We report that the mammalian mRNA cap methyltransferase, RNMT-RAM, promotes RNA Pol II transcription independent of mRNA capping and translation. In cells, sublethal suppression of RNMT-RAM reduces RNA Pol II occupancy, net mRNA synthesis, and pre-mRNA levels. Conversely, expression of RNMT-RAM increases transcription independent of cap methyltransferase activity. In isolated nuclei, recombinant RNMT-RAM stimulates transcriptional output; this requires the RAM RNA binding domain. RNMT-RAM interacts with nascent transcripts along their entire length and with transcription-associated factors including the RNA Pol II subunits SPT4, SPT6, and PAFc. Suppression of RNMT-RAM inhibits transcriptional markers including histone H2BK120 ubiquitination, H3K4 and H3K36 methylation, RNA Pol II CTD S5 and S2 phosphorylation, and PAFc recruitment. These findings suggest that multiple interactions among RNMT-RAM, RNA Pol II factors, and RNA along the transcription unit stimulate transcription.  •mRNA cap methyltransferase, RNMT-RAM, promotes RNA Pol II-dependent transcription•RNMT-RAM-dependent transcription is independent of mRNA cap methylation•RNMT-RAM binds to the entire length of pre-mRNA and to transcription-associated proteins•Significant loss of RNA Pol II gene occupancy is observed on RNMT-RAM suppression The mammalian mRNA cap methyltransferase, RNMT-RAM, prepares pre-mRNA for processing and translation and regulates expression of a subset of mRNAs. Here, Varshney et al. report that RNMT-RAM regulates transcription independent of mRNA cap methylation and translation. RNMT-RAM binds the full length of pre-mRNA and recruits proteins associated with transcription. S. cerevisiae cap methyltransferase, ABD1, is important for transcription of certain genes , linking guanosine to the first transcribed nucleotide via a 5′-to-5′-triphosphate linkage . This guS. cerevisiae, the cap methyltransferase ABD1 interacts directly with phosphorylated Serine 5 (phospho-S5) RNA Pol II C-terminal domain (CTD), whereas in S. pombe, the cap methyltransferase PCM1 is recruited in a complex with Cdk9/Pch1 (P-TEFb)  . The mam(P-TEFb) . RNMT ha(P-TEFb) . RAM als(P-TEFb) . The RAM(P-TEFb) .Here we report that RNMT-RAM functions independently of mRNA cap methylation to promote RNA Pol II-dependent transcription. Sublethal suppression of RNMT-RAM expression results in massively reduced RNA Pol II occupancy, reduced net mRNA synthesis, and reduced pre-mRNA levels. Increasing RNMT-RAM expression in cells increases transcription in a methyltransferase-independent manner. Furthermore, recombinant RNMT-RAM stimulates transcription in isolated nuclei, confirming that this mechanism is independent of mRNA capping, mRNA translation, and mRNA stability. RNMT-RAM associates with pre-mRNA along its entire length via interaction with RAM and interacts with several transcription-associated complexes. We propose that the human cap methyltransferase complex promotes transcription by RNA Pol II via multiple RNA and protein contacts along the transcription unit.3H-uridine for 30 min, which is converted to 3H-UTP and incorporated into nascent transcripts. mRNA was enriched by oligonucleotide (oligo)-dT affinity, and 3H-uridine incorporation was determined. Because the median half-life of mRNA is ∼10 hr, a 30 min uridine pulse predominantly measures transcription , which reduce expression of RNMT and RAM A Gonato. To focucription . Net mRNcription A. In a scription B. HA-RNMcription C. Furthecription . TherefoTo investigate whether RNMT-RAM has a direct impact on transcription, nuclear run-on assays were performed. Nuclear run-on measures nascent transcription from engaged polymerase in isolated nuclei, independent of mRNA capping, translation, and stability . In thesTo further evaluate the mechanism by which RNMT-RAM influences transcription, we required the identity of the genes most affected by its inhibition. RNA sequencing was performed on HeLa cells following RAM depletion for 36 hr in 5 biological replicates C–S2E. DeTo define the role of RAM in transcription, RNA Pol II occupancy on the genome was investigated using chromatin immunoprecipitation (ChIP). Following transfection with RAM siRNA or non-targeting control for 36 hr, RNA Pol II ChIP was performed on three biological replicates that exhibited a high correlation genome wide and at an individual gene level A and S4BAn alternative approach to investigating RNA Pol II occupancy is to identify RNA Pol II peaks throughout the genome, i.e., regions where the RNA Pol II ChIP reads are significantly enriched relative to the input sample. Peak calling identified 9,464 consensus peaks in control cells, whereas following RAM depletion, only 1,323 peaks were identified, an 86% reduction D and S7.RNMT-RAM suppression not only had a major impact on genome-wide RNA Pol II loading A–2E but 32P RNA labeling, RNMT-RNA and RAM-RNA complexes were visualized in SDS-PAGE as smears, migrating more slowly than RNMT (66 kDa) and RAM (14 kDa)  A. Protei(14 kDa) A. The 50in vitro to the aspargine- and arginine-rich (NR-rich) region (amino acids 56–91), and RAM 1–55 binds to RNMT (The dependency of RNMT-RNA binding on RAM was investigated. RNA binding to the RNMT lobe deletion mutant (Δ416–Δ456) was below the limit of detection B. This m to RNMT . To map  to RNMT D. Althou to RNMT . As expeTranscripts bound to HA-RNMT-RAM complexes were isolated from HeLa cells using CLIP and RNA fragments sequenced as in Genome coverage analysis of the 727,624 uniquely aligned RNMT-RAM CLIP reads pooled from the three replicates revealed that most aligned to intronic regions of pre-mRNA, as opposed to the 5′ localization expected of a 5′ cap methyltransferase B. Long nAlthough transcripts from 5,436 genes were identified as binding to RNMT (average log counts per million [logCPM] > 5), for further mechanistic analysis, the 684 transcripts found in the top quartile of all three RNMT-RAM CLIP replicates were designated as RNMT-enriched transcripts . AnalysiBecause RNMT-RAM binds to the full length of pre-mRNA and promotes transcription, we investigated whether it binds to transcription-associated proteins. SILAC (stable isotope labeling of amino acids in cell culture) media were used to differentially label cellular proteins, allowing comparative quantitation. HA-RNMT was immunoprecipitated from extracts of cells grown in R6K4 medium, and a mock IgG immunoprecipitation (IP) was performed on extracts of cells grown in R0K0 medium A. Mass sin vitro and in cells . PAFc binds directly to RNA Pol II across the gene and is involved in transcription initiation and elongation by interacting with transcriptional regulators and promoting many aspects of the transcription cycle . The mecin cells . The RNAin cells C. CTR9 iin cells F. The imin cells G. CTR9 rConsistent with its influence on PAFc recruitment, suppression of RAM expression resulted in suppression of hallmarks of PAFc function. PAFc co-operates with the Mediator complex to stimulate the mono-ubiquitination of histone H2B at lysine 120 (H2Bub), which is critical for histone H3 lysine 4 trimethylation (H3K4me3) . PAFc alBecause RNMT-RAM binds across the length of pre-mRNA, binds to transcriptional regulators, and promotes transcription, the relationship between RNMT-RAM transcript binding and RNA Pol II occupancy was investigated. Genome-wide, RNMT-RAM transcript binding correlated positively with RNA Pol II occupancy over associated gene loci B and 6C.Here, we report that RNMT-RAM promotes RNA Pol II-dependent transcription. The impact of RNMT-RAM on transcription is direct and independent of mRNA capping, mRNA stability, and mRNA translation; expression of MTD RNMT-RAM promotes net mRNA synthesis in cells, and RNMT-RAM promotes the output of engaged RNA Pol II in isolated nuclei . RNMT-RAHow does RNMT-RAM affect transcription? The activating subunit, RAM, has a key role. Previously, both the RNMT-activating domain (1–55) and the RNA binding domain (55–90) were demonstrated to be important for cell proliferation . HoweverOne feature of the impact of RNMT-RAM on RNA Pol II distribution is that it reduces RNA Pol II at the TSS and the gene body  and 6. RS. cerevisiae reported that many RNA Pol II-associated factors, including PAFc, crosslink to nascent RNA , 10% knockout serum replacement, 1% mem-NEAA, 1 mM sodium pyruvate, 0.1 mM 2-mercaptoethanol, and 100 U/mL recombinant Leukemia inhibitory factor (LIF). Transient transfections were performed using Lipofectamine 2000 (Thermo Fisher Scientific) with 4 μg pcDNA5, 2 μg pcDNA5-HA-RNMT, 4 μg pcDNA5-HA-RNMT Δ416–456, 2 μg pcDNA5 HA-RNMT MTD, 2 μg pcDNA5-HA-RNMT Δ1–120, and 1 μg pcDNA5-HA-RNMT 1–120 or 4 μg of pcDNA5 RAM-GFP for 48 hr. siRNA transfections were performed with 200 pmol RAM siRNA or non-targeting control  using RNAiMAX (Thermo Fisher Scientific) for 36 or 48 and 72 hr in HeLa and mESCs, respectively. siRAM1 and siRAM4 in HeLa cells are D-021286-01 and D-021286-04 (Dharmacon). The siRAM mixture used in mESCs is equimolar D-049592-01 and D-049592-03 (Dharmacon). HeLa and mESCs stably expressing RNMT were made with the pBMN retroviral vector system and pPyPCAGGS vector. Cells were treated with 3 μg/mL of actinomycin D or DMSO for RNA decay experiments.HeLa and HEK293 cells were cultured in DMEM and 10% fetal bovine serum at 37°C in 5% CO2 at 254 nm in Stratalinker 2400. Lysates were sonicated for five 30 s pulses in a Bioruptor (Diagenode) at low amplitude. Nuclease digestion with 5 μL Turbo DNase (Life Technologies) and 10 μL 1:500 (high) to 1:10,000 (low) dilution of RNase A  were performed in a thermomixer at 37°C for 3 min of shaking at 1,100 rpm. HA-RNMT was immunoprecipitated with 10 μL Pierce anti-HA magnetic beads for 2 hr at 4°C. IP RNA was labeled using the 32P-labeled 3′-RNA linker (5′-Pmn.GGAACCGUGGGCUCUUAAGGU-3′) prepared by kinase labeling and purification through Sephadex G-25 columns as in CLIP was performed according to Chromatin immunoprecipitation (ChIP) was performed using 2 μg anti-RNA Pol II or 5 μL CTR9 antibody with 25 μg HeLa chromatin as in Cell lysates were prepared in 50 mM Tris (pH 7.5), 50 mM sodium chloride, 1% Triton X-100, 270 mM sucrose, 1 mM sodium orthovanadate, 1 mM EGTA, 1 mM EDTA, 10 mM β-glycerol phosphate, 5 mM sodium pyrophosphate, 1 mM DTT, 10 μM leupeptin, 1 μM pepstatin, 0.1 mg/mL aprotinin, and phosphatase inhibitor (PI) 2 and PI3 cocktail (Sigma) and were precleared with 20 μL washed protein G Sepharose  for 30 min at 4°C on a rotating mixer. Resin was pelleted and supernatant was incubated with 2 μg antibody for 2 hr at 4°C. 1 μL RNase A  was added before IP where required. Protein G Sepharose beads were prepared by washing twice in lysis buffer, blocking with 1 mg/mL BSA at RT for 30 min, and washing thrice in lysis buffer. 20 μL of beads were added to each IP and incubated at 4°C for 1 hr on a rotating mixer. IPs were washed thrice with ice-cold lysis buffer without detergent and eluted with 2× Laemmli sample buffer by boiling at 70°C for 10 min before western blot analysis.6 cells plated the day before were lysed and washed in buffer containing 10 μM leupeptin, 1 μM pepstatin, 0.1 mg/mL aprotinin, 1:1,000 phosphatase inhibitor cocktail II and III, and 5 mM DTT (Sigma). Nuclei were stored in nucleus storage buffer that contained the same protease and phosphatase inhibitors, DTT, and 1 U/μL RNAase inhibitor (RNAsin). 5fM recombinant RNMT and/or RAM protein were added to nuclei from a 10 cm plate and incubated for 20–30 min at 30°C, with 1 mM ATP, 1 mM cytidine trisphosphate (CTP), 1 mM guanosine triphosphate (GTP), 0.5 mM BrUTP, 0.5 mM UTP, and 1 U/μL RNAsin. Recombinant RAM 1–90 was used, rather than full-length RAM (1–118), because the latter is unstable. Following nuclear run-on and RNA purification, Br-U IP was performed with more than 10 μg RNA . Following IP, RNA was resuspended in 30 μL, with 10 μL used for cDNA synthesis and PCR.Nuclear run-on was performed as in 3H-uridine (PerkinElmer) for 30 min 36–48 hr post-transfection with siRNA or pcDNA5-based constructs. Total RNA was purified with TRIzol (Thermo Fisher Scientific) and quantified using a Qubit RNA BR assay kit (Thermo Fisher Scientific). Poly(A) mRNA was purified from total RNA using the NEBNext poly(A) mRNA magnetic isolation module (NEB) or mRNA Direct (Ambion). 3H-uridine incorporation into mRNA was quantified by scintillation, counting equal amounts (40–70 ng) of purified poly(A) RNA.Subconfluent HeLa cells were incubated with 60 μM 5,6-RNA was extracted from HeLa cells using the TRIzol reagent (Thermo Fisher Scientific). Libraries were prepared with TruSeq Stranded Total RNA with the Ribo-Zero kit (Illumina). Sequencing was performed on the Illumina NextSeq platform. Reads were aligned using STAR software, and transcripts were quantified with the HTSeq package . LibraryRNA was isolated using TRIzol (Thermo Fisher Scientific). 100–200 ng RNA was used to prepare cDNA using the iScript cDNA synthesis kit (Bio-Rad). PCR was performed with the EvaGreen supermix (Bio-Rad) on the CFX384 Touch real-time PCR detection system (Bio-Rad). Primer sequences are in 2, and 10 mM KCl. Lysates were precleared with protein G Sepharose. Equal cell lysates from R0K0- and R6K4-labeled 293 HA-RNMT cells were affinity purified with murine IgG agarose (Sigma) and monoclonal anti-HA antibody-conjugated agarose (Sigma), respectively, and washed with lysis buffer; eluates were mixed at a 1:1 ratio. The mix was resolved by SDS-PAGE, and lanes were excised into five slices before in-gel tryptic digestion. Samples were analyzed on a LTQ-Orbitrap XL mass spectrometer (Thermo Fisher Scientific) coupled to a U3000 nano-LC system (Dionex). Data were analyzed with MaxQuant (v.1.0.13.13) with the MaxQuant Human database and Mascot search engine v.2.3.2 (Matrix Science) (HEK293 cells were cultured in R0K0 media with 84 mg/L L-arginine (Sigma) and 146 mg/L L-lysine (Sigma) or R4K4 media with an of equal concentration L-arginine 13C and l-Lysine 4,4,5,5-D4 (Cambridge Isotope Laboratories) for 5 cell doublings and were lysed in 10 mM HEPES, 15 mM MgClScience) . Data weScience) .


The prebiotic significance of laboratory experiments that study the interactions between oligomeric RNA and mineral species is difficult to know. Natural exemplars of specific minerals can differ widely depending on their provenance. While laboratory-generated samples of synthetic minerals can have controlled compositions, they are often viewed as "unnatural". Here, we show how trends in the interaction of RNA with natural mineral specimens, synthetic mineral specimens, and co-precipitated pairs of synthetic minerals, can make a persuasive case that the observed interactions reflect the composition of the minerals themselves, rather than their being simply examples of large molecules associating nonspecifically with large surfaces. Using this approach, we have discovered Periodic Table trends in the binding of oligomeric RNA to alkaline earth carbonate minerals and alkaline earth sulfate minerals, where those trends are the same when measured in natural and synthetic minerals. They are also validated by comparison of co-precipitated synthetic minerals. We also show differential binding of RNA to polymorphic forms of calcium carbonate, and the stabilization of bound RNA on aragonite. These have relevance to the prebiotic stabilization of RNA, where such carbonate minerals are expected to have been abundant, as they appear to be today on Mars. It has been nearly 70 years since Bernal broadly conjectured on possible roles of rocks and minerals in the assembly of complex organic species relevant to the origin of life . This th(i) Concentration. Whether they are delivered by meteorite or created on Earth, prebiotic organic molecules are expected to be dilute and, if concentrated, react unproductively with each other. Rock and mineral surfaces offer the opportunity to concentrate relevant species from dilute aqueous environments, perhaps without unproductive intermolecular reactions. Such adsorption as a concentration mechanism offers an alternative to evaporation in a desert environment. Mineral adsorption from a large ocean is an especially attractive alternative to desert evaporation for those who think that dry land was sparse on the early Earth .(ii) Productive catalysis. Concentration is itself a way of "catalyzing" bimolecular reactions. However, rocks and minerals have also been considered as sources of conventional catalysis, where species on the surface of the mineral stabilize a transition state with respect to adsorbed ground state species .More recently, and especially after the emergence of the "RNA first" hypothesis for the origin of Darwinism on Earth , rocks a(iii) As inhibitors of reactions. One key problem obstructing the assembly of prebiotically productive organic species is the well-known propensity of organic molecules, especially those containing carbonyl groups such as carbohydrates, to react further to yield unproductive "tars". Mineral species, especially if they are slightly soluble into an aqueous environment, have been proposed to prevent classes of unproductive reactions –8.(iv) Stabilizers. Many useful pre-biological polymers are subject to destruction by environmental forces, such as ultraviolet radiation and radioactivity. Adsorption of these onto mineral surfaces has been shown to slow that destruction , in some® prebiotic chemistry" that dominates the field. However, in addition to creating an opportunity, this complexity creates problems, both intrinsic and experimental. For every constructive reaction that might be catalyzed by a mineral, the potential exists for that mineral to catalyze a destructive reaction. Further, although a mineral (by definition) is a pure substance, real minerals invariably have non-canonical elements incorporated within them; these defects may easily be the reason why a natural mineral adsorbs organic molecules or has an interesting reactivity. Further, even with an ideally pure mineral, the catalysis of interest can occur in defects in its crystalline surface. All of these problems are difficult to manage in a controlled laboratory environment.As Hazen and Sverjensky remark , mineralHow are we to explore this new complexity as we accommodate those who "plead" for a role for mineralogy in models for the origin of life? Two approaches are possible. On one hand, we might build a collection of natural minerals, and then run experiments on them with biopolymers having prebiotic interest, such as RNA. Unfortunately, natural minerals vary in chemical composition from specimen to specimen, and certainly from locale to locale. This is obvious even to an amateur. For example, natural calcium phosphate (apatite), of possible prebiotic interest as a source of the phosphate essential to prebiotic RNA synthesis , has difAlternatively, reagents that have the components of those minerals, with exacting levels of purity, might be mixed in the appropriate ratio to create a synthetic mineral as a precipitate. Experiments might then be run on these synthetic minerals to study their interaction with biopolymers of interest, such as RNA. This approach has the advantage of offering exactly the kind of "controlled experiments" that chemists like. However, it is frequently criticized as being "artificial".Even if this problem were to be mitigated or ignored, general chemical physics intervenes. Solid phases with high surface areas, and precipitates in particular, are general adsorbents, especially for macromolecules. Therefore, it is difficult to know, if RNA (for example) adsorbs onto a surface, whether the adsorption is in any sense specific, or whether it is just a general manifestation of big molecules adsorbing to big surfaces.Here, we introduce a general strategy that mitigates some of these problems. The experiments measure the adsorbance of radiolabeled RNA onto binary inorganic species that have been obtained in two ways. In one, the species is precipitated as a synthetic mineral via a double decomposition reaction between the two mineral components. In the second, the mineral itself is obtained from a natural source, and the experiment measures the percentage of radiolabeled RNA bound to the natural mineral. In a third approach, two precipitated minerals are combined, and the partition of radiolabel RNA between the two is measured.This strategy then asks whether the trend in radiolabeled RNA adsorption is consistent across their various forms and presentations, especially within a set of minerals having a common anion  but differing in their cationic components . Here, we may even seek a Periodic Table trend, where adsorption changes consistently in a series of minerals as one of their elements is replaced by another element in a row or column of the Periodic Table.Underlying these experiments is the following rationale: If the same trends are observed both in precipitated synthetic minerals as well as in natural minerals, and if radiolabeled molecules are partitioned consistently between two mineral species precipitated together, the effects cannot easily be nonspecific as general adsorption of big biopolymers onto big surfaces.We report here the first cases where this rationale has been applied for RNA over a range of minerals. Surprisingly, some of these showed Periodic Table trends, in both their precipitated synthetic and natural forms. Further, we speculate that these trends can be accounted for by the changing size of the mineral lattice resulting from different ionic radii of different elements in a Periodic Table series.We examined first various binary carbonate minerals with Group II  cations. These are interesting not only because carbon dioxide is likely to have been an abundant component of an early Earth atmosphere, but also because alkaline earths form a well-known set of binary carbonates that include insoluble magnesium, calcium, strontium, and barium carbonates .The magnesite specimen was from Minas Gerias, Brazil; the calcite was a specimen of "Iceland spar". The strontianite was obtained from the Minerva Mine in Illinois , and the witherite was obtained from Cave in Rock, Illinois. The specimens were washed with hydrogen peroxide (30%) followed by water and then ethanol to remove potential organic surface contaminants. The samples were then dried in air while covered.32P labeled 83-mer RNA . This length was chosen because it is representative of lengths that Holliger, Joyce and others suggest is needed to initiate Darwinism [To flat surfaces of the cleaned mineral were added droplets of an aqueous (unbuffered) solution of 5’-arwinism –15. Altharwinism , these warwinism . This de2O to remove unbound RNA. Then, the amount of RNA bound was calculated by subtraction of counts per minute in the washes.After adsorption, the mineral surface was washed several times with HThe results are shown in Following the dual-approach rationale, we then asked whether the same results could be qualitatively observed with precipitated synthetic minerals . These results are collected in 3 2.71 g/cm3; MgCO3 2.96 g/cm3; SrCO3 3.5 g/cm3; BaCO3 4.29 g/cm3). The partition of RNA between each pair was then observed by pre-equilibration of the radiolabeled 83-mer RNA in a column with the two minerals, followed by dissection of the column and counting various slices within it. The labeled RNA partitioned as seen in the synthetic minerals precipitated individually: Ba > Sr > Ca > Mg.To complete the analysis, we then co-precipitated various pairs of synthetic carbonates by mixing the appropriate aqueous solutions of the alkali metal chlorides with an aqueous solution of sodium carbonate in a 1:1 ratio . These wBinary alkaline earth sulfates are fewer in mineral form, since the first member of the Periodic Table series  is quite soluble in water. However, with precipitated synthetic minerals, the same trend was observed, with barium sulfate binding RNA more than strontium sulfate, which bound more RNA than calcium sulfate . The corHowever, and as a limitation of this approach, many minerals that might be made in the laboratory have no known natural correlates that we can examine in parallel. For example, in the synthetic borate minerals, the barium species also binds most tightly. However, to our knowledge, no natural strontium or barium borate mineral has been reported. The natural calcium borate mineral that we tested (colemanite) bound 31% of the RNA presented to it. Further, although the magnesium borate mineral is known naturally as boracite, we were unable to get a precipitate of the synthetic mineral by mixing magnesium chloride and sodium borate .While a Periodic Table trend is easy to observe, there is no reason a priori why such a trend should exist. For example, one might speculate that RNA would adsorb better onto a surface if the pattern of anion and cation sites on that surface matches more closely the distances of the anionic sites (phosphates) on the RNA molecule. While one might expect different cations in a mineral would change the spacing of those sites, there is no reason why the heaviest cation would have sites that match RNA the best. Indeed, if this were the mechanism for different surfaces having different affinities for RNA, one might expect within a Periodic Table trend to have a mineral that maximally absorbs somewhere in the middle of the series, rather the end of the series.We may, in fact, see this in these data. For example, among the precipitated phosphates, the calcium species bound more RNA (93%) than the magnesium phosphate (64%), the strontium phosphate (84%), and barium phosphate (32%). While the calcium phosphate is well-known in the natural world in various forms (apatite), and while calcium is known to be replaced in natural minerals by strontium and barium to give species known as "strontium apatite" and "barium apatite", the strontium and barium forms are very seldom found in nature, and are not available for this kind of study.The same comments apply to vanadates and arsenates, which we examined because of their structural resemblance to phosphates –19. HereThe alkaline earth carbonates and sulfates make conveniently simple systems where the natural-synthetic combination analysis can be easily applied. Other classes of minerals are more difficult to manage for two classes of reasons.First, the cation(s) in the mineral may be redox active. Here, in a precipitation to give a synthetic mineral, the presence of oxygen can lead to a precipitate having the cation in mixed oxidation states.2PO4−, HPO42−, and PO43−) are, de facto, the buffering species in the precipitation experiments. Nevertheless, we collected data for a variety of natural species, including several that are not conveniently made by double decomposition reactions from water-dissolved salts. These are shown in Second, we cannot conveniently add a buffer to control the pH in an experiment that precipitates synthetic minerals; it would add an unnatural component into the system. This means that different anions with different protonation states  and zinc carbonate (smithsonite) were examined. Both adsorbed comparable amounts of RNA to their surfaces in the mineral specimens that were examined, 11% and 5% respectively. Comparable amounts of RNA adsorbed to each of the precipitated minerals . However, it was difficult to find a rationale to compare these numbers across the Periodic Table to numbers obtained with the alkaline earth carbonates.Finally, several silicates were examined for their ability to adsorb RNA. Silicates, of course, are represented by a very large number of minerals, and this work examined only a very small fraction of these. We recently reported work examining the adsorption and stabilization of RNA on opal .3, is a third metastable phase of CaCO3. It occurs much less commonly in nature because it is the least thermodynamically stable. It generally and rapidly transforms itself into one of the other two forms [3 that precipitates upon mixing CaCl2 and Na2CO3 in water at near-neutral pH and room temperature and pressure; absent contaminants [Another layer of complexity comes from the fact that the same set of atoms can form different crystal forms. For example, calcium carbonate can precipitate as calcite, aragonite, or vaterite. Calcite crystallizes in a trigonal space group; aragonite has an orthorhombic structure –25. Calcwo forms . Vateritaminants , aragoniaminants  and by p3 system, we obtained natural specimens of the mineral aragonite and calcite. Experiments consistently showed that aragonite adsorbed more radiolabeled RNA than calcite. To obtain a synthetic mineral by precipitation, we reasoned that if RNA prefers to bind to aragonite over calcite, then perhaps RNA would nucleate the formation of an aragonite precipitate over a calcite precipitate.To complete our analysis of the CaCO3 precipitated preferentially as aragonite in the presence of RNA, here isolated from Aspergillus. This was first observed when a solution of Na2CO3 (1 M) was mixed with a solution of CaCl2 (1 M) in the presence of 160 ng rRNA, with control experiments identical except for the absence of RNA. Both precipitates were stained with Feigl’s stain, with which aragonite is stained black, while calcite remains white  was spotted on five pieces of natural aragonite, washed with H2O to eliminate unbound RNA, placed dry in a thermoblock at increasing temperatures , and incubated for two hours. After incubation, RNA was eluted from aragonite with 1 M formic acid, purified, and loaded on denaturing PAGE with a set of control samples where RNA was treated the same way, but in aqueous phase . Interestingly, ≈70% of the RNA bound to aragonite remained full-length after incubation at 95 °C for 2 hours. In contrast, RNA treated the same way but in aqueous solution  amorphous materials. However, the size of their crystals is generally smaller than the size of crystals of minerals collected in the field.Where possible , replicas were done. However, the main point presented here is that the error is not the kind of "error" that can be analyzed by standard statistical methods. This requires that the error be "normally distributed". Here, the error problems come from systematic errors relating to the natural samples, as two different exemplars of the "same" mineral, or even two different portions of the same specimen, may in fact be of different composition and thus may give different results. They are not Boltzmann "normally" distributed, and adding standard deviations from multiple runs provides only the deceptive illusion of statistical support. In this work, we circumvented this problem by asking whether the trend in radiolabeled RNA adsorption is consistent across the various forms and presentations of a mineral.The most obvious limitation of this comparative approach comes from nature herself. The rarity of minerals having different elemental compositions determines their availability for these experiments. Some elemental compositions are simply not found in nature at all.Thus, an analysis that involves Group  comparison of transition metal minerals in the Periodic Table is not particularly sensible using this approach. For example, iron phosphate is a well-known mineral (vivianite) and it adsorbs RNA ≈12%, . However2O3, hematite, 30% in our experiments), but not to magnetite (Fe3O4) [Likewise, horizontal comparisons across the Periodic Table are problematic, even within transition metals. For example, we found that RNA binds to natural titanium dioxide  and iron(III) oxide .Some evidence suggests that the crystalline surface is important in this trend. For example, like witherite and strontianite, aragonite adsorbs RNA better than calcite. Further, the crystal structures of witherite and strontianite belong in the same family as the crystal structure of aragonite. Likewise, vaterite resembles aragonite in its crystal structure more than either resembles calcite. Together, these results suggest, at least at the level of hypothesis, that the molecular structure of RNA is more compatible with the surface of an orthorhombic carbonate crystal (the “aragonite group”) having cations arranged with pseudo-hexagonal symmetry, than with the trigonal crystals observed in calcite and magnesite.Here, the adsorbance of RNA on aragonite, and the ability of aragonite to nucleate the growth of aragonite and/or vaterite over the thermodynamically more stable calcite has potential prebiotic significance, as does the stabilization of RNA on these carbonates. All of these minerals are likely to have been present on early Earth. They are also known on Mars . Today, These results must remain tentative until reproduced by other laboratories, of course. We remain concerned that specific properties of natural minerals may differ with different sources, different impurities, different levels of success in cleaning their surfaces, and a thousand other variables that might influence these results . MitigatHowever, as a cautionary note, we point to the results  that werFor this study, a 83-nt long labeled RNA molecule was produced by in vitro transcription and isolated by gel electrophoresis. Its sequence was:32P-CGCUGUACGCAACACAAGGCUUAUGGUGUAUCCUCCUGGAUCACGUGUGGUACGUACUGUCCGAUUAUUUCUAAUCGGGAUAC-3’. Data suggest that this RNA may fold into a rod-like, stem-loop structure including three bulges separated by four stems .5’-2O, once briefly, and once overnight. All fractions were read at the scintillation counter by Cherenkov counting. Percent adsorption was calculated dividing the amount of radioactivity remained in the pellet by the sum of radioactivity in all the washes, multiplied by 100.Double decomposition reactions to obtain synthetic minerals by precipitation were prepared by mixing 100 μL each of 100 mM solutions; these gave the synthetic minerals used to collect the data given in In another set of experiments, pellets were lyophilized and weighed prior to the addition of labeled RNA . Unfortunately, this approach was not successful for comparative purposes, due to two opposite effects. In some cases , the facility with which precipitate minerals redissolved in aqueous RNA solutions prevented any possibility for measurements. In other cases, the increased generic adsorption of aqueous solutions by dry surfaces allowed the powdered minerals to retain all the RNA added regardless of the interactions specific to the mineral (data not shown).We also collected data for adsorption on precipitated minerals formed at different starting pHs, with values taken before mixing the salts, after the precipitate is formed (pH of the supernatant), and after the RNA is adsorbed (pH of the supernatant) (data not shown). The observations did not alter the conclusions of this paper and were thus omitted.2O, 30% H2O2, ddH2O, EtOH 99%. Minerals were then air-dried for about 30 min in a sterile environment.All natural minerals used where from the Benner collection . Prior t2O droplets of increasing sizes (10 μL to 100 μL) were used to wash the area were the RNA was spotted, until no radioactivity could be detected in the washes. All fractions were then read at the scintillation counter, along with 2 μL of the radioactive RNA originally used. The amount of RNA adsorbed was calculated by subtracting the cpm in all the washes from the cpm of the original 2 μL. In the case of vanadinite, the mineral piece was small enough to fit directly into a scintillation vial, allowing the direct measurement of the radioactivity bound to the piece. This compared to the subtration method showed that the latter was accurate to within ±5%.For each mineral, droplets (2 μL) containing ≈100 fmol of radiolabeled RNA were spotted on the surface and let adsorb for 45 min at room temperature. Macro-surface areas of the droplets were obtained with the program ImageJ (NIH). Subsequently, H2CO3. Two of the carbonates were then combined into a 5 mL chromatographic column. In the second, the chloride salts of two competing metal species were mixed first (1 mL 1 M each) and then let react with 2 mL of 1 M Na2CO3; in this method, the two minerals co-precipitated, allowing the formation of ternary carbonates that contained two metals together .To obtain carbonate columns, either of two methods was used. In the first, each carbonate was prepared separately by mixing 1 mL of a 1 M aqueous solution of the chloride (x = Mg/Ca/Sr/Ba) with 1 mL of 1 M Na32P-labeled RNA in 1 mL of H2O was added to the mixture. The RNA was allowed to interact with the minerals by 40 min tumbling at room temperature (rt). After this time, each column was set upright in a undisturbed environment for about 15–20 hours to allow the different minerals with different densities to separate.With either method, after formation of a precipitate, ≈1 pmol of 5’-Autoradiography of the RNA in the columns was obtained by setting a phosphorimager screen (BioRad) tightly against the row of columns in their rack, with the aid of paper clips and weights, for 2 hours in a dark room. Screens were scanned with a Personal Molecular Imager (PMI) phosphorimager (BioRad) and analyzed with the software QuantityOne (BioRad).After removing the supernatant, carbonate columns were then quickly frozen in liquid nitrogen, extruded from the plastic container by tapping, set against a sterile ruler, and quickly sliced into 0.5–1 cm slices. These were finally passed into clean tubes and radioactive counts in each slide were read at the scintillation counter with the Cherenkov method.2O. In the staining experiments, samples were obtained by double decomposition reaction by mixing aqueous solutions of Na2CO3  and CaCl2 . Samples were pelleted, supernatants discharged, and Feigl’s stain (400 µL) was added with vortexing.Feigl’s stain  is a solThe mixtures were then incubated at room temperature, where development of gray color was monitored for up to 3 days. Samples that turned gray generally started developing color after about 20 minutes, while samples that were unstained (white) remained such for the duration of the monitoring period.2O, 30% H2O2, ddH2O, and then EtOH (99%) to remove all organic species. The specimens were then air-dried under cover for about 30 min.For these experiments, five small clusters of aragonite were obtained from an original crystal cluster with the use of a hammer. These were extensively washed with tap water, ddH°C in a sterile environment for 2 hours. In parallel, the same amounts of RNA were incubated at the same temperatures in 1.5 mL low-binding test tubes.Droplets (2 μL) containing ≈100 fmol of radiolabeled RNA were spotted on the surface of each crystal. The material was allowed to adsorb with liquid evaporation and by incubating the mineral pieces at 25, 37, 55, 75, or 95 2O to eliminate formic acid. The residue was then dissolved in 95% formamide gel loading buffer for denaturing PAGE analysis . Gels were dried for 30 min at 80 °C before being exposed to a phosphorimager screen for quantitative autoradiography.After incubation, RNA adsorbed to aragonite surfaces, or adhering to the tubes’ plastic, was released by washing the surfaces with 100 mM aqueous formic acid (100 µL); the released RNA was recovered in new tubes. These samples were subjected to three cycles of evaporation and resuspension in ddHIdentification of synthetic minerals was conducted with a power X-ray diffractometer equipped with a copper target . All diffraction profiles were obtained at a step size of 0.01°, with a divergence and receiving slit of 1° and 0.3 mm, respectively.


Defective interfering (DI) viruses are natural antivirals made by nearly all viruses. They have a highly deleted genome (thus being non-infectious) and interfere with the replication of genetically related infectious viruses. We have produced the first potential therapeutic DI virus for the clinic by cloning an influenza A DI RNA (1/244) which was derived naturally from genome segment 1. This is highly effective in vivo, and has unexpectedly broad-spectrum activity with two different modes of action: inhibiting influenza A viruses through RNA interference, and all other (interferon-sensitive) respiratory viruses through stimulating interferon type I.We have investigated the RNA inhibitory mechanism(s) of DI 1/244 RNA. Ablation of initiation codons does not diminish interference showing that no protein product is required for protection. Further analysis indicated that 1/244 DI RNA interferes by replacing the cognate full-length segment 1 RNA in progeny virions, while interfering with the expression of genome segment 1, its cognate RNA, and genome RNAs 2 and 3, but not genome RNA 6, a representative of the non-polymerase genes.Our data contradict the dogma that a DI RNA only interferes with expression from its cognate full-length segment. There is reciprocity as cloned segment 2 and 3 DI RNAs inhibited expression of RNAs from a segment 1 target. These data demonstrate an unexpected complexity in the mechanism of interference by this cloned therapeutic DI RNA. Viral replication frequently generates progeny genomes that contain extensive deletions –4. This cis-acting signals required for replication and packaging into virus particles [The influenza A virus genome comprises 8 segments of single-stranded negative-sense RNA (vRNA) in the form of ribonucleoprotein (RNP) complexes, and one copy of each of these segments is required to make an infectious virus particle . Influenarticles –13. DI Rarticles , 15.Influenza virus genome replication commences with synthesis of positive-sense (cRNA) copies of the vRNAs of the infecting virus, and in turn these are used as templates for synthesis of new vRNAs . vRNA isSynthesis of influenza virus RNAs is carried out by a virus-encoded RNA-dependent RNA polymerase present within a RNP complex consisting of the vRNA or cRNA in association with the virus NP protein and the heterotrimeric virus RNA polymerase comprised of the PB1, PB2 and PA proteins. The latter are encoded by vRNA segments 2, 1 and 3, respectively. Mutations in PB2 and PA can decouple transcription (mRNA synthesis) from replication (vRNA and cRNA synthesis), such that a mutant polymerase is defective in replication but not in transcription or vice versa , 21. AltLittle progress has been made towards understanding the mechanism of interference by DI viruses. For snapback/copyback vesicular stomatitis virus (VSV) DI RNAs that have a high degree of double-strandedness, but not VSV DI RNAs generated by central deletion, the key factor in interference seems to be the extent of terminal complementarity of the DI RNA such that a longer DI RNA with more extensive terminal complementarity out-competes a shorter DI RNA of the same structure –35. In aMost studies of DI influenza virus-mediated interference to date have been carried out with naturally occurring preparations, and are compromised by the presence of mixtures of several different defective RNA sequences , 41. ThiBpiI and Eco31I sites of pPolI-220 [The plasmids encoding the 8 gene segments of the A/WSN strain of A/WS/33 H1N1) and plasmids expressing the polymerase proteins and NP , and theN1 and plPolI-220  such thaPolI-220  by RT-PCPolI-220 . A furth2 flask. Total cellular RNA was extracted with 2 ml Trizol reagent per sample (Invitrogen) on days 1, 2 and 3 after co-culture while tissue culture fluid was collected for virus titration and RNA extraction. Virions were purified by ultracentrifugation. RNA was extracted with phenol/chloroform, and then ethanol precipitated.Human 293T cells (American Type Culture Collection) were transfected as previously described. Briefly, for northern blot analysis, a well of 70% confluent 293T cells in a 12-well plate was transfected using TransIT LT1 transfection reagent (Mirus) with 8 PolI expression plasmids encoding virion sense RNA, and cDNA plasmids for expression of PB2, PB1, PA and NP proteins, with or without pPolI-244. The transfected cells were then incubated at 37 °C overnight before co-culture with MDCK cells (obtained from the American Type Culture Collection) in a 25 cm2). This is ‘active DI virus’. Longer UV irradiation (8 min) inactivates the protective activity in mice, but does not affect haemagglutinin or neuraminidase activities, and so controls for any immune system-stimulating or receptor-blocking effects of 1/244 DI virus particles (‘inactivated DI virus’).For preparation of virus stocks containing DI 1/244 and 1/244 KO DI RNA, virus in tissue culture fluids produced by transfection was passaged once in embryonated chicken’s eggs and allantoic fluids were harvested to produce a stock of virus. The virus produced in embryonated chicken’s eggs is a mixture of 1/244 DI virus or 1/244 AUG KO DI virus packaged in A/WSN virion proteins and infectious helper A/WSN strain of A/WS/33 (H1N1) virus. These were purified by differential centrifugation through sucrose, and resuspended in PBS. Stocks were standardized according to their haemagglutination titre and stored in liquid nitrogen. The DI virus stock was UV-irradiated to remove helper virus infectivity using a short burst (40 s) of UV irradiation at 253.7 nm (0.64 mW/cmv/v) formaldehyde, washed and blocked with 5% (w/v) milk powder in PBS. The infected cells were probed with a monoclonal antibody specific for the HA of A/WSN in PBS containing 0.1% Tween 20. After washing, goat anti-mouse IgG–alkaline phosphatase conjugate (Sigma) in TBS containing 0.1% Tween 20 was added, and infected cells were detected with nitrotetrazolium blue chloride/BCIP (Sigma) in Tris-buffered magnesium chloride and sodium chloride . The infectivity titre was determined by counting at least 50 positively stained cells (foci) at an appropriate dilution in each of the triplicate wells. The mean number of counts was determined to give a titre in focus-forming units (f.f.u.) ml−1.MDCK cell monolayers in 96-well plates were infected with supernatant containing rescued A/WSN as described previously . After 150 or 1000 f.f.u.), active DI virus alone, a mixture of A/WSN + active DI virus, or A/WSN + inactivated DI virus. The amounts of DI 1/244 used was the same as used in previous studies of protection from disease and mice were subsequently monitored for clinical disease according to our standard protocol [50) to determine their immune status. All work with animals was governed by a licence approved by the UK Home Office and complied with national regulations. Animal work was approved by the University of Warwick Animal Welfare and Ethical Review Board as required by the Animals (Scientific Procedures) Act (1986) governing animal experimentation in the UK. Animals culled for humane reasons were subjected to an approved procedure to minimise suffering.To assess the degree of in vivo protection afforded by DI virus, groups of 5 C3H/He-mg mice were each inoculated intranasally under light ether anaesthesia with A/WSN alone  were added and further incubated at 55 °C for 1 h. The reaction was terminated by heating at 95 °C for 5 min with gel loading dye II (Ambion). The transcription products were resolved on a 6% (w/v) polyacrylamide gel containing 7 M urea in TBE buffer and detected by phosphor imaging. The primers used, and the expected product sizes, are shown in Table Primer extension analysis was carried out as previously described , using pTen μg of total cellular RNA or 50% of the yield of purified virion RNA from each sample was analysed by glyoxal-agarose gel electrophoresis. Poly A-containing mRNA was selected using a GenElute Direct mRNA preparation kit (Sigma) according to the manufacturer’s instructions. Non-polyadenylated RNA that did not bind to the column during mRNA preparation, and therefore contained the viral cRNA, was retained for further study. The RNA was transferred onto Hybond-N membrane  overnight using 20× SSC. The membrane was then baked at 80 °C for 2 h and hybridized with digoxigenin (DIG)-labelled probes overnight. The full-length positive-sense DIG-labelled segment 1, segment 2 and segment 7 probes were transcribed in vitro in the presence of DIG-UTP (Roche) from PCR products containing a T7 promoter. The Roche system with a digoxigenin-specific FAb antibody fragment conjugated to alkaline phosphatase and the chemiluminescent CSPD substrate was used for detection. Blots were exposed to Fuji X-ray film until the desired density was achieved and bands were quantified by densitometry using ImageJ (NIH).Human 293T cells were transfected with the segment 1-GFP RNA expressing plasmid, plasmids expressing PB1, PB2, PA and NP proteins, and increasing amounts of an additional PolI plasmid expressing a DI RNA  or a full-length RNA . At 2 days post-transfection, the cultures were examined for GFP expression. Each dataset was replicated several times and all data were included in the calculations. Digital images of the cell monolayers were taken by phase-contrast and epifluorescence microscopy. Five field fluorescence images were randomly selected and analysed for the proportion of the visualised area expressing GFP using the HCImage software (Hamamatsu). The visualisation detects cells expressing a range of GFP levels to include those that may have been transfected with different levels of the reporter plasmids. A mean was calculated to give the percentage of the GFP positive area per monolayer.It has been shown that a truncated form of the flu A PB2 protein is capable of inducing type I interferon and an antiviral state . DI 1/24The mRNA transcribed from 1/244 DI RNA contains the translation start codon of the PB2 open reading frame 1 (ORF-1). This has the potential to encode a protein comprising the first 41 amino acid residues of PB2 fused to 21 amino acid residues translated from a different reading frame generated as a result of the deletion, making a protein of 62 residues in total Fig. . This puRNAs were harvested 48 h after transfection of 293T cells with plasmid encoding either the 1/244 DI RNA or the 1/244 AUG KO DI RNA together with plasmids expressing the PB2, PB1, PA and NP proteins. Primer extension analysis, using a mixture of primers to detect both positive- and negative-sense RNAs simultaneously Table .In our experience all stocks of influenza virus contain some level of a complex population of DI viruses, which makes examination of the activity of any individual transfected DI RNA in a cell culture infection model technically impossible. In such experiments it is necessary to use a high level of infectious virus which results in a prominent signal from the naturally occurring DI RNAs that obscures the signal from the transfected plasmid expressing 1/244 DI RNA (unpublished data). We have therefore used an established in vitro RNP reconstitution approach –55 to exTo determine whether the in vitro interference is also reflected in an infection model, we compared the abilities of 1/244 DI and 1/244 AUG KO DI RNA to protect mice from disease that normally follows challenge with influenza A/WSN (H1N1). Virus stocks containing either 1/244 DI or 1/244 AUG KO DI RNA were produced using reverse genetics as before  and miceIt was reported previously that the interference mediated by a DI virus occured at the level of packaging , and we To distinguish between the effects of DI RNA on viral RNA synthesis and RNA packaging, GFP expression from the Seg 1-GFP construct was determined as described above. Human 293T cells were transfected with Seg 1-GFP and plasmids expressing PB1, PB2, PA and NP proteins, and GFP fluorescence determined 2 days post-transfection. In addition, increasing amounts of a PolI plasmid expressing either DI 1/244 or an alternative DI RNA . However, 0.5 μg and 1 μg of 1/244 reduced the levels of mRNA and cRNA synthesised from segment 1 by >99% while levels of vRNA remained at 14%. Thus 1/244 DI RNA has a profound effect on all RNAs synthesised from the segment 1 target, but differentially targets positive-sense RNAs. To control for the specificity of action of the inhibiting RNA, either a segment1 (encoding the intact PB2 gene) or a segment 6 (encoding the NA gene) plasmid was transfected in the place of the DI RNA plasmid. Fig. 2 = 0.90; data not shown), confirming that fluorescence was a faithful marker of mRNA synthesis.Quantitation of the primer extension products showed that mRNA and cRNA levels were considerably more reduced than was vRNA in the presence of 0.1 μg to 0.5 μg of 1/244 plasmid DNA Fig. , b. AddiTo determine if 1/244 DI RNA-mediated inhibition of RNA synthesis is segment specific, the segment 1 target (Seg1-GFP) was replaced with a plasmid directing the synthesis of either full-length segment 2, segment 3 or segment 6 vRNAs. Fig. p = 0.0002), and to 4% with 1 μg plasmid  and negative-sense (vRNA) RNAs are distinct processes , 21. DatThese data provide the first description of a novel mechanism for preferential production of flu DI RNA compared to RNA production from the cognate genome segment. As the amount of transfected 1/244 DNA was increased there was a proportionate decrease in the mRNA, cRNA and vRNA synthesised by the segment 1 target RNA Fig.  whereas,The reduction of mRNA levels by 1/244 DI RNA, indicated by the expression of GFP, was not observed when full-length segments 4 or 6 were used as the target, indicating that 1/244 DI RNA does not interfere with all genome segments and appears to act selectively on the synthesis of positive-sense RNA from segments 1, 2 and 3. Others have shown that segments 1, 2 and 3 direct the synthesis of considerably lower levels of mRNA relative to vRNA compared with other genome segments, and it has been suggested that segments 1–3 mRNAs are produced by primary transcription rather than transcription from newly synthesised vRNA , 75. ThuThe ability of influenza DI RNAs to supplant their cognate genome segment during the packaging process explains their amplification in virus preparations. However, data presented here also indicate that the widely held view that interference results solely from the ability of the DI RNA to be replicated faster than the longer, cognate full-length RNA, while possibly a contributory factor, need not always be the only mechanism involved. Rather, the 1/244 DI RNA specifically targets the synthesis of full-length genome segments 1, 2 and 3. Further DI RNAs derived from segments 2 and 3 also inhibit RNA synthesis from full-length segment 1. Inhibition primarily affects segments 1–3 as RNA synthesis directed by segment 6 is inhibited to a significantly lesser degree. Taken together, the data suggest that interference depends upon a direct association between each DI RNA and segment 1. Since all virion and DI RNAs form RNP complexes with NP protein, we suggest that specific interactions reside in the DI RNA itself through an element, which might be a sequence, a motif comprising non-contiguous sequences, or a structural motif. This may be analogous to the association of genome segments through direct RNA: RNA interactions that have been described as a component of virus packaging , 78–83.The data presented here suggest there may be previously unknown interactions between full-length RNAs 1, 2 or 3 and their cognate DI RNAs, possibly mediated through yet to be identified recognition elements. These interactions appear to confer complex yet highly specific effects on viral transcription, and is therefore likely to be of evolutionary significance to the survival of the virus. This would suggest that DI RNAs are not a mere accident of replication, as frequently proposed, but may have evolved as a non-heritable means of negatively regulating virus infection. These findings take us closer to understanding the evolutionary significance of DI RNAs and their interactions with infectious virus, and nearer to understanding the action of antivirals based on DI RNAs.


It was first discovered in kinetoplastid protozoa followed by its reporting in a wide range of organisms. In plants, RNA editing occurs mostly by cytidine (C) to uridine (U) conversion in translated regions of organelle mRNAs and tends to modify affected codons restoring evolutionary conserved aminoacid residues. RNA editing has also been described in non-protein coding regions such as group II introns and structural RNAs. Despite its impact on organellar transcriptome and proteome complexity, current primary databases still do not provide a specific field for RNA editing events. To overcome these limitations, we developed REDIdb a specialized database for RNA editing modifications in plant organelles. Hereafter we describe its third release containing more than 26,000 events in a completely novel web interface to accommodate RNA editing in its genomics, biological and evolutionary context through whole genome maps and multiple sequence alignments. REDIdb is freely available at  RNA editing is an essential co/post transcriptional process able to expand transcriptome and proteome diversity in addition to alternative splicing. The term RNA editing was first introduced in 1986 to describe the addition and deletion of uridine nucleotides to and from mRNAs in trypanosome mitochondria  in inosine (I) in double RNA strands (dsRNAs) through the catalytic activity of the adenosine deaminase (ADAR) family of enzymes. To date, more than 4 million events have been collected and annotated in dedicated resources such as DARNED, RADAR, and REDIportal  to uridine (U) conversion particularly in translated regions of mRNAs, albeit the opposite event (U-to-C substitutions) has been observed in some taxa, especially in chloroplasts RNAs  and an appropriate field to unambiguously describe them is not provided. RNA editing modifications are often reported as misc_feature or even as simple exception notes. With the aim to overcome these limitations and create a cured catalog of plant RNA editing events, we developed the specialized REDIdb database. Its first release stored 9,964 modifications distributed over 706 different nucleotide sequences, increased to 11,897 in the following update.After 10 years of massively parallel sequencing, we present here REDIdb 3.0, an upgraded release that annotates 26,618 RNA editing events distributed among 281 organisms and 85 complete organellar genomes.All changes have been recovered from Genbank and literature using a semi-automated bioinformatics procedure in which each annotation has been manually checked to avoid redundancy or inconsistencies due to errors in flatfiles.The web-interface was totally restyled and developed using the latest computational technologies in the field of database querying and managing.Furthermore, many computational facilities have been integrated to improve the user experience and ensure continuous and future updates of the database. Indeed, REDIdb 3.0 accommodates RNA editing in its genomics, biological and evolutionary context through whole genome maps and multiple sequence alignments.Although a variety of RNA editing databases have been released such as DARNED  scripts. Each flatfile is screened for RNA editing features using the SeqIO parser included in the Biopython (1.68) module  to efficiently show large tables in html documents. Genome rendering, available for complete organellar genomes, has been developed in pure python, mimicking OGDraw graphics  module, a data access library to MySQL engine. The web interface, instead, is built in BootStrap (3.3.7), while data presentation is based on DataTables, an https://biojs.net/).Query results are dynamically generated using the CGI (common gateway interface) technology. Multiple sequence alignments of edited cDNAs and proteins have been generated by ClustalOmega  based on the powerful D3 JavaScript library for visualizing data using web standards (https://d3js.org/). Functional domains have been detected using InterPro engine . To preserve the full compatibility with previous database versions, accession numbers linked to old entries have been maintained unchanged.All REDIdb sequences including RNA editing events are identified by unique accession numbers , taking into account, as much as possible, eventual feedbacks from the users.CL conducted the bioinformatics analyses and wrote the first manuscript draft; EP and GP conceived the study and contributed to writing and revising the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


XIST RNA triggers the transformation of an active X chromosome into a condensed, inactive Barr body and therefore provides a unique window into transitions of higher-order chromosome architecture. Despite recent progress, how XIST RNA localizes and interacts with the X chromosome remains poorly understood. Genetic engineering of XIST into a trisomic autosome demonstrates remarkable capacity of XIST RNA to localize and comprehensively silence that autosome. Thus, XIST does not require X chromosome-specific sequences but operates on mechanisms available genome-wide. Prior results suggested XIST localization is controlled by attachment to the insoluble nuclear scaffold. Our recent work affirms that scaffold attachment factor A (SAF-A) is involved in anchoring XIST, but argues against the view that SAF-A provides a unimolecular bridge between RNA and the chromosome. Rather, we suggest that a complex meshwork of architectural proteins interact with XIST RNA. Parallel work studying the territory of actively transcribed chromosomes suggests that repeat-rich RNA ‘coats’ euchromatin and may impact chromosome architecture in a manner opposite of XIST. A model is discussed whereby RNA may not just recruit histone modifications, but more directly impact higher-order chromatin condensation via interaction with architectural proteins of the nucleus.This article is part of the themed issue ‘X-chromosome inactivation: a tribute to Mary Lyon’. The initial coating of the X-chromosome by XIST RNA is known to trigger a cascade of events that result in stable chromosome-wide silencing of transcription (a). The resulting inactive X chromosome (Xi) is depleted of RNA polymerase II, enriched with repressive histone and DNA modifications, and structurally reorganized, as reviewed in detail elsewhere . T. TXIST Ret al. [XIST RNA to Xi seems to occur only in certain transformed cell lines and pluripotent stem cells. While XIST localization in cancer cells and stem cells is clearly important, the majority of normal differentiated cells appear to have redundant or complex mechanisms to faithfully anchor XIST to Xi. Interestingly, irregular nuclear shape and compromised nuclear scaffolding has been observed in transformed and cancer cells [XIST is functionally compromised or mislocalized and the Barr body is not observable in some cancers [XIST RNA. Similarly, pluripotent stem cells, which also require SAF-A for XIST expression/localization, have less developed nuclear substructure and express very low levels of the nuclear matrix protein lamin A/C [The results from Kolpa et al.  demonstrer cells ,54. In f cancers ,56. Theramin A/C ,58. We sXIST RNA, are not restricted to the Xi, but are distributed widely over chromatin. Thus, it is plausible that this repertoire of scaffolding proteins could interact with other nuclear non-coding RNAs to modify chromatin architecture throughout the genome [XIST RNA exemplifies a broader paradigm for non-coding RNA function in nuclear organization.Finally, we note that SAF-A and other nuclear matrix proteins, including many reported to associate with e genome ,60. This5.in cis even after long-term inhibition of transcription and is unperturbed by high salt extraction and DNase digestion of chromatin (b), suggesting that like XIST, Cot-1 RNA is in tight association with a nuclear scaffold. Consistent with this idea, Cot-1 RNA signal is mislocalized in cells expressing dominant-negative mutants of SAF-A [Remarkably, abundant repeats including LINEs, SINEs, transposable elements and simple sequences, comprise over half the human genome, but their potential contribution to chromosome structure and regulation remains under studied. Rather than avoid analysis of repetitive sequences, our recent work intentionally targeted analysis of ‘junk’ that comprises the bulk of chromosomes. Using Cot-1 DNA  as a probe in RNA FISH experiments, we identified a remarkably abundant and stable association of repeat RNA with the interphase chromosome territory of active chromosomes in all human and mouse cell types tested . Cot-1 Rhromatin b, suggesof SAF-A . If and b), clearly indicating a preferential association with euchromatic regions. Cot-1 RNA is released from mitotic chromosomes (much like XIST) and resynthesized in G1 daughter cells after cell division. Inhibiting the resynthesis of these RNAs after cell division with transcriptional inhibitors prevents the reorganization and ‘opening up’ of nuclei. These, and other results suggest that Cot-1 RNA may be required to promote an active chromatin state. In support of this hypothesis, RNase treatment of nuclei is known to cause rapid ‘collapse’ of chromatin structure as judged by DAPI staining [XIST functions somehow to strip other RNAs or reorganize chromatin in a way that contributes to cytologically observable compaction of the Barr body.Cot-1 RNA, though broadly distributed in nuclei of all cell types tested, is conspicuously absent over constitutive and facultative heterochromatin  the retention of XIST RNA in a bright, localized nuclear territory even after removal of chromatin [XIST RNA localization in contrast to mild effects of SAF-A knockdown, consistent with dominant negative effects on other elements of the inter-connected scaffold [XIST RNA does not require chromosome-specific sequences, yet clearly and comprehensively recognizes the structure of the chromosome from which it is transcribed [This model does not preclude that hromatin , (ii) thhromatin , (iii) tscaffold , and  are widely distributed within mammalian nuclei, do these interact with other non-coding RNAs (e.g. repeat-rich RNAs), perhaps in a manner that differs from XIST RNA?Whatever the detailed mechanisms, it is intuitive that changes in condensation can be propagated across a large chromosome, and this likely involves repeating patterns of higher-order folding. Thus, interspersed repetitive sequences could well be involved in propagation of chromatin state. In addition, a recently recognized property of many RNA binding proteins suggests to us an additional concept of how they might propagate chromatin architecture. RNA binding proteins, including SAF-A, disproportionately contain low-complexity domains of polar, uncharged amino acids predicted to be ‘prion-like’ . Prion-l-binding , to locatructure . Since rtructure , it will7.XIST RNA described above strongly support the in vivo reality of some form of non-chromatin nuclear scaffold/matrix. However, since early studies reported that as much as 70% of nuclear RNA remains with the matrix fraction, a long-standing criticism has been that the extensive extraction procedures can precipitate RNAs on a proteinaceous residue. Hence, it will be important to use genomic approaches and nuclear fractionation techniques to establish whether RNAs isolated from the nuclear scaffold fraction represent a random precipitate or indeed a distinct population including a fundamentally important subset of long non-coding RNAs that are embedded with interphase chromosome structure.The model forwarded here is clearly influenced by the concept of an insoluble, non-chromatin nuclear scaffolding, also termed the nuclear matrix, which we recognize remains somewhat controversial or not fully established. However, this is a fundamentally important concept with profound implications for understanding epigenome regulation, since chromatin-associated RNA may act with nuclear scaffolding to modify chromatin architecture. We assert that properties of XIST, undoubtedly the principal paradigm for negative regulation of transcription by a non-coding RNA, details remain elusive or controversial; to our knowledge, no experiment or model system has definitively demonstrated the relative importance of the myriad of semi-redundant activities (a) downstream of XIST in repressing transcription. One of these mechanisms clearly seems to be recruitment of histone modifying enzymes. While control of individual gene transcription can be under ‘local’ control (e.g. histone modification at promoters), regulation of larger chromosomal regions may involve creation of nuclear domains, similar to the localization of inactive genes in the peripheral heterochromatic compartment [XIST RNA triggers (directly or indirectly) global architectural reorganization to form a silent nuclear compartment lacking Cot-1 RNAs and RNA polymerase II. Association of genes with this domain (directly abutting it or within the periphery) could contribute to their robust silencing, as we have discussed elsewhere [XIST, may modulate their surrounding environment to influence transcription through directly enacting architectural changes by embedding with and/or modifying RNA binding proteins in the nuclear scaffold that regulate the accessibility or utilization of nearby chromatin.Currently, the mechanisms of how chromosomal, or ‘architectural’, RNAs could then contribute to regulation of gene expression are unclear. As described here and elsewhere, even for tivities a downstrpartment  or adjacpartment . XIST RNlsewhere . We spec


Streptomycetes are soil-dwelling Gram-positive bacteria that are best known as the major producers of antibiotics used in the pharmaceutical industry. The evolution of exceptionally powerful transporter systems in streptomycetes has enabled their adaptation to the complex soil environment.Streptomyces species examined possesses a rich repertoire of from 761-1258 transport proteins, accounting for 10.2 to 13.7 % of each respective proteome. These transporters can be divided into seven functional classes and 171 transporter families. Among them, the ATP-binding Cassette (ABC) superfamily and the Major Facilitator Superfamily (MFS) represent more than 40 % of all the transport proteins in Streptomyces. They play important roles in both nutrient uptake and substrate secretion, especially in the efflux of drugs and toxicants. The evolutionary flexibility across eleven Streptomyces species is seen in the lineage-specific distribution of transport proteins in two major protein translocation pathways: the general secretory (Sec) pathway and the twin-arginine translocation (Tat) pathway.Our comparative genomic analyses revealed that each of the eleven Streptomyces species. These expansive transport systems are important mediators of the complex processes including nutrient uptake, concentration balance of elements, efflux of drugs and toxins, and the timely and orderly secretion of proteins. A better understanding of transport systems will allow enhanced optimization of production processes for both pharmaceutical and industrial applications of Streptomyces, which are widely used in antibiotic production and heterologous expression of recombinant proteins.Our results present a catalog of transport systems in eleven The online version of this article (doi:10.1186/s12864-016-2899-4) contains supplementary material, which is available to authorized users. Streptomyces is a group of soil-dwelling Gram-positive bacteria, which are well known for their ability to produce a broad array of secondary metabolites including antibiotics, antifungals, antiparasitic drugs, anticancer agents, immunosuppressants, and herbicides [Streptomyces coelicolor, there are more than 600 predicted transport proteins with a large proportion being the ATP-binding Cassette (ABC) and Major Facilitator Superfamily (MFS) transporters, which have been implicated in the transport of secondary metabolites including antibiotics [rbicides , 2. Theyrbicides , 4. In orbicides , 6. For ibiotics . In addiibiotics –10. ThesStreptomyces.Transporters are of critical importance to all living organisms in facilitating metabolism, intercellular communication, biological synthesis and reproduction. They are involved in the uptake of nutrients from the environment, the secretion of metabolites, the efflux of drugs and toxins, the maintenance of ion concentration gradient across membranes, the secretion of macromolecules, such as sugars, lipids, proteins and nucleic acids, signaling molecules, the translocation of membrane proteins, and so on . A TransStreptomyces species enables comprehensive analysis of the transport protein families in Streptomyces. In this study, we report a catalog and comparative genomic analysis of transporters in eleven Streptomyces species with complete genome sequences and annotations, including S. coelicolor (SCO), S. avermitilis (SAV), S. bingchenggensis (SBI), S. cattleya (SCAT), S. flavogriseus (SFLA), S. griseus (SGR), S. hygroscopicus (SHJG), S. scabiei (SCAB), S. sp. SirexAA-E (SACTE), S. venezuelae (SVEN) and S. violaceusniger (STRVI) [Streptomyces transporters, using the nomenclature in the TCDB. The class, transmembrane topology and substrate specificity of these transporters are investigated in detail. An improved understanding of Streptomyces transporters will bring new insights into the mechanisms underlying the unique and powerful secretion systems of secondary metabolites and proteins in this group of bacteria of enormous economic and biomedical significance.The availability of genomes from closely related  (STRVI) , 18–24. Streptomyces genomes to query the TCDB [S. bingchenggensis, which has the largest genome, and the largest number of protein-coding genes, has the largest number of transporters, whereas S. cattleya contains only 761 transporters, the lowest number and proportion of transporters among the eleven Streptomyces species.Strong material intake and secretion capacity powered by transport systems is an adaptive attribute of soil-dwelling bacteria . We usedthe TCDB , 25 usinStreptomyces transporters to identify the transmembrane segments (TMSs). The number of TMSs ranges from 0 to 24. The largest number of TMSs observed in a transporter in the eleven Streptomyces genomes varies from 16 to 24 , most of which are also ABC transporters. These topology patterns suggest that during the evolution of transporters in S. griseus, the “6 + 3” events may be more frequent than the typical “6 + 6” events observed in ten other Streptomyces species [The capacity of a transporter is often associated with the complexity and topology of its transmembrane region(s) where the major events of substrate uptake or output across the cell membranes take place. Using the TMHMM (TransMembrane prediction using Hidden Markov Models) algorithm , we perf species , 28.TablStreptomyces transporters fall into seven classes and 171 transporter families according to the TCDB system . This class of transporters plays important roles in various aspects of bacterial life cycle, especially in the import and export of secondary metabolites, and cation transportation.The Primary Active Transporters (Class 3) is the most abundant class of transporters in Streptomyces. 212-330 transporters in eleven Streptomyces genomes belong to this class, which account for 24.4 %-31.4 % of all the transporters. The porters in this class include uniporters, symporters and antiporters. The most abundant family, MFS, in Class 2 transporters has been implicated in drug efflux. Lineage-specificity is also observed in this class of transporters. For example, S. bingchenggensis possesses two Ion-gradient-driven Energizers (TC 2.C), while the other ten Streptomyces species only have Porters  (TC 2.A).Class 2 transporters, the electrochemical potential-driven transporters, are also widely found in Streptomyces. 22-34 channel/pore transporters are present in these eleven genomes, accounting for 2.3 %-3.2 % of all the transporters. The majority of these channel-type proteins are alpha-type channels (TC 1.A), which have been implicated in stress responses of Gram-positive bacteria, especially responses to osmotic pressure [Streptomyces; only S. bingchenggensis has a transport protein from this subclass.Class 1 transporters are not abundant, but are functionally important for pressure . A smallS. cattleya, which has the smallest repertoire of transporters among the eleven Streptomyces, does not seem to contain any Glc transporters; it remains unknown if it uses an alternative system. Only 12-21 members of the Class 4 transporters, the Transmembrane Electron Carriers, are found in Streptomyces. Two subclasses are present, including the Prokaryotic Molybdopterin-containing Oxidoreductase (PMO) family (TC 5.A.3) and the Prokaryotic Succinate Dehydrogenase (SDH) family (TC 5.A.4), which transfer electrons mainly by redox reactions. Class 8, the Accessory Factors Involved in Transport, is the least abundant transporter class (0.4 %-0.7 %) in Streptomyces.Classes 4, 5, and 8 are relatively less abundant. About 3.0 %-5.3 % of all the transport proteins are Class 4 transporters. Two major subclasses observed in Class 4 are the PTS Glucose-Glucoside (Glc) family (4.A.1) and the Fatty Acid Transporter (FAT) family (4.C.1), which are responsible for the transport of glucoses-glucosides and fatty acids, respectively. Notably, Streptomyces can be grouped into Class 9, an incompletely characterized class. While their exact physiological roles are yet to be elucidated, they might be involved in the transport of ions, implicated by their sequence similarities with the members of the HlyC/CorC (HCC) family (TC 9.A.40), and the Tripartite Zn2+ Transporter (TZT) family (TC 9.B.10).A significant number (60-75) of transporters in Streptomyces [Many of the 171 transporter families are involved in the transfer of ions, saccharides, amino acids, polypeptides, proteins, drugs, toxins and other compounds. The two most abundant and perhaps also the most important families are in the ABC (TC 3.A.1) and MFS (TC 2.A.1) superfamilies. They are responsible for the secretion of a wide array of antibiotics in ptomyces , 30.Streptomyces genomes are members of ABC superfamily. ABC transporters are characterized by a conserved ATP hydrolyzing domain for energy provision, pore-forming membrane-integrated domain(s), and a substrate-binding domain [32.7 %-47.5 % (249-597) of all the transport proteins in the eleven g domain , 32. TheStreptomyces genomes are specialized in the uptake of diverse nutrient substances. This intake system includes families of Carbohydrate Uptake Transporters  that transport saccharides, Polar Amino Acid Uptake Transporters and Hydrophobic Amino Acid Uptake Transporters  that transfer amino acids, Polyamine/Opine/ Phosphonate Uptake Transporters and Quaternary Amine Uptake Transporters  that transfer amine substances, Iron Chelate Uptake Transporters and Manganese/Zinc/Iron Chelate Uptake Transporters  that transfer metal ions.The 30 intake families (TC 3.A.1-3.A.33) that we identified in the Streptomyces efflux families are involved in the transport of macromolecular substances. These transporters are believed to be essential for Streptomyces due to their roles in drug efflux and protein secretion. The drug efflux system regulates various aspects of the response to drug compounds mediated by Drug Exporters , Drug Resistance ATPases , Macrolide Exporters (TC 3.A.1.122), β-Exotoxin I Exporters (TC 3.A.1.126), Multidrug Resistance Exporters (TC 3.A.201) and Pleiotropic Drug Resistance transporters (TC 3.A.1.205). Potent protein transport in Streptomyces is regulated by Protein/Peptide Exporters , Lipoprotein Translocases (TC 3.A.1.125), AmfS Peptide Exporters (TC 3.A.1.127), and SkfA Peptide Exporters (TC 3.A.1.128).Unlike the intake system, the 35 Streptomyces genomes. Streptomyces possesses 39 subfamilies of MFS transporters, including 20 intake systems, 13 efflux systems and 6 systems whose transport direction is unknown. The substances transported by the intake systems are mainly saccharides and organic acids.Unlike the ABC transporters, the MFS transporters are driven by an electrochemical potential formed by ion concentration gradients across the cytomembrane . There aStreptomyces, with varying mechanisms of action, including Drug:H+ Antiporters , Aromatic Compound/Drug Exporters (TC 2.A.1.32), Fosmidomycin Resistance transporters (TC 2.A.1.35), Acriflavin-sensitivity transporters (TC 2.A.1.36), and Microcin C51 Immunity Proteins (TC 2.A.1.61), to name a few.One of the most important roles of the MFS transporters is drug efflux . DiverseStreptomyces is evidenced by the broad scope of the substrates being transported. Figure Streptomyces, including carbon sources, drugs, toxicants, electrons, inorganic molecules, macromolecules, amino acids and derivatives, nucleotides and derivatives, vitamins, and accessory factors. The carbon source transporters are the most abundant, with their proportion of all the transport proteins ranging from 21.7 to 31.6 % in eleven genomes. Notably, the substrates of an average of 6.4 % of the transporters in Streptomyces genomes examined cannot be determined based on genomic analysis, and await advanced structural and biochemical characterization.The capacity of the complex and powerful transporter system in Streptomyces transporters can be divided into three classes, uptake, efflux and bidirectional, according to the direction of the substrates transported  and the Sec system (TC 3.A.5) [S. scabies, the transporters in the Tat pathway secrete several toxicity-associated proteins [Streptomyces genomes we looked at, lineage-specificity is clearly shown with respect to the copy number variation of these genes  , 9. The C 3.A.5) . In S. sproteins . While t TC 2.A.6 and the  TC 2.A.6 and the tA3 Fig. . The tatStreptomyces . The transporter classification and amino acid sequences of all classified transporters were downloaded from the TCDB database (http://www.tcdb.org/) [http://www.membranetransport.org/) which included the transporter classification data of S. coelicolor and S. avermitilis, and from the Transporter Inference Parser database [http://biocyc.org/), which identified transporter according to their function annotation and included the relevant data of S. coelicolor, S. avermitilis, S. griseus and S. scabies.The completed whole genome data of the eleven es Table , includidb.org/) . We alsodb.org/)   (http://pfam.xfam.org/) [Streptomyces transporters, with Pfam GA as the threshold. TMHMM (http://www.cbs.dtu.dk/services/TMHMM/) [Streptomyces transporters.The BLASTP search of all the proteins in eleven the TCDB , 25. Theam.org/)  was used/TMHMM/)  was usedStreptomyces transporters into families and subfamilies of homologous transporters according to the TC system [Streptomyces transporters were classified at the transporter family level. The transporters in superfamilies such as ABC and MFS were classified at the subfamily level.On the basis of the degree of similarities with known or predicted transporters in the TCDB, as well as the conserved domains and the number and location of TMSs, we further classified the C system . The TC C system , 12. MosStreptomyces transporter was predicted based on homology to functionally characterized transporters in the TCDB. Classification of a putative transporter into a family or subfamily according to the TC system allows for the prediction of substrate types and transport direction with confidence [The substrate and transport direction of each nfidence , 17, 41.Multiple sequence alignments were obtained using Clustal X 2.1 . Phyloge


Fine particulate matter (PM2.5) exposure, especially to its organic components, induces adverse health effects on the respiratory system. However, the molecular mechanisms have still not been fully elucidated. Long non-coding RNA (lncRNA) is involved in various physio-pathological processes. In this study, the roles of lncRNA were investigated to reveal the toxicology of PM2.5. Organic extracts of PM2.5 from Nanjing and Shanghai cities were adopted to treat human bronchial epithelial cell lines (BEAS-2B and A549). RNA sequencing showed that the lncRNA functioned as antisense RNA, intergenic RNA and pre-miRNA. The mRNA profiles were also altered after exposure. PM2.5 from Nanjing showed a more serious impact than that from Shanghai. In detail, higher expression of n405968 was positively related to the elevated mRNA levels of inflammatory factors (IL-6 and IL-8). Increasing levels of metastasis associated lung adenocarcinoma transcript 1 (MALAT1) were positively associated with the induced epithelial-mesenchymal transition (EMT) process. Similar response was observed between both cell lines. The higher content of polycyclic aromatic hydrocarbons (PAHs) is likely to contribute to higher toxicity of PM2.5 from Nanjing than that from Shanghai. Antagonism of aryl hydrocarbon receptor (AHR) or inhibition of CYP1A1 diminished the effects stimulated by PM2.5. Our results indicated that lncRNAs could be involved in the toxicology of PM2.5 through regulating the inflammation and EMT process. PM2.5 has adverse impacts on the respiratory system. Epidemiological studies have shown that PM2.5 is associated with abnormal lung function4, lung infection5, and lung cancer6. Long-term improvement of air quality was associated with better lung-function growth in children7. By contrast, toxicological data have been largely insufficient for understanding the molecular mechanisms. PM2.5 is composed of organic carbon (OC), elemental carbon (EC), water-soluble inorganic ions and metal elements. OC consists of various organic compounds, including PAHs, PCBs, phthalate esters, aldehydes, ketones, benzene and so on. Crustal elements  and toxic heavy metals  are both present in PM2.5. water-soluble inorganic ions contains NO3−, SO42−, NH4+, Cl−. The constituents of PM2.5 are regional and time specific10. The components of PM2.5 largely affected its toxic outcome11. The particle contributed to the toxicityin a way which is similar to that of nanomaterials13. In addition, organic chemicals adsorbed on the surface possess appreciable toxicity15.Serious air pollution, especially fine particulate matter , is a major health concern16. Growing numbers of lncRNAs are being continuously discovered, and they play important roles in cancers and other diseases19. The expression levels of MALAT1 (metastasis associated lung adenocarcinoma transcript 1) and AFAP1-AS1 (actin filament associated protein 1 antisense RNA 1) were both significantly up-regulated, whereas TUG1 (taurine-upregulated gene 1) and HMlincRNA 717 were both down-regulated in non-small cell lung cancer (NSCLC)20. The usefulness of lncRNA as a non-invasive marker is expected in lung disease21.Long noncoding RNA (lncRNA), which has more than 200 nucleotides in length, is commonly involved in physiological and pathological process23. Bisphenol-A and diethylstilbestrol could alter the expression of HOTAIR in vitro and in vivo24. LncRNA could be a potential indicator of the toxicity induced by chemical exposure25.LncRNA functions in response to environmental stimuli. MALAT1 and HOTAIR were both involved in the epithelial-mesenchymal transition (EMT) induced by cigarette smoke extract28. Differentially expressed lncRNAs were co-analyzed with their corresponding mRNAs. The expressions and phenotypes of several lncRNA-mRNA pairs related to the pathology of lung cancer were further studied upon exposure. The mediating role of the AHR signaling pathway was shown in the study and was also evidenced by the presence of 16 priority polycyclic aromatic hydrocarbons (PAHs) in PM2.5.To the best of our knowledge, lncRNA has not yet been considered in toxicological studies of PM2.5. In this study, organic extracts of PM2.5 from Nanjing and Shanghai cities were both adopted to treat both BEAS-2B and A549 cells. These two cell lines have been widely used in toxicological studies of pollutants on the respiratory systemTo ensure that transcriptional changes induced by exposure were related to RNA expression rather than cell viability, the effect of PM2.5 on cell viability was first determined by MTT assay  were higher than those in Shanghai (15.695 ng/m3). Total BaP equivalent concentrations in Nanjing were 53% higher than those in Shanghai.The levels of sixteen priority PAHs were determined, and their total concentrations are presented in Table Cells were co-treated with Nanjing PM2.5 and AHR antagonist or CYP1A1 inhibitor Fig. . Elevate29.The roles of lncRNAs in the toxicity of chemicals have been receiving attention recently. In this study, we detected the responses of lncRNAs to organic extracts of PM2.5 exposure in human bronchial epithelial cells. The lncRNA landscape in bronchial epithelial cells was obtained by RNA sequencing. LncRNA was widely involved in the toxicity of PM2.5. Previous studies of the toxicology of PM2.5 were all conducted at the level of DNA, mRNA, miRNA or protein. Our results provided a new layer of lncRNA to facilitate the understanding of PM2.5 toxicology. Differentially expressed lncRNAs were widely detected following exposure to PM2.5 from either city. Additionally, abnormal expression of lncRNA also occurred at the clinical level. Dysregulated lncRNA was widely observed in patients with lung diseases30. In our study, the levels of IL-6 and IL-8 both increased in BEAS-2B cells after PM2.5 exposure. PM2.5 exposure also triggered the release of IL-8 in A549 cells. However, the level of IL-6 stayed at low levels after exposure. Regulation of n405968 might be one of the ways by which PM2.5 triggers the inflammatory response in BEAS-2B cells rather than in A549 cells. MALAT1  is involved in the regulation of cell cycle, cell migration, and cancer metastasis22. In our study, elevated levels of MALAT1 were positively associated with EMT processes, as indicated by the down-regulation of E-cadherin, up-regulation of N-cadherin, as well as the invasive ability of cells. PM2.5 likely triggers the EMT process by enhancing the expression of MALAT1. AFAP1-AS1 and its sense transcript AFAP1 showed reverse transcript patterns upon NJ PM2.5 exposure in BEAS-2B cells. LncRNA could function as an antisense transcript of the coding genes. In our study, AFAP1-AS1 and its antisense transcript AFAP1 showed reverse expression patterns upon PM2.5 exposure. A large amount of antisense lncRNA was predicted in our study. LncRNA (n345347) is the antisense to HOXB. HOXB acts as the transcription factor, and it is involved in lung development and related diseases, such as small cell lung cancers and bronchopulmonary sequestration32.In our study, two types of lung epithelial cells (BEAS-2B and A549) were adopted to determine the effects of organic components of PM2.5 on lung cells. PM2.5 has no significant toxicity to the viability of both cells at lower dose of 5 μg/mL. Inflammation and EMT are both involved in the etiology of lung cancer. LncRNA n405968 is the host gene of microRNA-155 (MIR155), which participates in inflammation34. PM2.5 from Nanjing is predominantly from coal combustion whereas the major source is diesel and gasoline exhaust in Shanghai . Concentrations of total PAHs and the corresponding benzopyrene (BaP) equivalences were higher in Nanjing than in Shanghai. Although the content in PM is very small, PAHs were regarded as the primary toxic constituents of PM and contributed to a higher risk of lung cancer37. In accordance, more differentially expressed lncRNAs were detected upon exposure to PM2.5 from Nanjing than from Shanghai. Levels of secreted inflammatory factors and their invasion ability were higher after treatment with PM2.5 from Nanjing than that from Shanghai. PAHs functioned through AHR receptor, which is an important mediator of the toxicity of organic chemicals. In this study, we found that this receptor-mediated effect also existed in the regulation of lncRNA expressions by PM2.5. Antagonism of AHR or inhibition of CYP1A1 obviously diminished the influence of PM2.5 on lncRNA-mRNA expression pairs. AHR mediated the carcinogenic effect stimulated by pollutants38.Diverse sources of PM2.5 differ in their constituents and health outcomesIn conclusion, our study provided new information on the toxic mechanism by which PM2.5 exerted its adverse health impacts through affecting the expressions of lncRNAs. LncRNA was involved in the inflammatory reaction and in the EMT process of lung epithelial cells. The AHR-CYP1A1 pathway mediated the effects of PAH-bound PM2.5 on lncRNA. This molecule could be a valuable target for revealing the toxicological mechanism of PM2.5.3/min by a large-size volume active sampler . The average mass of PM2.5 in each membrane was 135.5 ± 37.6 mg and 121.2 ± 33.9 mg for Nanjing and Shanghai, respectively. One eighth of each membrane was used for solvent extraction of organic components. The remainder was dedicated to chemical analysis. For each city, samples from eight days were randomly pooled together for extraction. The membrane was cut into pieces, and then soxhlet extracted for 24 h in dichloromethane. The extract was dried under nitrogen sweeping. Then, the extracts were re-suspended at a stock concentration of 50 mg/mL in dimethyl sulfoxide (DMSO) to facilitate dissolution in cell media.PM2.5 was collected from Nanjing city  and Shanghai city  in the Yangtze River Delta region of China in Autumn 2014. The sampling period was on weekdays from November 6th to 17th. The climate parameters were as follows: average temperature  and barometric pressure . PM2.5 was captured on quartz filters  at a flow rate of 1.05 m2 humidified chamber at 37 °C. The human lung adenocarcinoma A549 cells (the Cell Bank of Type Culture Collection of Chinese Academy of Sciences) were maintained in DME/F12 (1:1) medium  supplemented with 10% FBS  under the same condition. Both cells were exposed to organic extract of PM2.5 from both cities at the designated doses. The control group received the same extract from a null membrane. The dose of organic extract (5 μg/mL) equaled 10–20 μg/m3 of total PM2.5. These PM2.5 concentrations are in the regimen of the World Health Organization (WHO) annual standard39. In addition, this dose did not induce significant cellular toxicity as measured by MTT assay  was obtained from the China Center for Type Culture Collection (CCTCC). The cells were cultured in DMEM (Dulbecco’s modified Eagle’s medium)  supplemented with 10% FBS in a 5% COvs the control group.Cell viability was determined by MTT  colorimetric assay. The cells were seeded in 96-well plates at a density of 6,000 per well. The serial exposure concentrations were 0, 5, 10, 25, 50, 75 and 100 μg/mL. After an exposure period of 48 h, MTT was added, and the optical density (OD) was measured at 550 nm by a SpectraMAX M5 microplate reader . Cell viability was calculated as the relative value of the exposed group Total RNA was obtained from cells by an RNA extraction kit , according to the manufacturer’s instructions. RNA quality and quantity were determined on a nanophotometer . Then, equal amounts from each of the four replicates were pooled together for RNA sequencing.Cufflinks40. Cuffcompare was utilized to distinguish known and novel lncRNA transcripts, based on UCSC hg19 and NONCODE V3.041. RNA expression levels were compared between different groups by Cuffdiff. RNAplex was adopted to predict antisense lncRNA-mRNA duplexes42. Pre-miRNA was predicted by miRBase43. The lncRNA family was predicted by the Rfam database44.The noncoding RNA and mRNA were enriched from total RNA of BEAS-2B cells by removing rRNA. Then, they were fragmented into short fragments (approximately 200–500 nt). The fragments were used to synthesize first-strand cDNA by a random hexamer primer. PCR amplification was then performed to construct a library. The library was sequenced using the Illumina HiSeqTM 2000 at the Beijing Genomics Institute. Raw sequencing data from the control, Nanjing and Shanghai PM2.5-exposed groups were assembled and predicted independently. Then, all of the data were merged together to remove the redundant transcripts and to optimize the structure of the transcripts. Transcript assemblies were generated using TM kit . The second PCR reaction was performed as follows: the thermal cycle of an initial denaturation step at 95 °C for 30 s, then 40 cycles at 95 °C for 5 s and 60 °C for 20 s on a Roche 480 instrument. A dissociation step was adopted to confirm the correct amplification of the target gene. Three replicates of each PCR reaction were performed for each tested gene. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used to normalize the expression levels of target genes. The relative expression levels of the tested genes were analyzed using the 2−ΔΔCt method45. The primer sequences are listed in supplementary information 1 Table First strand cDNA was synthesized from total RNA using the SYBR Premix Ex TaqThe culture medium was collected for ELISA after treatment of cells with PM2.5. Concentrations of both interleukin-6 (IL-6) and IL-8 were measured by ELISA following the manufacturer’s instructions .Transwell assay was used to examine the cell invasion using a Matrigel invasion chamber with polycarbonate membranes  coated with Matrigel (BD Biosciences). The invaded cells were fixed, stained, and counted by averaging ten fields under an Olympus CKX41 inverted microscope .46. The total BaP equivalent concentration was obtained by totaling the equivalence values of individual PAHs. The individual equivalence was calculated by multiplying the concentration by its BaP coefficient, according to the previous study47.The concentrations of sixteen priority PAHs were determined in PM2.5 by GC-MS/MS following our previous studysupplementary information 1supplementary information 2