Escherichia coli (E. coli) 16S rRNA at 10 fM, which corresponds to ~100–1000 colony forming units/mL (CFU/mL) depending on cellular rRNA levels. The development of a rapid, sensitive, and cost-effective nucleic acid detection platform is sought for the detection of pathogenic microbes in food, water and body fluids. Since 16S rRNA sequences are species specific and are present at high copy number in viable cells, these nucleic acids offer an attractive target for microbial pathogen detection schemes. Here, target 16S rRNA of E. coli at 10 fM concentration was detected against a total RNA background using a conceptually simple approach based on electromechanical signal transduction, whereby a step change reduction in ionic current through a pore indicates blockage by an electrophoretically mobilized bead-peptide nucleic acid probe conjugate hybridized to target nucleic acid. We investigated the concentration detection limit for bacterial species-specific 16S rRNA at 1 pM to 1 fM and found a limit of detection of 10 fM for our device, which is consistent with our previous finding with single-stranded DNA of similar length. In addition, no false positive responses were obtained with control RNA and no false negatives with target 16S rRNA present down to the limit of detection (LOD) of 10 fM. Thus, this detection scheme shows promise for integration into portable, low-cost systems for rapid detection of pathogenic microbes in food, water and body fluids.A PCR-free, optics-free device is used for the detection of  In recent years, demand for a binary  test also has become apparent for the rapid screening of food for pathogens such as shiga-toxin producing Escherichia coli (E. coli) or Listeria monocytogenes. Clearly, there is strong motivation for development of sensitive, robust and portable molecular diagnostic devices that can give rapid yes/no results for the presence of important bacterial pathogens in food, water and body fluids.Reportedly, 95% of deaths in the developing world are due to lack of access to proper diagnostics for infectious diseases . Even inEssentially, there exist two options for pathogen diagnosis by molecular means: immunoassays and detection of pathogen-specific nucleic acid (NA). However, many of the existing platforms for NA detection at sufficiently low concentration require amplification by PCR, fluorescent or enzymatic labels, and expensive instrumentation ,3. It isRecently, NA amplification-free electrochemical methods were demonstrated for the detection of specific RNA sequences in urine  and saliWhen detection schemes requiring NA amplification, optics, and reagents other than the capture probe are removed from consideration, the number of reported technologies is very small and includes cantilever-based methods and nanotube- or nanowire-based approaches  The cantDue to species-specific sequence variations, 16S ribosomal RNA (or the corresponding gene) commonly has been used as a pathogen-specific target in numerous detection schemes ,22,23; aE. coli strain, (ATCC 25922). This strain offers an attractive test system for our technology since its genome has been sequenced and studied thoroughly. Also, an optimal probe sequence complementary to its 16S rRNA has been extensively tested by other groups -3-ethylcarbodiimide (EDC) in MES buffer) and incubated at 50 °C for 45 min. Ten nanomoles of amine-functionalized PNA detection probes were added to the coupling buffer and incubated with the beads at 50 °C for two hours. mPEG-amine (100 mM) was added to the reaction mixture and incubated at 50 °C for one hour to cap most remaining carboxylic acid groups on the bead surfaces so as to reduce aggregation and nonspecific binding of nucleic acids to the beads. Subsequently, 100 mM ethanolamine was added to the bead suspension to cap residual carboxyl groups, and the preparation was incubated at 50 °C for an additional hour. The same procedure was repeated for coupling the universal PNA probes to the beads. Finally, the beads were washed four times in 0.4 × SSC buffer  and stored in PBS buffer  at 4 °C. Prior to hybridization, the zeta potentials of PNA-bead batches were measured in 1 mM KCl, 10 mM HEPES (pH 7.0) to confirm the near charge neutrality of the beads. The zeta potential after four washes for beads conjugated with E. coli-specific PNA and capped with PEG and ethanolamine was −3.75 mV, and −3.27 mV for beads coupled with the universal PNA probes. At this low zeta potential, the beads tended to aggregate despite the PEG capping, which prevented meaningful diameter measurement by dynamic light scattering.Fifty microliters of 3-µm-diameter, carboxylic acid-functionalized polystyrene microspheres at 1.69 × 10E. coli (ATCC 25922). Nutrient broth medium  was prepared for culturing Pseudomonas putida (ATCC 12633) and Pseudomonas fluorescens (ATCC 13525). After preparing the ATCC culture media, they were steam sterilized at 121 °C for 20 min. Bacterial culture plates for colony counting were prepared according to standard procedures (ATCC).Tryptic soy medium  was prepared for culturing E. coli (ATCC 25922) with 1 mL culture tryptic soy medium, diluting that into 10 mL culture medium followed by incubation at 37 °C and 240 rpm until log phase was reached. Then, 1 mL of this starter culture was diluted into 250 mL culture medium and incubated at 37 °C and 240 rpm until log phase. To assess log phase, the optical density was measured at a wavelength of 600 nm  at 30 min intervals, and the culture was removed from the incubator at an optical density of 0.4–0.6. Similar preparative steps were repeated for the two control Pseudomonas strains in nutrient broth medium at 26 °C and 240 rpm. Log phase cells from the three different bacterial cultures were diluted serially into sterile saline, plated and incubated overnight in preparation for later colony counts. Simultaneously, 1 mL of original culture was prepared for RNA extraction by centrifugation at 25,000× g for 15 min at 4 °C. After removing the supernatant, cell pellets were immediately processed for total RNA extraction as described below.A starter culture was prepared by mixing lyophilized Total RNA extraction and purification was performed using the PureLink RNA Mini Kit and PureLink DNase Set, and the total extracted RNA was eluted in 100 μL RNase-free purified water. After RNA extraction from all bacterial dilutions, 5 µL of total RNA from each sample was measured for concentration and integrity using an Agilent 2100 Bioanalyzer.6 PNA-beads were washed twice with wash buffer  Tween 20, pH 10) and once with hybridization buffer . Total RNA extracts from target and control bacteria were diluted serially to achieve the desired concentrations for device testing. RNA samples then were hybridized with PNA-beads functionalized with the target probe and the universal probe separately in 100 µL reaction volumes of hybridization buffer at 68 °C overnight.Prior to hybridization, 2.1 × 10After hybridization, samples were washed twice in wash buffer and resuspended in 100 µL of 1 mM KCl, 10 mM HEPES pH 7.0 prior to introduction into the device. Total RNA extracted from target and control bacteria incubated with the target PNA-beads and universal PNA-beads were injected into the large opening of the micropipette and a potential of 25 V was applied to direct the beads having bound NA to the micropipette tip, which serves at the “pore” in this incarnation of our system . More deE. coli and establish the limit of detection (LOD) of our detector in terms of the concentration of 16S rRNA. To assess the LOD of our system, extracted total RNA samples from approximately 109 CFU/mL were quantified and inspected using an Agilent Bioanalyzer 2100, serially diluted, and then introduced into our detector.The experiments were designed to investigate the accurate detection of the target E. coli was 364 ng/µL. The percentage of 16S rRNA was 18.2% of total RNA, thus the concentration of 16S rRNA was 66.3 ng/μL. Using an approximate molecular weight of 1500-base ssRNA , the molar concentration of 16S rRNA was calculated to be 137.8 nM. The Bioanalyzer measured the size of 16S rRNA to be 1494–1924 nucleotides. Also, it is essential to check the quality of the extracted RNA since RNA molecules are susceptible to degradation by RNAse present in the environment. The Bioanalyzer instrument measures the RNA integrity number based on evaluation of the 23S and 16S ribosomal ratios using an RNA integrity number algorithm. The integrity number of extracted total RNA from target E. coli was 9.2 (10 represents highest integrity and 0 represents lowest integrity), indicating that the extracted RNA was largely intact.The Bioanalyzer was used to measure RNA concentration, size, and integrity number using an on-chip gel electrophoresis technique. The concentration of total RNA extracted from the target Pseudomonas putida , the total extracted RNA concentration was 449 ng/µL and the percentage of 16S rRNA was 25.7%, giving a 16S rRNA concentration of 115.4 ng/μL. The size of the 16S rRNA was reported to be 1260–1914 nucleotides, and the RNA “integrity number” was 10. The calculated 16S rRNA concentration was 239 nM.The same measurements and calculations were repeated for RNA extracted from the two control bacteria. For the control, Pseudomonas fluorescens , was 261 ng/µL and the percentage of 16S rRNA was 24.5%, giving a 16S rRNA concentration of 64 ng/μL. The 16S rRNA size was 1294–1984 nucleotides and the RNA “integrity number” was 10. We calculated the 16S rRNA concentration to be 133 nM. The data reported by the gel chip electrophoresis analysis of extracted total RNA from target and control bacteria is shown in the The total extracted RNA concentration of the other control, 6 target and universal PNA-beads in high stringency conditions to reduce non-specific binding and enhance selectivity. These high stringency hybridization conditions were carefully chosen based on a study by Li et al. in which the same conditions were used for quantitative Dechlorosoma suillum rRNA detection by PNA molecular beacon assays [Following the RNA analysis, extracted total RNA samples were serially diluted, based on their 16S rRNA concentrations, to concentrations between 10 pM and 1 fM by dilution factors of 10. Diluted samples were then each hybridized separately with 2.1 × 10n assays . After hP. putida rRNA at 10 pM. Notably, target 16S rRNA at 10 fM was also detected in three separate experiments with no false negatives, and the controls incurred no false positives at this concentration. However, in the case of 1 fM target 16S rRNA, no detection events were observed with the detection probe or the universal probe. We concluded that the concentration detection limit of our sensor for 16S rRNA is ~10 fM. This limit is consistent with previous work with our sensor where we found a 10 fM LOD for 1613-base ssDNA, which corresponds to ~100–1000 CFU/mL (depending on the growth rate of the bacterium at the time of harvest and the corresponding rRNA levels within the cells) [Target 16S rRNA at concentrations ranging from 10 pM to 100 fM was detected by the sensor with no false negatives. Also, only a few transient blocks (<60 s) were observed in control experiments that otherwise gave no false positives. e cells) ,25. In tFuture work will focus on 16S rRNA detection against a more realistic background of microbial nucleic acid. Work is also underway to scale-down the detector to the nano-regime in pursuit of still lower limits of detection.E. coli (ATCC 25922) 16S rRNA at 10 fM, which corresponds to ~100–1000 CFU/mL. The detector, as configured for E. coli 16S rRNA, did not give a signal in the presence of total RNA extracted from two control bacterial species, Pseudomonas fluorescens (ATCC 13525) and Pseudomonas putida (ATCC 12633) whose 16S rRNA genes are closely related to the target E. coli. To fully realize its potential, the detector would be integrated into a device containing a sample preparation frontend including cell lysis , and DNA separation , which have been shown to accomplish these tasks together in typically 10–15 min [Our previously described, PCR-free, optics-free detector for nucleic acids of specific sequence was used successfully to detect  ATCC 126 whose 160–15 min . Integra


Drosophila. We focus on how comparative genomics approaches have aided in the identification of sites that are likely to be advantageous. The use of RNA editing as a mechanism to adapt to varying environmental conditions will also be reviewed.Adenosine-to-inosine (A-to-I) RNA editing is an important post-transcriptional modification that affects the information encoded from DNA to RNA to protein. RNA editing can generate a multitude of transcript isoforms and can potentially be used to optimize protein function in response to varying conditions. In light of this and the fact that millions of editing sites have been identified in many different species, it is interesting to examine the extent to which these sites have evolved to be functionally important. In this review, we discuss results pertaining to the evolution of RNA editing, specifically in humans, cephalopods, and  Transcriptional modifications contribute to transcriptome diversity and flexibility, without the need to make hard-wired mutations at the DNA level. One important transcriptional modification is adenosine-to-inosine (A-to-I) RNA editing, performed by adenosine deaminases acting on RNA (ADAR), a family of enzymes that bind to double-stranded RNA (dsRNA) and deaminate adenosine to form inosine . InosineRNA editing is a very dynamic process, with editing levels varying from 0% to 100%, which can potentially be affected by environmental stimuli ,6 and diDrosophila only has 1 ADAR, with 2 dsRNA binding domains and 1 deaminase domain [Drosophila editing substrates as well as rescue many of the phenotypes of Drosophila ADAR mutants [Drosophila ADAR most closely represent mammalian ADAR2.ADAR proteins exist in all metazoans. A common feature among all ADAR proteins is at least 1 N-terminal dsRNA binding domain and a C-terminal deaminase domain. e domain . Its proe domain . A study mutants . Taken tAlthough most studies examining RNA editing evolution and adaptation have focused on editing sites, it is worth discussing evolutionary analyses of the ADAR proteins themselves. A study examining human and primate ADAR sequences found evidence of purifying selection for all 3 ADAR proteins in both humans and primates, with ADAR1 being less evolutionarily constrained than ADAR2 and ADAR3 . Howevertrans-regulators of RNA editing, which may be shared among these different organisms. In regard to the specific sites that are edited and how they may affect protein sequences, the proportions of sites that are recoding (nonsynonymous) and non-recoding (synonymous or in noncoding regions) can vary from species to species  in the regions around conserved editing sites, which is likely needed to form dsRNA structures that are important for editing to occur. The function of a few specific sites in potassium Kv2 channels was also investigated by injecting edited and unedited isoforms in Xenopus oocytes. Interestingly, for a site common to all 3 cephalopod species studied, the unedited form of the channel had different closing rates between the species, while the edited form showed similar closing rates for all the species [Given this exciting result, it is natural to wonder how many of the thousands of other editing sites in cephalopods are involved in adaptation. A recent study has shed some light on the conservation of editing sites across the genomes of these organisms and characterized this editing in more detail. By analyzing editing levels in cephalopods , as well as molluscs (nautilus and sea hare), the authors found extensive conservation of nonsynonymous editing sites across the different cephalopod species . In addi species . Considetrans-regulatory factors of RNA editing  [Drosophila, the overexpression of the unedited form of ADAR is lethal [Drosophila as well [The octopus Kv1.1 RNA editing event highlights another important advantage of RNA editing: unlike hard-coded SNPs, RNA editing can affect only a fraction of transcripts and thus facilitate fine-tuning . One exasophila) , thus als lethal . Editings lethal , editing as well –60. As tAlu sequences [Acropora millepora, a basal metazoan with one of the most primitive nervous systems, was recently discovered to have over 500,000 A-to-I editing sites, with the majority located in noncoding regions [Alu elements, which have high sequence similarity, may be functionally important in increasing transcriptome diversity (Alus tend to be in neuronal genes [Alus are neurologically functional and RNA editing plays a critical role in generating the high level of variation needed to produce a complex neurological system. Another functional role of editing may be the repression of harmful innate immune responses activated by Alu editing. The removal of RNA editing by ADAR1 leads to a harmful interferon induction caused by dsRNA sensor activation [Alus can form long dsRNAs, there may be a need to suppress this interferon induction when Alus are expressed. Editing Alu sequences could have additional advantageous functions in primate evolution, for instance by creating new splice sites for exonization, regulating editing in nearby coding sequences, and modifying miRNA binding and gene expression if they are located in 3’UTRs [Alu RNAs may keep them in the nucleus, which could prevent them from interfering with protein translation [Alu editing sites, out of millions, are functionally important and involved in adaptation.As stated previously, the majority of RNA editing events in humans and primates do not appear to be precisely targeted to recoding sites like in the GluA2 receptor transcript but rather occur in large batches in regions of equences . Interes regions . This su regions . Despiteiversity . More coal genes , so it itivation . Becausen 3’UTRs . Anothernslation ,67. FurtAlu regions and seems to be nonadaptive overall suggests that most editing is not functionally important, although the function of noncoding and Alu RNA editing is an open area of research. Editing seems to play more of an adaptive role in Drosophila and cephalopods, which have many more nonsynonymous editing sites compared with humans; additionally, many of these sites show evolutionary constraint. Although some of these sites—mainly nonsynonymous sites in ion channels—have been shown to affect the biophysical properties of these proteins, it is unclear whether these editing levels change as an adaptive response. In cephalopods, we do have an example of a temperature-adaptive editing site [With the identification of thousands of new editing sites in many different species, we can begin to determine the functional importance of editing, whether editing plays a role in adaptation, and how editing might differ between these species. The overall neurological role of editing appears conserved because studies in a variety of species have found that at least some neuronal nonsynonymous editing events are likely important. However, the extent of this varies vastly across different species. In humans, a few nonsynonymous editing sites do affect protein function. However, the fact that most editing is in noncoding ing site . With exThe results discussed in this review provide valuable insights into the evolution and adaptation of RNA editing, but some details still need to be filled in. For example, which sites not only alter their levels in response to environmental conditions but serve a functional purpose in doing so? In other words, how many other sites are similar to the adaptive site in the octopus Kv1.1 channel, both in the octopus and other animals? Though efforts to answer this question have been described, it is perhaps most beneficial to examine how editing levels differ in organisms from diverse environments. It may be especially helpful to examine other poikilothermic species because their body temperatures are associated with the temperature of their surroundings. Environmental variables besides temperature may regulate editing as well. If the editing events that do change in response to the environment are in conserved or constrained regions—or are predicted to affect protein levels or function—then their functions could be examined in further experiments. Although there are various ways to test this, the development of the gene editing CRISPR/Cas9 system has provided a convenient way to mutate editing sites in vivo so that these sites are fully edited or unedited. If organisms with different editing levels at a particular site show some phenotypic difference, this suggests that the editing site is functionally important. Another area that would be interesting to investigate further is the regulatory mechanisms underlying adaptive RNA editing sites. As mentioned earlier, while genetics and environment are involved with modulating editing levels, likely through altering RNA structure, their role in RNA editing adaptation is not completely clear; it is possible that these mechanisms differ for various organisms and environments. One intriguing scenario in which both factors are involved is that when an organism is faced with a new environment, such as a change in temperature, a change in editing occurs as an initial acclimatization response. However, if that change in editing proves to be beneficial for the organism, a genetic mutation could potentially make that change in editing “permanent,” thus leading to adaptation. Measuring the editing levels of these adaptive sites at various temperatures and in organisms of different genetic backgrounds would provide greater insight into this area. With these results, the full extent to which RNA editing is involved in adaptation and the underlying mechanisms of this process can be determined.


We investigated the activity of the DLEs through functional assays in live zebrafish embryos. The lft1 DLE localizes fluorescently labeled RNA similarly to the ndr1/sqt DLE. Similar to the ndr1/sqt 3′UTR, the lft1 and lft2 3′UTRs are bound by the RNA-binding protein (RBP) and translational repressor, Y-box binding protein 1 (Ybx1), whereas deletions in the DLE abolish binding to Ybx1. Analysis of zebrafish ybx1 mutants shows that Ybx1 represses lefty1 translation in embryos. CRISPR/Cas9-mediated inactivation of human YBX1 also results in human NODAL translational de-repression, suggesting broader conservation of the DLE RNA element/Ybx1 RBP module in regulation of Nodal signaling. Our findings demonstrate translational co-regulation of components of a signaling pathway by an RNA element conserved in both sequence and structure and an RBP, revealing a ‘translational regulon’.In many organisms, transcriptional and post-transcriptional regulation of components of pathways or processes has been reported. However, to date, there are few reports of translational co-regulation of multiple components of a developmental signaling pathway. Here, we show that an RNA element which we previously identified as a dorsal localization element (DLE) in the 3′UTR of zebrafish  These pDrosophila form stem-loop structures: the bicoid localization element 1 (BLE1)  , and specific sequence motifs, to structural motifs, or combinations of sequence and structure motifs. AU-rich elements (AREs) are destabilizing sequences in short-lived RNAs. They are composed of repeats of an AUUUA pentamer in an AU context. More than 4000 human transcripts contain a putative ARE . The rec1 (BLE1) , the trarb mRNAs , the apiess mRNA , and thent (HLE) . The K10function ,10. Howefunction ,11. Elemfunction . More thfunction . Howeverfunction .nodal-related1/squint mRNA  contains an RNA element that directs maternal sqt RNA localization into one or two cells in four-cell stage embryos  3′UTR was made using PCR. All deletions were confirmed by sequencing.Mutations in the DLE were generated by site-directed mutagenesis in the context of a full-length sa42ybx1 fish were maintained at 28.5°C, and embryos obtained by natural mating using standard procedures in accordance with institutional animal care regulations at the University of Warwick. To block the function of maternal Ybx1, embryos from females homozygous for a temperature sensitive mutant allele (sa42ybx1) were incubated at 28.5°C until the second cell division, shifted to 23°C until the 1000-cell stage, and returned to 28.5°C until sphere or 30% epiboly stages  (Promega). Un-incorporated nucleotides were removed using a micro-bio-spin column (BioRad), and fluorescent RNAs were purified using phenol-chloroform and eluted in DEPC water. Quality of the RNAs was examined on a 1.5% agarose gel, and the labeling efficiency and concentration of the RNAs was determined using a Nanodrop UV spectrophotometer (Thermo Scientific).Plasmid constructs were linearized using Not1 enzyme (NEB) and purified. Subsequently, one μg aliquots of template were used in transcription reactions containing 2.5 μL of Alexa 488 fluorophore UTP (0.150 mM), SP6 polymerase (Promega), mAliquots of 20 pg total of each capped fluorescent mRNA were injected into embryos at the one-cell stage, and embryos were scored visually for localization at the 4-cell stage using a Nikon SMZ18 stereo-microscope. Embryos that were unfertilized or showed cleavage furrow abnormalities were discarded. To mitigate bias in analysis, two researchers independently scored the injected embryos.sqt RNA (Alexa 488 UTP-labeled) was co-injected with 10 pg red fluorescent lefty1 or lefty2 RNA (Alexa 546 UTP-labeled) into 1-cell stage wild type embryos of the TU (Tuebingen) strain. Embryos were mounted in 0.7% low melting agarose, and imaged at the 4-cell stage. Z-stacks in two channels  with a step-size of 5 μm were acquired through the whole embryo at 10× magnification on a Nikon, ECLIPSE Ni microscope equipped with a HAMAMATSU digital camera C11440, ORCA-Flash4.OLT. Maximum intensity projections were generated from the Z-stacks for both channels using Fiji software  orthologues from ENSEMBL , 0.6 μl 5× transcription buffer (Promega), 0.4 μl T3 polymerase (Promega), 0.2 μl RNasin (Promega), 2.5 mM rATP, rGTP and rCTP and 0.025 mM rUTP (Roche) at 37°C for 3 h. The reaction was stopped by adding 100 μl TE containing 30% glycerol and ∼0.01% Bromophenol Blue.Radioactively labeled probes for EMSAs were transcribed from PCR products with T3 RNA polymerase. Templates for probe synthesis were generated by PCR with an extended phage T3 RNA polymerase promoter (AATTAACCCTCACTAAAGGGAGAA) appended to the 5′ end of the forwards primer, and gel-purified . The reaction was made up to 7 μl with sterile water, incubated for 10 min at room temperature, following which 1 μl of RNA probe  was added. The reaction was incubated for 20 min and loaded onto the gel, electrophoresed at 25 mA, dried, and auto-radiographed. The stacking gel was 25 mM Tris pH 6.8, 3% 19:1 acrylamide:bisacrylamide and the resolving gel was 0.5× TBE , 6% 19:1 acrylamide:bisacrylamide. The anode buffer was 0.5× TBE and the cathode buffer was either 50 mM glycine, 6 mM Tris, 0.2 mM EDTA, pH 8 . Recombinant Ybx1 protein was affinity purified by the His tag using Ni-NTA agarose beads (Qiagen) according to the manufacturer's instruction. The buffer was exchanged to 20 mM HEPES (pH 7.5), 200 mM NaCl by using ultra centrifugal filters (Amicon) and protein was stored in aliquots at –80°C. For gel-shifts, 1 μl of purified protein was pre-incubated with 4 μl of 2× gel-shift buffer  to make extracts. Debris was pelleted by centrifugation , and supernatants were flash frozen in 50 μl aliquots in liquid N2. One hundred-nucleotide long probes spanning the 3′UTR of sqt or lft1 were synthesized and used in RNA gel-shift assays. Templates for the probes were generated by PCR with an extended phage T3 RNA polymerase promoter (AATTAACCCTCACTAAAGGGAGAA) appended to the 5′end of the forward primer, and gel-purified. Primers are listed in ybx1 mutant embryos, and used in gel shift assays.For preparing embryo lysates to detect binding activity sqt, lefty1, lefty2 and gapdh transcripts. The cycle numbers for the PCR reactions were: gapdh 25 cycles, sqt 28 cycles, lft1 29 cycles and lft2 28 cycles. The list of primers is included in the RNA immunoprecipitation was carried out using embryo lysates as described . Embryoslefty1-gfp and lefty2-gfp reporter mRNAs were synthesized from linearized plasmid using the mMessage mMachine SP6 kit . The GFP CDS was inserted into the full-length lefty cDNA sequence, in frame right after the 3′end of the respective lefty CDS  at the 1-cell stage as described , sphere (4 hpf at 28.5°C), 30% epiboly (5 hpf at 28.5°C), and shield (6 hpf at 28.5°C) stages.Capped synthetic escribed . Capped For quantitative analysis of Lefty expression, injections and temperature shifts to 23°C were done as described above, and the embryos were shifted back to 28.5°C at the 1000-cell stage  or homozygous mutant fathers  for the translation experiments, mutant fish were outcrossed to wild type fish of the AB strain. Homozygous ybx1 mutant fish were maintained in the TU (Tuebingen) background.To obtain embryos from homozygous mutant mothers (Mlft1-gfp RNA or lft1-ΔDLE-gfp RNA were co-injected with rhodamine dextran (injection control) into 1 cell stage AB wild-type embryos. GFP and rhodamine signals were analysed for a subset of injected embryos. The embryos were incubated at 28°C until 2.5 days post fertilization and imaged using a Nikon SMZ18 microscope equipped with a Nikon DS-Fi2 color camera controlled by NIS-Elements F 4.0 software. Imaged embryos were scored by the following categories of Nodal loss-of-function phenotypes: wild type, mild, moderate, or severe. Phenotypes were independently scored by two researchers.Aliquots of 25 pg of either YBX1 targeting CRISPR/Cas9 expression vector , anti-α-Tub1:5000 , anti-YBX1 1:5000 (Sigma).HEK293FT cells were transfected with 2 μg of plasmid at a low passage number in Opti MEM medium using Fugene reagent (Promega). After 48 h, the transfection medium was changed to DMEM with 1 μg/ml of puromycin (Gibco). After a further 24 h, the puromycin treated cells were allowed to recover in DMEM media and extracted DNA from the cells was sequenced to confirm mutations in the sqt is also present in other components of the pathway, we examined the 3′UTRs of Nodal pathway components. Transcripts for nodal-related2/cyclops , lefty1 (lft1) and lefty2 (lft2)  and lefty1 or lefty2 (red) RNAs into one-cell stage embryos and found that they co-localized to the same cells in four-cell stage embryos  ,32,33 con Figure  and B. Ts Figure . If the s Figure . These acyc RNA activity is different from that of sqt, lft1 and lft2 RNAs, we first examined the secondary structure of the region encompassing the cyc DLE, and compared it to the secondary structure of the sqt DLE  in embryos. Immediately after injection, both cyc and cycΔCA fluorescent RNAs are visible in the middle of the embryo. However, by the four-cell stage, cyc RNA is largely diffuse whereas cycΔCA RNA is localized in the majority of embryos   reduce localization to a similar extent as that seen with ΔGSL. Compensatory mutations that create a stem–loop even with a different sequence, e.g., by swapping the arms of the stem  Figure  and B. ME Figure . These rsqt RNA is bound via the DLE and translationally repressed by the nucleic acid binding protein Ybx1 to ensure that Ndr1/Sqt signaling is shut off in early zebrafish embryos. Because the lefty 3′UTRs behave similarly to sqt DLE in embryonic localization assays, we determined if the lefty DLEs are also bound by Ybx1. We tested if short (100 nucleotide) radiolabeled lft1 3′UTR probes are bound by factors in zebrafish embryo lysates in RNA gel-shift assays. Similar to a sqt 3′UTR probe harboring the DLE, a lft1 3′UTR probe (lft1.3) spanning the DLE is shifted by lysates from 1 cell stage and 1000 cell-stage embryos  3′UTR radio-active labeled probes spanning the DLE regions in the sqt, lft1 and lft2 3′UTRs  from 1-cell stage embryo lysates  with embryo lysates from 1-cell or 1000-cell embryos, followed by RT-PCR to detect lft1 and lft2  and RNA-IP using mouse IgG antibodies do not show any amplification product , as also with input (positive control) from both stages  showed precocious and elevated expression of Lft1-GFP  show weak GFP expression starting from the 1000 cell stage  . Even at late blastula / early gastrula stages  only a quarter of control embryos show strong GFP expression , whereas most ybx1 mutants manifest very strong Lefty reporter expression. Similarly, in embryos injected with lft2-gfp, we observed strong GFP expression even at the 1000 cell stage in ybx1 mutant embryos but not in controls, and the number of embryos expressing GFP increases in ybx1 mutants at sphere and epiboly stages  Figure . In cont) Figure  when thes Figure .ybx1 mutant embryos compared to control embryos at all stages examined . However, analysis of embryo morphology at later stages shows that lefty1-ΔDLE-gfp injected embryos manifest more severe phenotypes compared to embryos injected with lefty1-gfp RNA  is significantly increased in YBX1−/- cells compared to control cells  RNA secondary structures do not strictly correspond to the ger RNAs . Therefoger RNAs  are potesqt/nodal RNA localizes in eggs and early embryos  in the protein-coding region, that we reported previously   see ). At theqt/nodal . Therefolefty and nodal RNAs) as responsive to Ybx1 is a short AGCAC sequence motif followed by a hairpin. The sequence and length of the loop differs considerably between sqt, lft1 and lft2, and yet in vivo, these elements behave similarly. It therefore seems likely that the Ybx1-binding site comprises of the short sequence and stem region, and the variable loop region in the DLE likely does not contribute significantly towards Ybx1 recognition.Interestingly, the minimal DLE we identified  to over two stem-loops (h) to multiple stem loops (bcd)  contains A’-form helices important for its function  ,9,66. A function . Studiesfunction . The loc stages K to over ch other . In our in vitro binding with short oligonucleotides (SELEX) had predicted some YBX1 sites in mammalian cells  in the zebrafish genome. Therefore, it is likely that the Ybx1 orthologues carry out similar, conserved functions in the regulation of gene expression. Accordingly, we demonstrated conservation of the regulation of human NODAL RNA by the DLE/YBX1 module between zebrafish and humans by showing: (i) the presence of a DLE in human NODAL RNA, (ii) similar behavior of human NODAL RNA to sqt RNA in RNA localization assays, (iii) binding of recombinant zebrafish Ybx1 to a NODAL 3′UTR probe in gel shift assay and (iv) elevated translation of a human NODAL translation reporter after CRISPR/Cas9 mediated depletion of YBX1 protein in human cells.Our assay is also useful for testing heterologous UTRs for activity in zebrafish, as we showed for human  domains , with hiHuman NODAL has been implicated in the maintenance of ESC cell pluripotency and in tumor metastasis. This raises the exciting possibility that this mechanism of translational control might regulate Nodal signaling in different developmental, physiological, and pathological contexts in humans, and warrants further exploration. Notably, human YBX1 has been found in mRNPs in neurons, a cell type known for extensive RNA transport, subcellular localization and localized translation –74. FurtSupplementary DataClick here for additional data file.


Drosophila melanogaster, has proven to be a valuable tool in our understanding of fundamental biological processes, and has empowered our discoveries, particularly in the field of neuroscience. In recent years, Drosophila has emerged as a model organism for human neurodegenerative and neuromuscular disorders. In this review, we highlight a number of recent studies that utilized the Drosophila model to study repeat-expansion associated diseases (READs), such as polyglutamine diseases, fragile X-associated tremor/ataxia syndrome (FXTAS), myotonic dystrophy type 1 (DM1) and type 2 (DM2), and C9ORF72-associated amyotrophic lateral sclerosis/frontotemporal dementia (C9-ALS/FTD). Discoveries regarding the possible mechanisms of RNA toxicity will be focused here. These studies demonstrate Drosophila as an excellent in vivo model system that can reveal novel mechanistic insights into human disorders, providing the foundation for translational research and therapeutic development.For nearly a century, the fruit fly,  Drosophila melanogaster has been utilized as a model organism to study a number of human neurodegenerative and neuromuscular diseases , and order them from resource centers such as the Bloomington Drosophila Stock Center. In addition, due to the nature that the mobilization of the P-elements are typically imprecise, these fly lines provide a means to generate excision mutants . Usually, a GAL4 fly line expresses GAL4 under the control of a cell- or tissue-specific promoter. This can be achieved by enhancer-trap screens, or the fusion of identified promoters with the GAL4 gene and subsequently microinjecting the constructs into fly embryos to produce specific GAL4 driver lines  repeat expansion had already been identified as a mutational mechanism underlying several human disease  loss-of-function of wild-type protein, (2) gain-of-function of toxic RNAs, or (3) gain-of-function of toxic proteins  gene  and double-stranded RNAs (dsRNAs) containing the expanded repeats. Both of them would eventually lead to the generating of toxic repeat-containing small RNAs (sRNAs), which is the true culprit of this mechanism of RNA toxicity in READs. It is known that hairpin structures can be formed by single stranded CAG repeats in polyQ diseases and CGG repeats in FXTAS  translation. Normally, protein synthesis is initiated by the start codon ATG, which leads to the translation of mRNA. Ranum and colleagues have discovered a novel mechanism which repeat expansions in READs can bypass the need of a start codon and mediate RAN translation of the repeat-containing transcripts , split ends, and CG3249, were all subsequently identified as genetic modifiers of this degeneration, suggesting that RNA rather than expanded protein is the source of toxicity  It has been shown that gain-of-function RNA toxicity contributes to the pathology of these diseases, and (ii) there are established Drosophila models for studying the RNA toxicity of these diseases.Recent work in Huntingtin (HTT) gene , respectively  induced nuclear inclusions and degeneration, recapitulating the pathogenic features of polyQ diseases. These studies, for the first time, demonstrated that D. melanogaster can be a powerful model to study human neurodegenerative diseases. Subsequently, more Drosophila models of HD and SCA3 , and cause misregulation in alternative splicing  is the most common cause of inherited intellectual disability, often associated with autism spectrum disorders  or the native Drosophila dFMRP (dfmr1 gene) fully rescues all detectable disease-like phenotypes in the Drosophila FXS model . While FXS is mostly caused by the loss-of-function of wild-type FMRP, which results in neurodevelopmental and behavioral disorders characterized by mental retardation, autism, anxiety and mood instability, FXTAS is mostly caused by the CGG repeat expansion that produces gain-of-function RNA toxicity in the CNS, which results in a late-onset neurodegenerative disorder, characterized by progressive intention tremor, gait ataxia, parkinsonism, and cognitive decline  gene, which encodes a RNA-binding protein , also known as Lou Gehrig's disease, is an adult-onset neurodegenerative disease characterized by the progressive loss of motor neurons in the cortex, brainstem, and spinal cord. Symptoms include the loss of movement control, difficulty in speaking, swallowing, and eventually breathing, which leads to fatality Kurtzke, . ALS affC9ORF72 was found to be associated with ALS combined with frontotemporal dementia 30] with a reporter in the compound eye, which resulted in severe retinal degeneration  repeats of GGGGCC and compared it with pure GGGGCC repeats which can be RAN-translated into DPR proteins in Drosophila 36 results in the same amount of ribonuclear foci formation. However, the expression of all RO constructs, including RO (GGGGCC)36, RO (GGGGCC)108, and even RO (GGGGCC)288, had no detectable effect on the fly eye. In contrast, both pure (GGGGCC)36 and pure (GGGGCC)103 caused degeneration. These findings suggest that, although both RNA and DPR proteins may contribute to toxicity, degeneration was primarily attributable to DPR proteins 36 constructs resulted in degeneration, whereas glycine-alanine (GA)36 and proline-alanine (PA)36 had no detectable effect. Even at the repeat length of 100, same results were obtained for the above DPR proteins, indicating that high arginine-containing DPR protein is the major cause of neurodegeneration in C9-ALS/FTD , poly(GR), or poly(GP) DPR proteins by RAN translation. Consistent with an aforementioned study, they found that the arginine-containing poly(GR) to be highly toxic A consensus from all these studies was that the DPR proteins that cause toxicity in C9-ALS/FTD contain arginine. Arginine is a unique amino acid that has as many as five potential hydrogen-bond donors that are readily available to react with biological hydrogen-bond acceptors. In DNA-protein complexes, arginine residues are the most frequent hydrogen bond donors to backbone phosphate groups  were expressed in the fly eye  that are the major determinants of toxicity and degeneration in this disease.Drosophila model, the researchers found that this branching defects can be enhanced by the upregulation or suppressed by the downregulation of dfmr1 or orb2  have been demonstrated to disrupt foci formation and reduce RNA toxicity in both DM1 and C9-ALS/FTD (Mulders et al., SUPT4H1, the human ortholog of Spt4, decreased sense and antisense RNA foci and DPR protein production in C9-ALS/FTD patient-derived cells (Kramer et al., The newest trend in the designing of small molecules is driving toward compounds with multiple modes of actions in combating the pathogenesis of the disease. Zimmerman and colleagues have reported a type of small molecule can target DM1 pathology by acting as transcription inhibitors, by inhibiting aberrant protein binding to toxic RNA, and by acting as RNase mimics to degrade the toxic RNA (Nguyen et al., Drosophila as an excellent model organism for the studying of READs. The studies mentioned above have revealed comprehensive information regarding the mechanisms of neurodegeneration in READs, demonstrating the elegant use of the fly models in pioneering discoveries of RNA toxicity. Despite the fact that protein toxicity is a major factor that contributes to the pathogenesis of many READs, we are now certain that RNA toxicity also plays a major role in inducing neural and muscle degeneration via a number of possible ways, including but not limited to the impairment of alternative splicing, the promotion of bidirectional transcription, the induction of nucleolar stress and disruption of nucleocytoplasmic transport, the generation of sRNA from hairpins and dsRNA, and RAN translation of repeat-containing peptides. Although the formation of visible ribonuclear foci is still a hallmark of pathogenesis, more, and more evidence is indicating that the initial emergence of foci does not equal to immediate detectable toxicity. The foci do not initiate the disease process per se. In fact, the formation of foci could possibly be a healthy response to temporarily sequester the toxic RNA. Although in the long run, foci may still contribute to the progression of the disease by sequestering RNA-binding proteins, and gradually lead to degeneration.In this review, we highlighted Drosophila research to ascend to the next level. For instance, Liu and colleagues have developed an improved RNA injection-based CRISPR/Cas9 system that is highly efficient for creating desired mutagenesis in the Drosophila genome with a mutagenesis induction rate up to 88% (Bassett et al., Drosophila geneticists as well. Online databases such as FlyBase has even introduced “Human Disease Model Reports” recently, further facilitating the studies of human diseases using the fruit fly (Millburn et al., Apart from the traditional advantages in genetics, the recent advances in CRISPR/Cas9 system and bioinformatics have become a powerful driving force that will surely aid Drosophila is a useful in vivo model system that can reveal novel mechanistic insights into human disorders, providing the foundation for translational research and therapeutic development. Looking into the future, we anticipate the tiny fruit fly to continue playing an important role in gene and drug discoveries in the ongoing battle between humans and neurodegenerative and neuromuscular diseases.In conclusion, ACK wrote the manuscript. HYEC contributed to the writing of 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.


The three prime untranslated region (3′-UTR) is known to play a pivotal role in modulating gene expression by determining the fate of mRNA. Many crucial developmental events, such as mammalian spermatogenesis, tissue patterning, sex determination and neurogenesis, rely heavily on post-transcriptional regulation by the 3′-UTR. However, 3′-UTR biology seems to be a relatively untapped field, with only limited tools and 3′-UTR resources available. To elucidate the regulatory mechanisms of the 3′-UTR on gene expression, firstly the 3′-UTR sequences must be identified. Current 3′-UTR mining tools, such as GETUTR, 3USS and UTRscan, all depend on a well-annotated reference genome or curated 3′-UTR sequences, which hinders their application on a myriad of non-model organisms where the genomes are not available. To address these issues, the establishment of an NGS-based, automated pipeline is urgently needed for genome-wide 3′-UTR prediction in the absence of reference genomes.ExUTR, a novel NGS-based pipeline to predict and retrieve 3′-UTR sequences from RNA-Seq experiments, particularly designed for non-model species lacking well-annotated genomes. This pipeline integrates cutting-edge bioinformatics tools, databases (Uniprot and UTRdb) and novel in-house Perl scripts, implementing a fully automated workflow. By taking transcriptome assemblies as inputs, this pipeline identifies 3′-UTR signals based primarily on the intrinsic features of transcripts, and outputs predicted 3′-UTR candidates together with associated annotations. In addition, ExUTR only requires minimal computational resources, which facilitates its implementation on a standard desktop computer with reasonable runtime, making it affordable to use for most laboratories. We also demonstrate the functionality and extensibility of this pipeline using publically available RNA-Seq data from both model and non-model species, and further validate the accuracy of predicted 3′-UTR using both well-characterized 3′-UTR resources and 3P–Seq data.Here, we propose ExUTR is a practical and powerful workflow that enables rapid genome-wide 3′-UTR discovery from NGS data. The candidates predicted through this pipeline will further advance the study of miRNA target prediction, cis elements in 3′-UTR and the evolution and biology of 3′-UTRs. Being independent of a well-annotated reference genome will dramatically expand its application to much broader research area, encompassing all species for which RNA-Seq is available.The online version of this article (10.1186/s12864-017-4241-1) contains supplementary material, which is available to authorized users. In eukaryotes, the 3′-UTR has considerable variation in number and length across taxa, with higher level organisms typically having more and longer 3′-UTRs than lower eukaryotes [The three prime untranslated region 3′-UTR) is the regulatory noncoding section of an mRNA, which plays a crucial role in mediating temporal and spatial gene expression , 2. Strus (PASs) , 3. Thes′-UTR is karyotes . In addikaryotes , 5. Theskaryotes , 6, 7. TC. elegans using Roche/454 pyrosequencing, and Xia et al. [The past ten years has witnessed tremendous advancements in ‘Omic’ technologies, with high-throughput sequencing revolutionizing the field of molecular biology . RNA-Seqa et al.  studied ExUTR, which enables genome-wide identification of 3′-UTR without references and annotations from massive RNA-Seq experiments.The UTRdb , EnsemblExUTR uses a genome-wide approach for the prediction of a 3′-UTR landscape from RNA-Seq experiments, irrespective of the availability of well-assembled reference genomes. Using this pipeline, we successfully obtained a large number of 3′-UTR candidates from both reference-based and de novo transcriptome assemblies of model and non-model mammals. A large overlap was observed between the 3′-UTR candidates and the curated 3′-UTR resources, and analyses of 3P–Seq data and their corresponding RNA-Seq data indicate the accuracy and reliability of the ExUTR pipeline. More importantly, ExUTR outweighs other current methods as it can correctly predict 3′-UTRs from de novo assembled transcriptomes, even in the absence of well-annotated genomes of the relevant species. Therefore, ExUTR can be used in RNA-Seq transcriptomic studies across diverse tissue types, developmental stages and physiological conditions, for both model organisms and non-model species, enabling a wider phylogenomic perspective on 3′-UTR biology.ExUTR is a Linux-based pipeline implementing a fully integrative analysis workflow, designed to incorporate core in-house Perl scripts as well as free third-party software tools and databases. Standard input and output formats, such as FASTQ, FASTA and BLAST output format, are used to facilitate the modularity between different software packages, allowing users to run certain modules only as needed. Compared to existing tools that depend mainly on well-annotated genomes, the design of ExUTR enables the prediction of 3′-UTRs based solely on the intrinsic signals of assembled transcripts, in the absence of reference genomes and related annotations. The architecture of ExUTR contains three steps, consisting of 1) Transcriptome assembly; 2) ORF prediction; 3) 3′-UTR sequence retrieval. Each step is described in detail below, and the workflow of the pipeline is visualized in Fig. https://github.com/huangzixia/ExUTR.ExUTR predicts 3′-UTR sequences based mainly on the intrinsic signals of the transcripts, the acquisition of a robust transcriptome assembly is optimal. Therefore, full-length transcripts are required to be constructed from RNA-Seq data using either reference-based methods or de novo methods. This whole process can be simply achieved by orchestrating highly standardized third-party software packages (see below), which can flexibly meet diversified needs from users due to varied experimental designs. Here, we suggest a procedure to generate the high-quality transcriptome assembly for 3′-UTR prediction.Since In general, prior to transcriptome assembly, adaptor sequences and low-quality regions are removed from raw FASTQ reads. This can be done by several popular quality-inspection tools such as Cutadapt , NGS QC The pipeline suggested in Step One allows users to easily customize the combination of third-party programs and their associated parameters to generate high-quality assemblies, due to varied demands. For the users who have already obtained robust assemblies, the whole step could be skipped by directly importing transcripts into Step Two for open reading frame (ORF) prediction and annotation.ExUTR, we employ a self-predicting method to extrapolate the ORFs in transcripts. In brief, for unstranded RNA-Seq experiments, transcripts are factitiously translated into protein sequences in all six possible reading frames, three in each of forward and reverse directions. This is required as both forward and reverse complementary transcripts will be produced during assembly. As it is well justified that the longest reading frame is most likely to be used in translation [Since the 3′-UTR immediately starts from the stop codon of a transcript, it is necessary that the structure of the sequence should be investigated, especially the open reading frame (ORF) which is crucial to determine the position of the 3′-UTR in a transcript. In nslation , we selenslation . For eacnslation , BLAST r3UTR_orf.pl script  together with a tab-delimited, CSV-compatible sheet containing 3′-UTR annotation.All these steps regarding 3′-UTR prediction, sequence retrieval and validation are carried out by the ExUTR, we employed publically available RNA-Seq data from both model and non-model mammals . Therefore, a higher number of 3′-UTRs were expected from human, cow and mouse transcriptomes . However, attention must also be paid to those candidates that have not been well characterized, in that they are likely to be potential novel 3′-UTR. It is commonly accepted that the 3′-UTR sequences, many of which are regarded as species-specific, evolve much faster than protein coding regions and have not been fully recorded yet [Using ies Fig. . This ismes Fig. . Notablymes Fig. . The larmes Fig. . In addirded yet . Thus, fP < 0.01)  Fig. . This ph01) Fig. . TypicalExUTR pipeline is its ability to predict 3′-UTR from massive transcripts without well-annotated reference genomes. To assess its application on de novo assemblies, we compared the whole set of 3′-UTR predicted from both reference-based and de novo methods for each species. We observed that a number of common 3′-UTRs were predicted by both reference-based and de novo methods while a proportion of unique 3′-UTRs were recovered by respective methods  data publically available : Linux.Programming language: Perl & Bash.Other requirements: BioPerl module.License: GNU GPLv2.Any restrictions to use by non-academics: None.Additional file 1:The detailed methods for the analyses of the demonstrated RNA-Seq data. (DOCX 149 kb)


Candida albicans, one of the most prevalent human pathogenic fungi, usually act as a commensal on skin and superficial mucosal, but has been found to cause candidiasis in immunosuppression patients. Previously, we have revealed the up-regulation of A-to-I RNA editing activity in response to different types of influenza virus infections. The current work is designed to study the effect of microorganism/fungi infection on the activity of A-to-I RNA editing in infected hosts.Adenosine-to-Inosine (A-to-I) RNA editing is catalyzed by the adenosine deaminase acting on RNA (ADAR) family of enzymes, which induces alterations in mRNA sequence. It has been shown that A-to-I RNA editing events are of significance in the cell’s innate immunity and cellular response to viral infections. However, whether RNA editing plays a role in cellular response to microorganism/fungi infection has not been determined. C. albicans infection. Eighty nine thousand six hundred forty eight and 60,872 A-to-I editing sites were detected in normal OKF6 and HUVEC cells, respectively. They were validated against the RNA editing databases, DARNED, RADAR, and REDIportal with 50, 80, and 80% success rates, respectively. While over 95% editing sites were detected in Alu regions, among the rest of the editing sites in non repetitive regions, the majority was located in introns and UTRs. The distributions of A-to-I editing activity and editing depth were analyzed during the course of C. albicans infection. While the normalized editing levels of common editing sites exhibited a significant increase, especially in Alu regions, no significant change in the expression of ADAR1 or ADAR2 was observed. Second, we performed further analysis on data from in vivo mouse study with C. albicans infection. One thousand one hundred thirty three and 955 A-to-I editing sites were identified in mouse tongue and kidney tissues, respectively. The number of A-to-I editing events was much smaller than in human epithelial or endothelial cells, due to the lack of Alu elements in mouse genome. Furthermore, during the course of C. albicans infection we observed stable level of A-to-I editing activity in 131 and 190 common editing sites in the mouse tongue and kidney tissues, and found no significant change in ADAR1 or ADAR2 expression .We first detected and characterized the A-to-I RNA editing events in oral epithelial cells (OKF6) and primary human umbilical vein endothelial cells (HUVEC), under normal growth condition or with C. albicans infection of human epithelial and endothelial cells led to the up-regulation of A-to-I editing activities, through a mechanism different from that of viral infections in human hosts. However, the in vivo mouse model with C. albicans infection did not show significant changes in A-to-I editing activities in tongue and kidney tissues. The different results in the mouse model were likely due to the presence of more complex in vivo environments, e.g. circulation and mixed cell types.This work represents the first comprehensive analysis of A-to-I RNA editome in human epithelial and endothelial cells. The online version of this article (10.1186/s12864-017-4374-2) contains supplementary material, which is available to authorized users. A-to-I RNA editing is a hydrolytic deamination reaction at the C6 position of adenine base occurring on double stranded RNAs, which is catalyzed by the adenosine deaminases acting on RNA (ADAR) family of enzymes , 2. InosRoles of A-to-I RNA editing were shown to be involved in innate immune response in virus-infected hosts, which displayed either antiviral or proviral activities by inducing changes that affect how viruses interact with their hosts , 14. TheCandida albicans is one of the most prevalent human pathogenic fungi. It grows as yeast or filamentous cells and can cause the infection candidiasis in humans, which was reported as the 4th leading cause [C. albicans in human hosts [C. albicans and the host immune response [C. albicans growing and coexisting with a human host without causing any symptoms of disease, is very much dependent on the immunological cross-talk between C. albicans and the human innate immune system. Alterations in the host can replace commensal factors with virulence attributes once the pathogenicity is induced. As a result, fungal cells adhering to the host epithelial cells begins invasion by host endocytosis, then active penetration and directs damage follows. Whether RNA editing plays a role in C. albicans-host immune system cross talk as a part of the innate immune response system remains unknown. In the current study, to investigate the A-to-I RNA editing events in C. albicans infections, a big-data analysis was performed using community RNA-seq data sets generated from human endothelial cells and oral epithelial cells during in vitro infection with 2 C. albicans stains, WO1 or SC5314 and from tongue and kidney tissues from mouse model of hematogenously disseminated candidiasis (HDC) [C. albicans infection was implicated with in vitro infection data.ng cause  of bloodng cause , 22. Manresponse –27. The is (HDC) . This woC. albicans infection on RNA editing of human epithelial and endothelial cells, we first analyzed the RNA-editing activities in human epithelial cells (OKF6 cell line) and endothelial cells (HUVEC cell line), which have no reported studies to date. We had 487 and 300 million paired-end RNA-seq reads (2X 100 bp) for OKF6 and HUVEC, respectively, for which the mapped reads were 409 and 268 million  and primary human umbilical vein endothelial cells (HUVEC cell line) infected with SC5314 or WO1 strains and collected at 1.5, 5, and 8 h post infection . The coneq reads X 100 bp We applied a pipeline modified from previous studies –31 to idAs shown in Fig. Comparison of the editing sites identified in two cell types Fig. , 22,741 C. albicans infection on A-to-I RNA editing in human epithelial and endothelial cells, we investigated the changes in editing activities during the course of infection. When mean editing level was used to measure the changes of editing activity  [Being the most prevalent human pathogenic fungi, is (HDC) .From our analysis, 89,648 and 60,872 A-to-I RNA editing events were identified for OKF6 and HUVEC cells, respectively. This represents the first comprehensive analysis of RNA editing editome in human epithelial and endothelial cells. The validity of these events was confirmed by cross-checking against the annotated RNA editing databases, DARNED, RADAR, and REDIportal. The significant fraction of newly identified A-to-I RNA editing events are interesting, as they represent editing sites specific to human epithelial and endothelial cells. Previously, functions of A-to-I RNA editing mediated by ADARs were discovered and documented mostly in neuronal receptors , and ionC. albicans infection, we measured their activities using the mean editing level of all events and the normalized editing levels of common editing sites across all time points. While the former displayed a slight upward trend of RNA editing activity during the course of infection, the latter showed a significant increase of RNA editing levels for sites located in the three different regions, Alu, Nonre, and Renonalu  under accession number GSE56093 and GSE67688 [C. albicans were as described [RNA-seq data for GSE67688 , 44. Celescribed , 44. Prehttps://broadinstitute.github.io/picard/). Unmapped reads and those with mapping quality score lower than 20 were removed by Samtools  [For RNA-seq data processing and mapping, our pipeline was modified from previous work –31, 45, 4 –q 20) .The protocol we used to call A-to-I RNA editing sites was derived from that described previously. In brief, the RNA-seq data mapped to reference genomes were subject to variant calling by the GATK analysis tool . The calEditing level is defined for an editing site as the number of reads with the edited base divided by the number of total reads mapped on the site. Normalized editing level is defined as the ratios of the editing levels at different time points to the maximum editing level for a specific editing site .We used CAVA  (that prLevel of expressed genes in RPKM (Reads Per Kilobase per Million mapped reads) was estimated from RNA-seq mapping results as described . BrieflyR package Venn Diagram  was usedAdditional file 1:The data sets information of collected RNA seq sample. (XLS 36 kb)Additional file 2:RNA editing sites of OKF6 and HUVEC cell lines. (XLS 29 kb)Additional file 3:Mouse organ or tissues RNA-seq mapping results. (XLS 13269 kb)Additional file 4:Cell line RNA-editing Sites overlap with database. (XLS 27 kb)Additional file 5:The number of RNA editing sites in OKF6 and HUVEC infection with different time point and strains. (XLSX 9 kb)Additional file 6:Editing level of RNA editing sites in OKF6 and HUVEC infection with different time point and strains. (XLS 28 kb)Additional file 7:Editing level of RNA editing sites locate in stable expression genes. (XLSX 9 kb)Additional file 8:Editing level of RNA editing sites locate in differentially expression genes. (XLSX 8 kb)Additional file 9:Correlation test between the genes expression of ADAR and the normalized editing level of common editing sites. (XLS 30 kb)Additional file 10:Mouse organ or tissues RNA-seq mapping results. (XLS 36 kb)Additional file 11:RNA editing sites of mouse organ or tissues. (XLS 215 kb)Additional file 12:C. albicans. (XLS 26 kb)The number of RNA editing sites with mapped bases in mouse model infection with Additional file 13:Distribution of RNA editing level of mouse organ or tissue. (XLS 27 kb)Additional file 14:Correlation test between the genes expression of Adar and the normalized editing level of common editing sites in mouse. (XLS 28 kb)


Drugs and vaccines based on mRNA and RNA viruses show great potential and direct translation in the cytoplasm eliminates chromosomal integration. Limitations are associated with delivery and stability issues related to RNA degradation. Clinical trials on RNA-based drugs have been conducted in various disease areas. Likewise, RNA-based vaccines for viral infections and various cancers have been subjected to preclinical and clinical studies. RNA delivery and stability improvements include RNA structure modifications, targeting dendritic cells and employing self-amplifying RNA. Single-stranded RNA viruses possess self-amplifying RNA, which can provide extreme RNA replication in the cytoplasm to support RNA-based drug and vaccine development. Although oligonucleotide-based approaches have demonstrated potential, the focus here is on mRNA- and RNA virus-based methods. Drug development has suffered from inefficiency, side effects and high costs. For this reason novel approaches for drug discovery are of great importance. RNA-based methods provide the advantage of targeting ‘production’ of drugs to diseased cells and vaccines to immune response-stimulating cells. RNA drugs have demonstrated therapeutic efficacy in eye and heart diseases and in various cancers in clinical trials. Likewise, RNA-based vaccines have provided protection against challenges with lethal doses of viruses such as Ebola and cancer cells in animal models. Modern drug discovery has suffered from finding improved and/or novel drugs due to inefficient delivery issues, drug efficacy and safety. In addition to classic approaches developing small molecule-based drugs and biotherapeutics, employing nucleic acids for therapeutic applications have become of potential interest ,2. In thDue to the sensitivity of single-stranded RNA (ssRNA) to degradation multiple efforts have been made to improve the stability of RNA molecules . These i7G) Cap plays an important role in RNA stability [in vitro transcription of RNA a problem has been the incorporation of cap analogs in reverse orientation resulting in an inability to efficiently transcribe mRNAs [in vitro transcribed RNA [The 5′ 7-methylguanosine triphosphate  and 3′-dephospho-coenzyme A (dpCoA) have been found in both Gram-negative and -positive bacteria. NAD+, reduced NAD+  and dpCoA have been postulated to be added to RNA after initiation of transcription similar to cap analogs [+, NADH and dpCoA are incorporated into RNA during the transcription initiation phase [de novo transcription initiation by cellular RNA polymerase (RNAP). Furthermore, both bacterial RNAP and eukaryotic RNAP II incorporate NCIN caps. The efficiency of NCIN capping is related to the promoter DNA sequences at and upstream of the transcription initiation site. Additionally, NCIN capping takes place in vivo with functional consequences. These findings might be important in future attempts to stabilize RNA for optimized delivery and expression.It was recently discovered that some bacterial RNA species possess a 5′-end structure similar to cap in eukaryotic RNA . Particu analogs . Howeveron phase . They we7G cap sequences by binding the PABP [in vitro transcribed RNA after transcription. The drawback of applying recombinant poly(A) polymerase is the generation of poly(A) tails of various lengths. In contrast, as mRNAs transcribed from DNA templates generate poly(A) tails of a defined length it is the preferred approach [Another approach to stabilize RNA molecules is by engineering the poly(A) tail at the 3′ end of mRNAs . It has the PABP . PABP hathe PABP . Poly(A)approach .in vitro transcription is between 120 and 150 nucleotides [In the context of poly(A) tail engineering, it has been demonstrated that the increase in poly(A) tail length generates enhanced efficiency of polysome formation, which also impacts the level of protein expression . Based oleotides ,21–22.Untranslated regions (UTRs) have been demonstrated to play important roles in post-transcriptional regulation of gene expression. These include modulation of mRNA transport from the nucleus and translation efficiency , subcellin vitro transcription of mRNA due to the presence of important regulatory elements. Incorporation of alpha-globin 3′ end UTRs has been demonstrated to stabilize mRNA [β-globin 5′ end and 3′ end UTRs provide improved translation efficiency [Globin UTRs have been applied for optimizing RNA for in vitro transcription followed by RNA electroporation of autologous T cells [The introduction of UTRs from the 5′ and 3′ ends can optimize ize mRNA ,29. Moreficiency . Globin  T cells  and intr T cells . Additio T cells .in vitro transcribed RNA [in vitro transcribed mRNA containing modified pseudouridine showed enhanced RNA stability and translation [in vitro transcribed RNA [in vitro transcribed mRNA was applied to remove dsRNA contaminants, which resulted in reduced production of type 1 IFN and pro-inflammatory cytokines [In attempts to improve the therapeutic properties of RNA, incorporation of natural nucleosides during RNA post-translational processing has proven useful for providing reduced immunogenicity of ibed RNA . For insnslation ,35. Howenslation . This apytokines The difficulties in achieving efficient delivery of RNA have seriously hampered the application of RNA for drug and vaccine development. For this reason, a variety of approaches have been evaluated for improved RNA delivery including optimized injection strategies, gene gun-based administration, protamine condensation, RNA adjuvants and encapsulation of RNA in nanoparticles consisting of polymers and liposomes .in vivo reporter gene expression in mice [The simplest application comprises of administration of naked mRNA by intramuscular injection, originally demonstrated by  in mice . Further in mice . Althoug in mice ,40, but  in mice ,42.In vitro transcribed mRNA coated in gold particles can penetrate cell membranes [melanocyte self-antigen TRP2 linked to EGFP (enhanced green fluorescent protein) elicited antigen-specific cellular and humoral responses and provided protection against B16 melanoma lung metastases [Several approaches have been evaluated to improve delivery including direct injection into the cytoplasm by application of gene gun-based delivery . In vitrembranes . In a motastases . Anothertastases . Protamitastases . Moreovetastases .CD40L, CD70, OX40L, GITR and CD83 into the mRNA to further increase the immunogenicity [In many vaccine applications it has been demonstrated that addition of adjuvants can substantially boost immune responses . It has genicity .in vivo mRNA delivery has been achieved generating strong immune response in animal models [OVA mRNA molecules encapsulated in DOTAP liposomes were injected intradermally into mouse ear pinnae, which provided protection against subcutaneous tumor challenges with EG7-OVA cells [OVA mRNA with GM-CSF mRNA resulted in enhanced CTL responses. In contrast, co-distribution of CD80 and IL-2 did not show this effect. In another approach, histidylated lipids have been optimized for mRNA delivery [melanoma-associated antigen MART1 mRNA histidylated lipopolyplexes formulated with a PEGylated derivative of histidylated polylysine and L-histidine-ethylamide liposomes demonstrated specific and significant protection against progression of B16F10 melanoma tumors. Enhanced anti-B16 responses were obtained by using a formulation containing both MART1 and MART1-LAMP1 mRNAs.In attempts to improve delivery and stability of RNA several encapsulation approaches have been evaluated . In thisl models ,63. For VA cells . Potentidelivery . Systemiin vivo. However, early observations indicated that DCs were only poorly transfected by lipoplexes [tumor-associated antigens (TAAs) mRNA or total tumor RNA [TAA mRNAs can be applied directly for vaccine strategies without the need of utilizing patient-specific tumor cells or antigens [TAA mRNAs have generated stimulation of antitumor responses [prostate-specific antigen (PSA) TAA mRNAs were administered to prostate cancer patients, which elicited a PSA-specific T-cell response and a significant decrease in PSA levels in six of seven patients [CEA mRNA transfected DCs showed good tolerance in pancreatic cancer patients although antitumor responses were obtained in only six out of 24 patients [MART1 mRNA-loaded Man(11)-LPR100.DCs are professional antigen-presenting cells, which play an important role in stimulation of immune responses . Targetipoplexes . Nanoparpoplexes . Relatedumor RNA . DCs traantigens . The disesponses . For inspatients . Moreovepatients ,73. In apatients . IntraveThe approach of using total tumor RNA from cancer patients has been evaluated in clinical settings for brain , lung 7, renal ,77 cancessRNA viruses have been frequently applied for vaccine development and cancer immunotherapy due to their capacity of RNA self amplification . Among RnsP1–4, responsible for highly efficient RNA replication, provides extreme amplification of RNA molecules, which due to the positive polarity can be directly translated in the cytoplasm. Both replication-deficient and -proficient alphavirus vectors have been engineered for gene delivery in vitro and in vivo. Moreover, the vectors can be applied in various forms such as recombinant viral particles, naked replicon RNA or layered RNA-DNA vectors [Alphaviruses belong to the family of togaviruses, possess a ssRNA genome of positive polarity and contain an envelope structure . The non vectors . Likewis vectors . In cont vectors ,84 and m vectors  viruses Self-amplifying RNA virus vectors have been frequently used for development of vaccines against infectious diseases and cancer . For insSFV-LacZ RNA provided tumor protection in mice challenged with CT26 colon tumors [in vivo. Furthermore, in attempts to prevent the host immune defense induced by intermediate dsRNA of self-amplifying RNA viruses, nonreplicative mRNAs encoding vaccinia virus immune evasion proteins E3, K3 and B18 were codelivered [Related to cancer immunotherapy, a large number of studies have confirmed that self-amplifying RNA viruses can generate tumor regression, prolonged survival and even tumor protection in animal models for various cancers . For insn tumors . It was n tumors . Studiesn tumors . Encapsuelivered . This ap7 or 0.36 × 108 IU of VEE-PSMA particles. Although no PSMA-specific cellular immune responses or clinical benefit were obtained, generation of neutralizing antibodies indicated that doses used in the study were suboptimal. Liposome encapsulated SFV particles expressing IL-12 have also been subjected to a Phase I clinical study in melanoma and kidney carcinoma patients [9 particles per m2.Furthermore, a Phase I clinical trial was carried out with VEE particles expressing the prostate-specific membrane antigen (PSMA) in patients with metastatic castration-resistant prostate cancer . No toxipatients . IntraveSeveral RNA-based drugs have entered clinical trials . These cde novo induction of antigen-specific immune responses and potent expansion of pre-existing immunity [Recent development of RNA-based vaccines has focused on lipid-nanoparticle encapsulation of RNA and applications of self-amplifying RNA virus vectors. For example, nucleoside-modified Zika virus prM and E glycoprotein RNA molecules have been encapsulated in lipid-nanoparticles . A singlimmunity .RNA-based biopharmaceuticals and vaccines represent a relatively new approach in drug discovery . HoweverToday, there are still several issues related to toxicity and drug delivery that needs to be addressed for RNA-based drugs and vaccines, but recent current development related to RNA stability and delivery methods based on encapsulated RNA molecules and self-amplifying RNA viruses will improve the possibility to make this approach a valid alternative for future medicine developmentRNA stability has been improved by addition of cap analogs and engineering of polyA tails5′ and 3′ end UTRs and chemical modifications provide improvement on RNA translation and productionRNA delivery has been optimized by RNA encapsulation and application of self-amplifying RNA virusesRNA-based drugs and vaccines have demonstrated therapeutic efficacy in preclinical animal models and in clinical trials


The ATP domain of DDX3 and DSBH domain of ALKBH5 were indispensable to their interaction with each other. Furthermore, DDX3 could modulate the demethylation of mRNAs. We also showed that DDX3 regulated the methylation status of microRNAs and there was an interaction between DDX3 and AGO2. The dynamics of m6A RNA modification is still a field demanding further investigation, and here, we add a link by showing that RNA demethylation can be regulated by proteins such as DDX3.DDX3 is a member of the family of DEAD-box RNA helicases. DDX3 is a multifaceted helicase and plays essential roles in key biological processes such as cell cycle, stress response, apoptosis, and RNA metabolism. In this study, we found that DDX3 interacted with ALKBH5, an m DEAD- (Asp-Glu-Ala-Asp-) box (DDX) proteins are the largest family of RNA helicases . This fa6A) RNA methylation is the most prevalent and enriched modification of both coding and noncoding RNAs [6A accounts for about 50% of total methylated ribonucleotides and is present in 0.1%–0.4% of all adenosines in total cellular RNAs [6A affects nuclear retention, pre-mRNA splicing, stability of mRNAs, and stability of small RNAs [6A around splicing sites and by inhibiting the binding of serine- and arginine-rich splicing factor 2 (SRSF2) [6A at the 5′ UTR (untranslated region) improves cap-independent translation [6A mark and facilitates ribosome loading [The 6-methyladenosine  . The prenslation , and eIF loading .6A in mRNAs affects cell differentiation and the expression of numerous genes including transcription factors [6A affects the differentiation of preadipocytes during adipogenesis [6A demethylation of a key pluripotency factor, NANOG, by ALKBH5 [Mettl3 (methyltransferase-like 3) and Mettl14 (methyltransferase-like 14) leads to low levels of m6A and reduced self-renewal of mouse ES cells [m factors . For insogenesis , 35. Expy ALKBH5 . Demethyy ALKBH5 . The depES cells .6A modification is posttranscriptionally installed by a multicomponent methyltransferase complex with at least three core proteins, namely, METTL3, METTL14, and Wilms' tumor 1-associating protein (WTAP) [6A demethylases, FTO and alkylated DNA repair protein AlkB homolog 5 (ALKBH5), demonstrate that the m6A RNA modification can be erased [The mn (WTAP) , 39. In e erased , 40.6A demethylation of RNAs. Since the dynamics of m6A RNA modification was largely elusive, here, we demonstrated that RNA demethylation could be subjected to regulation.In this study, we have showed that DDX3 and ALKBH5 interacted with each other and DDX3 could modulate the m2 incubator. All plasmid or siRNA transfections were carried out using Lipofectamine 2000 (Invitrogen) according to the manufacturer's protocol.HEK293T and HeLa cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 1% antibiotics and 10% fetal bovine serum (FBS). Cells were cultured at 37°C in a humidified 5% (vol/vol) COhttps://doi.org/10.1155/2017/8596135.All plasmids were constructed with recombinant method and/or restriction enzyme digestion and ligation. The full-length and all deletion constructs for DDX3 were cloned into p3xFLAG-Myc-CMV vector (Sigma-Aldrich). The full length and all deletion constructs for methyltransferases and demethylases were cloned into pKH3-HA vector. All constructs were confirmed by sequencing. Primer sequences used along with other oligo information were included in Supplementary Table S1 available online at 2 for 2 min in PBS. The cells were incubated on ice in modified RIPA lysis buffer ) for 20 min and rotated at 4°C for 10 min. Then, lysate was sonicated typically for 5 min at 30% amplitude, 3 sec pulses followed by 6 sec rest period. The lysates were clarified by centrifugation for 20 min at 14,000 rpm at 4°C. Fifty (50 μl) of Dynabeads Protein G magnetic particles (Invitrogen) were resuspended, 500 μl of lysis buffer. Then, 1.5 μg of respective antibodies was added to the beads and incubated on a rotating wheel at room temperature for 30 min. Beads were precipitated by magnet and finally resuspended again in cleared cell lysates and incubated for 2 h at 4°C. In the last step, the beads were resuspended in 50 μl of lysis buffer and subjected to Western blots.The cells were washed twice with ice-cold PBS and cross linked with UV 12000 J/cmThe proteins either from cell lyses or isolated from the IP beads were subjected to SDS-PAGE and then transferred on to a polyvinylidene difluoride (PVDF) membrane. Following blocking the membrane was probed using the corresponding primary antibodies overnight at 4°C. HA-Tag (C29F4) rabbit monoclonal, monoclonal anti-FLAG M2 (Sigma-Aldrich), mouse DDX3 (C-4) mAb, rabbit polyclonal anti-ALKBH5 (Sigma-Aldrich), and mouse anti-GAPDH antibody  were used. After washing, the membrane was incubated with secondary horseradish peroxidase-conjugated goat anti-rabbit IgG  or anti-mouse IgG antibodies for 2 h at room temperature. Enhanced chemiluminescence substrates (EMD Millipore) were then applied, and signals were detected using a chemiluminescence imaging system .Total RNA from cells was isolated using TRIzol reagent (Invitrogen) according to the manufacturer's protocol. The RNA concentration was determined by spectrophotometer. cDNA for qRT-PCR was synthesized from total RNA by the GoScript Reverse Transcription System (Promega) according to the supplied protocol using random hexamer primer and oligo dT. qPCR was performed with the GO Taq qPCR Master Mix (Promega) on the PikoReal Real-time PCR System (Thermo Scientific) according to the standard procedures.MicroRNA and poly(A) mRNA from cells were extracted using the mirVana™ miRNA Isolation Kit and PolyAT tract mRNA Isolation System (Promega), respectively, according to the manufacturer's protocol.190–293 to DDX3211–402 on a Rosetta web server (http://rosie.rosettacommons.org/docking2). The parameters used for docking are set to default [190–293 to DDX3211–402 was assigned an ID (33161) or can be accessed with the following link http://rosie.rosettacommons.org/docking2/viewjob/33161. Model with the best score was analyzed with PyMOL software . Structures used for modelling are obtained from protein data bank, accession numbers are 2I4I for DDX3211–402 [190–293 [The complex modelling was performed by docking ALKBH5 default –43. Dock3211–402  and 4061[190–293 .6A dot-blot was performed on a Bio-Dot Apparatus (Bio-Rad Laboratories Inc.). In brief, the RNA samples were denatured and spotted to nitrocellulose membrane under vacuum. After UV cross-linking, the membranes were baked at 80°C for 1 hr, and methylene blue staining was used to examine equal RNA loading. For detecting m6A levels, rabbit anti-m6A antibody (Millipore Sigma) was diluted with 1 : 500 in 0.1% TBST and 5% nonfat dry milk and incubated with the membranes overnight (4°C). Following extensive washing with 0.1% TBST, the blot was incubated with horseradish peroxidase- (HRP-) conjugated anti-rabbit IgG secondary antibody for 2 h at 25°C. The membrane was washed again with 0.1% TBST and visualized by ECL Western Blotting Detection Kit (Thermo Scientific). Dots were quantified with imageJ.The m3 per well. Cells were then transfected with siRNAs or scrambled control. MTT reagents were added at indicated time points. Four hours later, the supernatant was removed, and DMSO was added to dissolve the blue precipitates. The number of live cells was determined by the OD value, which was measured by a plate reader .Cell viability was measured with the MTT Cell Proliferation and Cytotoxicity Detection Kit  according to the manufacturer's recommendations. Cells in 96-well plates were plated at a density of 2 × 106A methyltransferases and demethylases. We constructed expression plasmids of METTL3, METTL14, WTAP, FTO, and ALKBH5. We cotransfected HEK293T cells with individual expression plasmids together with DDX3 expression plasmid  of DDX3 and the DSBH domain (AA 191–292) of ALKBH5 are responsible for their interaction (Figures DDX3 plays diverse cellular functions by interacting with different proteins through its different domains. In the present study, we describe a new role for DDX3 in RNA demethylation by physical interaction with the momain AA 1–292 of 6A RNA methylation is known to affect cell renewal and differentiation [α- and HIF-2α-dependent way, which eventually leads to enrichment of BCSCs in the hypoxic tumors [Mettl13 or Mettl14 reduced self-renewal of mouse ESCs [mntiation , 35. In c tumors . On the use ESCs .6A epitranscriptome and the functional relevance and physiological roles of DDX3 in the context of m6A modification.In this study, we have identified DDX3 as a partner of ALKBH5 and AGO2 to regulate the demethylation of mRNAs and miRNAs. Further studies would help to elucidate how these interactions contribute to regulated dynamics of m6A demethylase. We found ATP domain of DDX3 and DSBH domain of ALKBH5 to be indispensable to their interaction with each other. Furthermore, DDX3 could modulate the demethylation of mRNAs. We also found the interaction between DDX3 and AGO2, and DDX3 could modulate the demethylation of miRNAs. The dynamics of m6A RNA modification was largely elusive, and here, we showed that RNA demethylation could be regulated by proteins such as DDX3.In this study, we found that DDX3 interacted with ALKBH5, an RNA mTab. S1. Oligos used in the study.