Hyaloperonospora arabidopsidis co-opts the host cell's nuclear import machinery. We use HaRxL106 as a probe to determine redundant and specific functions of importin-α paralogs from Arabidopsis thaliana. A crystal structure of the importin-α3/MOS6 armadillo repeat domain suggests that five of the six Arabidopsis importin-αs expressed in rosette leaves have an almost identical NLS-binding site. Comparison of the importin-α binding affinities of HaRxL106 and other cargos in vitro and in plant cells suggests that relatively small affinity differences in vitro affect the rate of transport complex formation in vivo. Our results suggest that cargo affinity for importin-α, sequence variation at the importin-α NLS-binding sites and tissue-specific expression levels of importin-αs determine formation of cargo/importin-α transport complexes in plant cells.Importin-αs are essential adapter proteins that recruit cytoplasmic proteins destined for active nuclear import to the nuclear transport machinery. Cargo proteins interact with the importin-α armadillo repeat domain via nuclear localization sequences (NLSs), short amino acids motifs enriched in Lys and Arg residues. Plant genomes typically encode several importin-α paralogs that can have both specific and partially redundant functions. Although some cargos are preferentially imported by a distinct importin-α it remains unknown how this specificity is generated and to what extent cargos compete for binding to nuclear transport receptors. Here we report that the effector protein HaRxL106 from the oomycete pathogen  Coordination of nuclear and cytoplasmic events is mediated by nuclear pore complexes (NPCs) that span the nuclear envelope. Low-molecular-weight compounds such as solutes and proteins with a molecular weight of <40–60 kDa can traverse NPCs by passive diffusion Stewart, . Proteinet al., et al., 10–12[K/R]3/5)  effector AvrBs3 by the pepper Bs3 gene  revealed that 33% show entirely nuclear localization  whereas effector HaRxL106 interacts with MOS6, importin-α1, -α2 and -α4. However, results from directed protein–protein interaction assays might not predict with certainty the formation of specific cargo/importin-α complexes in plant cells.A subcellular localization screen of effector candidates from the Arabidopsis downy mildew pathogen Hpa effector HaRxL106 binds to the MOS6 armadillo repeat domain via a bipartite NLS with low micro-molar affinity, which is in the range of binding affinities that has been determined for other cargo/importin-α interactions , showed entirely nuclear localization when transiently expressed in in vitro, we generated E. coli expression constructs for the HaRxL106 effector domain , an HaRxL106ΔC version of the same domain (amino acids 46–227) and a truncated version of MOS6 lacking its N-terminal IBB domain. We purified all proteins from the soluble fraction of E. coli crude extracts via an N-terminal His6 tag and tested for direct protein–protein interactions by separating protein mixtures on an analytical size exclusion chromatography column . Out of these, importin-α1,-α2 and -α4 had the highest expression levels, followed by importin-α9, -α6 and -α3/MOS6  expression construct and co-expressed this fusion protein with YFPN-tagged cargo proteins in epidermal cells of N. benthamiana. Apart from an YFP signal in the nucleoplasm, which we observed for all BiFC pairs tested and therefore might result from spontaneous association of the YFP N- and C-terminal halves, we found that co-expression of YFPN–HaRxL106 with MOS6–YFPC resulted in speckles at the nuclear rim  and SAP11 (11 kDa) co-opt the importin-α/β pathway for efficient nuclear import  was obtained from NASC. Transgenic Arabidopsis plants expressing YFP– and RFP–HaRxL106 were generated by transforming ecotype Col-0 with A. tumefaciens strain GV3101 pMP90RK carrying pENS-YFP–HaRxL106 and A. tumefaciens strain GV3101 pMP90 carrying pH7WGR2–HaRxL106, respectively , pH 7.5] at a ratio of 2 ml buffer per 1 g leaf material. The extracts were centrifuged at 17 000 g 4°C 20 min and the supernatant was either boiled in sodium dodecyl sulphate (SDS) sample buffer for western blots or used for co-IPs. For western blots protein samples were separated by SDS-PAGE and electro-blotted onto polyvinylidene difluoride membrane. Antibodies used were α-HA 3F10 , α-GFP 210-PS-1GP , α-RFP-biotin ab34771 . For co-IPs a fraction of the supernatant was saved as ‘input’ sample and 20 μl GFP-beads  or HA-beads (Sigma) were added to 1.4 ml of the remaining supernatant. The samples were incubated on a rotating wheel at 4°C for 2 h followed by collecting the beads by centrifugation at 1200 g and 4°C for 1.5 min. The beads were washed 3–4 times with 1 ml extraction buffer and then boiled in SDS sample buffer to elute protein from the beads.Protein extracts were prepared by grinding http://www.malvern.com) in high gain mode at 25°C with all proteins diluted in buffer 20 mm HEPES, 150 mm NaCl, pH 7.5. His6–ΔIBB–MOS6 protein was pipetted into the sample chamber at 43–54 μm concentration and was titrated with His6-tagged HaRxL106, HaRxL106ΔC or SAP11 at concentrations between 320 and 940 μm. Two microlitre injections with 120 sec pause intervals were performed up to a cumulative volume of 38 μl. Binding isotherms were fitted to the integrated calorimetric data using Origin software . Control reactions titrating buffer into ΔIBBMOS6 showed that the heat of dilution was <0.1 Kcal mol−1 of injectant and therefore comparable with the values obtained at the end point of each titration. At least one technical replicate for each ITC experiment was performed and gave similar results.ITC experiments were performed using a MicroCal 205 calorimeter  with the following excitation wavelengths: YFP, 516 nm; RFP, 561 nm.http://www.gelifesciences.com) in 50 mm HEPES, 150 mm NaCl, pH 7.0. His6–ΔIBB–MOS6 protein was diluted to a concentration of 2 mg ml−1 and incubated with a 1 m excess of either His6–HaRxL106 or His6–HaRxL106ΔC for 1 h at 4°C. The samples were centrifuged at 17 000 g 4°C 20 min and 0.5 ml of the cleared supernatant was loaded on the column. The column was eluted at a flow rate of 0.5 ml min−1 with two column volumes of buffer and 0.5 ml fractions were analysed by SDS-PAGE.Analytical size exclusion chromatography was performed using a Superdex 200 HR 10/30 column (GE Healthcare, For a list of oligo-nucleotides and plasmids used in this study see


Contact between humans and the marine environment is increasing, but the capacity of communities to adapt to human presence remains largely unknown. The popularization of SCUBA diving has added a new dimension to human impacts in aquatic systems and, although individual-level impacts have been identified, cumulative effects on ecosystem function and community-wide responses are unclear. In principle, habituation may mitigate the consequences of human presence on the biology of an individual and allow the quick resumption of its ecological roles, but this has not been documented in aquatic systems. Here, we investigate the short-term impact of human presence and the long-term habituation potential of reef-fish communities to recreational SCUBA divers by studying symbiotic cleaning interactions on coral reefs with differing levels of historical contact with divers. We show that incidences of human contact result in a smaller decline in ecosystem function and more rapid resumption of baseline services on a reef in Utila, Honduras that has heavy historical levels of SCUBA diver presence, compared to an un-dived reef site in the Cayos Cochinos Marine Protected Area (CCMPA). Nonetheless, despite the generally smaller change in ecosystem function and decades of regular contact with divers, cleaning behavior is suppressed by >50% at Utila when divers are present. We hypothesize that community-wide habituation of reef fish is not fully achievable and may be biologically restricted to only partial habituation. Differential responses to human presence impacts the interpretation and execution of behavioral research where SCUBA is the predominant means of data collection, and provides an important rationale for future research investigating the interplay between human presence, ecosystem function, and community structure on coral reefs. Human presence can have a major impact on animal behavior and through this have significant consequences for ecosystem health and function. Habituation and tolerance mitigate the consequences of this impact on the biology of individuals and their ecological roles by determining when, if, and how organisms resume natural behaviors , 2. WhilThe majority of studies of animal behavior involve direct human observation. Minimally, this entails passive human presence, relying on the assumption that observers have a neutral impact on the behavior of interest. These qualifications can be exceedingly difficult to meet underwater. Often SCUBA divers appear suddenly and are suspended in the water column, looming over the benthos. SCUBA traditionally relies on noisy open-circuit regulators that emit low-frequency sound waves detectable up to 200m away , 16, andServices provided mutualistically are an integral part of the complex web of interactions in many communities and help maintain ecosystem health and function. On coral reefs, cleaning interactions are classic interspecific mutualisms between cleaners  and a community of client fish that span the taxonomic spectrum –29. The Bartholomea annulata and Pederson’s cleaner shrimp Ancylomenes pedersoni form ecologically important cleaning stations. These stations are small and stationary, easy to locate, and service members of at least 20 families of reef fish [B. annulata stations as an indicator of community-level behavioral stability to investigate how SCUBA diver presence impacts the provision of this service in the focal ecosystems. We use (i) traditional visual diver observations (direct presence), (ii) remote videography with divers nearby (ambient presence), and (iii) remote videography alone (diver absence) to quantify reef fish cleaning frequency and duration of temporary cleaning disruption. We then explore whether prolonged historical exposure to diver presence leads to community-wide habituation by comparing two separate but similar reef sites in Honduras which have experienced different extremes of historical diving pressure.On Caribbean coral reefs, the corkscrew sea anemone eef fish , 29, 31.We use this approach to demonstrate that complete habituation of reef fish to diver presence may be unattainable at the community level, regardless of the frequency and intensity of historical human contact and the opportunity for habituation it brings. Instead, we suggest that reef fish communities represent a mosaic of individuals that vary in their habituation levels and tolerance to divers. Our results bear on the interpretation of past ethological studies where direct human observation using SCUBA was the predominant form of data collection, and provide an important null hypothesis for future research on fish behavior and the impact of human presence on ecosystem function on coral reefs. The habituation potential of reef fish communities thus raises an important consideration that the ability to tolerate human presence and to maintain natural behaviors during episodes of diver contact could provide a competitive advantage to certain individuals and species, acting as a novel cue for natural selection and potentially sculpting reef fish communities in the future.We focus on two coral reef sites in the Bay Islands, Honduras. Coral View reef on the island of Utila (16°05’17.87”N 86°54’38.56”W) is a sloping fringing-reef extending from ca. 3–30m. It has been dived heavily and regularly from shore by recreational SCUBA divers for more than 20 years, and serves as the house reef for the adjacent Coral View Dive Resort. The site sees low level recreational boat traffic and snorkelers make regular visits alongside SCUBA divers. The second reef site is an unnamed reef in the Cayos Cochinos Marine Protected Area  and is a sloping fringing-reef extending ca. 1.5–20m which, to the best of our knowledge, has never previously been dived. This reef is unbuoyed and unmarked in an area with heavy historical restrictions on diving geared towards clearly identified dives sites, and consultation with park managers at the Honduran Coral Reef Foundation (HCRF) confirmed no diving is known to have occurred there. Additionally, reef development within CCMPA is most extensive along the northern edge of the archipelago, where the majority of mooring buoys and dive sites are located. Thus, access to our study site is restricted by HCRF and is geographically separate from any recreational or scientific diving that occurs within CCMPA. Some infrequent boat traffic  occurs close to the reef site, but no other historical contact with humans has taken place.B. annulata. All reef site assessments were conducted in tandem with cleaning observations, but were completed during the mid-morning or afternoon in order to avoid direct interference with diver observations and camera deployments at dawn. Assessments were completed in fewer than five dives at the study sites themselves, and so led to minimal human contact, especially when compared to the intense historical contact at Utila. Variations in key reef characteristics were statistically tested using independent t-tests in the software package SPSS [Both reef systems were surveyed for key ecosystem indicator criteria to assess their suitability for comparison in this study. These assessments all involved randomly placed 50m transects conducted at 6–8m depth in triplicate (n = 3) at six sub-sites within each system to provide local averages and to account for transiency in fish populations. Broad benthic community health was quantified through percentage live scleractinian coral cover, with data collected by point intercept using 0.25m intervals. Fish abundance and species richness were quantified using underwater visual census (UVC), with transect dimensions of 5 x 5m. Belt transects of 5m width were used to quantify density of age SPSS .B. annulata between 4–9m depth hosting A. pedersoni were tagged, measured, and mapped to avoid duplication, and the number of shrimps recorded. Each station was then randomly assigned to one of two observational treatments: diver observed  or video observed . Diver observations lasted 20 min, after a 5 min acclimation period, from 3–5m distance [At each study site, all distance , 27 usindistance . Video odistance . AlthougWhile the above analysis tested the effect of a SCUBA diver positioned directly in front of a cleaning station, ambient diver presence (defined here as a diver on a reef but > 10m from a cleaning station) may also have an effect on cleaning rates. In theory, divers may have a “halo-effect,” disrupting ecosystem function within an unknown radius, which may decrease in effect the further away a diver becomes. In our example, habituation to diver presence would reduce this halo-effect, allowing for cleaning rates to resume to ‘diver absence’ baseline rates even while divers remain in the water. To assess this halo-effect, videos at each station were separated into two treatments to provide separate measures of cleaning behavior in response to variation in diver presence: 1) ambient diver presence, and 2) diver absence. During ambient presence treatments, divers remained nearby on the reef (>10m from cleaning station); their open-circuit SCUBA was audible on recordings. Ambient presence video data were analyzed 5 minutes into each recording to provide an analogous “acclimation” time to mimic direct observation treatments. Diver absence videos were analyzed 60 minutes into each recording as above. Thus, data from each station represented repeated measures and significance between treatments was determined at each reef site independently using Wilcoxon signed-rank tests (non-parametric paired t-test).n = 11; CCMPA n = 15). Only cleaning stations where cleaning interactions were previously recorded could be used for this analysis, thus reducing our sample size. Statistical significance was assessed using non-parametric Wilcoxon signed-rank tests (non-parametric paired t-test) as above.To provide a third measure of ecosystem function disruption in response to diver presence, we recorded the interval of time (minutes) from camera deployment until the first cleaning interaction. As above, the comparison of these intervals was conducted in a repeated measures design using two treatments at each reef site: 1) ambient diver presence and 2) diver absence. Divers remained on the reef (> 10m from cleaning station) during ambient presence treatments; cameras were re-deployed at the same station by free-divers who immediately exited the water for diver absence treatments. This design tested the hypothesis that habituation shortens the avoidance response to human presence and permits a more rapid return to natural ecosystem functioning. This component used footage from both reef sites . Species from 18 fish families were documented at these sites  representing the potential reef fish client pool  (t(34) = 51.35, p < 0.01). There was no significant difference in anemone size  or group size of A. pedersoni  between stations used across treatments. Importantly, total cleaning rates recorded via video observations (diver absence) were not significantly different between sites , indicating a similar natural baseline cleaning frequency, and validating our later comparisons of cleaning behavior in response to diver presence.Despite major differences in historical diver presence, we find no significant differences between the two reef systems with respect to scleractinian coral cover , fish species richness , or ent pool . Total f-1. Divers at CCMPA only recorded six cleaning interactions from four client families at 31 directly observed cleaning stations  and Acanthuridae (surgeonfish) appear over represented at our cleaning stations relative to their underlying abundance. Only Gobidae (gobies) appear underrepresented relative to its underlying abundance , and a significant interaction effect between reef site and treatment . Bonferroni-corrected pairwise comparisons revealed a gradient of significant diver impacts across all treatments (-1), and significantly less than diver absence  and video (diver absence) treatments revealed a significant overall test result  than with ambient diver presence  both. Some species may be inherently more tolerant of SCUBA divers, while individual learned experience in a naturally intolerant species could trump this intolerance and lead to habituation. Alternatively, or in conjunction, habituation may be similar to flight initiation distance (FID), which appears to be a function of body size, taxonomic ID, group size, and trophic level , 33, 34.Nested within the concept of individual “tolerance” is also individual variation in risk-taking behavior. Trade offs between activity level and resource use, especially in the face of predation pressure, can shape community dynamics , 36. HerA logical extension of the conclusion that reef fish communities do not fully habituate to diver presence would suggest that the direct presence of SCUBA divers is sufficiently threatening to tip the balance against the benefit of parasite removal: fish forgo the procurement of cleaning services and accept the cost of reduced fitness as a result. Cleaning symbioses are well known to improve the fitness of client fish , 30, andLastly, conclusions drawn from animal behavior studies involving direct human observations depend on the assumption that observers have a neutral impact on the behavior of interest. These observations minimally entail passive human presence, and based on our findings we encourage a reduction in reliance on traditional observational approaches to studying animals using SCUBA and the incorporation of sites with historically high levels of diver presence wherever possible. Considering our data, direct diver observations alone would have significantly underestimated the natural cleaning rate at both sites and led to a falsely assumed, albeit statistically supported, inference of a difference in cleaning rate between CCMPA and Utila that video observations show to be incorrect. This would have led to false conclusions about ecosystem function and to a misconception of the factors controlling and structuring these interactions.Remote videography may not be suitable or practically applied to every question and diver presence may not impact all aspects of behavior. Regardless, the potential impact of divers should be incorporated more thoroughly within the context of experimental design, and our hypothesis that reef fish communities cannot fully habituate to SCUBA diver presence will provide an important null hypothesis for future research. Our results suggest that novel SCUBA diver presence on coral reef ecosystems will amplify disturbances associated with pressures such as coastal development and will retard the recovery of baseline ecosystem function, although repeated contact will ultimately lead to a reduction in the extent of this negative impact through partial habituation. However, the balance between the negative impacts of diver presence and the wide-ranging direct economic and conservation benefits of ecotourism remains to be seen, and a long-term net gain to the ecosystem is ultimately feasible. Regardless, our findings alter the way we think about interactions between humans and the marine environment, and suggest that unseen mechanisms are at play at the community level when coral reef ecosystems come into contact with SCUBA divers.S1 TableData included are from SCUBA diver collected and remote video collected cleaning interactions at Cayos Cochinos Marine Protected Area (CCMPA) and Utila.(PDF)Click here for additional data file.


RNA-Seq provides remarkable power in the area of biomarkers discovery and disease characterization. Two crucial steps that affect RNA-Seq experiment results are Library Sample Preparation (LSP) and Bioinformatics Analysis (BA). This work describes an evaluation of the combined effect of LSP methods and BA tools in the detection of splice variants.Different LSPs  allowed the detection of a large common set of splice variants. However, each LSP also detected a small set of unique transcripts that are characterized by a low coverage and/or FPKM. This effect was particularly evident using the low input RNA NuGEN v2 protocol.A benchmark dataset, in which synthetic reads as well as reads generated from standard (Illumina TruSeq 100) and low input (NuGEN) LSPs were spiked-in was used to evaluate the effect of LSP on the statistical detection of alternative splicing events (AltDE). Statistical detection of AltDE was done using as prototypes for splice variant-quantification Cuffdiff2 and RSEM-EBSeq. As prototype for exon-level analysis DEXSeq was used. Exon-level analysis performed slightly better than splice variant-quantification approaches, although at most only 50% of the spiked-in transcripts was detected. The performances of both splice variant-quantification and exon-level analysis improved when raising the number of input reads.Data, derived from NuGEN v2, were not the ideal input for AltDE, especially when the exon-level approach was used. We observed that both splice variant-quantification and exon-level analysis performances were strongly dependent on the number of input reads. Moreover, the ribosomal RNA depletion protocol was less sensitive in detecting splicing variants, possibly due to the significant percentage of the reads mapping to non-coding transcripts. Recently, several developments in RNA-Seq methods have provided an advance in the complete characterization of RNA molecules . These d+ selection versus ribosomal depletion, on splice variant detection. Furthermore, we compared NuGEN low input protocol with standard TruSeq protocol using BA tools for splice variant-quantification , and this step requires an accurate selection of the bioinformatics and statistical analysis techniques to be used. The approaches used to quantify known transcripts, i.e. transcripts annotated on the reference genome, and not yet characterized transcripts, i.e. transcripts not associated with an annotation on the reference genome, are different and characterized by different criticalities . In thisCuffdiff  and RSEMCuffdiff ,13) and Cuffdiff ).+  was used as reference to simplify the comparisons among LSPs. This was because 100 ng of total RNA input material represents the RNA quantity that can be at best obtained from a wide range of biological samples, e.g. animal models, biopsies, FACS sorted cell populations etc. Furthermore, it represents one of the cheaper RNA-Seq experiments available on the market.We analysed low input RNA (NuGEN) LSP and standard/high input RNA LSP (Illumina TrueSeq and Epicentre ScriptSeq). Sequencing data generated using TrueSeq unstranded PolyA+ selection and ribosomal RNA depletion affect splice variants detection. Specifically, we analysed the LSP effect on splice variants coverage/fragment per kilobase of exon per million reads mapped (FPKM), exons and exon-exon junctions counts. Total RNA, extracted from the mouse dendritic cell line D1 [+ selection, but for NuGEN v2 and TruSeq stranded LSP, which was also used in association with the ribo-zero ribosomal RNA depletion . For each experimental condition  80 million paired-end reads were collected. The 80 million reads for each condition were assembled combining multiple runs , low 0.5-2 ng) input protocols, PolyA00 ng, lo ng inputReads were mapped against the mouse genome version 9 (mm9). At least 83% of all reads generated by any of the LSPs could be mapped to the reference genome Table . The per+ RNA input in TruSeq unstranded libraries might be due to transcript quantification sensitivity. Transcripts quantification was done using Cufflinks, because Steijger et al. [+ mRNAs represented a tiny subset of the total input RNA, the increment in input material from 100 ng to 1000 ng was not enough to bring low expressed splice variants in the range of sensitivity of Cufflinks for transcript quantification, given as threshold FPKM > 0.1.Ts100 and ts1000, which were the two libraries prepared with TrueSeq unstranded using respectively 100 ng and 1000 ng of input material, allowed the detection of nearly the same number of transcripts . A possible explanation for the lack of an apparent advantage in using high versus low, PolyAr et al.  showed tr et al. . Thus, iIt was notable that the increase of input material affected the overall library yield . NuGEN protocol using 0.5 ng of input total RNA (nu05) had a very limited ability  and REM-EBSeq for the NU dataset Figure .The experiments performed on the synthetic dataset revealed inferior detection efficiency for all methods Figure . The besIn this work we present the first comparative evaluation of the combined effect of Library Sample Preparation and Bioinformatics Analysis on alternative splicing detection.+ enrichment , showed a similar behaviour for common detected transcripts. The transcripts detection was significantly impaired for the low expressed transcripts when comparing ribosomal depletion versus PolyA+ selection protocols.Library Sample Preparations using as starting material at least 100 ng of total RNA and undergoing PolyATranscripts that were specifically detected only by a single LSP showed poor coverage and they were probably not very informative for splice variant detection, because of the non-uniform coverage at exon-level. As for NuGEN low input protocol, the number of LSP-specific transcripts increased with the rise of the amount of total RNA input. However, those LSP-specific transcripts were characterized by low coverage and in general by very low exon-level counts. FPKM estimation for those transcripts could be misleading, since it had a very similar behaviour to that observed for the transcripts in common with the LSP based on the TruSeq protocol. Nu05, nu2 and nu100 showed a lower coverage with respect to ts100 for the common transcripts as well as for the exon count. The experiments on the benchmark datasets revealed that the lower exon counts generated from NU datasets (20/40/80NU) negatively affected the ability of exon-level based approach (DEXSeq) to detect alternative splicing events. On the other side, when a high number of input reads was used and the preparation was done using the TruSeq protocol (e.g. 80TS dataset), the exon-level based approach provided the best results. Exon-level analysis provided also the best compromise between detection and false discovery rate using the TruSeq protocol.https://genome.ucsc.edu/ENCODE/protocols/dataStandards/ENCODE_RNAseq_Standards_V1.0.pdf), would have been 76-bp paired-end sequencing, since the average insert size in the majority of LSP is 150-bp. The use of longer reads could be particularly useful in approaches like TopHat2/Cufflinks, where read splitting is used to detect intron-exon junctions.All our experiments were based on 50-bp paired-end sequences. This choice was a compromise between the performance of the experiment and the sequencing costs. However, the optimal solution, as suggested in the 2011 ENCODE white paper on RNA-seq . ERCC RNA Spike in Control Mix 1 (Ambion) was added to D1 total RNA as a first step of each protocol. 2). Two additional rounds of purification of the cDNA libraries were done using Agencourt Ampure XP SPRI beads (Beckman Courter) to remove > 600 bp double stranded cDNA. 3). The length distribution of the cDNA libraries was monitored using DNA 1000 kits on the Agilent Bioanalyzer. 4). Libraries were subjected to an indexed PE sequencing run of 2x51 cycles on an Illumina HiSeq 2000.+ containing RNA molecules were purified using polyT oligo-attached magnetic beads. Thermal fragmentation followed after two rounds of enrichment for PolyA+ mRNA. cDNA was synthesized using reverse transcriptase (Superscript II) and random primers. This was followed by second strand cDNA synthesis, end repair process, adenylation of 3' ends and ligation of the adapters. The products were then purified and enriched with 12 cycles of PCR to create the cDNA library.Illumina TruSeq RNA Sample Preparation kit version 1 (Low Sample Protocol) was used with slight modifications. Briefly, PolyATotal RNA was processed for cDNA synthesis using Ovation RNA-Seq system version 2 (NuGEN Technologies) according to the manufacturer's protocol. Briefly, first strand cDNA was prepared from total RNA using a unique first strand DNA/RNA chimeric primer mix and reverse transcriptase (RT). The primers have a DNA portion that hybridizes either to the 5' portion of the poly(A) sequence or randomly across the transcript. A DNA/RNA heteroduplex double-stranded cDNA was generated by fragmentation of the mRNA within the cDNA/mRNA complex, allowing the DNA polymerase to synthesize a second strand. The DNA then underwent SPIA amplification. SPIA cDNA were sheared to get a size range of 25 bp to 400 bp with the bulk of the material at 150 bp. This was done by sonication (Covaris model S2) with duty cycle 10, intensity 5 and cycle/burst 100 for 300 s. 100 ng and 500 ng of the sheared DNA were used for library preparation using the Encore Rapid Library Systems (NuGEN Technologies) according to manufacturer's protocol; the fragmented DNA underwent end repair, adaptor ligation (with 6 bases indexing tags), and final repair to produce the final library. 4 µl of each purified library underwent 10 cycles of PCR amplification using Illumina TruSeq PCR reagents.+ containing mRNA molecules were selected using RiboMinus™ Eukaryote kit (Life Technologies) for RNA-Seq according to manufacturer's instructions. The RNA samples were chemically fragmented using the StarScript Reverse Transcriptase Buffer and the cDNA Synthesis Primer was annealed to the RNA. 5′ end-tagged cDNA  was produced by random-primed cDNA synthesis. This was followed by 3′-Terminal Tagging of the cDNA using the Terminal-Tagging Oligo (TTO) which randomly annealed to the cDNA, including to the 3′ end of the cDNA and served as template for the extension of the cDNA by DNA polymerase. The resulting di-tagged were purified using Qiagen MinElute PCR Purification Kit. Enrichment of the purified di-tagged cDNAs was done with 12 cycles of PCR.PolyAIllumina TruSeq Stranded Total RNA Sample Preparation kit (Low Sample Protocol) was used with slight modifications. The removal of ribosomal RNA was done using Ribo-Zero Gold rRNA removal beads (Epicentre), which deplete samples of both cytoplasmic and mitochondrial ribosomal RNA. After depletion, the RNA was purified and fragmented using divalent cations and thermal fragmentation. First strand cDNA synthesis was performed using reverse transcriptase (Superscript II) and random primers. This was followed by second strand cDNA synthesis using DNA Polymerase I and RNase H and dUTP in place of dTTP. Libraries were prepared as described above for the TruSeq RNA protocol except for the end repair process.Illumina TruSeq Stranded mRNA Sample Preparation kit (Low Sample Protocol) was used with slight modifications. Briefly, polyA containing mRNA molecules were selected using polyT oligo-attached magnetic beads. Fragmentation and library preparation was done as described above for the TruSeq Stranded Total RNA protocol.The common background of the spikes-in dataset was made using paired-end reads generated preparing, with the TruSeq unstranded protocol, 5 libraries, starting with 1000 ng of total RNA extracted from the D1 cell (C1-C5), and 5 libraries starting with 100 ng of total RNA D1 cells (T1-T5)  for genes having more than one expressed splice variant. Specifically, we selected, as putative targets for spike-in, only those set of splice variant that were expressed both in C and T datasets. This restriction was meant to avoid those cases in which the alternative splicing detection problem reduces to differential gene expression identification.5 elements having a mean equal to the number of reads to be spiked-in, e.g. 100, and a standard deviation 10 times smaller than the mean. For example, we decided to spike-in 100 reads in the 5 replicated of ts100 background (C1-C5), thus 5 random values from a normal distribution of 105 elements with mean 100 and standard deviation 10 were chosen  and used to define the number of spike-in reads to be placed in each of the 5 replicates. The required synthetic paired-end reads 2x51nt were constructed to guarantee a uniform distribution both at exon and exon-exon junction level. Reads were then associated with a quality score of 40 and used to generate fastq files. Scripts used to generate the synthetic data set are available upon request.Out of the 2970 transcripts 58 were selected, with a similar procedure as above and for each transcript we spiked-in a specific number of reads   using deNone.CM generated spike-in datasets, LJ made all LSPs, PM performed NGS data QC and mapping, CF executed the bioinformatics comparisons between different LSPs, BM executed the bioinformatics comparisons between different LSP, DS supervised the bioinformatics analysis and revised the paper, CRA investigated the effects of LSPs on BA and wrote the paper, ZF supervised the sample preparations and wrote the paper, CRA and ZF supervised the present work.List of datasets generated with different LSPs on the same total RNA sampleClick here for filereproducibility between different sequencing runs. The datasets used to compare different LSPs were generated combining different sequencing run. Using deepTools webserver  we correClick here for fileEffect of total RNA input on library yield. The yield of library is shown with respect to the increment of input total RNA. TrueSeq protocol has a narrow range for the optimal library yield that is about 200 ng (blue dots). The increment of input total RNA for NuGEN protocol resulted in an increment on library yield. The overall yield is dependent also on the amount of cDNA used in the second step of the library preparation .Click here for fileBackground Paired-end reads datasets.Click here for file2 fold change associated to the 84 differentially expressed exons. The two dataset are very similar and even though few exons are detected as differentially expressed their log2 fold change difference is negligible.Exon-level analysis of ts100 and ts1000 dataset used as background for the construction of the spike-in dataset. A) Number of detectable exons, i.e. at least 1 reads mapped of an exon, with respect to the increase of total number of reads. The number of exons detectable by ts100 and ts1000 is very similar, although, over 50 millions reads, ts1000 seems to catch few more exons with respect to ts100. B) Exon-level differential expression calculated comparing the 5 technical replicates from ts100, used as background in T1÷T5, with respect to the 5 technical replicates of ts1000, used as background in C1÷C5. In red are shown the 84 exons detected as differentially expressed between the two groups, FDR ≤ 0.1. In the inset box is shown the distribution of the logClick here for fileEndogenous and spikes-in counts.Click here for fileSynthetic spikes-in data.Click here for fileEffect of different library normalization procedures in Cuffdiff analysis. Cuffdiff offers the possibility to use three types of library normalization: FPKM, geometic and quartile. Differentially expressed transcripts detected using 80TS dataset. A) 80TS spikes-in. B) Synthetic spikes-in.Click here for file10(FPKM). B) log10(coverage). Transcripts having in ts100 or tss at least FPKM ≥ 0.1; for level of FPKM lower than 0.1 the value in the plot was set by default to -2. The overall data shows a linear relation both for FPKM and for coverage. Red arrows highlight transcripts that are not correlated in expression in the two LSPs.Scatter plot of ts100 versus tss. A) logClick here for file


An adsorption affinity assay and saturation curve illustrated the preferential HALNP deposition density for precise therapeutic loading. (PLL/SPS)2.5 capping layer on top of the deposited HALNP monolayer further facilitated complete nanoparticle immobilization, cell adhesion, and provided nanoparticle confinement for controlled linear release profiles of the nanocarrier and encapsulated cargo. To our knowledge, this is the first study to demonstrate the successful embedment of a translatable lipid based nanocarrier into a substrate that allows for temporal and spatial release of both hydrophobic and hydrophilic drugs. Specifically, we have utilized our platform to deliver chemotherapeutic drug Doxorubicin from PEM confined HALNPs. Overall, we believe the development of our HALNP embedded PEM system is significant and will catalyze the usage of substrate mediated delivery platforms in biomedical applications.The ability to control the spatial distribution and temporal release of a therapeutic remains a central challenge for biomedical research. Here, we report the development and optimization of a novel substrate mediated therapeutic delivery system comprising of hyaluronic acid covalently functionalized liposomes (HALNPs) embedded into polyelectrolyte multilayer (PEM) platform via ionic stabilization. The PEM platform was constructed from sequential deposition of Poly-L-Lysine (PLL) and Poly(Sodium styrene sulfonate) (SPS) “(PLL/SPS) The engineering of drug delivery platforms facilitating spatial and temporal release of a therapeutic is one of the key challenges in biomedical research that can ultimately lead to society-wide improvement in disease management. In recent years, substrate mediated delivery of cargo has shown great promise in applications including drug and gene eluding films/scaffolds1235710In an effort to develop such sophisticated engineered surfaces, substrates such as polyelectrolyte multilayers (PEMs) have pioneered the implementation of substrate-mediated delivery in biomedical applications13141516171819202122232729Liposomes have become the most widely researched, tested, and commercialized nanocarrier system. Numerous products currently on the market such as Epaxal37394244Despite the salient features of PEMs and liposomes, the current research on liposome-PEM systems is extremely limited in both scope and application. Currently only few studies have analyzed the physical mechanism of liposome adsorption onto PEMs424.5”. We chose 4.5 bilayers because other studies have demonstrated that a similar PEM system with 4.5 bilayers produced a smooth and homogenous film4843134.5 base platform signifying an ideal surface for nanoparticle deposition . To determine the extent of the interaction between the HALNPs and PLL for potential ionic driven adsorption, we mixed the two components in PBS and measured their zeta potential and hydrodynamic diameter . From th4 and PLL (PLL/SPS)4.5 topped PEM platforms and validated the selective deposition of negatively charged HALNP on positively charged PLL (4 films using capillary force lithography (CFL) and exposed fluorescently labeled HALNPs onto these films. As observed in 4.5 films demonstrated that the HALNPs were not completely immobilized and were partially mobile on the PEM surface after 10 minutes  in order to develop our substrate mediated delivery platform is shown in rged PLL . To furt minutes . As a reh 10 min  and 30 mh 10 min . Atomic h 10 min . This il4 and (PLL/SPS)4.5 over a lipid range of 0.1 to 1000 μg/cm2 utilizing analogous deposition conditions described previously. By analyzing the percent difference in lipid adhesion between the PLL and SPS topped platforms over the range of lipid concentration, we estimated a minimum and maximum lipid concentration of 1.6 μg/cm2 and 31.6 μg/cm2 is necessary to overcome kinetically hindered deposition and reduction in embedment efficiency due to limited surface area, respectively ectively . This prectively . An expoNext we determined the release kinetics of the HALNPs from the PEM system both with and without the capping layer . We demo2.5 capping layer (HALNP-PEM). For this experiment, we used patient derived 21MT-1 metastatic breast cancer cells isolated from the metastatic pleural effusion as a model system. We performed a time course study via flow cytometry to determine the kinetics of cell uptake of the fluorescently tagged HALNPs when delivered in the bolus form compared to using the HALNP-PEM platform platform . At 12 hTo investigate the release mechanism of the HALNPs from the PEM platform, we employed fluorescent microscopy at different time intervals . We obse4.5 base with capping layer (HALNPDOX-PEM) (50) was 0.191 ± 0.030 μg/ml, 0.136 ± 0.025 μg/ml, and 0.197 ± 0.024 μg/ml for free DOX, HALNPDOX Bolus, and HALNPDOX-PEM delivery, respectively. The HALNPDOX achieved higher potency due to the potentially faster uptake by the cancer cells compared to free DOX and HALNPDOX-PEM. We also hypothesize that the HALNPDOX-PEM platform has a higher LC50 value compared to HALNPDOX bolus because at 24 hours not all HALNPs immobilized within the PEM films are released. As a control, a potency assay on empty HALNPs (no DOX) was performed to validate that the lipid nanocarrier is a non-toxic delivery system  within the HALNP-PEM platform. In addition to widespread implementation for cancer treatment, DOX was also chosen due to its extremely low molecular weight (MW) and subsequently high diffusivity with the rational that if DOX could be successfully encapsulated and embedded in a PEM platform for therapeutic purposes, virtually any hydrophilic drug could be delivered following this procedure. To compare the release mechanism and efficacy of the drug delivery, we performed a potency assay (MTT assay) on 21MT-1 metastatic breast cancer cells comparing three distinct delivery methods: 1) free DOX, 2) bolus delivery of DOX encapsulated in the aqueous core of HALNP nanoparticles (HALNPDOX Bolus), and 3) HALNPDOX embedded into the (PLL/SPS)DOX-PEM) , Table 2y system . In summWe next investigated the release profile of DOX from the HALNPDOX-PEM with and without the capping layer. We monitored the release of DOX in both the supernatant and the PEM platform via drug intrinsic fluorescence as a function of time . We obseTo decipher the mechanism of HALNP release and subsequent intracellular delivery from the HALNP-PEM platform, we employed our ability to successfully pattern HALNPs to probe the spatial uptake of the nanocarrier. The principle aim of the experiment was to determine if preferential uptake will occur with 21MT-1 breast cancer cells seeded on top of the HALNP patterns compared to the cells attached on the non-HALNP areas. CFL was used to pattern HALNPs tagged with green fluorescence atop PEM base films. 21MT-1 cell adhesion to the HALNP patterned PEM system with and without the capping layer  signifieWe also performed this experiment with DOX encapsulated inside the patterned HALNP-PEM system to determine if the local delivery of an active therapeutic was also possible with our liposome embedded PEM platform. We first adsorbed HALNPDOX onto the patterned PLL followed by subsequent addition of the capping layer. 12 hours after seeding of 21MT-1 cells, we observed a local uptake of the HALNPDOX particles dependent upon the nanoparticle deposition pattern . We selevia this route37383944Surface-mediated drug delivery involves the administering of therapeutics to adhering or suspension cells from either implantable devices or from tissue engineering scaffolds. Recently, liposomes have gained attention as a promising tool for targeted drug delivery to various parts of the body including tumors373839Here, we report the development of a novel substrate mediated therapeutic delivery system comprising of liposomal nanoparticles (LNPs) covalently functionalized with hyaluronic acid (HALNPs) and embedded within a PEM platform (HALNP-PEM) via ionic interactions. Both affinity assay and saturation kinetic curves were performed to determine the optimum HALNP embedment conditions and the maximum HALNP embedment capacity per unit area. A capping layer of 2.5 additional bilayers of (PLL/SPS) on top of the HALNP monolayer was employed for complete nanoparticle immobilization, support of cell adhesion, and increased control over the release profile of both the HALNP carrier and any therapeutic encapsulated in the nanocarrier. We directly compared bolus HALNP and the HALNP embedded within the PEM films to quantify the result of liposome confinement on intracellular uptake kinetics in patient derived metastatic breast cancer cells (21MT-1) isolated from the metastatic pleural effusion. This experiment validated the sustained release profile of over 60 hours for the HALNP-PEM system as compared to the burst release kinetics of the bolus HALNPs. In addition, we chose Doxorubicin (DOX) as a model hydrophilic cargo to verify the structural integrity of the particles following deposition and to probe the therapeutic index of the HALNP-PEM platform.capped), the time scale of release was significantly different in these two scenarios. For the cell-free experiment, at the 60 hour time point less than 10% of the total HALNP loaded into the PEM was released by diffusion/film swelling alone. However, for the cell containing experiment the flow cytometry data indicates that at the 60 hour time point all particles have been released in order to achieve the percent population positive for HALNP uptake seen in the bolus delivery at the 12 hour time point (100% uptake). In order for both of these events to hold true, there must be a strong driving force for HALNP release mediated by the cells themselves. To decipher this HALNP release mechanism from the PEM, we employed capillary force lithography (CFL) to pattern either fluorescently conjugated HALNPs or DOX encapsulated within HALNPs into our PEM platform. This time course study revealed the local cell-dependent uptake of HALNPs from the HALNP-PEM system - only cells above the patterned region took up the fluorescent nanoparticles. These findings further strengthened our hypothesis that release is driven by a cell-dependent uptake mechanism.We also analyzed the mechanism of HALNP uptake from the PEM platform. While controlled release of the HALNP was observed in both a cell containing , and a cThis study has systematically demonstrated the successful embedment of a proven translatable lipid based nanocarrier, HALNPs, into a PEM system via ionic interaction. This HALNP-PEM platform was capable of local and sustained delivery of a hydrophobic cargo in the form of fluorescently tagged cholesterol and a hydrophilic cargo in the form of the widely utilized chemotherapeutic, Doxorubicin. We modeled the maximum loading capacity of HALNPs per unit area for precise loading of any therapeutic cargo type, probed the HALNP uptake mechanism from the PEM system via implementation of the soft lithography technique CFL and found a cell mediated uptake process, and validated the ability of the HALNP system to retain the efficacy of an active therapeutic, DOX, all while controlling the spatiotemporal release profile.Overall, the development of our HALNP embedded PEM system will catalyze the usage of substrate mediated delivery platforms in biomedical applications, including the treatment of an array of disease models. For example, the current treatment method for Glioblastoma Multiforme (GBM) is maximal surgical resection of the cancerous tissue, radiotherapy, and chemotherapy5653***Please refer to the supplemental information for the full Materials and Methods.A polyelectrolyte multilayer (PEM) base structure of PLL and SPS was constructed by sequential adsorption of the polyelectrolytes on top of an oxygen plasma treated surface (glass slide or tissue culture plate). Multilamellar vesicles (MLVs) composed of PC, DPPE, and CHOL in a (3:1:1) molar ratio were created by the traditional dry film method as previously reported594.5 and SPS (PLL/SPS)4.5 topped base layers fabricated in a 12 well plate for three hours at room temperature. Following incubation, the surfaces were washed 3× with 20 mM HEPES and visualized with an Axiovert 40 CFL Zeiss  Inverted Microscope with fluorescent filter, and the images were captured with a Progres C3 Jenoptick camera. Following deposition of the HALNPs, a capping layer consisting of an addition 2.5 bilayers of PLL and SPS, (PLL/SPS)2.5, was added on top of the anionic nanoparticle monolayer following previously described adsorption protocol with the sole procedure change of using 20 mM HEPES for wash steps in place of DI water. The capping layer was added sequentially by hand in an aseptic environment to ensure sterile conditions.In order to track the adsorption of HALNPs, 0.15 mass % of Top Fluor fluorescently conjugated cholesterol was added to the initial lipid stock to effectively tag the nanoparticles. Then, to initially determine if the HA-LNPs could successfully adsorb onto a PEM surface, 180 μg lipid/cm^2 was incubated with both PLL (PLL/SPS)4.5, while the other was coated with an SPS topped layer (PLL/SPS)4. Then, a range HALNP concentration, 1 to 800 μg/ml, was incubated with the 48 well plates for 3 hours at room temperature covered in foil. Following this incubation time, the supernatants from each well were read against the standard curve to determine concentration of un-attached nanoparticles, and the percent difference between the PLL and SPS topped substrate wells  was used to determine the affinity of the HALNPs for that particular surface. The concentration of embedded particles deposited on the plate was also measured by reading the 48 well plate in a Biotek Synergy 2 Multi-Mode Plate Reader  to confirm conservation of mass . To determine the HALNP saturation conditions of the PLL topped base platform, the 1) supernatant and 2) nanoparticle embedded plate data from the affinity assay was used to determine the exact amount of mass loaded per area as a function of the nanoparticle loading concentration. From this information, the saturation point of HALNPs was determined from a nonlinear regression analysis via Sigma Plot Software .To evaluate the loading of HALNPs into the PEM platform a dual affinity and saturation curve assay was systematically implemented to determine optimum and saturated deposition conditions. First, a standard curve of known fluorescent HALNPs mass (termed lipid mass) was used to create a fluorescent standard curve. Then, two 48 well plates were coated with a PEM base layer. One plate was coated with a PLL topped layer (PLL/SPS)4.5. This base platform was then UV sterilized overnight. Filter sterilized HALNPs with 0.15% Top Fluor fluorescent cholesterol were then deposited on the PLL surface applying saturation conditions, followed by the addition of the capping layer . Each well was then seeded with 170,000 21MT-1 cells and placed in the incubator overnight to promote cell attachment. At 12, 24, and 36 hrs. The cells were washed 3× with sterile 1× PBS, trypsinized, and transferred to flow cytometry tubes. Theses samples were then analyzed for fluorescence in the green channel  against control cells. Both per cell fluorescence and total % population FITC positive information was measured. Additionally, bolus form HALNPs, free nanoparticles not bound to a substrate, were also incubated with 21MT-1 cells in an analogous manner (same seeding density of cells and concentration of 20.05 μg HALNPs per well) to compare the substrate mediated and bolus form intracellular delivery of HALNPs as a function of time.Flow cytometry was performed using a FACSCantoII from Becton Dickenson . Flow cytometry was specifically employed because it gives both per cell and population wide assessment of nanoparticle uptake via gating the analyzed cells into either a FITC positive (positive for HALNP uptake) or a FITC negative (no HALNP uptake) domain. A 12 well culture plate was first plasma treated and deposited with a PEM base of (PLL/SPS)50) was determined utilizing the MTT 2,5 diphenyl Tetrazolium Bromide) assay kit from Life Technologies . This classical colorimetric assay assesses cell health as a function of the mitochondrial conversion of MTT salt to Formazan. A two-step procedure was implemented for precise loading of DOX into the HALNPs and then into the HALNP-PEM platform. First a solution of 500 μg of DOX in 0.05× PBS was used to rehydrate a lyophilized vial of HALNPs and excess (un-encapsulated) DOX was removed using ultracentrifugation . A standard curve of known DOX concentrations was employed to determine the total amount of DOX encapsulated in the HALNP sample (“HALNPDOX”). The initial vial of HALNPs  concentration was known, thus both the total amount of encapsulated DOX and lipid were fully determined. Secondly, the HALNPDOX particles were adsorbed onto the (PLL/SPS)4.5 base platform ensuring the amount of lipid required to achieve a specified dosage of DOX was below the saturation conditions of the lipid as seen in 4.5 base platform. In addition, DLS and Zeta potential analysis of the HALNPDOX particles was analyzed to ensure the encapsulation of the DOX did not significantly alter with the size or charge of the HALNPs (and therefore the adsorption process). The (PLL/SPS)2.5 capping layer was added immediately after the HALNPDOX adsorption.The DOX concentration lethal to 50% of the 21MT-1 cells (LC4.5(HALNPDOX)(PLL/SPS)2.5], 2) Bolus form HALNPDOX, and 3) free non-encapsulated DOX. After a 24 hours incubation time between the three systems and the 21MT-1 cells, the media was aspirated and 5 mg/ml MTT working solution was added and incubated for 2 hours at 37 °C. Cells were then lysed with lysis buffer (acidified IPA) and the absorbance was measured at 570 and 620 nm using a Beckman Coulter AD340 plate reader . Percent viability was determined by normalization of the 570/620 absorbance ratio to the control untreated cells and positive control dead cells.21MT-1 cells were seeded at a density of 32,000 cells/well in three 48 well plates with different DOX configurations: 1) PEM platform with embedded HALNPDOX [(PLL/SPS)***Please refer to the supplemental information for detailed methods.How to cite this article: Hayward, S. L. et al. Ionic Driven Embedment of Hyaluronic Acid Coated Liposomes in Polyelectrolyte Multilayer Films for Local Therapeutic Delivery. Sci. Rep.5, 14683; doi: 10.1038/srep14683 (2015).


Post-transcriptional regulation of HIV-1 gene expression is largely governed by the activities of the viral Rev protein. In this minireview, the multiple post-transcriptional activities of Rev in the export of partially spliced and unspliced HIV-1 RNAs from the nucleus to the cytoplasm, in the translation of HIV-1 transcripts, and in the packaging of viral genomic RNAs are reviewed in brief.  The genome of the human immunodeficiency virus, HIV-1, encodes three classes of viral transcripts: 9 kb unspliced RNA, 4 kb partially spliced RNA, and 1.8 kb fully spliced RNA. The 9 kb RNA codes for Gag and GagPol proteins, while the 4 kb RNA generates the Env, Vif, Vpr, and Vpu proteins, and the 1.8 kb moiety produces Rev, Tat, and Nef . Full leHIV-1 splicing generally proceeds sub-optimally ,5. A numRev is a 116 amino acid protein. Like Tat, Rev is an RNA-binding protein containing a stretch of highly basic arginine residues. While Tat binds to the ~80 nucleotide TAR RNA hairpin motif which is contained in all classes of HIV-1 RNAs, Rev binds to the ~350 nucleotide stem-loop-rich RRE (Rev‑responsive element) RNA motif , that exists in unspliced/partially spliced viral RNAs [i.e., Rev). Rev binds RRE-RNA and docks it to the cellular export protein, CRM1 (chromosome region maintenance 1) [Eukaryotic cells typically retain unspliced and incompletely spliced RNAs in the nucleus while allowing intron-less, fully-spliced, RNAs to exit the nucleus. Because retroviruses use unspliced full‑length 9 kb viral RNA as the genomic RNA for progeny virions, as well as to encode Gag and GagPol proteins, and partially spliced 4 kb RNA (in the case of HIV-1) to synthesize Env, Vif, Vpr, and Vpu proteins, these viruses must develop mechanism(s) that actively release such RNAs from the nucleus into the cytoplasm. Retroviruses achieve this function through two major means. Genetically simpler retroviruses like Mason-Pfizer monkey virus (MPMV) have a ral RNAs . CTE‑conral RNAs ,22,23 (Fnance 1) ,25. The It should be emphasized that besides the RRE-sequence, unspliced and partially spliced HIV-1 RNAs contain several short instability sequence elements (INS) ,27. WhilIn addition to CRM1, Rev—RRE RNA interaction relevant to nuclear-cytoplasmic transport likely involves several other host cell co-factors. These include Ran (Ras-related nuclear protein), FG -repeat nucleoporins, RIP (Rev-interacting protein)/RAB (Rev/Rex activation domain-binding protein) ,34, and cis-RNA element specifying CRM1-mediated transport from the nucleus into the cytoplasm. The RNA-cap structure appears to be an additional component in the regulated transport of HIV-1 unspliced/partially spliced RNAs from the nucleus to the cytoplasm. The human cellular RNA methyltransferase, PIMT (peroxisome proliferator-activated protein with methyltransferase domain), was recently shown to enhance Rev-mediated export of HIV-1 RNA . PIMT istrans [Several early reports identified a role for Rev in promoting the translation of RRE-containing HIV‑1 mRNAs ,45. Moletrans .For HIV-1 and other retroviral RNAs , an emeThe full length unspliced HIV-1 RNA encodes the Gag-Pol proteins, but it is also the genomic RNA packaged into virions. A number of events impact the packaging of genomic viral RNA into virions. These include the appropriate exit from the nucleus of unspliced HIV-1 RNA, the dimerization of the genomic RNA, and Gag-RNA interaction in the cytoplasm prior to particle assembly and budding at the plasma membrane. As discussed above, Rev is pivotally important for the nuclear export of unspliced full length HIV-1 RNA from the nucleus. Recently a new activity for Rev/RRE in the packaging of genomic HIV-1 RNA has been broached. cis 5' untranslated region (UTR) ~120 bp (ψ) signal [It is conventionally held that the ) signal  is impor) signal ,53,54,55) signal ,53. For ) signal ,54, sugg) signal . To date, the study of HIV‐1 Rev has contributed to our understanding of intricate RNA-protein interactions that underlie RNA transport, translation and virion packaging. What was once considered as perhaps three discrete events now appear to be fully connected. Which pathway an RRE-containing RNA uses to exit the nucleus is apparently consequential to how it is destined for translation and packaging. If that RNA enters the polyribosome for translation then such choice excludes its utility for virion packaging. In the overall picture, Rev interaction with different cellular cofactors is also important in orchestrating the interconnected balance between roles in transport, translation, or packaging.


NanoString’s Prosigna™ Breast Cancer Prognostic Gene Signature Assay is based on the PAM50 gene expression signature. The test outputs a risk of recurrence (ROR) score, risk category, and intrinsic subtype . The studies described here were designed to validate the analytical performance of the test on the nCounter Analysis System across multiple laboratories.Analytical precision was measured by testing five breast tumor RNA samples across 3 sites. Reproducibility was measured by testing replicate tissue sections from 43 FFPE breast tumor blocks across 3 sites following independent pathology review at each site. The RNA input range was validated by comparing assay results at the extremes of the specified range to the nominal RNA input level. Interference was evaluated by including non-tumor tissue into the test.The measured standard deviation (SD) was less than 1 ROR unit within the analytical precision study and the measured total SD was 2.9 ROR units within the reproducibility study. The ROR scores for RNA inputs at the extremes of the range were the same as those at the nominal input level. Assay results were stable in the presence of moderate amounts of surrounding non-tumor tissue (<70% by area).The analytical performance of NanoString’s Prosigna assay has been validated using FFPE breast tumor specimens across multiple clinical testing laboratories. Molecular biomarkers have played an increasingly important role in identifying cancer patients with different prognostic outcomes and in predicting response to chemotherapy -3. MolecThe PAM50 gene signature measures the expression levels of 50 genes in a surgically resected breast cancer sample to classify a tumor as one of four intrinsic subtypes  , which hRecently, requirements for demonstrating utility of a tumor biomarker were established that include not only clinical validity, but also analytical reproducibility and robustness ,23. The 2 per slide, with tumor cellularity ≥ 10%. Non-tumor tissue from outside the circled area is removed by macrodissection of the corresponding unstained slides. RNA was extracted from slide mounted breast tissue sections using a RNA extraction kit manufactured by Roche to NanoString’s specifications [2, whereas 3 slides were input when the tumor surface measured 4-99 mm2. Following extraction of total RNA and removal of genomic DNA, RNA was eluted (30 μL volume) and tested to ensure it met the specifications for concentration (≥ 12.5 ng/ μL) and purity (OD 260/280 nm 1.7-2.5).The tissue input for the Prosigna assay was FFPE tissue that had been previously diagnosed to contain viable invasive breast carcinoma. The breast tumor tissue must be classified by a pathologist as invasive carcinoma . A pathologist reviews an H&E stain of a slide mounted tumor section to identify and circle the region of viable invasive breast carcinoma. The tumor surface area on the H&E stained section must be ≥ 4 mmications . For RNAin vitro transcribed RNA covering an approximately 1000 fold RNA concentration range (0.125 – 128 fM) and corresponding probes [in vitro transcribed RNA of the 58 targets for qualification and normalization purposes.The NanoString Prosigna assay  measuresg probes ,30. NegaExtracted RNA samples meeting quality and concentration specifications were hybridized (without reverse transcription or amplification) to capture and reporter probes for the measured genes and assay controls. The multiplexed hybridizations are carried out in a single-tube for 15 – 21 hrs at 65°C using 125 – 500 ng RNA . After hybridization, the target-probe complexes were processed on the nCounter Analysis System. Test sample data must meet a minimum threshold for expression of normalizing genes to ensure that the assay signal is high enough for the algorithm to produce precise results. The linearity of the positive control target titration and the non-specific background from negative control probes included in each assay is used to determine whether each assay performed within specification. Since the test is designed to be run in local molecular pathology labs, all quality thresholds are applied automatically to the data by embedded software; any failing metric causes an assay failure notice which prevents output of a Prosigna assay result. For samples meeting all quality thresholds, a clinically validated algorithm is used to determine the intrinsic subtype and ROR score, which are prognostic indicators of risk of distant recurrence of breast cancer ,21. The Operators for these studies were required to undergo training procedures to demonstrate proficiency, equivalent to what will be used to train users in molecular pathology laboratories for the decentralized test. Each site was given an overview of the NanoString technology and Prosigna assay procedures followed by an in-lab exercise where users were trained and qualified on tissue processing and assay procedures . Briefly, each user extracted RNA from three FFPE breast tumor tissue samples to demonstrate proficiency in tissue processing, and each user processed four prototypical breast tumor RNA samples  along with a negative control sample to demonstrate proficiency on the nCounter Analysis System.The analytical studies described herein were performed using pre-specified SOPs, statistical analysis plans and acceptance criteria using clinical-grade reagents, instrumentation, and software formatted such that no comparison of results between test centers could even be possible until the study was completed.in vitro diagnostic devices outlined in EP05-A2 [The RNA Precision study assessed the reproducibility of the Prosigna assay using a common template of purified RNA, thereby isolating the device-specific components of analytical validity from variables associated with tissue processing. The experimental design for analytically validating the precision of the assay from RNA was based on Clinical Laboratory and Standards Institute (CLSI) guidelines for the evaluation of precision of  EP05-A2 . This deFive pooled breast tumor RNA samples were generated from archived FFPE breast tumor tissue samples containing viable invasive breast carcinoma, to comprise a sample set representing each intrinsic breast cancer subtype and risk classification group Table . Since tSingle use aliquots of each pooled breast tumor RNA sample and three reagent lots were distributed to each of the three testing sites to complete the following testing scheme Figure . Each ofUpon completion of the study the blinded data were collected from each site and merged with the expected test result and study variables  associated with each unique sample ID. The prospectively defined analysis plan was then executed on the merged analysis dataset.The pre-specified primary aim of the RNA precision validation was to demonstrate that there was no significant differences for the continuous ROR score assay output across the three testing sites.The following variance components model was used to characterize the sources of variability:ROR Score = site + operator + lot + run + within-runwhere all components were treated as random components, and the RNA assay component of variation was defined as the sum of all these components. Variance components were estimated using the R procedure “lmer”. To test whether sites were significantly different, the following versions of the above model were fitted:ROR Score = site + operator + lot + run + within-run& ROR Score = operator + lot + run + within-runwhere site was now treated as fixed and all other components were treated as random. A likelihood ratio test with 2-degrees of freedom was performed using the fitted models to determine whether the effect of site was significant (α = 0.05). A similar analysis was performed for the assay reagent lots.For each of the 5 pooled samples, the classifications into the 4 intrinsic subtype categories  were summarized using frequency tables.The reproducibility study assessed the analytical validity of the Prosigna assay, including all steps involving in clinical lab implementation (i.e. tissue handling and RNA isolation SOPs as well as the device-specific assay steps), using a common set of breast cancer tissue samples.The experimental design for analytically validating the reproducibility from tissue was based on CLSI guidelines for the evaluation of precision of in vitro diagnostic devices outlined in CLSI EP05-A2. This design allows for the measurement of variability between and within a number of assay variables including testing site, FFPE sample block, operator, reagent lot, and assay run.A set of 43 banked FFPE breast tumor blocks from hormone receptor positive breast cancer patients with confirmed invasive breast carcinoma was selected from the biobank at Washington University at St. Louis for this reproducibility validation study. The sample collection and conduct of this study were conducted in compliance with the study protocols and local IRB procedures. One FFPE block for each case was selected using the following criteria:1. Every case should represent a unique breast cancer patient2. All must be primary breast cancers3. All are pathology confirmed invasive ductal or lobular carcinoma, a mixtures of these types, or classified as no special type4. All are hormone receptor positive (ER + or PgR+) breast cancer5. All must have a recorded tumor size6. FFPE blocks should be < 10 years old2 tumor area (1 slide/test) and 4 - 100 mm2 tumor area (3 slides/test)7. A minimum of 10 cases each of ≥ 100 mm2 and at least 10 cases contain 4 - 99 mm2 tumor area was implemented to validate the number of slides required for the assay. The blocks were not prescreened with the assay prior to inclusion, but it was anticipated that the 43 samples would cover a broad range of ROR scores representative of the intended use population, including both node-negative and node-positive patients, and each risk classification group. Seventeen tissue samples were from node-negative patients, 6 from node-positive patients and 20 were from patients whose regional lymph node status was provided by the biobank as NX.The criterion that at least 10 cases contain ≥ 100 mmFor reproducibility testing Figure , three sFor each tissue sample, a test run consisting of macrodissection, RNA extraction, and testing with the Prosigna assay was performed by a single operator at each site following the provided standard operating procedures. Each operator performed a minimum of four test runs consisting of up to 10 tissue samples per run. Each batch of tissue samples required a minimum run time of 3 days from tissue processing to result. Isolated RNA that met the quantity and quality specifications from each of the slide mounted sections was tested twice in separate assay runs. Different lots of RNA isolation kit reagents were used at each site, and a single lot of the Prosigna assay kit was used at all three sites.The test results for all samples remained blinded to all personnel at all sites until the study was complete. Upon completion of the study the blinded Prosigna assay data were collected from each site and merged with the expected test result and study variables  associated with each unique sample ID. The prospectively defined analysis plan was then executed on the merged analysis dataset.The pre-specified primary aim of the tissue reproducibility validation was to demonstrate the Prosigna assay is highly reproducible, when combining all sources of variation. For this study, “highly reproducible” was defined as a total standard deviation (SD) of less than 4.3 ROR units. The value of <4.3 was chosen because if two samples have true ROR scores that differ by 10 units, a total SD of 4.3 means that 95% of the time the higher of the two will still have a higher individual observed ROR score. A change of 10 ROR units corresponds to an average change in 10-year distant recurrence free survival of 7% and 6% for node negative and node positive patients respectively .The following variance components model was used to characterize the sources of variability:where FFPE Block was treated as a fixed component, and site and section were treated as random components. The “site” term measured the systematic site-specific variation that was constant across all tissue samples . The tissue section component measures random variation that differed as a function of review/processing or within FFPE block variation. The error term was derived from the duplicate RNA samples and estimated the combination of run-to-run and within-run variance. Variance components were estimated using the R procedure “lmer”. In the above model, the variance components were estimated from a combined analysis of all FFPE blocks after verifying that were no systematic changes in tissue-specific variation as a function of ROR score.Measurement = FFPE Block + site + tissue section + errorThe tissue and RNA isolation components were estimated using the reproducibility validation and the assay components were estimated using the RNA precision validation. The total variability, where Additional categorical analyses were performed using two classifications:•3 risk-categories  using both the node-negative and node-positive cutoffs,•4 intrinsic subtype categories RNA from each tissue sample was tested twice at each site so there are 4 possible comparisons between sites for each tissue sample leading to a total number of possible comparisons of 4*number of tissue samples. For each of the two classification schemes (risk category or subtype), the pair-wise concordance between sites was estimated as the fraction of all possible comparisons that were concordant and an exact-type 95% confidence interval was calculated.In addition, a post hoc analysis compared the normalized gene expression from the 50 classifier genes between the tissue replicates from all valid specimens tested at each site using a linear regression and correlation analysisThirteen FFPE breast tumor blocks containing pathologically-confirmed infiltrating ductal carcinoma were obtained and RNA was extracted from multiple slide mounted tissue sections from each block using the defined procedure Figure . The indThe pre-specified primary aim of the RNA input study was to demonstrate the Prosigna assay results were unchanged at the extremes of the assay specification range (125 and 500 ng RNA) regardless of the assay reagent kit lot used. For each kit lot, the test statistic was the average difference between the mean ROR score at a given input level where the average is across the n different samples. In this equation, ivalence . The inpFor each pooled sample a linear regression and correlation analysis was also performed between each replicate at each RNA input level and one of the two replicates run at 250 ng of RNA. The difference in the ROR score (ΔROR) from the nominal RNA input level (250 ng) for each replicate at each RNA input level was calculated by subtracting the ROR score calculated from one of the two replicates run at 250 ng from ROR scores calculated at the other input levels. Additionally, the ΔROR was calculated and linear regression and correlation analyses were also performed between the two replicates at 250 ng. The mean ΔROR, slope, intercept, and correlation values  were calculated using the pairwise comparisons for all passing samples at each input level for both kit lots.For the no-target (water) samples, the percentage of samples failing the minimum threshold for expression of normalizing genes was calculated. All no-target samples were required to give a failing test result.Twenty three FFPE breast tumor blocks were obtained containing pathologically-confirmed infiltrating ductal carcinoma microscopically-assessed to have 10 – 95% of the total tissue area containing normal/non-tumor tissue. Pathologists identified additional tumor interferents  within or near the margins of the tumor in ten of the 23 blocks.For each FFPE breast tumor block, H&E stained slides were prepared and up to nine unstained sections were cut and mounted on slides. For the inclusion of the interferent, the sections were processed according to the assay procedure with the exception that identified normal/non-tumor tissue or any additional interferents were included in the isolation (“non-macrodissected slides”). For the macrodissection where the non-tumor and other interferents were removed, three or  three and six slides were processed according to the Prosigna assay protocol.The change in ROR (ΔROR) due to the interferent was calculated using the ROR score from the non-macrodissected slides minus the ROR score from the macrodissected slides Figure . For theThe precision of the Prosigna assay starting from RNA was assessed with 5 pooled breast tumor RNA samples each tested 36 times at each of the three sites. There were no individual test samples that failed the pre-specified data QC metrics in the software so the analysis includes 540 results from 54 valid runs. For all five tumor RNA samples, the total SD was less than 1 ROR unit on a 0 - 100 scale Table , and theThe distribution of measured ROR scores for each of the five pooled RNA samples was also examined across the three lots, six users and three test sites. The range of ROR scores for the 108 independent measurements was ≤4 units for each of the 5 sample pools . One hundred percent (100%) of samples passing tissue review and RNA isolation specifications yielded passing results from the Prosigna assay.The call rate for the 43 tissue samples evaluated was 95%, 93%, and 100% for sites 1, 2, and 3 respectively. Forty samples yielded results at all sites (RNA isolation of one sample at one site required repeating). One tissue sample yielded results at 2 sites, and 2 samples yielded results at a single site, while the other sites did not obtain sufficient RNA to perform the assay for these samples. The measured tumor surface area for 4/5 RNA isolation failures was very small  of ROR scores Figure  and all Table The total SD including all source of variation (tissue and RNA processing variability) was 2.9 indicating that the Prosigna assay can measure a difference between two ROR scores of 6.75 with 95% confidence.The site-to-site concordances for the two categorical classifications are shown in Table •One sample had duplicate Luminal A results at one site and duplicate Luminal B results at each of the other two sites.•One specimen had duplicate Luminal A results at one site, duplicate HER2-enriched results at another site and one each of Luminal A and HER2-enriched at the third site.The average intercept, slope, and Pearson’s correlation of the pair-wise comparisons between sites are reported with the 95% confidence interval (Table The average ROR score for the tested samples covered a broad range (20 – 82) and all intrinsic subtypes – including 5 Luminal A, 4 Luminal B, 3 HER2-enriched and 1 Basal-like sample  were completely contained within -3 and 3 ROR units. The ROR scores at 125 and 500 ng RNA were therefore equivalent to those at the target input concentration of 250 ng for each of the two reagent kit lots tested meeting the primary objective of the study. Of note, when characterizing the RNA levels outside of the assay specification, the ROR scores at 62.5 ng RNA were not equivalent (with an upper confidence limit at 3.26) to those at the target input concentration of 250 ng for one of the two lots tested. This illustrates the importance of performing the assay according to the defined procedure.When the lots were combined the normalized gene expression values and ROR scores were consistent to those at the target input concentration of 250 ng within and even outside the RNA input limit specifications Table . CharactOut of 23 samples six were Luminal A, seven were Luminal B, two were HER2-enriched, and eight were Basal-like. The average ROR score for the 23 samples covered a broad range (10 – 83),  estimate of a patient’s risk of recurrence Figure . EliminaBreast cancer gene expression testing has been the subject of many studies demonstrating its capacity to stratify breast cancers by prognostic risk ,16,32,33Analytical reproducibility is a requirement for the implementation of all diagnostic tests, but it is especially critical for decentralized tests given the challenges of maintaining reproducibility across pathologists, technical operators, and instrumentation. However, decentralized tests also have many advantages over Laboratory Developed Tests that are performed at single central laboratories. By avoiding the need for shipping tissues, turnaround times and costs are reduced. The capacity for the laboratory physician to interact directly with the treating physician greatly aids medical care, for example in facilitating appropriate prioritization of critical specimens, explaining equivocal or unexpected results, and quickly recognizing inadequate specimens and what can be done to get a result helpful to the patient as soon as possible. Although first generation breast cancer prognostic tests were performed in central labs , second The Prosigna assay was tested across a range of RNA mass inputs that is consistent with what will be expected in a clinical lab setting. The assay is robust across that range, similar to what has been reported with other multigene breast cancer tests ,39. AddiThe observation of biased subtype calls and ROR scores with the inclusion of non-tumor tissue is consistent with a prior study , howeverThe precision and reproducibility of the Prosigna assay, estimated from repeat measurements of pooled tumor RNA sample(s) and de-identified patient tissue samples across multiple testing sites is similar  to what was previously reported for centralized lab tests ,38. Thesin vitro diagnostic test. Training of the assay workflow  takes less than one week. The pre-specified SOPs are easy to follow and the procedure of RNA extraction and Prosigna assay are straightforward. All operators, most of whom were naïve users to the Prosigna assay, were able to pass the training procedures on the first attempt, before executing the pre-specified study protocols. Although overnight incubations are required during RNA extraction and RNA – probe hybridization, the incubation temperature is constant, and hands-on time requirements for the whole experiment are very limited. Furthermore, the analyses for subtype call and ROR score are simplified and controlled by integrating the algorithm into the software for raw data processing, reducing the potential for human error in data cleaning and analysis.Our experience of implementing the nCounter platform in our CLIA-certified hospital laboratory environments proved to be straightforward, confirming the simplicity of the assay and its suitability as an The FDA cleared and CE marked Prosigna assay based on the PAM50 gene expression signature has recently been shown to predict the risk of distant recurrence in women with hormone receptor positive early stage breast cancer treated with five years of endocrine therapy ,20. ThisROR: Risk of recurrence; SD: Standard deviation; FDA: US Food and Drug Administration; CLSI: Clinical Laboratory and Standards Institute; EGAPP: Evaluation of genomic applications in practice and prevention; FFPE: Formalin-fixed paraffin-embedded.TN disclosed that he is one of the holders of the patents on which the Prosigna Assay is based and is a co-founder of Bioclassifier, LLC which licenses the PAM50 algorithm to NanoString Technologies, Inc. JS, SF, BW, ND, and MM all disclose that they are employees of and shareholders in NanoString Technologies. CS is a paid consultant of NanoString Technologies, Inc. All other authors had no disclosures to report.TN, JS, BW, CS, SF, SL contributed to the study design and protocols and drafted the manuscript. CS, JS, and BW performed statistical analysis and presentation of data. DG performed tissue review for the tissue reproducibility study and the tissue interference study. ND, MM, and GB performed the assay for the RNA precision study. ND and GB performed the assay for the tissue reproducibility study. ND performed the assay for the RNA input and tissue interferents studies. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/14/177/prepubSite to site ROR sample means. Mean ROR scores were calculated for each pooled RNA sample, and likelihood ratio test for significance was performed to test for differences between sites. There were no significant differences in the results observed across sites for the five pooled RNA samples tested. All p-values were well above 0.05 for the likelihood ratio test of significance of site with 2 degrees of freedom for each pooled RNA sample. The differences in means between sites were all less than 0.5 ROR units on a 0-100 scale.Click here for fileWithin site gene expression comparisons from the tissue reproducibility study. Pairwise correlations, slopes, and intercepts of normalized 50 genes for replicate RNA Hybridizations from the tissue reproducibility study. The average intercept, slope, and Pearson’s correlation of the pair-wise comparisons are reported with the 95% confidence interval.Click here for fileHierarchical clustering of all samples from the tissue reproducibility study. Clustering analysis (using a Pearson’s distance metric and average linkage) was performed on the median centered normalized, Log2 transformed and scaled sample data to further characterize the gene expression in the tissue samples. The tissue sample and RNA sample replicates were always only clustered together and the node heights are almost imperceptibly low (indicating highly correlated gene expression).Click here for fileAverage ROR Score for the 13 unique tumor RNA samples within the RNA Input Study. Data are colored by the intrinsic subtype result at 250 ng of RNA.Click here for fileROR Score for the 23 unique macrodissected tumor samples. Data are colored by the intrinsic subtype result for each tissue. For tissues with multiple isolations the subtype result illustrated was from the macrodissection with the most number of slides processed.Click here for file


Arabidopsis thaliana’s DRB2 protein results in a significant increase in the population of RNA polymerase IV (p4) dependent siRNAs, which are involved in the RNA-directed DNA methylation (RdDM) process.In plants and animals, a large number of double-stranded RNA binding proteins (DRBs) have been shown to act as non-catalytic cofactors of DICERs and to participate in the biogenesis of small RNAs involved in RNA silencing. We have previously shown that the loss of in vivo but that drb2 mutants do not have a significant variation in TE DNA methylation.Surprisingly, despite this observation, we show in this work that DRB2 is part of a high molecular weight complex that does not involve RdDM actors but several chromatin regulator proteins, such as MSI4, PRMT4B and HDA19. We show that DRB2 can bind transposable element (TE) transcripts We propose that DRB2 is part of a repressive epigenetic regulator complex involved in a negative feedback loop, adjusting epigenetic state to transcription level at TE loci, in parallel of the RdDM pathway. Loss of DRB2 would mainly result in an increased production of TE transcripts, readily converted in p4-siRNAs by the RdDM machinery.The online version of this article (doi:10.1186/s12870-015-0455-z) contains supplementary material, which is available to authorized users. RNA recognition by proteins is based on a number of specialized amino acid modules that interact with the structure and/or the primary sequence of their RNA targets. The double-stranded RNA binding motif (DSRM) is an example of such module. The DSRM is an evolutionary conserved 65 to 68 amino acids region that can adopt a typical α − β − β − β − α fold with the most conserved residues mainly located in its C-terminal part -3. DSRMsArabidopsis thaliana, including four Dicer-Like (DCL) and five double-stranded RNA binding (DRBs) proteins   during 60 minutes at 4°C with gentle rotation. Constant volumes of the crude extract were removed from the master tube at given time points and treated with 50 mMTris pH 7.5 to stop the reaction. Each tube corresponding to a time point was halved and one half was treated in 4X Laemmli buffer containing 3% β-mercaptoethanol and the other half in 4X buffer without β-mercaptoethanol. Extracts were analysed by western blot.DRB2-FlagHA crude extract were obtained by resuspension in 500 mM HEPES pH7.5, 150 mM NaCl, 0.1% Igepal, 5 mM MgClDNA from inflorescences was extracted using the DNeasy plant minikit (Qiagen) following the manufacturer’s protocol. DNA was digested with methylation sensitive HaeIII enzyme (GGCC) and AtSN1/SB4-8, which contains 3 different HaeIII sites was amplified. Disappearance or reduced levels of a fragment after digestion with a given enzyme indicate loss of methylation at that site. RDRP is used as a loading control as it does not harbour any HaeIII sites.ddm1 or ddm1/drb2 seedlings segregating from a single ddm1−/− drb2+/− plant. 2 μg of RNA were DNase treated using the Promega RQ1 kit following manufacturer’s recommendations. 0.2 μg of treated RNA was reverse transcribed using PrimeScript RT reagent kit  in a final volume of 10 μl using a mix of oligodT and random hexanucleotides as preconized by the manufacturer. One μl of cDNA was used for amplification, using the Eco Real-Time system (Illumina) and SYBR Premix Ex Taq II (Tli RnaseH Plus) (Takara) in a final volume of 15 μl. Each value then represents a mean of three independent biological replicates and standard error of the mean is applied after normalisation to Actin2 gene expression. Primers can be found in Additional file Total RNA was extracted as described above from 3 pools of 2 three weeks old Agrobacteriums harbouring the MSI4-eGFP and DRB2-FlagHA binary plasmids were grown under the appropriate selection until O.D (600 nm) reached 0.8. 5 ml were pelleted by centrifugation and resuspended in 10 mM MES, 10 mM MgSO4, and 100 μM acetosyringone. Equal volumes of each solution was mixed and 5 week old leaves were infiltrated. Plants were maintained in 16 h-light/8 h-dark conditions for 55 hours before IP was performed as described.The Input and eluted RNA from DRB2-FlagHA IPs were labelled with radioactive pCp using the T4 RNA ligase from Ambion (AM2140), following the manufacturer’s protocol. 100 ng of total RNA and half the elution volume were labelled with 3 μl pCp in a final volume of 20 μl overnight at 4°C. RNA was then phenol extracted and precipitated in absolute ethanol, 0.3 M sodium acetate and 20 μg of glycogen. Pellets were washed in 80% ethanol and resuspended in 20 μl of loading buffer  and secondary structures were removed by boiling for 5 minutes. Resulting samples were loaded in either 6% or 15% acrylamide (19:1 acrylamide:bis acrylamide) 8 M urea, 0,5X TBE gel and separated by electrophoresis. 6% Acrylamide gels were further dryed for one hour at 80°C under vacuum. Exposure is performed as described in the northern blot section.