B, being supplied in cis.Another important question, which can hardly be addressed experimentally, is the possibility of re-initiation of minus-strand synthesis inside the replication vesicle. While theoretically it is feasible that the replicative machinery re-initiates minus-strand synthesis on newly produced plus-strands inside the replication compartment (poutk), they can also exert tight control over protein translation. Newly synthesized RNA can either be exported to the cytoplasm where it can be used for another round of protein translation , or it accumulates within the replication vesicles; there, however, it cannot be used as a template for minus strand synthesis due to the above described reasons.The observed relative shortage of active polymerase within the replication vesicles and the lack of internal re-initiation consequently prevents an exponential amplification of the viral RNA within the replicative compartment. Replication vesicles thus attenuate the rate of viral replication by limiting the availability of the factors required for minus-strand initiation. At the same time, depending on the export rate of newly synthesized plus-strand RNA from the replication vesicles  and the generation of new RNA (replication), therefore constitutes an important additional level of control over the virus' replication kinetics. This high degree of controllability might be one reason for the evolutionary success of membranous replicative structures, as basically formed by all positive strand RNA viruses. In case of HCV, it allows for sustained low-level replication as is required for the establishment of persistence, mainly by restricting availability of the required proteins within the replicative compartment.HF) in our model has been found to be a requisite for the attainment of a steady state and its concentration was a determinant regulating plateau levels of viral RNA and protein between the two differently permissive cell lines. For that reason, we now systematically assessed the impact of different availabilities of HF onto steady-state levels of viral RNA and protein. For HCV RNA levels, this analysis showed a linear correlation with HF concentrations in the range tested: the more abundant HF was, the more RNA replication took place. Interestingly, however, polyprotein levels exhibited a saturation behavior, reaching a plateau for HF concentrations above 20 “molecules”  , HF availability is limiting the formation of replication vesicles (HF levels of around 50 “molecules”), in contrast, rapid formation of replication vesicles occurs with an associated rapid increase in viral RNA levels. However, ribosome availability (iboR) then becomes limiting for protein translation  . To undevesicles . Therefonslation , explainnslation . AccordiHF, surprisingly the host translation machinery is the bottleneck in high permissive cells.Interestingly, these findings suggested that the actual mechanisms governing the establishing of the steady state in low permissive and high permissive cells are different. While in low permissive cells the formation of replication vesicles is the limiting step due to a lack of host factor HF in our model sufficed to explain the observed difference in HCV replication dynamics between high and low permissive cells, it was intriguing to identify the biological nature of this factor. For that purpose, we set out to compare gene expression profiles of Huh-7 cells of different passage number or clonal origin that we had found to exhibit substantially different permissiveness for HCV RNA replication HF as suggested by the above analysis . These analyses mainly identified metabolic processes such as lipid metabolism and cell growth and proliferation, which is in line with the notion of HCV RNA replication requiring proliferating cells for efficient replication, at least in Huh-7 cells As differential abundance of the host factor (or host process)  compare , we fitt compare . We coulHF candidate genes was too large to be functionally validated individually within this study, we surveyed previously published data on HCV host factors, including a manually curated database of HCV-host interactions  and cytosolic NS proteins (cytE) to be taken up into the RC.In the present study, we have developed a mathematical model of the intracellular steps of HCV replication. In contrast to previous models HF) as a new species in our model, and required viral RNA (in the form CT) and NS protein (cytE) to form a complex with it in order to allow for the initiation of negative strand RNA synthesis and the formation of the RC. Assumption of different concentrations of this host factor then was sufficient to very accurately explain the differences in RNA replication permissiveness in the two cell lines. This final model therefore completely satisfied all experimental observations and could also correctly predict the effects of targeted perturbations during extensive validation experiments.While these model extensions greatly enhanced the fitting quality to the data of a single cell line, we then identified that step in the model, at which an altered kinetic rate could explain the dynamics found in the second cell line as well. For this purpose, we tested a series of hypotheses, fitting the model simultaneously to the two differently permissive cell lines and allowing only those parameters to differ that would be influenced by the host cell in the respective hypothesis. By this approach, we could exclude various processes, e.g. differences in translation efficiency, altered cytoplasmic RNA degradation or different RNA synthesis rates within replication complexes. It is also biologically plausible, that these processes do not differ between the two examined Huh-7 cells lines and therefore cannot explain the observed differences in permissiveness; both, translation and RNA degradation have been shown before to be comparable across different Huh-7 cells We then used the calibrated and validated model to further study individual steps of the viral lifecycle. Sensitivity analysis was applied to pinpoint the most influential steps, perturbation of which would lead to the greatest impact on replication dynamics and yield. A very interesting first finding was that once steady state replication has been reached, the system proved to be relatively robust towards perturbation of individual sub-steps of replication. The degradation rate of RNA inside the RC was the most sensitive parameter under these conditions, and had a significantly higher influence than all other rates. This parameter, however, can hardly be influenced biologically or therapeutically. Very likely, this robustness is key to HCV's prevailing in the face of cellular stress- and innate immune responses In contrast, at an early time point after introduction of HCV genomes into the cell, the system was found to be substantially more fragile with respect to the number of sensitive parameters. This suggests that therapeutic intervention with HCV replication by DAAs would be most efficient in newly infected cells, emphasizing the potential of such drugs for the prevention of reinfection upon liver transplantation. The processes found to be most sensitive during the early phase of replication were polyprotein translation as well as the RNA polymerization rate of NS5B. Of note, polyprotein cleavage by the viral NS3/4A protease was surprisingly little influential. This, however, has been described before, e.g. in a study examining the role of cyclophilin A for HCV replication cytE) and a host factor (HF), and replication complexes (ipR) would mainly depend on cT (and possibly cytE and HF) and the availability of vesicles. In effect, formation of productive replicative vesicles would again depend on those three species, CT, cytE and HF and should in principle be compatible with our simplified one-step model.One simplification that we accepted in developing the model is that the formation of the membranous vesicles is modeled as one step  togetherOn similar lines, for reasons of simplicity, our model considers only one single, large replication compartment. This assumption is clearly not correct, as numerous sites of virus induced convoluted membrane structures have been observed in HCV replicating cells Curiously, a central result of our study was the conclusion that the assumption of a key host factor was essential to fit our model to the dynamics of RNA replication. This factor was important to explain RNA replication in Huh-7 cells, but might not be as limiting in other HCV permissive cells, e.g. primary human hepatocytes. Moreover, in a physiological setting, restrictions in other steps of the viral life cycle, e.g. sub-threshold receptor levels during entry HF was sufficient to model the varying RNA replication efficiencies in different Huh-7 populations, we performed gene expression profiling to identify genes potentially defining permissiveness. While our analysis identified 355 genes, whose expression correlated with the degree of permissiveness of the respective cell line, there were no single factors or well-defined pathways that stood out significantly. In order to test the limiting nature of these identified factors for HCV RNA replication, one would have to individually overexpress those genes in low permissive cells and assay for an enhancement in HCV replication. Whereas this was beyond the capacity of our current study, we made use of extensive publicly available data on cellular interaction partners of HCV /condition(s) pcytμ , e.g. upon stress or under conditions of an activated immune response, the system would cross a threshold and replication would die off inevitably. Therefore, to compensate for such a lack of protection of the replication machinery, HCV would have to develop a completely different amplification strategy, most likely involving a much higher rate of RNA synthesis in order to maintain sustained replication. This, very likely, would not be compatible with low-level, low profile replication as required for persistence in vivo situation, particle formation, as was also suggested by another modeling approach Already during model development, but also throughout our model analyses, the formation and function of the membranous replication compartment was found to be crucial for successful viral HCV replication. Previous literature as well as our model analysis imply that membrane alterations serve at least three distinct purposes. For one, they provide a protected environment for RNA replication, shielding this very sensitive process from the host cell degradative machinery as also shown experimentally before We provide the first comprehensive modeling of the entire RNA replication cycle of a positive strand RNA virus, from the onset of RNA replication to steady state levels. However, membranous replication sites are a hallmark of all positive strand RNA viruses with very different replication strategies. In case of HCV the membranous replication compartment seems to have a rather limiting role in virus RNA replication, probably contributing to viral persistence and chronic disease. In contrast, most positive RNA viruses replicate fast, cause acute diseases and are cleared by the immune system (e.g. the closely related flaviviruses such as Dengue or West Nile virus). Interestingly, in the related group of pestiviruses, pairs of viral isolates have been found, replicating either in a non-cytopathic/persistent or in a cytopathic/acute manner Another obvious yet intriguing direction into which our presented modeling approach could be developed, is extending it to comprise the full infectious virus life cycle, including particle production and secretion, receptor binding and cell entry. In fact, two very recent publications studied RNA replication kinetics upon HCV infection low passage refers to naïve Huh-7 cells, passaged less than 30 times in our laboratory, see also Binder et al. All cells were maintained in supplemented Dulbecco's modified Eagle medium (DMEM) as described previously 341PI-Luc/NS3-3′/JFH1/5′Con (5′-NTR exchange) and pFK-I341PI-Luc/NS3-3′/JFH1/XCon (3′-NTR exchange) 341PI-Luc/NS3-3′/Con1/ET/∂g. In vitro transcription of HCV replicons was performed as described previously For kinetic analyses of HCV RNA replication, the genotype 2a (JFH1 isolate) constructs pFKi389LucNS3-3′_dg_JFH (wild-type) and pFKi389LucNS3-3′_dg_JFH/ΔGDD (replication deficient) in vitro transcribed HCV RNA by electroporation as described previously 6 cells were transfected with 10 µg of HCV RNA, corresponding samples were pooled and cells were seeded into 6-well plates for luciferase assays as described above or into 10 cm cell-culture dishes at a density of 4×106 cells per plate (2×106 cells/plate for time points 48 h and 72 h) for RNA preparation and Northern blotting. For the 0 h RNA sample, 4×106 cells were washed twice with DMEM directly after electroporation, pelleted and lysed in guanidinium isothiocyanate. Other samples were lysed at the indicated time points  and lysates were stored at −80°C until further processing.Cells were transfected with For determination of HCV replication by luciferase activity measurement, all samples of one experiment were frozen at −80°C upon harvesting and thawed simultaneously prior to luciferase detection. Measurements were performed as described in Binder et al. in vitro transcription of plasmid pBSK-JFH1/6273-3′ in vitro transcripts of known quantity, which were loaded onto the same gel. Cross-hybridization of minus-strand probes with the plus-strand standard was observed to a low extent and corrected for.RNA preparation and Northern blotting were performed according to established procedures www.ingenuity.com).Permissiveness of eight Huh-7 derived cell-lines was assessed using a standard luciferase replication assay as described above. Total cellular RNA of untransfected cells was then isolated by Trizol extraction according to the manufacturer's protocol , and gene expression was measured using the Affymetrix Human Genome U133 Plus 2.0 platform. Data were normalized in R/Bioconductor using RMA normalization. Genes were filtered using the variance-based (IQR) filter in nsFilter, and log2 fold-changes between high and low permissive cells were computed. We then fitted a linear model to the data, predicting replication efficiency in the eight cell lines from the corresponding gene expression values. ANOVA was used to assess statistical significance of individual genes. Hit selection was done using a relatively low threshold of 0.2 on the p-value and a log fold-change of at least 0.3, corresponding to a change in expression of approximately 25%. Resulting genes were intersected with published RNAi screening We developed a mathematical model using ordinary differential equations based on mass action kinetics. The model is subdivided into two compartments: 1) initial RNA processing, translation into the polyprotein and polyprotein processing (cleavage) occur in the cytoplasm, and 2) viral genome replication takes place inside of the replication compartment. A graphical summary of the model is shown in CytoplasmpunpR  is assumed constant, the number of ribosomes available for translation is given by ibototR – CT, and it is not necessary to introduce a separate equation for ribosomes. Unprocessed and processed RNAs punpR and pcytR degrade with rate constants punpμ and pcytμ, respectively  proteins cytE  as well as a cellular factor HF to form a replication complex within the induced vesicular membrane structure. This cis interaction of pcytR and translated NS proteins is realized in the model by requiring active translation complexes cT instead of free pcytR for the formation of replication complexes. The host factor HF catalyzes the formation of IpR, at the rate Pink. Once IpR is formed, ribosomes are freed again at rate Pink. This leads to the ternary reaction C+ECyt+HF→RIp+RIboT, simultaneously describing formation of the replication compartments and initiation of minus strand RNA synthesis, compare also supplementary HF is freed again when IpR degrades or upon completion of minus strand synthesis. As the total number of host factor molecules in the cell is assumed constant, we can replace HF by HF(0) – RIp, where HF(0) is the total number of HF molecules in the cell. Lastly, since we use a luciferase readout to measure polyprotein concentration, we furthermore include a luciferase marker L in the model, which is produced at the same rate as the polyprotein (2k), however does not require further processing and degrades with rate Lμ  with scalar y and input vector x = , the first order sensitivity index with respect to ix is the expected amount of variance that would be removed from the total output variance, if we knew the true value of ix, divided by the total unconditional variance:iS is a measure of the relative importance of the individual variable ix in driving the uncertainty in the output y. In contrast, the total sensitivity index with respect to a variable ix measures the residual output variance if only ix were left free to vary over its uncertainty range, and all other parameters were known:TiS is a measure of how important a parameter is in determining the output variance, either singularly or in combination with other parameters. To assess the significance of obtained indices, eFast furthermore calculates the first and total order sensitivity index for a dummy parameter that is not part of the model. Indices that are not significantly larger than this dummy parameter index should not be considered different from zero Global sensitivity analysis was performed using the extended Fourier Amplitude Sensitivity Test (eFAST) Figure S1et al.Fit of the original model by Dahari (EPS)Click here for additional data file.Figure S2χ2 and Akaike Information Criterion (AIC) values compared. Line colors indicate polyprotein (black), plus-strand RNA (red) and minus-strand RNA (blue).Quantitative assessment of alternative models to explain differences observed in HP and LP cells. Alternative models were set up to explain observed data, assuming that cells differ in (A) the initial RNA processing, (B) different numbers of ribosomes available for RNA translation, (C) different RNA degradation rates in the cytoplasm, (D) different polyprotein translation rates, (E) different rates of formation of the replication compartment, (F) different RNA synthesis rates inside the replication vesicles, (G) different RNA degradation rates inside the replication vesicles, and (H) different export rates of newly synthesized RNA into the cytoplasm. Models were fitted to the experimental data, and resulting (EPS)Click here for additional data file.Figure S3Comparison of activatory with consumed host factor (HF) model. The left plots show the model predictions in the high permissive cell line, the right plot shows the predictions for the low permissive cell line. Upper panels: activatory (enzymatic) HF model, lower panels: consumed HF model.(EPS)Click here for additional data file.Figure S4Global sensitivity analysis of the replication model. Sensitivity analysis was performed using the extended Fourier Amplitude Test (eFAST) at  4 hours and  72 hours. Shown are eFast total order sensitivity indices for  plus strand RNA,  minus strand RNA, and  viral protein. These total sensitivity indices account for first and higher order sensitivities involving each of the parameters indicated. The dashed horizontal lines are sensitivities of a negative control parameter that does not occur in any of the equations, and are thus a measure of background variability of the sensitivity estimation procedure. Sensitivities lower or equal to the dashed line should not be considered as significantly different from zero.(EPS)Click here for additional data file.Figure S5RCμ in the replication compartment. Note the transition at RCμ = 0.4, where a switch occurs from low-level persistent replication to complete clearance of the infection.The figure shows attained steady state levels of positive strand RNA, for different values of the RNA degradation rate (EPS)Click here for additional data file.Figure S6c+Ecyt+HF→RIp+RiboT by two binary reactions, assuming that cT and cytE bind first, forming an intermediate complex C in a reversible reaction with rates ak (forward reaction) and bk (backward reaction), that then irreversibly reacts with HF to yield IpR and iboR with rate ck. The figure in panel (A) was obtained by fixing parameter ak to 3e-4, varying parameter bk between 1 and 200, and then optimizing parameter ck to fit the experimental data. The plot shows that increases in bk can be compensated by increases in ck, rendering the model practically non-identifiable. Panels (B) and (C) show the obtained fits to the data for bk = 10 and bk = 200, with associated values ck = 0.41 and ck = 7.64, respectively, and χ2 values of 3.55 and 2.45, respectively. Blue line and points: (-) RNA, Red: (-) RNA, Black: Viral Polyprotein (Luciferase).The figure replaces the ternary interaction (EPS)Click here for additional data file.Figure S7Ten different runs for each high and low permissive cells, using a stochastic solver (implicit tau method) to make simulations with our calibrated replication model. Individual runs show a very similar behavior to the deterministic ordinary differential equation model, indicating that stochastic effects to not play a major role in determining the overall dynamics of the model.(EPS)Click here for additional data file.Figure S8Ecytμ and Lμ, which are highly correlated, however, the value for Ecytμ is known from literature and is not calibrated using the data Lμ identifiable. Similarly, the high correlation seen between ibototR and 2k as well as the high correlation between 0HF and 4mk are unproblematic, as parameters 2k and 4mk were set based on literature data Poutk und pcytμ, is unproblematic, as an additional constraint pcytμ = 2 Tcμ on pcytμ is used in the parameter estimation.Correlation between model parameters from identifiability analysis using SensSB (EPS)Click here for additional data file.Table S1Differentially expressed genes between the eight cell lines analyzed. The first column is the gene name, the second column the corresponding Affymetrix ID. The logfc column is the logarithm of the fold expression change between the high and low permissive cells, whereas the p-value is computed from an analysis of variance of the full panel of all eight cell lines.(XLS)Click here for additional data file.Table S2Differentially expressed genes between the eight cell lines analyzed, showing the 355 genes with log-fold change >0.3 or <−0.3 between high and low permissive cells, and p-value<0.2 in correlation analysis with permissiveness over all 8 cell lines. The table gives log fold-changes for all 8 cell lines, as well as p-value of correlation for all genes.(XLS)Click here for additional data file.Table S3www.ingenuity.com). The functional analysis identified the biological functions that were most significant to the data set. Molecules from the dataset that met the p-value<0.2 and log fold-change >0.3 or <−0.3 criteria were associated with the biological functions in the Ingenuity Knowledge Base. Right-tailed Fisher's exact test was used to calculate a p-value determining the probability that each biological function assigned to the data set is due to chance alone. Shown are annotations for category and cellular function, together with p-value, number and names of respective molecules.Annotation of 355 differentially expressed genes correlating with permissiveness to cellular function categories. Analysis was done using IP Click here for additional data file.Table S4www.ingenuity.com). Canonical pathway analysis identified the pathways from the IPA library of canonical pathways that were most significant to the data set. Molecules from the data set that met the p-value<0.2 and log fold-change >0.3 or <−0.3 criteria and were associated with a canonical pathway in the Ingenuity Knowledge Base were considered for the analysis. The significance of the association between the data set and the canonical pathway was measured in 2 ways: 1) A ratio of the number of molecules from the data set that map to the pathway divided by the total number of molecules that map to the canonical pathway is displayed. 2) Fisher's exact test was used to calculate a p-value determining the probability that the association between the genes in the dataset and the canonical pathway is explained by chance alone.Annotation of 255 differentially expressed genes correlating with permissiveness to canonical pathways. Analysis was done using IP Click here for additional data file.Table S5HF).Parameter estimates for the HCV replication model (consumed (PDF)Click here for additional data file.Text S1The supplementary text contains additional information on gene expression analysis, model development and parameter estimation, and model analysis.(PDF)Click here for additional data file.


Arabidopsis, exosome regulates expression of a “hidden” transcriptome layer from centromeric, pericentromeric, and other heterochromatic loci that are also controlled by small (sm)RNA-based de novo DNA methylation (RdDM). However, the relationship between exosome and smRNAs in gene silencing in Arabidopsis remains unexplored. To investigate whether exosome interacts with RdDM, we profiled Arabidopsis smRNAs by deep sequencing in exosome and RdDM mutants and also analyzed RdDM-controlled loci. We found that exosome loss had a very minor effect on global smRNA populations, suggesting that, in contrast to fission yeast, in Arabidopsis the exosome does not control the spurious entry of RNAs into smRNA pathways. Exosome defects resulted in decreased histone H3K9 dimethylation at RdDM-controlled loci, without affecting smRNAs or DNA methylation. Exosome also exhibits a strong genetic interaction with RNA Pol V, but not Pol IV, and physically associates with transcripts produced from the scaffold RNAs generating region. We also show that two Arabidopsis rrp6 homologues act in gene silencing. Our data suggest that Arabidopsis exosome may act in parallel with RdDM in gene silencing, by epigenetic effects on chromatin structure, not through siRNAs or DNA methylation.The exosome functions throughout eukaryotic RNA metabolism and has a prominent role in gene silencing in yeast. In  Arabidopsis exosome complex in such mechanisms. Evolutionarily conserved from archaea to humans, the exosome is a stable complex of RNase-like and RNA binding proteins that plays a central role in RNA metabolism in eukaryotes. Depletion of the exosome allows some repetitive sequences to escape from silencing. Most of these transcripts emanate from centromeric and pericentromeric chromosomal regions and other heterochromatic loci, and many derive from repetitive and transposable elements. In plants, TEs are targeted for de novo DNA methylation by smRNA–mediated pathways. However, we found that exosome depletion has only minor effects on smRNA populations that are acting in the main silencing mechanism in Arabidopsis, siRNAs–dependent DNA methylation RdDM. Instead, exosome depletion affects histone H3K9 dimethylation, an epigenetic mark that affects chromatin structure and thus alters transcription. Our data suggest that the exosome collaborates in gene silencing, likely acting in a parallel pathway to other mechanisms. We also propose that the Arabidopsis exosome may coordinate the transcriptional interplay of different RNA polymerases to modulate repression of some repetitive sequences.To maintain genomic stability and prevent expansion of invasive genomic sequences such as transposable elements (TEs), eukaryotes have evolved defensive mechanisms to control them. Here, we examine the role of the  High-throughput analyses have revealed that eukaryotic genomes are pervasively transcribed in vitroThe exosome complex plays a central role in RNA metabolism in eukaryotes. Evolutionarily conserved from archaea to humans, the exosome is a stable complex of RNase-like and RNA binding proteins that catalyzes 3′ to 5′ processing and decay of various RNA substrates de novo methylation of cytosines in all sequence contexts at the region of siRNA–DNA or siRNA-RNA sequence homology. This silencing pathway requires two plant-specific RNA polymerases, Pol IV and Pol V, specializing in transcriptional gene silencing (TGS) RdDM induces S. pombe RNA Pol II carries out the functions attributed to Pol IV and Pol V in plants, therefore, it generates both siRNA precursors and scaffold transcripts to which siRNAs bind at loci that are subject to siRNA-mediated silencing. Exosome defects in S. pombe were reported to result in the loss of transcriptional silencing from centromeric, silent mating type, and telomeric loci S. pombe, in the absence of exosome-mediated degradation, abundant aberrant RNA species enter the RNAi pathway and interfere with heterochromatic silencing through competition for RNAi biogenesis machinery, resulting in the dramatic decrease in centromeric siRNAs The model system in which siRNA-mediated silencing is the best understood mechanistically is fission yeast. In To answer these questions we employed next-generation sequencing to profile populations of smRNAs in exosome-depleted plants, and in mutants of RdDM pathway genes. Unexpectedly, we found that loss of the exosome subunits had little effect on the global populations of smRNAs and had no affect on the level of DNA methylation in examined RdDM loci; rather, it resulted in a reduction of histone H3K9 dimethylation. We propose that the Arabidopsis exosome may coordinate the transcriptional interplay of RNA polymerases Pol II, Pol V and Pol IV, to achieve the appropriate level of transcriptional repression of heterochromatic loci.Previously, we found that the majority of transcripts upregulated in RRP4 and RRP41 exosome depletion mutants originate from the promoters, repeats, intergenic, and siRNA generating regions RRP4 and RRP41 are lethal; therefore, we used inducible RNA-interference (iRNAi) constructs to reduce RRP4 and RRP41. The seedlings of RRP4 (rrp4-i) or RRP41 (rrp41-i) transgenic plants grown on estradiol-containing medium to induce the RNAi constructs subsequently exhibit a growth arrest . Profiling silencer sequences by size and by first nucleotide revealed that the majority of the silencer sequences are 21, 22 and 24 nt and start with 5′U or 5′A . Both methods of normalization produced similar results; therefore, only data normalized using filtered reads (FLR) are presented graphically in this study.We first examined the smRNAs from the U or 5′A , suggested reads . Each liA. thaliana range from 21 to 24 nt. Our libraries were constructed using 15–32 nt smRNAs; therefore, we were able to detect any effect exosome depletion might have on smRNA metabolism. We found that exosome defect does not lead to changes in smRNAs in the 15–19 nt and 26–32 nt categories (data not shown). Importantly, the group of 20–25 nt smRNAs, which contains the majority of functional smRNAs, was present in similar proportions, although with minor variations, relative to the number of total reads in the libraries of both of exosome depletion mutants and in their corresponding non-induced lines, and constituted about half of total smRNAs mapped to the genome . This approach also allowed us to confirm that smRNAs observed in exosome depletion lines are siRNAs produced by components of the RdDM pathway and not short RNA degradation products accumulated in the absence of functional exoribonucleolytic complex.In Arabidopsis, repeats and TEs are silenced by siRNAs through RdDM; therefore, we examined the effect of exosome loss on 20–25 nt smRNAs corresponding specifically to TEs and repeats. Surprisingly, we saw no changes in the groups of smRNAs mapped to tandem repeats (TR), inverted repeats (IR), dispersed repeats (DR) or the group of TEs in both exosome mutants . The divnrpd1, nrpe1, dcl3 and rdr2 with rrp41-i iRNAi knock-down line did not exacerbate the phenotypes of single exosome depletion mutants  corresponds to the transcript identical to the sequence of the DNA top strand (and therefore produced from the bottom DNA strand), and the bottom transcript is identical to the sequence of DNA bottom strand. The scaffold RNAs were reported to correspond to region B top strand rrp41 iRNAi/nrpe1 double mutants relative to nrpe1 and rrp41 iRNAi single mutants, while no change was observed in rrp41 iRNAi/nrpd1 double mutants  and dimethylated histone H3 lysine 9 (H3K9me2) are hallmarks of heterochromatin, and silencing of solo LTR, AtSN1 and IGN5 loci also involves histone modifications nrpd1 and nrpe1 mutants, which affect Pol IV and Pol V, respectively. We found that RRP41 depletion also led to a decrease in H3K9me2 but less than observed in nrpd1 and nrpe1 mutants , and the alleles of the rrp6l2-2 and rrp6l3-1 are SALK alleles (Col-0 ecotype). To control for effects of ecotype, we compared the amount of region A transcript in rrp6l3-1, rrp6l2-2, rrp6l1-2/rrp6l2-2 mutants to Col-0 wild type plants, and rrp6l1-2, rrp6l1-2/rrp6l2-2 mutants to Ws ecotype plants  and by the subunit Rrp6, which is substoichiometric, nuclear-specific, and not essential for viability. Degradation of e plants .rrp6l1-2 and rrp6l2-2 mutants exhibited increased accumulation of transcripts produced from region A. As would be expected based on cytoplasmic localization of RRP6L3 protein, no effect was observed in rrp6l3-1 mutants. To our surprise, we observed a dramatic derepression of region A in rrp6l1-2/rrp6l2-2 double mutants, suggesting that both RRP6L1 and RRP6L2 proteins are involved in the silencing of this region and might have a redundant function in this process.We found that, similar to depletion of the core subunits RRP4 and RRP41, rrp6l1-2, rrp6l2-2, and rrp6l1-2/rrp6l2-2 double mutants. We found that methylation was not affected in these mutants regardless of the extent of derepression of the region , which binds to H3K9me2 heterochromatin and propagates H3K9me2 spreading, being titrated by an excess of heterochromatic RNA rrp41 iRNAi/nrpe1 double mutant did not exhibit any additive or synergistic effect on the loss of H3K9me2 relative to respective single mutants as well, suggesting that the loss of H3K9me2 observed in the exosome mutants is unlikely to result from an unknown functional homolog of Arabidopsis HP1 simply titrating an excess of ncRNA off chromatin, as reported in fission yeast.We also observed that the exosome physically associates with the polyadenylated ncRNA transcripts from scaffold producing regions. We could not reliably crosslink the exosome to the DNA of the target locus by ChIP (data not shown), although this could simply reflect the difficulty of reliably crosslinking proteins to DNA through RNA, or it could mean that the exosome binds to the transcripts after they are released from the locus and that exosome-mediated regulation of the transcripts may be important for maintenance of chromatin structure around the locus. H3K9 dimethylation was reported to be disturbed and lost when isolated Arabidopsis nuclei were treated with RNase A rrp41-i and rrp4-i mutants in solo LTR and AtSN1 suggest that the exosome is involved in regulation of either processing or level of RNA from these loci , RNA Pol V , RDR2 , and DCL3  mutants were described previously rrp41 iRNAi/nrpd1-3, rrp41 iRNAi/nrpe1-11, rrp4 iRNAi/nrpd1-3,and rrp4 iRNAi/nrpe1-11 double mutants were obtained by crossing of rrp41 iRNAi and rrp4 iRNAi with nrpd1/nrpe1-11 line. rrp41 iRNAi/dcl3-1, rrp41 iRNAi/rdr2-1 double mutants were obtained by crossing.iRNAi lines of exosome subunits RRP4 and RRP41, RNA Pol IV  according to the manufacturer's protocol. The total RNA sample was used for sequencing library construction using the Small RNA sample Prep v1.5 kit and TruSeq Small RNA Sample Prep kit  according to the manufacturer's instructions. The smRNA libraries were sequenced using the Illumina Genetic Analyzer II  and Illumina HiSeq 2000  according to the manufacturer's instructions. HiSeq 2000 sequencing reads were demultiplexed using Casava v 1.8  before further bioinformatic analysisArabidopsis lines used in this study carried iRNAi cassette transgenes used for inactivation of either RRP4 or RRP41 exosome subunit genes iRNAi transgenes were filtered out from each library and libraries were analyzed separately to ensure accurate interpretations. The remaining smRNA reads, termed FLR for filtered reads, were used for further analysis.Data processing was done using available tools and custom in-house UNIX shell programming 6 . Both methods of normalizations were compared and found to produce results which lead to identical interpretations, therefore, only data analyzed using filtered reads are presented in this study.Each library was normalized either to the total number of mapped non-redundant reads or to the total number of non-redundant filtered reads (FLR), multiplied by 10Classification of small RNAs was performed by BEDTools (v2.10.0) The small RNA reads with 20 to 25 nt length were calculated and plotted versus the sum of their normalized reads per million (rpm). The relative frequencies of each 5′ terminal nucleotide of the small RNAs were calculated , S2  andRepetitive genomic features were classified using TAIR9 Tandem Repeat Finder (version 4.04) For analysis of locus-specific expression of smRNAs , the expressed normalized reads per million (rpm) were calculated for respective genomic locus and locus-specific datasets were plotted for comparisons.t, primers listed in Actin2 (At3g18780) as endogenous reference. Polyacrylamide Northern Blot analyses were performed as described Total RNA was isolated from 7-day-old seedlings using the Trizol method. For RT-qPCR, 1–4 µg of total RNA digested with DNase I (Fermentas) was reverse transcribed 1 hour either at 50°C (for oligo-dT primer) or 55°C (for specific primers) using 60–100 units SuperScript III Reverse Transcriptase (Invitrogen). Transcripts were quantified by RT-qPCR using the comparative threshold cycle method (ΔΔCGenomic DNA was isolated from 7-day-old seedlings using a DNeasy kit (QIAGEN). The methylation analysis using DNA sensitive methylation enzymes was followed as described ChIP was performed as described RIP assays were performed by adapting an existing protocol Figure S1iRNAi silencer sequences produced by rrp4-i and rrp41-i cassettes in response to estradiol treatment.  20–25 nt smRNAs corresponding RRP4 in rrp4-i (A) and corresponding and to RRP4 in rrp41-i (B) depletion mutants profiled based on the length of the reads.  20–25 nt smRNAs produced from in rrp4-i (C) and rrp4-i (D) depletion mutants profiled based on both their length and the terminal 5′ nucleotide. The major silencer sequences are 5′U and 5′A smRNA species.(TIF)Click here for additional data file.Figure S2rrp4-i, RRP41, rrp41-i, RRP41/nrpd1, rrp41 iRNAi/nrpd1, RRP4 iRNAi/nrpe1 and rrp41 iRNAi/nrpd1 mutants.miRNA families, miR-158a, miR-158b, miR-860, miR-823, miR-841, miR-5561 and variations in sequence length. miRNA families miR-158a, miR-158b, miR-860, miR-823, miR-841, and miR-5561 and variations in sequence length in each family. smRNAs mapped to matching mature miR-158, miR-860, miR-823, miR-841, and miR-5561 sequences (TIF)Click here for additional data file.Figure S3rrp4-i, rrp41-i exosome depletion lines and RdDM mutants. All locus-specific datasets of 20–25 nt smRNAs are plotted versus the sum of their normalized reads per million (rpm).  RT-PCR analysis of RRP6L1 and RRP6L mRNA expression in rrp6L1 and rrp6L2 insertion mutants.Effects of exosome deletion, RdDM, and other mutants. (A) Diagrams of IGN5, REG 3 and REG 4 genomic loci, based on analysis of transcription units by Wierzbicki et al. (2008) (TIF)Click here for additional data file.Table S1p4-i, RRP41, and rrp41-i plants.Summary of smRNA sequence reads in the libraries of RRP4, rr(XLS)Click here for additional data file.Table S2nrpd1, rrp41-i/nrpd1, RRP41/nrpe1, rrp41-i/nrpe1, RRP41/rdr2, rrp41-i/rdr2, RRP41/dcl3 and rrp41-i/dcl3 plants.Summary of smRNA sequence reads in the libraries of RRP41/(XLS)Click here for additional data file.Table S3p4-i, RRP41, and rrp41-i mutant plants.Expression profiling of known mature miRNAs in the libraries of RRP4, rr(XLS)Click here for additional data file.Table S4Oligonucleotides used in this study.(XLS)Click here for additional data file.


The human TAR RNA-binding protein, TRBP, was first identified and cloned based on its high affinity binding to the small hairpin trans-activation responsive (TAR) RNA of HIV-1. TRBP has more recently been found to be a constituent of the RNA-induced silencing complex (RISC) serving as a Dicer co-factor in the processing of the ~70 nucleotide pre-microRNAs(miRNAs) to 21-25 nucleotide mature miRNAs.Using co-immunoprecipitation and protein-identification by mass spectrometry, we characterized intracellular proteins that complex with TRBP. These interacting proteins include those that have been described to act in protein synthesis, RNA modifications and processing, DNA transcription, and cell proliferation.Our findings provide a proteome of factors that may cooperate with TRBP in activities such as miRNA processing and in RNA interference by the RISC complex. MicroRNAs (miRNAs) are important regulators of cellular development, differentiation, proliferation, apoptosis, metabolism, and oncogenesis -4. CelluTRBP contains two double-stranded RNA binding domains (RBD)  and was e.g. ABCF1, EEF1A2, EIF2AK2 and EIF2S1) (Table Figure Although previous reports implicated TRBP as active primarily in the cytoplasm, our mass spectrometry results (Additional file Recently, TRBP truncating mutations were found in human cancers with microsatellite instability . EvidencTRBP: TAR-RNA binding protein 2; RISC: RNA-induced silencing complex; TAR: trans-activation responsive element.The authors declare that they have no competing interests.YHC constructed the TRBP expression plasmid, performed co-immunoprecipitations, prepared MS samples, analyzed the results; OJS provided mass spectrometry analyses; KTJ conceived of the study. YHC and KTJ prepared the manuscript. All authors read and approved the final manuscript.Supplementary Table S1. Pubmed ID, gene name, functional description, hits in proteomics study and alternative symbol for TRBP-interacting factors.Click here for fileSupplementary Materials and Methods.Click here for file


TERT intron 6 (>1 kb from exon–intron junctions). At least nine repeats are required for generating the major non-functional ‘minus beta’ isoform, which skips exons 7 and 8. RNA:RNA pairing between the repeats and the pre-mRNA might bring exons 6 and 9 closer, thereby promoting exon skipping. To demonstrate this, we show that mutations within the repeat that abolish exon skipping are corrected by compensatory mutations in the pre-mRNA. This study thus identifies RNA:RNA pairing by repetitive sequences as a novel form of alternative splicing regulation in a gene crucial for cancer survival and sheds new light on functional roles for short repetitive sequences embedded deep within introns throughout the genome.Telomerase adds telomeric repeats onto chromosome ends and is almost universally upregulated in human cancers. Here we demonstrate that RNA:RNA pairing regulates splicing of the catalytic subunit of human telomerase (TERT). Human alleles contain a variable number of 38 bp repeats within  Telomerase activity can be regulated by alternative splicing of its catalytic subunit TERT. Here, Wong et al. demonstrate that TERT splicing is regulated via RNA:RNA pairing of repetitive intronic sequences with the pre-mRNA, thus revealing a new function for conserved elements embedded within introns. Alternative splicing of pre-mRNA allows for the generation of vast protein diversity from a limited amount of genetic information56810Dscam in Drosophila melanogaster and related species)SF1, zinc finger protein 162:ZFM162) is the only other mammalian gene for which RNA:RNA pairing has been demonstrated. Previously, we have identified a region within intron 6 of hTERT pre-mRNA that contains a variable number of 38 bp repeats, that lies >1 kb from exon–intron junctions, and is only conserved in old but not new world primates or other mammals. This block of repeats in intron 6 is necessary to promote exons 7 and 8 skipping, therefore producing the major non-functional ‘minus beta’ TERT isoform. Although once believed to be ‘junk’ DNA, variable number of tandem repeats (VNTRs) dispersed throughout the genome have been found to have several diverse biological functionsRNA:RNA pairing is a splicing mechanism used for mutually exclusive exon choice in a few insects . For example, although the crystal structures are related, there is virtually no sequence conservation between the Altogether, our results suggest that a minimum of nine 38 bp repeats is necessary for RNA:RNA pairing in human telomerase to either change the proximity of exon 6 and 9 splice junctions and/or expose the necessary docking sites for splicing factors or the spliceosome for splice site selection. We have demonstrated the first example of RNA:RNA pairing using a relatively non-conserved repetitive element regulating exon skipping in a mammalian gene. The prevalence of similar regulation in other genes remains to be determined. Manipulating the factors regulating RNA:RNA pairing and the splicing of telomerase may provide additional therapeutic approaches for inhibiting telomerase activity in cancer cells.2 in 4:1 DMEM:Medium 199, containing 10% calf serum .HeLa cervical carcinoma cells were cultured at 37 °C in 5% COThe Flp-In System  was used to generate HeLa cells (purchased from American Type Cell Culture (ATCC)) with a stably integrated FRT site. The population of transfected cells were subcloned and individual clones were screened for a single FRT insertion site by southern blotting. Expression level of the FRT site was determined by beta galactosidase staining. Three Hela clones  were used to confirm the results observed. Data for Hela clone no. 8 is shown in TERT minigene was previously constructed by inserting portions of the human telomerase sequences into the pcDNA5/FRT expression vector . The sequence of the minigene is available upon request. Pre-existing unique restriction sites in the minigene were used to replace the wild-type sections with the sections containing compensatory mutations to avoid additional sequence changes. Section 1 was flanked by a 5′ MluI site and a 3′ PacI site. Section 3 was flanked by a 5′ FspAI site and a 3′ BssHII site. Section 4 was flanked by a 5′ BssHII site and a 3′ SphI site . A phI site .hTERT minigene using Taq polymerase PCR . The reaction was initially denatured at 94 °C for 3 min, then denatured at 94 °C for 30 s, annealed at 61 °C for 30 s and extended at 72 °C for 1 min for 30 cycles, with a final extension at 72 °C for 10 min. The PCR product was resolved on a 5% denaturing polyacrylamide gel and visualized at 650 nm.Using the RNeasy Plus Mini Kit , RNA was extracted from Hela cells stably expressing the minigene. Complementary DNA was made using Biorad iScript. A minigene specific forward primer 5′-CTGGCTAACTAGAGAACCCACTGC-3′ and a cy5-labelled reverse primer 5′- AGGCTGCAGAGCAGCGTGGAGAGG-3′ were used to examine the splicing of the M.S.W., J.W.S. and W.E.W. designed the study. M.S.W. performed the experiments and prepared the manuscript. J.W.S. and W.E.W. supervised the data analysis and edited the manuscript.How to cite this article: Wong, M. S. et al. Regulation of human telomerase splicing by RNA:RNA pairing. Nat. Commun. 5:3306 doi: 10.1038/ncomms4306 (2014).