RNA is central to the COVID-19 pandemic—it shapes how the SARS Coronavirus 2 (SARS-CoV-2) behaves, and how researchers investigate and fight it. However, RNA has received relatively little attention in the history and philosophy of the life sciences. By analysing RNA biology in more detail, philosophers and historians of science could gain new and powerful tools to assess the current pandemic, and the biological sciences more generally. The history and philosophy of the life sciences (HPLS) offers powerful resources for the analysis of, and response to, the COVID-19 pandemic.However, there are also key elements of the pandemic that to date have gained little attention in HPLS. One of these elements is ribonucleic acid (RNA). In what follows I will look at two reasons why RNA matters for the pandemic: its role in viral behaviour and its importance as a tool in biomedical research. Given the central role of RNA, developing a history and philosophy of this molecule will provide HPLS scholars with important tools to analyse the unfolding of the pandemic and science’s response to it. Such a history and philosophy of RNA will also enable scholars to build a more detailed understanding of the contemporary life sciences, as RNA is becoming a more central element in biological theory and practice Darnell, . It alsoThe first and probably most prominent reason why RNA matters for the COVID-19 pandemic can be found in the genomics of SARS-CoV-2: like many other viruses—and unlike plants, animals, or bacteria—the genome of this virus consists of an RNA molecule.At the heart of these differences is the fact that RNA, in contrast to DNA, can form intricate three-dimensional structures on its own, ranging from simple hairpins to more elaborate higher-order arrangements.The three-dimensional folding of RNA genomes raises new methodological and theoretical challenges. The structures have been shown to have functional relevance for all stages of the viral life cycle and virologists now often talk of two layers of information that viral genomes carry, the sequence layer and the structural layer  when they describe RNA viruses . A second reason is that RNA has transformed how researchers study and intervene in biological systems. If HPLS can develop a more detailed understanding of these RNA-related transformations in research practice, it will also further our understanding of how scientists analyse and tackle the current pandemic.guide for a range of DNA- or RNA-modifying enzymes, targeting them to specific sites on cellular or viral genomes.Underlying the growing power of RNA as a research tool is again the fact that RNA polynucleotides can fold into three-dimensional structures. This opens a broader spectrum of interactions with both DNA and proteins, making RNA a unique structural glue: rather than serving as a simple messenger that connects DNA with the protein translation machinery – an important but limited role that RNA was demoted to for decades in biological theory – RNA now emerges as a structurally complex The discovery of these “non-coding” RNAs  has re-shaped how scientists understand the working of cells, the development of organisms, and the dynamics of evolution. Over recent years, non-coding RNAs have been identified as key players in processes such as the regulation of gene expression, genome stability, carcinogenesis, or anti-viral defence  and also for novel gene editing tools, such as CRISPR-Cas9.5The idea of using RNA to re-program biological processes is also used in the development of novel vaccines . Tracking and understanding these changes in research practice and in biological theory will represent an important task for HPLS, not only to build a stronger response to the COVID-19 pandemic, but also to build a better understanding of the modern life sciences more generally.


High-throughput processing of the separated cells is enabled using microfluidics platforms. However, dissociation of tissue results in loss of information about cell location and morphology and potentially alters the transcriptome. An alternative approach for collecting RNA from single cells is to re-purpose the electrophysiological technique of patch clamp recording. A hollow patch pipette is attached to individual cells, enabling the recording of electrical activity, after which the cytoplasm may be extracted for single cell RNA-Seq (“Patch-Seq”). Since the tissue is not disaggregated, the location of cells is readily determined, and the morphology of the cells is maintained, making possible the correlation of single cell transcriptomes with cell location, morphology and electrophysiology. Recent Patch-Seq studies utilizes PCR amplification to increase amount of nucleic acid material to the level required for current sequencing technologies. PCR is prone to create biased libraries – especially with the extremely high degrees of exponential amplification required for single cell amounts of RNA. We compared a PCR-based approach with linear amplifications and demonstrate that aRNA amplification ( The introduction of RNA sequencing (RNA-Seq) expanded the toolset for characterization of neurons; however, the original RNA-Seq techniques were not able to perform expression profiling of individual cells, limiting our ability to investigate cellular heterogeneity. We7, or using microfluidic devices such as those employed in Drop-Seq8 or the Chromium controller (10x Genomics)9. Unfortunately, the treatments used to dissociate tissues lead to loss of complex morphology of individual neurons, eliminate location information, destroy cell connectivity, and irreversibly change the electrophysiological properties of neurons. Furthermore, dissociation may trigger changes in the transcription profile of cells11.Most of such approaches involve tissue dissociation to produce a single cell suspension, followed by sorting of the cells by fluorescence-activated cell sorting (FACS)1. This approach has been used by others (Patch-Seq)12, utilizing the SMARTer technique13, in which PCR amplification is used to produce sufficient amounts of DNA for sequencing.Patch clamp was invented to record cellular electrical activity, which is often specific to a given cell or neuron type; however, whether or not electrophysiological recording is performed, the patch clamp pipettes can be used to collect individual cells or their contents from within intact or sliced tissue. Cells may be chosen for study based on their morphology, electrophysiological properties and/or location in the tissue or organ. We developed a protocol using patch clamp for collection of cellular contents followed by RNA-Seq15. These challenges led us to investigate alternative linear amplifications methods of DNA or RNA, which are less prone to such bias16. Two major approaches are available for linear amplification of nucleic acids: (a) isothermal DNA amplification, which is implemented in Ovation® RNA-Seq System V2  kits, and (b) aRNA amplification, which utilizes in vitro transcription (IVT)17.Whole transcriptome sequencing of single cell material requires amplification of minute amounts of RNA/cDNA – typically in the range of 10 pg, or even less. If PCR amplification is employed and the number of thermal cycles is large, biased amplification of certain RNA species becomes noticeable, for example, favoring cDNA fragments of shorter length or of lower GC content8 and CEL218 protocols, outperformed the PCR-based protocols. CEL-Seq and CEL2 require multiplexing, involving use of barcoded primers to enable parallel processing steps. However, the benefits of multiplexing diminish, and complexity of the protocol becomes an unnecessary complication with lower numbers of samples, such as for electrophysiological patch clamp measurements, when every single cell is collected through a lengthy process.Support for the utility of linear amplification methods for single cell analysis came from an analysis using ERCC  spike-in controls, which showed that aRNA amplification used for single cell transcriptome applications, such as the CEL-Seq19.In this study we focused on protocols that can be applied to single cell or sub-single cell material and that do not require multiplexing. We chose the Ovation® RNA-Seq System V2 kit for linear DNA amplification, and the aRNA method for linear RNA amplification. The NuGEN kit was originally designed for 500 pg input RNA (the amount of RNA in ~50 cells), thus we modified the protocol to work with single cell RNA amountsin vitro transcription of cDNAs for linear RNA amplification, as first described in 199220. Several modifications of this technique had been described previously22. We made further adjustments and compared our modified aRNA protocols with the NuGEN and SMARTer protocols with nucleic acid material extracted from single cells using patch clamp technique.The aRNA method uses 4; in fact, collection is usually incomplete, as RNA in the nucleus and in the branches of neurons may not be collected. Therefore, we also tested robustness of amplification using only 5 pg of input RNA.In order to compare the efficiency of the different protocols, we used a standard input of 10 pg (similar to the amount in single cells) of Universal Human Reference RNA . Patch clamp collection of cells does not yield identical amounts of RNAin vitro transcription protocol and SMARTer protocol19 and single cell data transcriptomes collected from embryonic brain neurons using Fluidigm C1 which utilizes the SMARTer method23. We, in addition, performed the original in vitro transcription protocol and SMARTer protocol side by side with our modified aRNA protocol  and transcriptome mapping ratio calculated as a percentage of mapped reads which mapped to the transcriptome, excluding both rRNA  and mtRNA . The third metric was gene model discovery rate, assessed as the number of genes with more than 5 mapped reads detected per 3 million mapped reads. A Recent single cell RNA-Seq (scRNA-Seq) study indicated that more than one million reads are required to analyze the variance in expression2. We used 3 million as our baseline for our analysis to increase the gene complexity because we are targeting highly complex neuronal cells24.We used 5 metrics to assess the RNA-Seq data of amplified products. The first two metrics were reproducibility, measured as the Pearson coefficient of correlation of expression profiles between technical replicates, and accuracy, measured as a correlation between gene expression measured in 10 pg or 5 pg samples as compared to bulk RNA-Seq of UHR RNA. Prior to assessing reproducibility and accuracy metrics the samples were normalized by downsampling to 3 million mapped reads.The other two metrics, which were applied only to UHR data, assess 22 was developed for single cell applications and consists of 3 rounds of linear amplification cycles. The protocol employs column purification of nucleic acids (cDNA or aRNA at different stages of the protocol), and RNA ethanol precipitation between the cycles. Column purification and alcohol precipitation are prone to loss of nucleic acid26, which could be especially detrimental when the initial amount of material is minute. To address this potential issue, we replaced column purification of nucleic acids and ethanol precipitation with magnetic bead purification , which increases effective concentration of template, resulting in higher yield Fig. . Decreas19. Samples with less than 3 million mapped reads were removed from the analysis and the remaining samples were downsampled to 3 million mapped reads to normalize the data, increasing consistency. Data quality was evaluated using the five metrics described above. A comparison indicates that the modifications we introduced to the aRNA method substantially increased data quality in every metric for 10 pg input material , and the reproducibility of the modified aRNA protocol was practically identical with the SMARTer protocol (0.76 vs 0.78) Fig. . However19 protocol for single cells followed by NuGEN Ovation® Rapid Library Systems , (333 single cells). We also processed cells using original aRNA protocol but only few cells (5 cells) successfully generated single cell libraries. We compared our single cell RNA-Seq data from these methods.We used patch clamp to collect cytoplasmic material from 1,013 single cells from different layers of embryonic brain Fig. . We then23 (GSE67835) processed by the Fluidigm C1 instrument utilizing the SMARTer protocol followed by the Nextera library preparation. We processed raw data from all three methods  through the same analytical pipeline in Partek Flow. Again, we downsampled each sample to 3 million mapped reads, after removal of those with fewer reads.To expand the comparison, we also accessed publicly available raw data (FASTQ files) from 466 single cells dissociated from fetal and adult brainAmounts of RNA extracted from single cells by patch clamp can be highly variable resulting in high variation in amount of produced aRNA. We excluded samples (n = 135) that were failed to yield aRNA compared to negative control (water), which were run in parallel. In addition, there were a few outliers with an abnormally high library concentration (>100 ng/µl) (n = 2). We excluded these libraries for analyses as they had a large amount of dimer sequences and these failed the QC due to low mapping rate. Lastly, samples (n = 14) producing library amount lower than 200 ng (the minimum amount required for sequencing by Illumina protocol), were also excluded.total mapping rate, transcriptomic mapping ratio and gene model discovery rate. The total mapping rate was similar between SMARTer (62%) and modified aRNA methods (63%) and much lower with the NuGEN approach (24%), as a result of spurious amplification (as seen in UHR samples). The Transcriptomic mapping ratio was similar with SMARTer and modified aRNA protocols (62%), whereas it was lower with the NuGEN protocol (42%) . The SMARTer and original aRNA approaches detected fewer genes , and NuGEN detected the lowest number of genes (608).Single cell RNA-Seq data were analyzed using following metrics: methods 6% and mucin vivo or in relatively intact tissue slices, obviating the need to disperse tissues to separate cells, which previous work has shown can alter gene expression.Using patch clamp to extract single cell material for RNA sequencing offers multiple advantages over approaches that involve tissue dissociation to isolate single cells. Patch clamp provides the opportunity to record location, morphology and electrophysiology of a single cell for correlation with the expression profile of the same cell. Patch clamp can be used for recording In order to work with the minute amount of extracted single cell RNA, which often is less than the total cellular RNA content, we improved the efficiency of the procedures for RNA collection, handling and amplification. The protocols for patch clamp and collection of cytoplasm are critical, as they determine the amount of input RNA and how well it is preserved. For instance, we sliced brain samples under HEPA-filtered air hood to eliminate contamination of material during collection of cytoplasm from single cells. Additional precautions included using an RNase inhibitor in our intracellular solution, and flash-freezing the collected cytoplasm in lysis buffer in liquid LN2, immediately after mixing, to reduce potential RNA degradation.Using reference RNA, we compared RNA sequence quality across protocols that variously employ PCR amplification, linear DNA amplification and linear RNA amplification. These protocols were then evaluated using measures of gene model discovery rate, precision, accuracy and resistance to amplification of parasitic, non-transcriptomic fragments. In our hands, linear RNA amplification based on the modified aRNA method produced the most robust results, was the most sensitive, and consistently produced higher quality data in comparison to PCR amplification.We then tested the methods on RNA extracted from single neuronal cells from fetal human brain and compared our results with publicly available single cell RNA-Seq data. These analyses found that methods based on either linear amplification or PCR are sensitive enough and produce consistently meaningful results with single cell material. When data quality was compared, however, it becomes clear that the modified aRNA method, based on IVT, has a higher mapping rate, transcriptome ratio and gene model discovery rate, than the PCR method, confirming conclusions from comparison of these methods using UHR.et al.18 developed a method utilizing only one aRNA amplification cycle, by taking advantage of the increased initial cDNA amount due to multiplexing single cell samples prior to aRNA amplification. Further incremental improvements in the methodology, such as high-yield RNA transcriptases  or sequencing library methods that require less input cDNA  may result in decreased number of linear amplification rounds to one, even without multiplexing.Reducing the number of rounds of RNA amplification may be a key factor contributing to the improved performance of the modified aRNA method. Every additional round requires RNA purification, cDNA synthesis and aRNA amplification, and every step is prone to variable loss of nucleic acids, increasing noise and bias. Recognizing this problem, Hashimshony The universal human reference RNA  was purchased and dissolved in RNase-free water , according to manufacturer’s recommendations to 1 µg/µl stock solution. Working solutions with concentration 10 pg/µl were made by serial 1:10 dilutions and kept in low-binding microcentrifuge tubes at −80 degrees not more than two weeks.Deidentified tissues from adult human brains were acquired from medical waste after written informed consent was received from adults undergoing neurosurgery. Deidentified human fetal tissue was obtained following informed consent from parents, from elective terminations. Collection of this tissue was performed under approval by the Institutional Review Boards of both Children’s Hospital Los Angeles and the Keck School of Medicine of the University of Southern California. University of Southern California institutional review board approvals HS-12-00474 and HS-13-00399 were obtained for this work. All methods were performed in accordance with the relevant guidelines and regulations.2 1 mM; MgATP 4 mM; GTP 0.3 mM; phosphocreatine 8 mM; HEPES 10 mM; EGTA 11 mM; pH 7.25 and 300 mOsm) containing recombinant RNase inhibitor (Clontech) (0.4 U/µl); and mounted in a standard patch pipette holder, connected to an automatic pressure control unit . Under microscopic visualization, the patch pipette tip was maneuvered to the vicinity of a neuron, while positive pipette pressure (25–50 mmHg) was maintained. Once the pipette touched the cell membrane, gentle suction (−15 to −30 mmHg) was applied to enable formation of a giga-seal. We made the whole-cell configuration  by application of strong negative pressure to remove small patch of membrane. The content was expelled into a PCR tube containing 5 µl of lysis buffer  by breaking the end of pipette tip and applying positive pressure (25–50 mmHg). The cellular material was centrifuged followed by 1 or 2 freeze thaw cycles to assure complete lysis of the material. For every cell we recorded metadata containing information about morphology, electrophysiological recording, location in tissue, details of the method used for RNA processing and library preparation along with details about batch of processing.The embryonic brains from second trimester were sliced in a HEPA filter protected fume hood with VT1000 vibratome (Leica) into 400um slices. Neurons in tissue slices from resected embryonic brain were visually identified under illumination with an infrared Dodt gradient contrast system (homebuild). Patch pipettes  were filled with intracellular solution  were diluted in water to final volume of 5 µl. Alternatively, a whole volume of single cell lysate (5 µl) was used as a starting material for the protocol.To the 5 ul of each sample, we added: 4.9 µl of RNase free water, 2.4 µl of 5x First Strand buffer , 1.2 µl of dNTPs , 0.45 µl of 100 mM DTT  and 0.3 µl of dt-T7 oligo . This entire mixture was incubated for 5 minutes at 70 degrees to denature the RNA, then immediately placed on ice and the first strand enzyme mix was added: 0.3 µl of Rnasin , 0.45 µl of Superscript III , 1 µl of RNAse-free water. First strand synthesis was performed at 42 °C for 30 minutes, followed by 70 °C for 15 minutes.To the 9.35 ul of the above reaction, we added second strand mix , 0.75 µl of dNTP mix, 1 µl of DNA polymerase I , 0.25 µl of RNase H , 8.26 µl of RNase-free water) and incubated for exactly 2 hours at 16 °C. After incubation, 1 µl of T4 DNA polymerase  was added and samples incubated for an additional 10 minutes at 16 °C. Double stranded DNA was then purified using 52 µl of Agencourt XP RNAclean beads  according to manufacturer’s protocol and eluted in 4 µl of water.Reagents from Ambion MEGAscript® T7 Transcription Kit  were used for aRNA amplification. The 1 µl of each ATP, GTP, CTP, TTP, 10X reaction buffer, 10x Enzyme mix were added to each sample followed by incubation at 37 °C for 14 hours. Amplified RNA was purified with 18 µl of Agencourt RNACleanXP Beads and eluted in 4 µl of water.After addition of 1 µl of Random primers , samples were incubated at 70 °C for 10 minutes. First strand synthesis was performed by adding first strand mastermix , 1 μl of Superscript III) and successive incubations at RT (25 °C) for 10 minutes, 42 °C for 30 minutes, 95 °C for 5 minutes.1 µl of dt-T7 oligo (10 ng/µl) was added to the above 10 ul first strand reaction for each sample. After 5 min of incubation at 70 °C, second strand master mix was added  and samples were incubated exactly 2 hours at 16 °C. After incubation, 1 µl of T4 DNA polymerase  was added and samples incubated for an additional 10 minutes at 16 °C. Samples were then immediately purified with 70 µl of Agencourt RNACleanXP Beads and eluted in 4 µl of water.In vitro transcription was performed the same way as in the first round, and samples were purified with Agencourt RNAClean XP beads. RNA samples were eluted in 50 µl of RNase-free water and the amount of aRNA was measured using Agilent RNA ScreenTape  on an Agilent 2200 Tapestation.For bulk RNA samples (100 ng of UHR) and 10 pg UHR samples processed with modified aRNA protocol libraries were generated using Illumina Truseq Stranded mRNA Library Preparation kit . Samples from aRNA (round 2) were dried with SpeedVac  into low volume (less than 1ul) and added to the Elute-Prime-2 stage of the TruSeq Stranded mRNA protocol  to bypass the poly-A mRNA selection process. Rest of the steps were performed, following the manufacturer’s protocol using TruSeq RNA Single Indexes Set A and B .19.For samples processed with Ovation® RNA-Seq System V2 , sequencing libraries were made using NuGEN Encore Rapid Library system  according to the protocol previously reportedSize distribution of sequencing libraries was assessed by Agilent D1000 Screen Tape (5067-5582) on an Agilent 2200 Tapestation. Library concentrations were measured by KAPA Library Quantification Kits for Illumina® Platforms . 24 libraries were multiplexed and sequenced on an Illumina HiSeq 2500 System on Rapid flowcells (GD-402-4001) using HiSeq Rapid SBS Kit V2 with 100 bp single-ended reads.Sequencing reads were trimmed from both ends based on quality score using filter module in Partek Flow with settings of 25 minimum read length. The trimmed reads were mapped to human genome (hg38) and transcriptome (GENCODE Genes - release 26) using STAR aligner (STAR - 2.5.3a) within Partek Flow pipeline . Total mapping rate was measured as part of standard Post Alignment QA/QC modules in Partek Flow. Read counts were quantified for each sample using STAR transcript/gene expression quantification function, and the transcriptome mapping ratio was taken from Quantification summary. For further analysis BAM files were downsampled to 3,000,000 mapped reads using the SAMtools module in Partek Flow. Samples with less than 3,000,000 mapped reads were excluded. The reads were assigned to genes using htseq module within Partek Flow. In addition, number of reads was normalized with quantile normalization prior to comparison. For every sample we calculated the number of genes detected per 3,000,000 mapped reads (defined as genes with more than 5 uniquely mapped reads), Pearson coefficients of correlation between gene expression of technical replicates and between each sample and bulk RNA. For comparison with in-house SMARTer and original aRNA protocol, we compared the gene discovery rate with samples downsampled to 100,000 reads as there was limited number of reads on these samples.Supplementary Information.


C9orf72 gene represents the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar dementia (FTLD). Since the discovery one of the non-exclusive mechanisms of expanded hexanucleotide G4C2 repeats involved in ALS and FTLD is RNA toxicity, which involves accumulation of pathological sense and antisense RNA transcripts. Formed RNA foci sequester RNA-binding proteins, causing their mislocalization and, thus, diminishing their biological function. Therefore, structures adopted by pathological RNA transcripts could have a key role in pathogenesis of ALS and FTLD. Utilizing NMR spectroscopy and complementary methods, we examined structures adopted by both guanine-rich sense and cytosine-rich antisense RNA oligonucleotides with four hexanucleotide repeats. While both oligonucleotides tend to form dimers and hairpins, the equilibrium of these structures differs with antisense oligonucleotide being more sensitive to changes in pH and sense oligonucleotide to temperature. In the presence of K+ ions, guanine-rich sense RNA oligonucleotide also adopts secondary structures called G-quadruplexes. Here, we also observed, for the first time, that antisense RNA oligonucleotide forms i-motifs under specific conditions. Moreover, simultaneous presence of sense and antisense RNA oligonucleotides promotes formation of heterodimer. Studied structural diversity of sense and antisense RNA transcripts not only further depicts the complex nature of neurodegenerative diseases but also reveals potential targets for drug design in treatment of ALS and FTLD.The hexanucleotide expansion GGGGCC located in  Academic Editor: C9orf72 gene on chromosome 9p21 represents the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar dementia (FTLD)—two fatal neurodegenerative diseases with progressive loss of motor neurons in the brain and spinal cord . Af. Af1H-NMas 25 °C . In CD sas 25 °C . 2D DOSYspectrum .2C4)4 and r(G4C2)4 with samples of individual RNA oligonucleotides on 20% native PAGE at pH 6.0 and 5 °C reveals a slow migrating band at approximately 40 bp, which corresponds to heterodimer (Comparison of equimolar mixture of r(Gerodimer . Low-int2C4)4 and r(G4C2)4 was exposed to low pH and to the presence of K+ ions that could induce structural changes leading to formation of i-motifs and G-quadruplexes, respectively. However, lowering of pH did not result in any significant difference of imino signals in 1H-NMR spectrum 4 forms a homodimer with 16 Watson-Crick G-C base pairs. Homodimer was previously observed for closely related oligonucleotide r((C4G2)3C4) for which the crystal structure revealed 12 Watson-Crick G-C base pairs and 6 hemi-protonated C+-C base pairs [+-C base pairs and CCCG loop. This process was also observed in the presence of 10% w/v PEG, which was used to simulate molecular crowding conditions. The formation of hairpin adopted by r(C4G2)4 was previously proposed only through data obtained with CD spectra [1H-NMR spectra of r(G2C4)4 at pH 4.5 and 5 °C reveal the presence of imino signal at δ 15.75 ppm. According to its chemical shift value, this signal could correspond to imino protons of hemi-protonated C+-C base pairs within i-motif.Here we have shown that sense and antisense RNA oligonucleotides containing four hexanucleotide repeats of pathological repeat expansion of se pairs . Lowerin spectra . Althoug4C2)4 forms two structures, homodimer and hairpin, at pH 6.0 and 25 °C. Their equilibrium populations are temperature-dependent. Lowering the temperature favors the formation of homodimer and stabilization of its inherent G-G base pairs in N1 carbonyl symmetric geometry. Both structures are stable within narrow pH range between 5.0 and 6.0. While further decrease in pH leads to disappearance of all imino signals, increase to pH 7.0 favors homodimer. In contrast to cytosine-rich antisense oligonucleotide r(G2C4)4, guanine-rich sense oligonucleotide r(G4C2)4 has the potential to form secondary structures called G-quadruplexes. Several studies have shown that G-quadruplexes adopted by pathological RNA molecules influence many essential cell processes such as translation, splicing, nuclear transport and function of RNA-binding proteins [4C2)4 was observed in the presence of K+ ions, which are the most abundant in cells. Even in the presence of 100 mM K+ ions for more than one week, r(G4C2)4 does not form G-quadruplexes. Only after annealing, during which homodimer and hairpin are almost completely unfolded, a group of new signals corresponding to imino protons of guanines involved in G-quartets appears at 25 °C, thus indicating the presence of G-quadruplexes. Latter observations are in agreement with recent spectroscopic data combined with chemical and enzymatic probing of r(G4C2) repeat expansion, which together suggest temperature-dependent equilibrium between stable hairpins formed at 37 °C and G-quadruplexes adopted at higher annealing temperatures [4C2)4 in the presence of 200 mM K+ ions at pH 7.4 were observed previously [1H-NMR and CD spectroscopy together with results from UV melting experiments imply that oligonucleotide r((G4C2)3GGGGC) in the presence of 40 mM KCl forms a unimolecular four-stacked parallel-stranded G-quadruplex with propeller loops [4C2GGGG) at 100 mM K+ ions adopt a dimeric three-stacked parallel-stranded G-quadruplex with propeller loops [Previous studies of RNA dimers showed that G-G base pairs are the most stable compared to other mismatches . Howeverproteins ,25,26,27proteins . To appreratures . Diffusieviously . Additioer loops . FurtherC9orf72 gene occurs on both DNA strands leading to production of sense and antisense RNA transcripts, which are simultaneously present in the nucleus. Moreover, RNA foci of sense and antisense RNA transcripts appear to colocalize meaning that the presence of complementary strand could influence the structures formed independently by individual RNA transcripts [1H-NMR and CD spectra as well as PAGE indicate that sense and antisense RNA oligonucleotides form a heterodimer, while structures adopted by individual RNA oligonucleotide are still present as minor species. Exposure of heterodimer to physiological concentration of K+ ions as well as its annealing did not lead to G-quadruplex formation. In both cases, additional G-C base pairs within the heterodimer were stabilized, which implies that heterodimer is the favorable structure under these conditions. When equimolar mixture of DNA oligonucleotides d(G2C4)8 and d(G4C2)8 in solution with 100 mM K+ and low pH was exposed to PEG, signals of imino protons involved in G-quartets, G-C base pairs and hemi-protonated C+-C base pairs within i-motifs were observed [2C4)4 and r(G4C2)4 does not undergo structural changes at pH 4.5 and formation of hemi-protonated C+-C base pairs associated with i-motif. Even in the presence of 100 mM K+ ions at pH 4.5, the heterodimer maintained its structure. The effect of low pH and K+ ions on heterodimer was also observed in the presence of PEG. While 100 mM K+ ion concentration in the presence of 10% w/v PEG at pH 7.0 did not promote G-quadruplex formation, the reduction of pH to 4.5 under these conditions led to destabilization of certain G-C base pairs and most likely stabilization of N1-carbonyl G-G base pairs resulting in formation of heterodimer with one base pair shift.Transcription of the nscripts ,31. In t4C2 repeat RNA G-quadruplexes, which ultimately prolonged the life expectancy of ALS/FTLD fruit fly model [4C2)8 and porphyrin TMPyP4 has shown that TMPyP4 distorts the formation of G-quadruplex, which prevents binding of proteins associated with ALS such as hnRNPA1 [C9orf72 repeat expansion associated with ALS and FTLD demands further comprehensive studies as structures adopted by RNA transcripts act not only as important promoters of RNA toxicity-dependent mechanism but also present promising therapeutic targets in ALS, FTLD and other repeat-associated neurodegenerative diseases.Finally, described RNA structures and dynamic equilibria they are involved in could represent potential drug targets for modulation of DPRs and RNA toxicity-dependent mechanism. Structures of sense and antisense RNA transcripts could play a key role in translation where the production of DPRs could be initiated or repressed through formation of protein-binding hairpins, i-motifs and G-quadruplexes . Recentlly model . In addi hnRNPA1 . Self-di hnRNPA1 ,34. ASOs hnRNPA1 ,36 In on hnRNPA1 . Althoug2C4)4 and r(G4C2)4 were purchased from Eurogentec . After addition of 5 mL of 2 M LiCl samples were heated to 90 °C for 5 min. Samples were extensively dialyzed against autoclaved H2O through four 30 min cycles and then concentrated using an Amicon ultrafilter . Following the overnight lyophilization, samples were diluted in 90% of autoclaved H2O and 10% of 2H2O. The concentrations of the 300 μL NMR samples were 0.9, 0.7 and 0.3 mM for r(G2C4)4 and 1.1, 0.7, 0.3, 0.2, 0.1 and 0.05 mM for r(G4C2)4. In the case of equimolar mixing experiments, equimolar quantities of r(G2C4)4 and r(G4C2)4 were diluted to final oligonucleotide concentration of 0.2 mM per sense and antisense strand. Samples of r(G4C2)4 and mixed RNA oligonucleotides were also heated to 90 °C for 5 min prior to annealing. For molecular crowding experiments, sample of r(G2C4)4 and mixed RNA oligonucleotides were diluted in 10% w/v PEG (8000 MW)  in autoclaved H2O to 0.3 and 0.2 mM oligonucleotide concentration per strand, respectively. The pH of samples was adjusted by the addition of 0.1 mM LiOH or HCl.Reverse-phase HPLC purified RNA oligonucleotides r(G2O was used for baseline correction. For CD spectroscopy the sample of r(G2C4)4 was measured immediately after dilution in autoclaved H2O at 5, 25, and 37 °C, at pH 7.0. Measurements at different pHs were performed in autoclaved H2O at 25 °C and pH 4.5, 5.0, 5.5, 6.0, 6.5 and 7.0. The pH of samples was adjusted by the addition of 0.1 mM LiOH or HCl. CD spectra of equimolar mixture of r(G2C4)4 and r(G4C2)4 in autoclaved H2O before and after annealing were recorded at 25 °C. CD measurements were carried out on a sample of r(G2C4)4 and mixed RNA oligonucleotides diluted to concentration of 100 μM per sense and antisense strand.CD spectra were acquired on Applied Photophysics Chirascan CD spectrometer using a 0.1 cm path length quartz cell. The wavelength was varied from 200 to 320 nm with 0.5 nm per step. Time-per-point was set to 0.5 s. A blank containing only autoclaved H2C4)4 and r(G4C2)4 were performed on a Varian Cary 100 Bio UV-VIS spectrometer (Varian Inc.) equipped with a thermoelectric temperature controller. UV melting experiments were performed on samples diluted to 2.6, 3.0, 7.7, 9.3 and 20 μM oligonucleotide concentration per strand in 20 mM lithium cacodylate buffer at pH 5.0 and 7.0. Each sample was placed in 1, 0.5 or 0.1 cm path-length quartz cell. Mineral oil and a tightly fixed cuvette cap were used to seal the quartz cells in order to prevent evaporation and sample loss due to high temperatures. To prevent condensation at lower temperatures a stream of nitrogen was applied throughout the measurements. Folding/unfolding processes were followed between 20 and 90 °C by measuring absorbance at 260 nm. Data were acquired using scan rate of 0.1 °C min−1. Temperature of half transition (T1/2) for homodimer was determined using first derivative method, while temperature of half transition for hairpin was estimated from obtained melting curves.Melting experiments of r(G2C4)4, r(G4C2)4 and equimolar mixture of r(G2C4)4 and r(G4C2)4 was performed on 20% polyacrylamide gel (5 °C at 100 V) in 1×TBE (pH 6.0) buffer. Samples for native PAGE were prepared by mixing 5 or 10 μL of stock or diluted RNA oligonucleotide solution, 5 μL of loading buffer 15% Ficoll and diluted to 20 μL with autoclaved H2O. The approximate band size was determined by using the GeneRuler Ultra Low range DNA Ladder . The samples were treated at room temperature overnight before loading, except for the equimolar mixture sample, which was heated to 90 °C and cooled slowly at room temperature prior to loading. Following the overnight electrophoresis, the gel was stained with Stains-All gel stain solution (Sigma Aldrich).Native PAGE of r in autoclaved H2O. In experiments with K+ ions 3M KCl was diluted into the sample up to the desired concentration of K+ ions. 1D and 2D NMR spectra were processed and analysed using VNMRJ (Varian Inc.), MestReNova and Sparky (UCSF) software. All NMR spectra were acquired on 600 MHz NMR spectrometer unless stated otherwise. DSS  was used as external reference in all NMR spectra.NMR spectra were obtained with Agilent Technologies VNMRS 800 MHz and DD2 600 MHz NMR spectrometers equipped with triple resonance cold probe. Standard 1D 4C2)4 sense and cytosine rich r(G2C4)4 antisense RNA oligonucleotides associated with ALS and FTLD. Under conditions approaching physiological relevance, both sense and antisense RNA oligonucleotides form dimers and hairpins. Equilibrium of structures adopted by antisense RNA oligonucleotides is pH-dependent, hence lowering the pH favours hairpin formation. On the other hand, equilibrium of structures adopted by sense RNA oligonucleotides is temperature-dependent owing to prevalent formation of dimer at lower temperatures. Furthermore, sense RNA oligonucleotides form structurally different G-quadruplexes in the presence of KCl and antisense RNA oligonucleotides indicate i-motif formation at low pH and temperature. Simultaneous presence of both RNA oligonucleotides revealed formation of heterodimers although structures adopted by individual RNA oligonucleotides seem to persist in the minority. Adopted structures of pathological RNA oligonucleotides could play a key role in sequestering RNA binding proteins and, therefore, represent an important biological target of drug design in the combat against ALS and FTLD.Exploiting NMR and complementary methods allowed determination of structures adopted by guanine rich r(G


Spodoptera litura have not been determined. In this study, we identified two CHS genes, SlCHS1 and SlCHS2, which encode proteins with 1565 and 1520 amino acid residues, respectively. Transcriptional analysis suggested that SlCHS1 has a high expression level in the integument whereas SlCHS2 showed the highest expression level in the midgut. During S. litura growth and development, SlCHS1 and SlCHS2 were both predominantly expressed in the fourth-instar larval stage. In addition, the expression of SlCHS1 and SlCHS2 could be induced by 20-hydroxyecdysone (20E). Silencing of SlCHS1 by RNA interference significantly inhibited the pupation and molting of S. litura larvae (RNAi), while knockdown of SlCHS2 had no significant effects on the S. litura phenotype. These results may provide a new molecular target for control of S. litura.Chitin is one the main components of the insect cuticle, and chitin synthase (CHS) is an important enzyme required for chitin formation. CHS has been characterized in various insect species, but the structure and biochemical properties in  N-acetyl-d-glucosamine (GlcNAc) and is the second most abundant biological polymer after cellulose in nature. Chitin has been detected in a wide variety of organisms, ranging from the simplest algae to fungi, nematodes and arthropods [N-acetyl glucosamine subunits by chitin synthase to produce chitin microfibrils [N-acetyltransferase (GNPNA), phosphoglucosamine mutase (PAGM) and UDP-N-acetylglucosamine pyrophosphorylase (UAP). The last step is the chitin biosynthetic pathway catalyzed by chitin synthase (CHS), which can catalyze the transfer of sugar moieties from activated sugar donors to specific acceptors [Chitin is a linear amino polysaccharide polymer made up of β-1,4-thropods . In insethropods ,4. The bthropods . Howeverofibrils . In addicceptors .CHS cDNA sequences have been characterized in several insect species, including Drosophila melanogaster [Manduca sexta [Tribolium castaneum [Locusta migratoria [Spodopetra frugiperda [Diaphorina citri [CHS1 is exclusively expressed in the epidermis underlying the cuticular exoskeleton and related ectodermal cells such as tracheal cells, while CHS2 is highly expressed in the midgut, and its coding enzyme is responsible for the synthesis of PM-associated chitin [Chitin synthase  is an integral membrane glycosyltransferase that is essential for chitin chain polymerization and deposition in insect chitinous structures . CHS is nogaster , Manducaca sexta , Triboligratoria , Spodopeugiperda , and Diana citri . In insena citri . CHS1 isCHS in different species. Chen et al. revealed that the cuticle of Spodoptera exigua larvae was disordered and that the epithelial walls did not expand uniformly after silencing CHSA [Leptinotarsa decemlineata, knockdown of CHSAa, CHSAb and CHSB in second- and fourth-instar larvae lowered chitin contents in whole body and integument samples and thinned tracheal taenidia [Spodoptera litura. RNAi refers to highly conserved cellular mechanisms in which Argonaute (Ago) family proteins bind small RNAs to trigger the degradation of longer RNAs through the sequence complementarity [Caenorhabditis elegans by Fire et al., gene knockdown through RNAi induced by double stranded RNA (dsRNA) has been widely applied for the management of insect pests [In recent years, RNA interference (RNAi) has been widely used to research the functions of ing CHSA . In Lepttaenidia . Howeverentarity . Deliverentarity ,21. Sincct pests .Spodoptera litura (Lepidoptera: Noctuidae) is an important herbivorous pest responsible for widespread economic damage to numerous field vegetables and ornamental plants in tropical and subtropical regions [S. litura is primarily achieved through the application of various chemical insecticides. However, S. litura has evolved high resistance to every class of pesticides used against it [S. litura shows a high level of resistance to spinosad, indoxacarb, and methoxyfenozide [S. litura have developed resistance to multiple insecticides in South Asia, including chlorpyrifos, β-cypermethrin and methomyl [S. litura. Chitin is found in insect exoskeletons, but is not found in vertebrates [S. litura. regions . At presainst it . Shad etfenozide . In addimethomyl . Therefotebrates . TherefoSlCHS1 and SlCHS2) were identified from the genome database of S. litura, and their spatial and temporal expression profiles were analyzed. In addition, the expression levels of SlCHS1 and SlCHS2 can be induced by 20E. Furthermore, silencing of SlCHS1 influences S. litura larvae pupation and molting. However, silencing of SlCHS2 has no significant influence for molting of S. litura. Herein we explore whether these genes can provide useful information on CHSs as targets for the identification of novel insecticides?In the current study, two cDNAs encoding chitin synthase , Gannan Normal University, Ganzhou, China. Larvae were reared in culture dishes on an artificial diet at 27 °C and 70%–75% relative humidity, with a photoperiod of 12 h light and 12 h dark, until they became adult moths. The main components of the artificial diet include corn starch, soybean flour, agar, yeast powder, sorbic acid and cholesterol. All male and female adults were placed in a plastic case, and moderate hydromel was added to keep the plural adults alive. The produced eggs were reared based on the above conditions. S. litura were collected at different developmental stages, including larvae, pupae and adults. Moreover, the first day of sixth-instar larvae were dissected to obtain various tissues, including the integument, head, Malpighian tubule, fat body and midgut. The midgut was cleaned using precooled DEPC-water to remove the remaining food debris, and stored at −80 °C.20E treatment was performed according to a previous report with some modifications . In brieSlCHS1 and SlCHS2, S. litura total RNA was extracted from different tissues of sixth-instar larvae  and at different developmental stages  using the animal tissue total RNA kit . RNA concentration and purity were assayed using a NanoDrop2000 spectrophotometer  at absorbance ratios of A260/230 and A260/280. The integrity of total RNA was confirmed using standard agarose gel electrophoresis with ethidium bromide (EB) staining. Total RNA was reverse-transcribed in a 20 μL reaction system using a Fast 1st strand cDNA Synthesis kit (with gDNase) (Simgen) according to the manufacturer’s instructions. In brief, 2.0 μL of 5 × gRNA buffer and 1 μg of total RNA were mixed, and then RNase-free water was added to reach 10 μL, which was then incubated at 42 °C for 3 min. Afterward, 4 μL of 5 × RT buffer and 2 μL of RT enzyme mix were added and incubated at 95 °C for 3 min. The cDNA was stored at −20 °C for later use.To analyze the spatiotemporal expression patterns of S. litura, a TBLASTIN search of the S. litura genome database (https://www.ncbi.nlm.nih.gov/genome/14271?genome_assembly_id = 350050) was performed using the amino acid sequences of D. melanogaster CHS1 (NM_079509.3), D. melanogaster CHS2 (NM_079485.4), M. sexta CHS1 (AY062175.2) and M. sexta CHS2 (AY821560.1) as queries. This resulted in the identification of two cDNA sequence that we have named as SlCHS1 and SlCHS2. To confirm the correctness of the two candidate CHS sequences, reverse transcription PCR (RT-PCR) was carried out to amplify the full-length ORF sequence using gene-specific primers  was predicted according to the ORF finder tool (https://www.ncbi.nlm.nih.gov/orffinfer/). The molecular weight (MW) and isogenic point (pI) of SlCHS1 and SlCHS2 were calculated using ExPASy (http://web.expasy.org/compute_pi). The signal peptides of SlCHS1 and SlCHS2 were predicted using SignalP 4.1 Server . The functional domain was predicted by using SMART software (http://smart.embl-heidelberg.de/). The membrane-spanning domain was predicted by TMHMM Server v. 2.0 (http://www.cbs.dtu.dk/services/TMHMM/). The phylogenetic tree was constructed with MEGA 7.0 software using the neighbor-joining method with 1000-fold bootstrap resampling. Protein sequences from different insect species were obtained from GenBank (http://www.ncbi.nlm.nih.gov/) and used in the phylogenetic analysis . Relative expression levels were calculated by using the 2−∆∆Ct method. There were three biological replicates and three technique replicates for each sample. The reference gene chosen for analysis of SlCHS1 and SlCHS2 in different tissues and different developmental stages was glyceraldehyde-3-phosphate dehydrogenase (GAPDH). All data were analyzed using a one-way analysis of variance (ANOVA) and Tukey’s test.RT-qPCR was performed to analyze the relative expression levels of software . The 20-SlCHS1 and SlCHS2 was synthesized using the T7 RioMAX Express RNAi System  based on the manufacturer’s instructions. The forward and reverse primers were designed to amplify 439 bp and 421 bp for SlCHS1 and SlCHS2, respectively , and the midgut and integument were collected at 24 h and stored at -80 °C. After 24 h, all live insects were collected to isolate total RNA and synthesize cDNA. The effect of dsSlCHS1 and dsSlCHS2 on gene expression was evaluated by RT-qPCR. A total of three biological replicates were used for each experiment. All data were analyzed using ANOVA and Tukey’s test.dsRNA targeting ectively . GFP dsRSlCHS1 and SlCHS2 were identified from S. litura. The cDNA sequence of SlCHS1 (GenBank accession number: XM_022964624.1) contains an ORF of 4698 bp encoding a protein of 1565 amino acid residues with a predicted MW of 178.1 kDa and a pI of 6.56. SlCHS2 (GenBank accession number: XM_022821184.1) contains an ORF of 4563 bp encoding a protein of 1520 amino acid residues with a predicted MW of 174.1 kDa and a pI of 5.83. In terms of protein structure, SlCHS1 contains three domains, including an N-terminal domain (residues 1-561) with nine transmembrane helices, a putative catalytic domain (residues 562-900) and a C-terminal domain (residues 901-1565) with an additional seven transmembrane helices  reduced after injection of 500 ng/μL dsRNA at 24 h compared with the dsGFP control treatment -cytochrome P450 reductases (SlCPRs) increased larval mortality by 34.6% (LC15 dose) and 53.5% (LC50 dose) by RNAi [S. litura cytochrome P450 monooxygenases 321B1 (SlCYP321B1) further increased mortality by 25.6% and 38.9% when fifth-instar larvae were exposed to chlorpyrifos and β-cypermethrin [S. litura genome database. The structural domain analysis suggested that both SlCHS1 and SlCHS2 contain 16 transmembrane helices  transcript was preferentially expressed in the epidermis [SlCHS1 was confirmed in the integument and midgut. Therefore, we speculated that SlCHS1 may play a critical role in the process of cuticle formation. The insect midgut epithelium is commonly lined by an invertebrate-unique structure, the peritrophic matrix (PM), which facilitates the digestion of food and the protection of the gut epithelium [SlCHS2 may be involved in chitin formation in PM. In T. castaneum, the CHSB gene is the major or sole contributor to PM chitin synthesis [SlCHS1 and SlCHS2 showed high expression in the fourth-instar larval stage /ultraspiracle (USP) [SlCHS1 and SlCHS2 expression levels can be induced after 20E treatment  . In insele (USP) ,44. In treatment . In a pre to 20E . In Bactvariants . Based oS. litura larvae were used for RNAi analysis. The results showed that the transformation of larval–pupae and pupae–adults was disrupted after silencing of SlCHS1, while S. litura larvae showed no obvious change after silencing of SlCHS2 , while TcCHS2-specific RNAi had no effect on metamorphosis [S. exigua, the cuticle was disordered, and the epithelial walls of larval tracheae did not expand uniformly after injection of dsSeCHSA [SlCHS1 was mainly involved in the formation of epidermal structure and that SlCHS2 was associated with PM formation.RNAi has already proven its usefulness in functional genomics research in insects, but it also has considerable potential for the control of pest insects . For thef SlCHS2 . In T. corphosis . In S. edsSeCHSA . These rSlCHS1 and SlCHS2 were identified based on genome database of S. litura. RT-qPCR analysis suggested that SlCHS1 and SlCHS2 were highly expressed in the integument and midgut, respectively. Developmental stage expression analysis showed that both SlCHS1 and SlCHS2 were predominantly expressed in the fourth-instar larval stage. In addition, the expression of SlCHS1 and SlCHS2 could be induced by 20E. Furthermore, silencing of SlCHS1 significantly inhibited the pupation and molting of S. litura larvae by RNAi. These results suggest that SlCHS1 and SlCHS2 were involved in the formation of chitin in integument and peritrophic matrix (PM), respectively. In our following study, we will design some inhibitors directly targeted at S. litura chitin synthases and combined with RNAi to control S. litura.Two cDNA sequences of


We will highlight the latest insights into the mechanisms of how these posttranscriptional modifications influence tumour development, maintenance, and progression. Finally, we will summarize the latest advances on the development of small molecule inhibitors that target specific writers or erasers to rewind the epitranscriptome of a cancer cell and their therapeutic potential.RNA modifications have recently emerged as critical posttranscriptional regulators of gene expression programmes. Significant advances have been made in understanding the functional role of RNA modifications in regulating coding and non-coding RNA processing and function, which in turn thoroughly shape distinct gene expression programmes. They affect diverse biological processes, and the correct deposition of many of these modifications is required for normal development. Alterations of their deposition are implicated in several diseases, including cancer. In this Review, we focus on the occurrence of N For example, TET2 and ALKBH1 mutations have been associated to lymphoblastic and myeloid leukaemia [5C in RNA too, raising the question as to whether defects of RNA hydroxymethylation could be also linked to cancer.5-hydroxymethylation writers have been found altered in cancer too. eukaemia –212. TETeukaemia , TET2 iseukaemia , and TETeukaemia . While mTo date no specific cytosine-5 RNA methyltransferase inhibitor has been developed. Yet, given that several drugs designed to interfere with cytosine-5 methylation of DNA rely on the use of chemical analogues of cytosine, they may well interfere with RNA methylation , 216. InIn line with the notion that inhibition of tRNA methylases may contribute to chemotherapy resistance, silencing of other tRNA methyltransferases such as the 7-guanosine methylase METTL1 which also methylates tRNAs at the variable loop of several tRNAs has shown to increase sensitivity of cancer cells to 5’FU .Pseudouridine (Ψ), the C5-glycoside isomer of uridine, was the first posttranscriptional modification discovered and is one of the most abundant modifications of RNA , 219. DePseudouridine was initially detected on yeast tRNAs and rRNA , 225, an53UUCNANNC60 sequence in most tRNAs, and on the three-dimensional structure of the TΨC loop [Pseudouridylation can be achieved through two distinct mechanisms, namely RNA-independent and RNA-dependent pseudouridylation. The RNA-dependent mechanism relies on RNA–protein complexes known as box H/ACA small ribonucleoproteins (snoRNPs), which consist of a box H/ACA snoRNA and four core proteins; dyskerin , non-histone protein 2 (Nhp2), nucleolar protein 10 (Nop10) and glycine-arginine-rich protein 1 (Gar1). In H/ACA ncRNA is responsible for substrate recognition through complementary base-pairing interactions with the RNA substrate, and the catalytic activity is provided by DKC1 . In addition, the study showed that the presence of Ψ at position 8 of this novel class of small ncRNAs was required for efficient biding to polyadenylate-binding protein 1 (PABPC1), an integral component of the 5′ cap translation initiation complex, resulting in displacement of PABPC1 from capped mRNAS and reprograming translation [In tRNAs, pseudouridylation is found at the anticodon stem and loop, at the D stem and in a conserved at the Ψ loop, position 55, all of which stabilize its tertiary structure and are important for codon–anticodon base-pairing  Fig. 4)4). Novelnslation . Thus, tThe role of Ψ on other RNAs remains still poorly understood. For instance, TERC is also pseudouridylated , where tIn sum, Ψ deposition may confer different molecular properties to the modified RNAs, which changes their fate or activity. While most recent studies are focused on Ψ modifications in mRNA, yet its role on mRNA is still unclear. Thus, further studies will be required to fully understand the molecular role of Ψ on mRNAs. Ψ is highly abundant on other RNA types such as snRNA, rRNA or tRNA, and whose role has been studied for many years. Yet, recent findings are still revealing novel and unexpected functions, such as the role of Ψ on tRNA fragments, and thus indicating that other functions are still to be discovered.DKC1 inactivating mutations [1acp3Ψ modification of rRNA found in colorectal carcinoma, which affects the direct interaction of tRNA with ribosomal P site, altering and deregulating translation in cancer cells [One of the most studied disease linked to defects in pseudouridylation is the X-linked Dyskeratosis Congenita (X-DC), associated to utations . Dyskeriutations . X-DC isutations . Patientutations . DKC1 alutations , breast utations , 257, coutations , 259, luutations , 260, prutations , head anutations , 263, glutations , HCCs [2utations , and speutations , 267 or utations –270  too [6A sites, where the cytidine deaminase APOBEC1 is fused to the m6A-binding YTH domain [5C detection, bisulphite-RNA sequencing is the gold standard, yet the reproducibility is low, especially in low abundant and unstable RNA such as mRNAs [EpiNano algorithms which identifies m6A from RNA reads with an overall accuracy of ~90%, open new avenues to explore additional RNA modifications in the future [To achieve this, we first need to develop system-wide methods and tools for rapid and quantitative detection of RNA modifications. Most of the stablished methods rely on next-generation sequencing and, as such, they are typically blind to nucleotide modifications. Consequently, indirect methods are required that are based on immunoprecipitation techniques using specific antibodies, or enzymatic methods and chemical labelling and unique base-modification properties of RNA pairing . These m6Am) too . In fact6Am) too . To overH domain . Yet, thas mRNAs . The laras mRNAs . Yet, caas mRNAs , 156. Reas mRNAs , 26, 221as mRNAs . RNA modas mRNAs . Or by cas mRNAs . Althougas mRNAs , yet cure future .HIF1A mRNA is stabilized by m6A deposition, while in melanoma cells high levels of HIF1α protein are maintained by modifications at the U34 wobble position of tRNAs [Nsun2 deletion are caused by decrease methylation of tRNAs, or other RNA substrates may as well contribute to the observed phenotype [The dynamic expression patterns of writer, reader and eraser proteins complicate the identification of the precise functional consequences of aberrant deposition of modifications on RNA metabolism. Thus uncovering the complete repertoire of cellular RNA substrates and the writers, readers, and erasers will unveil how the intricate network of epitranscriptomic events can converge into similar cellular processes and showing how their unbalanced deposition may lead to pathologies. For example, of tRNAs , 143. Fuof tRNAs . In addihenotype , 182.5C, leads to perturbed accumulation of tRNA fragments, a novel class of functional ncRNAs whose role is associated with aberrant protein synthesis rates and reprograming of the translational machinery in tissue and cancer stem cell populations [We start to appreciate the wide range of functional consequences of the aberrant deposition of RNA modifications in human diseases including cancer. For example, aberrant deposition of tRNA modifications, including Ψ and mulations , 230. Thulations  and highulations . Yet, ouulations . More imulations .6A, m5C or Ψ to cancer. The extensive number of RNA modifications that constitute the epitranscriptome and the reversible nature of epitranscriptomic aberrations hold promise to the emergence of a promising field of epitranscriptomic therapy, which is already making progress with the recent development of effective inhibitors against m6A modifying proteins. In the last years, several seminal studies have shed light onto the diagnostic and therapeutic potential of targeting the cancer epitranscriptomic code [6A. The inconsistent evidence may reflect the precise functional outcome of the RNA modification on each modified RNA type, their crosstalk with other active signalling processes and the dynamic nature of the epitranscriptome. Identifying accurate epitranscriptome biomarkers and defining the oncogenic or tumour suppressive effect of a given aberrant modification within a specific cellular context, cell type, cellular proliferative capacity or tumour microenvironment will guide finding the exact molecular targets to develop selective and effective therapies for a given tumour type.Recent advancements in the rapidly evolving field of epitranscriptomics have linked the reprogramming of components of the epitranscriptomic machinery including writers, erasers or readers of the mmic code , 33, 317mic code . In addi6A erasers, yet none of them have reached clinical stages [5C methylases, azacytidine and decitabine (5-aza2′-deoxycytidine), which are cytidine analogues and can inhibit any cytosine-5 methylase, have been approved for clinical use in haematological malignancies [Though aberrant expression of several methylases and Ψ synthetases has now been described in cancer, it remains unclear whether they could be efficient targets for cancer therapy. Thus, the precise contribution of methylases and Ψ synthetases to tumour initiation, growth, metastasis and resistance need to be further investigated. It remains unclear the RNA target specificity of each enzyme and how specific modified targets can contribute to the malignant phenotype. In addition, little is known on the dynamics of the deposition of these modifications, their erasers and how the binding to their readers influence the RNA metabolism and tumour cell fate. Yet, the availability of the 3D structure of most of these enzymes and the fact that potent and selective inhibitors have been found , it is rl stages . For m5Cgnancies . However6A, m5C or Ψ regulate cell survival to stress in many settings and stem cell functions, targeting them represent a very promising opportunity to specifically target these cells populations and reduce chemoresistance and recurrence. Whether other epitranscriptomics changes can lead to drug resistance is not yet understood. This clearly opens the opportunity to explore novel avenues to develop diagnostic tools based on epitranscriptomic signatures that allow for better patient stratification. In addition, combinatorial therapeutic strategies with the potential to re-establish the normal epitranscriptomic landscape or inhibit survival signalling pathways are promising strategies to specifically eliminate cancer stem cells. Combinatorial therapies that target independent pathways may be a better option as the possibilities for the development of tumour drug resistance may be more limited. Understanding the machineries and factors that introduce, remove, or read RNA modifications will allow the development of novel drugs with pharmaceutical value, not only for cancer but other complex human pathologies that have been linked to aberrant deposition of RNA modifications such as diabetes, neurological, immune and mitochondrial-linked disorders [Treatment failure in certain settings has been attributed to the presence of sub-populations of cancer stem cells or persister cells which are intrinsically resistant to many therapeutic approaches . Given tisorders .


Although primary androgen deprivation therapy resulted in tumour regression, unfortunately, majority of prostate cancer progress to a lethal castration‐resistant prostate cancer, finally die to metastasis. The mutual feedback between AKT and AR pathways plays a vital role in the progression and metastasis of prostate cancer. Therefore, the treatment of a single factor will eventually inevitably lead to failure. Therefore, better understanding of the molecular mechanisms underlying metastasis is critical to the development of new and more effective therapeutic agents. In this study, we created prostate cancer CWR22rv1 cells with the double knockout of Akt1 and Akt2 genes through CRISPR/Cas9 method to investigate the effect of Akt in metastasis of prostate cancer. It was found that knockout of Akt1/2 resulted in markedly reduced metastasis in vitro and in vivo, and appeared to interfere AR nuclear translocation through regulating downstream regulatory factor, FOXO proteins. It suggests that some downstream regulatory factors in the AKT and AR interaction network play a vital role in prostate cancer metastasis and are potential targeting molecules for prostate cancer metastasis treatment. The knockout studies of mouse embryos have shown that simultaneous knockout of Akt1 and Akt2 can cause embryo death. Although Akt1 knockout alone does not kill the embryo, it suppresses tumorigenesis in Pten ± mice.Although previous studies carried out in various species and cell types have provided the foundation for our current understanding of the Akt and its associated signal pathway network, the functions and mediators that actually act to control CaP development and progression are not yet well defined. At present, the CRISPR/Cas9 method has rapidly swept the entire scientific research field and has become a revolutionary technology for DNA mutation and editing. In this study, we knockout Akt1 and Akt2 genes in CaP CWR22rv1 cells to explore the importance of Akt in CaP metastasis and its regulated downstream factors in order to find specific targets.22.12.Human prostate cancer cell lines of CWR22Rv1 were purchased from the Chinese Academy of Cell Bank. CWR22Rv1 cells were cultured in RPMI 1640 media supplemented with 10% FBS and incubated at 37°C in a humidified incubator containing 5% CO2.2Use software to design hAKT‐gRNA sequences. hAKT1‐gRNA‐F1:CACCGAAGGTGCGTTCGATGACAG and hAKT1‐gRNA‐R1:AAACCTGTCATCGAA CGCACCTTC; hAKT2‐gRNA‐F1: CACCGTCTCGTCTGGAGAATCCACG and hAKT2‐gRNA‐R1: AAACCGTGGATTCTCCAGACGAGAC, and then insert into the px300 plasmid to construct the px300‐HAkt‐gRNA plasmid. The CaP CWR22rv1 cells were simultaneously transfected with px330‐AKT1, px330‐AKT2 and plko plasmid with puromycin tag, then puromycin screened, and monoclonal cells were isolated and sequenced.2.36 Akt1 and Akt2 knockout (marked as Akt1/2 DKO) CWR22rv1 cells mixed Matrigel. The viability of the cells was >90% as determined by trypan blue exclusion. Tumour volume (cubic millimetres) was measured using a calliper, applying the formula [volume = length × width × width/2] for approximating the volume of a spheroid. Tumour burden per mouse was calculated by accumulating the tumour volume thrice weekly. After the tumour volume is >4 cm3, the animals were killed; lymph node and lungs were examined for visible metastatic foci after H&E staining. All animal protocols were approved by the Institutional Animal Care and Use Committee of Shenzhen Biochemical Institute.3‐week‐old BALB/c nude mice  were injected s.c. into the flank regions with 2 × 102.4Cell lysates were generated, and Western blotting with Akt1, Akt2, FOXO1, FOXO3, FOXO4, AktpT308, FOXOpT24/32 and GAPDH antibodies were carried out as described previously.2.5Modified Matrigel‐based Boyden chamber assays were performed as previously described.2.6−ΔCt method was used to calculate the expression of genes.Total RNA was isolated using TRIzol Reagent (Sigma) and was reverse transcripted into cDNA using Revert Aid First Strand cDNA Synthesis Kit (Thermo Scientific). Real‐time PCR was performed using SYBR premix EX Taq (TaKaRa) and analysed with CFX96 Real‐Time System (Bio‐Rad). Real‐time primer sequences were listed in Table 2.7t test. All statistical analyses were performed by using SPSS 16.0 software program. P < .05 was considered statistically significant.Statistical significances between groups were determined by two‐tailed Student's 33.1To interrogate the importance of Akt in CaP metastasis, our study directly targeted to exon 3 of the Akt1 and Akt2 genes Figure A,B and w3.23, the mice were killed and the related organs were harvested and analysed for metastasis. None of the animals examined in this study were overt metastases in local draining lymph nodes, kidney and lung of the Akt1/2 DKO and control groups. Furthermore, micrometastases were evaluated with human Alu sequence by qPCR. Samples harvested from Akt1/2 DKO groups presented with significantly decreased incidence of metastases to lungs compared with the control group (P < .001)  could affect spontaneous metastasis in vivo in a subcutaneous xenograft model using CWR22rv1/Akt1/2 DKO cells and control cells in nude mice. When the primary tumours were grown in the mice exceeded 4 cm) Figure . These o) Figure .3.3In order to further explore the mechanism of Akt affecting CaP metastasis, we used RNA sequencing technology to analyse the effect of Akt1/2 knockout on transcriptional RNA of CWR22rv1 cells. This analysis identified 2156 genes whose expression changed ≥ 1.5‐fold Figure , which aOf these, bioinformatic analysis and literature search revealed a bunch of genes are AR‐regulated genes Figure  and invo3.4As we know, after Akt is activated by phosphorylation at Thr308, it regulates cell proliferation, survival, motility and metastasis through phosphorylating a host of downstream mediators, including GSK3β, FOXOs and mTOR. To evaluate the effects of gene disruption on known Akt‐signalling pathway, we starved cells of serum overnight and then stimulated them with foetal bovine serum (FBS). Striking changes in the levels of phosphorylated Akt and FOXOs were observed after FBS was added to control cells Figure .To determine whether increased activity of FOXOs contributes to the decreased invasive capacity of Akt1/2 DKO CWR22rv1 cells, Akt1/2 DKO cells transfected with siRNA against FOXOs were tested for invasiveness. The results demonstrated that knockdown of FOXOs Figure  rescues 3.5In addition to transcription factor activity, FOXO can affect the function of other proteins through protein‐protein interactions, such as FOXO1 can directly bind to AR and inhibit its activity to affect the progression of prostate cancer,4In mammals, the FOXO family consists of four members , of which FOXO6 exists only in the brain, so the other three proteins are the major peripheral proteins.At present, the development of anti‐tumour drugs is mainly focused on inhibiting major tumour‐related genes, such as AR and Akt. Many inhibitors targeting these two genes have been developed and entered clinical validation,In short, it suggests that some downstream regulatory factors in the AKT and AR interaction network play a vital role in prostate cancer metastasis and are potential targeting molecules for prostate cancer metastasis treatment.The authors declare that there are no conflicts of interest.Bing Su: Conceptualization ; Writing‐original draft (lead). Lijuan Zhang: Investigation (supporting); Methodology (supporting). Wenfang Zhuang: Data curation (lead). Wei Zhang: Conceptualization (supporting); Writing‐original draft (supporting). Xiaofan Chen: Investigation (lead); Methodology (lead).Fig S1Click here for additional data file.Table S1Click here for additional data file.


RNA‐catalyzed RNA ligation is widely believed to be a key reaction for primordial biology. However, since typical chemical routes towards activating RNA substrates are incompatible with ribozyme catalysis, it remains unclear how prebiotic systems generated and sustained pools of activated building blocks needed to form increasingly larger and complex RNA. Herein, we demonstrate in situ activation of RNA substrates under reaction conditions amenable to catalysis by the hairpin ribozyme. We found that diamidophosphate (DAP) and imidazole drive the formation of 2′,3′‐cyclic phosphate RNA mono‐ and oligonucleotides from monophosphorylated precursors in frozen water‐ice. This long‐lived activation enables iterative enzymatic assembly of long RNAs. Our results provide a plausible scenario for the generation of higher‐energy substrates required to fuel ribozyme‐catalyzed RNA synthesis in the absence of a highly evolved metabolism. A plausible scenario for the prebiotic generation of activated RNA substrates under mild aqueous conditions is presented. Using water‐soluble diamidophosphate, in situ production of 2′,3′‐cyclic phosphate‐activated oligoribonucleotides and their subsequent ligation by a ribozyme can be achieved. Modern cells have evolved elaborate metabolic networks in mild aqueous conditions to ensure their self‐preservation and to sustain their pool of activated building blocks. In contrast, it remains unclear how analogous (re‐)activation of building blocks in primitive precursor systems could have been possible without access to sophisticated protein enzymes. Given a likely central role of RNA catalysts (ribozymes) during the Origin of Life,In the laboratory, RNA synthesis by ribozymes is achieved through the ligation of pre‐activated RNA substrates. For example, some artificial ribozymes obtained by in vitro selection utilize 5′‐triphosphate activation chemistry for phosphodiester formation.3, 4 TDiamidophosphate (DAP) was recently identified as a promising water‐stable and prebiotically plausible reagent to phosphorylate biological building blocks such as nucleic acids, amino acids, and lipid precursors.12, 132+, which are required for efficient >P formation under aqueous conditions, are incompatible with half‐lives of ribozymes in water.However, high levels of metal ions such as MgHere, we demonstrate RNA activation by DAP under conditions that are amenable to the catalytic activity of >P‐dependent ribozymes. In particular, we found that the concentrating and preserving environment of water‐ice enables efficient formation of >P‐activated RNA. The activated RNA can be used in situ for RNA ligation by derivatives of the naturally occurring hairpin ribozyme (HPz), which serves as a versatile model system for prebiotic RNA ligation.18, 1931P NMR . Among the three activators tested in this study, imidazole‐containing reactions were faster  and yielded the highest amounts of >Ps  with DAP and imidazole (or its derivatives 2‐amino‐ and 2‐methylimidazole) at −20 °C and monitored the reactions using ion‐exchange liquid chromatography . In solution, DAP reacts with phosphates to form the corresponding amidopyrophosphate. This intermediate forms the 2′,3′‐cyclophosphate in the presence of a 2′‐OH group, with the amidophosphate as the leaving group Scheme .[12] I Figures , S13–S16 Figures . After 7Having shown that DAP can be used as an efficient activation agent for N>P formation in frozen water‐ice systems, we sought to explore whether DAP can also activate oligonucleotides to fuel ribozyme‐catalyzed ligation reactions. Previous studies have shown that HPzs are capable of efficient >P‐dependent in‐ice ligation of RNA substrates.cis‐ligating version of the HPz  generated with ethyl‐3‐(3′‐dimethylaminopropyl)‐carbodiimide (EDC), with which circa 88 % self‐ligation was observed via urea‐PAGE in reaction conditions used in previous HPz in‐ice studies .sub‐P, the non‐activated 3′‐monophosphate RNA.To monitor the coupled activation–ligation reactions, we developed a reporter electrophoretic mobility shift assay (EMSA) based on a 2+, and imidazole for their potential to enable coupled activation–ligation reactions. We identified the inclusion of 5 mm DAP, 5 mm Mg2+, and 5 mm imidazole as optimal conditions thus far for the combined activation and ligation of the RNA substrate sub‐P , and used these conditions in all further experiments. We also noted an increased ligation yield at 50 mm DAP in the absence of imidazole. We presumed that the increased concentration of DAP, especially in eutectic ice, compensates for the absence of imidazole. However, increasing both DAP and imidazole concentrations to 50 mm resulted in a lower ligation yield compared to 5 mm . This result may be due to the high DAP and imidazole concentrations that reduce the concentration‐by‐freezing efficiency for the RNA components. The higher initial concentration of the two components reduces the amount of water‐ice necessary to reach the molal equilibrium concentrations of the unfrozen phase.Following the implementation of the reporter EMSA, we sought to test the efficacy of the coupled DAP activation–ligation reactions under various conditions. Specifically, we tested a range of concentrations of DAP, MgKa=6.952 group of DAP (pKa≈5.5).We then explored the pH dependency of the activation–ligation reaction. We speculated that a highly acidic environment may decrease the availability of the DAP‐activating agent imidazole pKa=6.95 and S22.−1; UMP, 12 g L−1; AMP, 8 g L−1; GMP, 8 g L−1)Ka values of the 2′‐OH group, between 13.22 and 13.47,We initially observed a considerable influence of the base moiety on DAP‐mediated phosphate activation for the four mononucleotides Figure . Therefom 2MI or 2AI instead of IMI  and found that ligation yields were considerably lower (approximately 25 % after 84 days) in the presence of 2AI or 2MI compared to IMI (approximately 55 %). The lower pKa of IMI  and 2‐aminoimidazole (2AI) are close analogues of imidazole (IMI) that are considered as superior 5′‐leaving groups for non‐enzymatic copying of RNA templates.33, 34Next, we examined the performance of DAP compared to the prebiotically implausible carbodiimide EDC, which is typically used to generate >Ps. Intriguingly, the long‐term kinetics of RNA activation under in situ ribozyme catalysis‐compatible conditions showed higher ligation yields with DAP compared to EDC Figure . While oFinally, we investigated whether DAP activation also allows multiple ligations to produce long RNAs, which is a prerequisite for the formation of more complex RNA molecules. To this end, we probed our one‐pot activation/ligation scheme for concatenation reactions of RNA oligomers  should be addressed to the authors.SupplementaryClick here for additional data file.


Arthropod-borne viruses are diverse pathogens and include the emerging virus chikungunya virus, which is associated with human disease. Through genetic screening, we found that the conserved RNA binding protein DDX56 is antiviral against chikungunya virus in insects and humans. DDX56 relocalizes from the nucleus to the cytoplasm, where it binds to a stem-loop in the viral genome and destabilizes incoming genomes. Thus, DDX56 is an evolutionarily conserved antiviral factor that controls alphavirus infection. Drosophila and human cells. Furthermore, we found that DDX56 also controls the emerging alphavirus chikungunya virus (CHIKV) through an interferon-independent mechanism. Using cross-linking immunoprecipitation (CLIP-Seq), we identified a predicted stem-loop on the viral genomic RNA bound by DDX56. Mechanistically, we found that DDX56 levels increase in the cytoplasm during CHIKV infection. In the cytoplasm, DDX56 impacts the earliest step in the viral replication cycle by binding and destabilizing the incoming viral genomic RNA, thereby attenuating infection. Thus, DDX56 is a conserved antiviral RNA binding protein that controls alphavirus infection.DEAD box RNA helicases regulate diverse facets of RNA biology. Proteins of this family carry out essential cellular functions, and emerging literature is revealing additional roles in immune defense. Using RNA interference screening, we identified an evolutionarily conserved antiviral role for the helicase DDX56 against the alphavirus Sindbis virus (SINV), a mosquito-transmitted pathogen that infects humans. Depletion of DDX56 enhanced infection in  Innate immune defenses rely on the recognition of invading pathogens by the host cell. In the case of RNA viruses, recognition is largely driven by RNA binding proteins that recognize foreign structures on viral RNAs . Key senDEAD box helicases recognize the phosphate backbone of RNA rather than the nucleotide bases, suggesting that they bind RNAs with a sequence-independent but structure-dependent mechanism . AlthougIn addition to the canonical cytoplasmic RNA sensors RIG-I and MDA5, emerging data suggest that many DEAD box helicases also have roles in viral RNA recognition and cellular antiviral responses. DDX1, DDX21, DHX36, and the adapter protein TRIF form a complex in the cytosol, which binds double-stranded RNA (dsRNA), contributing to interferon (IFN) induction –9. DDX41–Alphaviruses are a large group of enveloped single-stranded, positive-sense RNA viruses of about 11 to 12 kb. Alphaviruses that infect humans are transmitted by vector mosquitoes and include Sindbis virus (SINV) and chikungunya virus (CHIKV). SINV is the prototypical alphavirus, and it typically causes mild infection in humans. After initial outbreaks in Africa, SINV spread to Europe in the 1960s and now has seroprevalence of around 5% in Finland , 14 and Different alphaviruses replicate using the same general mechanisms . Virions18–Drosophila cell culture and found that Hlc (dDDX56) is antiviral against SINV. DDX56 is a DEAD box helicase with known nucleolar localization and a role in ribosome biogenesis  screen of conserved DEAD box helicases in ogenesis , but a rWe found that DDX56 is dispensable for interferon induction, leading us to hypothesize that DDX56 binds viral RNA to control infection more directly. We used cross-linking immunoprecipitation (CLIP-Seq) to immunoprecipitate RNAs bound by DDX56 during CHIKV infection of human cells. Strikingly, we found that approximately 10% of the DDX56-bound RNAs were viral sequences. Moreover, we observed a strong enrichment of DDX56 binding within the coding region of the CHIKV RNA-dependent RNA polymerase . This peak is adjacent to a highly conserved hairpin motif that promotes the translation of the RdRp, a step required to launch productive infection , 22. ThiDrosophila DL1 cells and infected these cells with a green fluorescent protein (GFP) reporter strain of Sindbis virus (SINV-GFP); 3 days postinfection, levels were quantified using microscopy with automated image analysis. We used dsRNA against the GFP reporter as a positive control for knockdown and a nontargeting dsRNA as a negative control. We identified antiviral helicases using a cutoff of >2-fold increase in infection; of the 22 DEAD box helicases in our panel, six exceeded this threshold, and five of those had no impact on cell survival  to knock down a panel of conserved DEAD box helicases  in Droso10.1128/mBio.02623-20.1FIG S1Drosophila RNA helicase genes was depleted by RNAi in DL1 cells. (A) Cells were infected with SINV-GFP at day 3 and fixed at 24 h postinfection. Percent infection was quantified by automated microscopy and expressed as fold change in percent infection compared to a nontargeting control. Graph shows mean and SEM from three replicates. (B) Quantification of cell numbers from panel A, quantified (number of nuclei) relative to a nontargeting control. Mean and SEM from three replicates are shown. (C) Representative microscopy images from panel A. Signal from SINV-GFP reporter virus is displayed in green, and Hoechst nuclear counterstain is in blue. (D) U2OS cells were transfected with pooled or individual siRNAs to DDX56. Infection with CHIKV was assessed by automated microscopy. Graph shows mean and SEM; *, P < 0.05. (E) U2OS cells were transfected with “control, IFIT1, or DDX56” siRNAs, and infection with CHIKV was assessed by RT-qPCR normalized to GAPDH and by automated microscope. Graphs show mean and SEM. *, P < 0.05; **, P < 0.01. Download FIG S1, PDF file, 0.1 MB.DDX56 is antiviral. (A and B) A panel of Copyright © 2020 Taschuk et al.2020Taschuk et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Drosophila helicases, using the human osteosarcoma cell line U2OS, as it is readily infected by many arthropod-borne viruses (arboviruses) and has an intact interferon system. We used short interfering RNA (siRNA) transfection to knock down DEAD box helicase genes and evaluated the effect on SINV-GFP infection by reverse transcription-quantitative PCR (RT-qPCR) and microscopy  as well as the unrelated negative-sense RNA virus vesicular stomatitis virus (VSV). We found that DDX56 was antiviral against both alphaviruses but not VSV using both automated microscopy and RT-qPCR to quantify viral infection in U2OS cells  and E anThe role of type I interferons and interferon-stimulated genes (ISGs) in controlling alphavirus infection is well established , 23, 24.DDX56 is a nucleolar protein that has been implicated in ribosome biogenesis . Viral iAF490259) using STAR  Arc diagram of structure prediction from Copyright © 2020 Taschuk et al.2020Taschuk et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mBio.02623-20.5TABLE S1Table S1, XLS file, 0.3 MB.CLIP-Seq coverage depth on CHIKV genome showing positive-strand alignments. Per-base coverage depth of CLIP-Seq reads on CHIKV genome; positive-strand alignments are reported as positive values (CHIKV_coverage_plus.xlsx). Data shown are related to Copyright © 2020 Taschuk et al.2020Taschuk et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mBio.02623-20.6TABLE S2Table S2, XLS file, 0.3 MB.CLIP-Seq coverage depth on CHIKV genome showing negative-strand alignments. Per-base coverage depth of CLIP-Seq reads on CHIKV genome; negative-strand alignments are reported as negative values (CHIKV_coverage_minus.xlsx). Data shown are related to Copyright © 2020 Taschuk et al.2020Taschuk et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the AF490259). We also observed greater coverage depth at the 3′ end of the genome than at the 5′ end, corresponding to the subgenomic RNA, and very few reads mapping to the antigenome . The relative enrichment of 3′-end coverage and lack of antigenome sequences may be related to the relative abundance of these RNA species during infection; very little antigenome is produced relative to genome, while subgenomic RNA is produced in excess  before shifting cells to 37°C to allow infection to proceed. We collected RNA every 4 h from control and DDX56-depleted cells and quantified viral RNA by RT-qPCR. Levels of viral RNA were similar in control and DDX56-depleted cells at t = 0 and t = 4 h, suggesting that binding and entry are not impacted. The antiviral effect of DDX56 becomes evident between 4 and 8 h postinfection, which is the time frame when viral RNA synthesis is launching, and becomes more pronounced as replication proceeds  translation suggests that DDX56 binding impacts early steps in the replication cycle, as translation of nonstructural proteins is required for initial viral RNA synthesis. To determine how early in infection the DDX56 antiviral phenotype became apparent, we measured viral RNA levels over time in synchronized infections. CHIKV was prebound to cells at 4°C, and unbound virions were removed  . TogetheOnce CHIKV enters and uncoats in the cytoplasm, the incoming RNA genome must be translated to launch infection. Because we observed decreases in viral RNA production and in the percentage of cells that were productively infected , as well10.1128/mBio.02623-20.4FIG S4n = 3. *, P < 0.05 by unpaired t test on half-life values from three independent experiments. (B) Schematic of DDX56 overexpression constructs. Download FIG S4, PDF file, 0.1 MB.Multiple pathways impact CHIKV stability. (A) U2OS cells were transfected with the control or with siRNAs targeting RNA decay pathways, and viral RNA stability was analyzed as in Copyright © 2020 Taschuk et al.2020Taschuk et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the There are various RNA decay pathways in cells. We performed initial studies to determine which RNA decay pathways may contribute to DDX56-mediated destabilization of CHIKV genomes. To this end, we tested three pathways: the 5′ to 3′ decay machinery (Xrn1), the 3′ to 5′ decay machinery , and the nonsense decay machinery (Upf1) , 33. ForDDX56 is a nucleolar resident protein that we found relocalizes during CHIKV infection. DDX56 contains lysine- and arginine-rich motifs in its C-terminal sequence, which have been identified as putative nuclear localization signals (NLS) . To inveTo assess the importance of DDX56 localization for its effect on the CHIKV RNA stem-loop, we cotransfected WT or cytoplasm-localized DDX56 along with a reporter plasmid containing the CHIKV nsP4 gene that includes the stem-loop. The levels of the mRNA reporter were reduced in cells expressing cytoplasmic DDX56 compared to those of wild-type, nucleolar DDX56 . This deDEAD box helicases play fundamental roles in all aspects of cellular RNA metabolism. This requires interaction of the DEAD box domain with distinct RNAs to carry out these diverse functions. Our knowledge of the interactions of DEAD box helicases with viral RNAs is incomplete. Given that these helicases bind structural features of RNAs and viral RNAs are known to be highly structured, we and others have begun to characterize these interactions. Our genetic screening identified three DEAD box RNA helicases, DDX56, DDX23, and DDX46, as antiviral against alphaviruses in both arthropod and human cells. We focused on DDX56, which we found is antiviral against both SINV and CHIKV early in infection. While DDX56 is predominantly nucleolar, a fraction is also present in the cytoplasm where viral RNA resides, and cytoplasmic DDX56 levels increase during the early phases of infection. This led us to discover that DDX56 directly binds cytoplasmic viral RNA. We found that DDX56 interaction with the incoming viral genome decreases the stability of viral RNA, preventing this RNA from efficient translation and launching of infection.In addition to finding an interaction between DDX56 and CHIKV RNA, CLIP-Seq allowed us to determine specific features of this interaction. First, we found a dearth of binding to the negative-sense antigenome. While this viral RNA species is indeed the least prevalent, the conspicuous lack of signal suggests that this RNA is protected from RNA binding proteins, and it has been shown that Semliki Forest virus (SFV) antigenomes are sequestered in membrane spherules capable of protecting them from experimental RNase treatment . Second,We found that DDX56 impacts the stability of the incoming viral genome, with CHIKV RNA exhibiting a longer half-life in cells depleted for DDX56. Furthermore, overexpression of a cytoplasmic-localized DDX56 construct resulted in lower levels of CHIKV nsP4 mRNA expressed from a reporter. Taken together with our observations of increased cytoplasmic localization of DDX56 during CHIKV infection and our CLIP-Seq findings that DDX56 binds specifically to CHIKV RNA in the nsP4 gene, we hypothesize a model where DDX56 in the cytoplasm interacts with the nsP4 region of the CHIKV genome, leading to destabilization of the viral RNA. Because a fraction of DDX56 is present in the cytoplasm at baseline, this interaction may begin as soon as CHIKV enters a cell and uncoats. However, accumulation of DDX56 in the cytoplasm over the course of CHIKV infection suggests that as infection proceeds, more DDX56 becomes available for cytoplasmic interaction with viral RNA.Viruses must evade RNA degradation pathways or coopt stabilizing factors in order to replicate. Stability factors such as HuR can interact directly with viral RNA and promote replication, including binding the 3′ untranslated region of SINV . ConversWhen DEAD box helicases bind RNA, they can serve as platforms for recruiting additional effectors or driving assembly of ribonucleoprotein complexes to promote RNA degradation. DDX17 (P72) serves as a cofactor for ZAP to promote degradation of viral sequences by the exosome and also interacts with the exosome in an RNA-dependent manner in the absence of ZAP . For splWhile DDX56 is largely nucleolar, consistent with its role in rRNA biogenesis, we found that a subset of DDX56 accumulates in the cytoplasm upon CHIKV infection. Viral infection is known to impact many aspects of cellular function, and we and others have observed that localization of RNA-binding proteins and nucleolar factors is disrupted during alphavirus infection, including DDX56, nucleolin, NPM1, and fibrillarin , 48. IntOur studies also found that two other nuclear helicases, DDX23 and DDX46, were antiviral in both insect and human cells. DDX23 and DDX46 are part of the core splicing machinery . A recenin vivo are thought to be at low multiplicities of infection, the presence of DDX56 increases the likelihood that an incoming viral genome will be degraded rather than translated. Thus, DDX56 targeting of viral RNA may lead to clearance of infection from a cell prior to the launch of viral replication. Future studies will investigate whether we can manipulate this pathway by increasing the levels of cytoplasmic DDX56 or increasing the efficiency of viral RNA degradation to enhance cellular defenses against alphavirus infection.Overall, our data describe a novel role for DDX56 as an antiviral factor. Binding of viral RNA by this host protein as it enters the cytoplasm leads to the destabilization and loss of the infectious RNA. Since infections Drosophila melanogaster DL1 cells were obtained from DRSC and maintained at 25°C in Schneider’s Drosophila medium (Gibco) containing 10% fetal bovine serum (FBS), l-glutamine (Gibco), and penicillin-streptomycin (Gibco). U2OS cells  and HeLa cells were obtained from ATCC and maintained at 37°C in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco) supplemented with 10% FBS, l-glutamine (Gibco), and penicillin-streptomycin (Gibco).CHIKV isolate PC-08  was obtasiRNAs for DDX56 and controls were obtained from Ambion (SilencerSelect) or Sigma  and collected 16 to 20 hpi, with CHIKV  and collected 20 to 24 hpi, and with VSV  and collected 10 hpi. Infections were monitored by microscopy or RT-qPCR assays. For biochemical and sequencing studies, cells were infected with CHIKV at an MOI of 5.t tests.At the indicated time points, cells were washed once with phosphate-buffered saline (PBS) and collected in TRIzol  for RNA extraction according to the manufacturer’s instructions or fixed with 4% formaldehyde for 10 min for quantification by microscopy. Cells were stained for immunofluorescence imaging using anti-CHIKV ascites V-548-701-562 (ATCC VR-1241AF) at 1:300 or CHK-265 (gift from M. Diamond) at 1:500 and anti-mouse 488 and Hoechst 33342  at a final concentration of 5 μg/ml. SINV-GFP and VSV-GFP reporter viruses were counterstained with Hoechst. Automated imaging and image analysis  were used to quantify cell number and percent infection where 3 sites per well were averaged, and at least 3 independent experiments were used for statistical analysis using For CHIKV replication time courses, U2OS cells were infected at an MOI of 10, and infection was synchronized by allowing viruses to bind to cells at 4°C for 1 h. Cells were washed twice with PBS to remove unbound virus, and plates were transferred to 37°C in complete culture medium. At the indicated time points, samples were washed once with PBS and collected in TRIzol. RNA extraction was performed using a column-based kit  with in-column DNase treatment .t test.To assess stability of entering viral genomes, U2OS cultures were infected at an MOI of 50; infection was synchronized by allowing viruses to bind to cells at 4°C for 1 h, washing twice with PBS to remove unbound virus, and transferring cells to 37°C with complete medium containing cycloheximide at 20 μg/ml . At each time point, cells were digested with 0.25% trypsin to remove surface-bound virions and washed once with PBS before collection in TRIzol  for RNA extraction using a column-based kit  with in-column DNase treatment . Viral RNA half-life was calculated by nonlinear regression, and half-lives from 3 experiments were compared using an unpaired two-tailed TC method (t test on log2(fold change). Primer sequences are listed in RNA samples were reverse transcribed using MMLV RT  and random hexamers according to the manufacturer’s instructions. Diluted cDNA was used for SYBR-based qPCR . Relative quantification was calculated according to the ΔΔT method . SignifiAB_10750607; Bethyl), glyceraldehyde-3-phosphate dehydrogenase , lamin B1 , tubulin , CHIKV nsP1 , CHIKV nsP2 , and CHIKV nsP3 .Cells were collected by trypsinization and lysed in radioimmunoprecipitation assay (RIPA) buffer supplemented with protease inhibitors. Samples were run on Tris-glycine SDS-PAGE gels and transferred to polyvinylidene difluoride (PVDF) membranes (Immobilon) for Western blot detection. Blots were exposed with chemiluminescent regents and imaged with a charge-coupled device (CCD)-based detection system (Amersham). The following antibodies were used: DDX56  or rabbit IgG control  antibodies bound to Dynabeads protein A beads . A 32P-labeled RNA adapter was ligated to the 3′ end of RNA, and samples were run on an SDS-PAGE gel and transferred to nitrocellulose membrane. 32P signal from the adapter-labeled RNA-protein complexes was used as a guide to excise appropriate regions of the membrane and extract RNA. A 5′ RNA adapter containing a degenerate barcode was ligated to the extracted RNA. Samples were reverse transcribed and PCR amplified to prepare the sequencing library, using primers that added specific index sequences for each sample to allow multiplexing of sequencing runs. Samples were pooled and sequenced on Illumina HiSeq at the University of Pennsylvania Next Generation Sequencing Core. Results represent three independent sets of infections and immunoprecipitations.CLIP-Seq was performed with a protocol modified from Vourekas and Mourelatos . U2OS ceAF490259.3) appended and treated as an additional chromosome. To exclude cross-contaminating signal from our analysis of viral reads, reads aligning to the CHIKV genome were further filtered to exclude any reads that matched in sequence, barcode, and mapping location between samples within each replicate. CLIP Tool Kit tag2collapse was then used to collapse PCR duplicates that mapped to the same genomic coordinates. Analyses were run on the Penn Medicine Academic Computing Services High-Performance Computing System, and results were visualized in R and IGV. Figures were generated in R. Software packages used are listed in Raw FastQ reads were trimmed using cutadapt  and Trim10.1128/mBio.02623-20.8TABLE S4Table S4, DOCX file, 0.1 MB.Analysis and visualization software. List of software tools and R packages used in data analysis and figure generation; includes references and version information. Download Copyright © 2020 Taschuk et al.2020Taschuk et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the BC001235) was obtained from the MGC ORFeome collection. An HA tag was added to the C-terminal end of the DDX56 open reading frame by PCR amplification using Phusion polymerase and recircularization using T4 DNA ligase. Site-directed mutagenesis of the DDX56 cDNA sequence was performed in the same way. A cGFP-nsP4-Flag reporter construct was constructed by inserting nsP4-Flag amplified from CHIKV replicon pJM40  into the plasmid pCRII-TOPO CMV-cGFP-SV40  (AF490259.3) and 5763 to 6019 of SINV hrsp (accession number J02363).Secondary structures of viral RNA sequences were predicted using default parameters for RNAfold on the Vienna RNA webserver based on Vienna version 2.4.13 . ConservM045798) . Regions