Adenovirus Virus-Associated (VA) RNAs are the first discovered viral noncoding RNAs. By mimicking double-stranded RNAs (dsRNAs), the exceptionally abundant, multifunctional VA RNAs sabotage host machineries that sense, transport, process, or edit dsRNAs. How VA-I suppresses PKR activation despite its strong dsRNA character, and inhibits the crucial antiviral kinase to promote viral translation, remains largely unknown. Here, we report a 2.7 Å crystal structure of VA-I RNA. The acutely bent VA-I features an unusually structured apical loop, a wobble-enriched, coaxially stacked apical and tetra-stems necessary and sufficient for PKR inhibition, and a central domain pseudoknot that resembles codon-anticodon interactions and prevents PKR activation by VA-I. These global and local structural features collectively define VA-I as an archetypal PKR inhibitor made of RNA. The study provides molecular insights into how viruses circumnavigate cellular rules of self vs non-self RNAs to not only escape, but further compromise host innate immunity. Adenovirus Virus-Associated (VA) noncoding RNAs interfere with the host system by mimicking double stranded RNAs. Here, the authors report a 2.7 Å crystal structure of VA-I RNA providing an understanding of its function. At least one VA RNA exists in all known adenovirus serotypes and most (~80%) contain both3. The multifunctional VA RNAs interfere with essentially all host systems that interface with double-stranded RNAs (dsRNAs), from their sensing by protein kinase R (PKR), export by Exportin-5, processing by Dicer, editing by ADAR, to activation of oligoadenylate synthetases (OAS), etc4. Dicer-processed terminal strands of VA are further assembled into functional RISC complexes that may target additional host systems6. Collectively, VA RNAs contribute ~60 fold to viral titers and confer adenoviruses general resistance to interferon-mediated antiviral defense7.Adenoviruses infect the respiratory system and the gastrointestinal and urinary tracts and can be life-threatening for immunocompromised patients. Late in infection, adenoviruses produce two extraordinarily abundant  induces PKR autophosphorylation and activation of its latent kinase activity12. Activated PKR phosphorylates the alpha subunit of eukaryotic translation initiation factor 2 (eIF2α) to block cap-dependent translation initiation and production of new viral particles13. Indeed, PKR-deficient mice are exceptionally susceptible to intranasal infection by vesicular stomatitis and influenza viruses14. To counter the debilitating restriction by PKR, nearly all known viruses have evolved protein or RNA antagonists to evade or inactivate PKR. The constant tug-of-war with viral antagonists has driven intense positive selection of PKR in vertebrates15. Besides manipulation by viral elements, PKR activity is subject to tight control by host protein factors including NF9016, TRBP17, ADAR118, etc. Furthermore, endogenous, highly structured RNAs also modulate PKR activity, such as snoRNAs19, the IFN-γ and TNF-α pre-mRNAs21, nc886 RNA22, etc. Recently, additional endogenous RNAs have been shown to directly associate with PKR, including nuclear transposable elements and mitochondrial dsRNAs proposed to mediate mitochondria–cytosol communication, especially during stress23.PKR is a central component of the interferon response and the best-characterized target of VA RNAs. PKR recognizes dsRNAs produced during viral replication or bidirectional transcription, and exerts antiviral and antiproliferative effects through signaling pathways including NF-kB, TNF, STATs, p53, etc24. It is comprised of an elongated apical stem loop, a central domain proposed to contain a pseudoknot, and a terminal stem thought to be dispensable for PKR binding and inhibition  analyses of VA-I uncovered a number of unusual features of this RNA. The apical domain is exceptionally thermostable , causing it to migrate much slower on denaturing Urea-PAGE and to resist denaturation by up to 6 M urea. Yet, structural-probing analyses revealed sensitivity of this region to both ssRNA-specific and dsRNA-specific RNases 29. This led to the proposal that the VA-I apical region exists as two structurally different, functionally distinct conformers29. However, it is perplexing how a single RNA region is both extraordinarily stable and yet fluid enough to diverge into two distinct conformations. The structurally complex central domain has been long proposed to contain a 3-bp pseudoknot between Loop 8 and Loop 10, based on covariation of the loop sequences25. Yet its functional importance for PKR regulation has remained unclear. UV-melting analysis of the central domain revealed an unusual sensitivity to solution pH, the structural basis of which remains unknown30. At the junction between the apical and central domains is an essentially invariant tetrastem structure  and codon–anticodon interactions. Such resemblances shed light on the evolutionary origin of VA RNAs and may hint at undescribed functions of VA-I’s central domain.To address these open questions and understand how viral RNAs employ unique tertiary structures to confound and defeat host immune proteins, we determined a 2.7 Å crystal structure of the apical and central domains of VA-I RNA of adenovirus serotype 2 Fig. . This RN33. The SAXS envelopes of VA-I generally showed obtuse angles between the apical and terminal stems, while the crystal structure captures VA-I in an acutely bent “V” shape  that form a continuous stack. In the strand opposite the cytosines, A66 makes a single sugar edge-Hoogsteen hydrogen bond to the second cytosine (C68) in the stack, completing a robust apical structure. Unexpectedly, although single-stranded, the C67–C69 stack assumes essentially the same helical structure as in an A-form duplex  strongly compromised it  that contains four interspersed wobble pairs  and C93 , it is reasonable to speculate that the sequence of the tetrastem may serve to capture dsRBM2 which presumably binds to the proximal end of the stack.At the proximal end of the apical stem, A41 and U92 do not form ssRNA linkers as previously proposedion Fig. , while cC93 Fig. . C93 notC93 Fig. . The briC93 Fig. . In siliC93 Fig. . In contC93 Fig. . ConversC93 Fig. . AdditioConsistent with the functional importance of the tetrastem, the apical stem loop alone (26 stacked layers) exhibited minimal activity towards PKR Fig. . Remarka3+ reactivity30. Interestingly, A123 employs two hydrogen bonds from its N1 and N3 to G106’s 2'-OH and 2-exocyclic amine, respectively 6 to tandem G·U pairs that has been used as a general strategy to obtain phase information for RNA crystals44.Next we examined potential metal-binding sites on VA-I. Specific Mgsed Fig. . Both adm 7 Fig. . We propair Fig. . G·U or 33. Our structure further revealed that the pseudoknot is reinforced by stacking with G106 on top and extensive A-minor interactions with A36 , whereas SAXS envelopes generally showed obtuse angles between the apical and terminal stems58, and the nc886 RNA22, a recently described human noncoding RNA that inhibits PKR, are similarly enriched for wobble pairs. Thus, the convergent use of wobble pairs in at least three PKR-inhibitory RNAs may reflect a general strategy to construct a pseudo-activator—an inhibitory device that is disguised as an activator. dsRNAs interspersed with wobble pairs, as well as other helical imperfections, may sufficiently mimic ideal dsRNAs to recruit and trap their target enzymes such as PKR and OAS1, but employ subtle structural or chemical deviations to misalign multimerization interfaces or to distort catalytic geometries, ultimately leading to non-activation or inhibition.One of the prominent local features of the VA-I structure is the over-representation of wobble base pairs that populate the apical stem, terminal stem, and even Stem 7 Fig. . Interes41. Given the extreme abundance of VA RNAs, there is little incentive to create the strongest possible PKR inhibitor. Thus, priority was given to guard against inadvertent activation by misfolded or degraded viral RNAs, which can lead to catastrophic restriction of the viruses. It is clear that the wild-type VA-I is not optimized for maximized PKR-inhibitory activity, as a number of substitutions that we and others tested actually enhanced PKR inhibition. In addition to their effects on PKR and OAS1, the wobble pairs may also impact other targets of VA RNAs. The A+54·C79 wobble and another potential A+5·C151 wobble pair in the terminal stem likely constitute preferred ADAR-editing sites and may contribute to its capture or inhibition. Therefore, the evolution of VA sequence and structure was likely constrained by selective pressures from multiple host targets, each with distinct RNA-binding specificities. The simultaneous selection of convergent and divergent features produced an extant multifunctional RNA that contains an amalgam of balanced features and characteristics4.It was shown that the wobble base pairs allowed VA RNAs to escape OAS1 and PKR activation at the expense of inhibitory potency against PKR32, an outcome counter to VA-I’s intended function. There are at least three possible, mutually non-exclusive mechanisms by which the central domain prevents PKR activation. First, the central domain pseudoknot may function to constrain the overall shape or flexibility of VA-I to block PKR activation. The multiple targets and functions of VA-I likely required a longer structural platform than its PKR-inhibitory core (mini-VA) can provide. The extant longer VA-I scaffold was proposed to have originated from concatenated host tRNA genes and our observed structural similarities to tRNAs support this notion3. Such longer RNA platforms, especially those that are near-linear in architecture or contain flexible regions, carry substantial risks of activating PKR or other dsRNA sensors. This is exacerbated by PKR’s ability to straighten bent, bulged, and other non-contiguous dsRNA helical segments and stack them into extended platforms that generally activate PKR59. For host RNAs, such risks are mitigated by shielding by RNA-binding proteins or editing by ADAR, etc.18. For VA-I, the pseudoknot appears to limit its conformational freedom and generate an acute bend in the dsRNA trajectory, thus antagonizing PKR’s structure-remodeling activity to avoid induced activation. This notion is consistent with the lowest temperature factors of this region 61, and 4-thio-U crosslinking between an ssRNA–dsRNA activator and the basic patch60. The precise mechanisms by which the VA-I central domain blocks PKR activation await structural and biochemical analyses of PKR–VA-I complexes. It is further possible that the VA-I central domain uses its unique 3D structure to execute pro-viral functions that have not been well-characterized, such as the inhibition of RNA helicases.In contrast to its auxiliary role in PKR inhibition through facilitating RNA folding, the central domain pseudoknot is crucial for preventing PKR activation by a full-length VA-I. This is evidenced by the observation that pseudoknot disruptions consistently converted VA-I into robust PKR activators62. The 60-nt mini-VA RNA not only exhibits full inhibitory activity against PKR to boost protein translation, but also will permit simultaneous use of therapeutic small hairpin RNAs (shRNAs) for gene silencing, as it lacks the regions that target Dicer and Exportin-5. Conversely, if normal translation activity is desired, a truncated VA-I RNA with a shortened apical stem will likely only target the RNAi machinery while sparing PKR and translation. Ectopic expression of VA RNAs down-regulates PKR activity and can be employed to counteract excessive activation of the interferon response and to protect tissues from apoptosis and inflammation. VA-I also holds exceptional promise as a versatile vehicle to exogenously express and fold RNA fragments of interest similar to the widely used tRNA scaffold in which a cargo RNA is inserted in the ASL63. The extraordinary potency of VA transcription, proven cytoplasmic delivery and stability, and built-in suppression of dsRNA cargo-triggered immune response, make for an ideal RNA-expression platform.The structural elucidation of VA-I RNA informs the development and optimization of VA RNAs as valuable tools in biotechnology applications, adenovirus-based gene therapies, and vaccine development, etc. Co-transfection of VA genes in mammalian cells (e.g. the Promega pAdVantage vector) overcomes PKR-mediated translation inhibition and dramatically increases protein expression by 10–70 fold65 and inform the design of functional RNA devices that elicit specific desired cellular responses.As the quintessential viral noncoding RNA, the specific structural features of the multifunctional adenovirus VA-I provide a starting point and reference to understand a growing collection of highly structured endogenous  and exogenous  RNA modulators of host proteins, such as PKR, OAS1, and others. Comparative analyses of exemplary activating and inhibitory RNAs are expected to clarify the principles of self vs. non-self RNA discrimination47. For crystallization, 4.0–8.0 mg/mL (112–223 μM) VA-I RNA in RNA folding buffer consisting of 10 mM Tris–HCl (pH 7.5), 50 mM KCl, 5 mM MgCl2, and 1 mM spermine was mixed 1:1 with a reservoir solution comprised of 50 mM sodium cacodylate (pH 6.5) and 28–30% 2-methyl-2,4-pentanediol (MPD) in a hanging-drop vapor diffusion tray. Single, obelisk crystals grow in 1–2 days and reach maximum dimensions of ~500 × 50 × 30 μm3 after 18–21 days. RNA crystals were cryo-protected in a synthetic mother liquor that contains 40% MPD and vitrified in liquid nitrogen at 100 K. Crystals exhibited the symmetry of space group P4122 and unit cell dimensions are shown in Table The adenovirus serotype 2 VA-I RNA was modified by removing the functionally dispensable terminal stem and loop 2 (nucleotides 1–31 and 132–161) and appending a 6-bp stem  from 0.2 to 0.8 in the highest resolution shells. Ir substructure was readily identified by SHELX67 and Phenix.HySS68, and refined by Phenix.Autosol, producing a figure of merit (FOM) of 0.56. RNA helices and nucleosides were clearly visible in the resulting experimental electron density map. Model building was performed in Coot69, as guided by iteratively generated MR-SAD maps using an evolving model. Finally, the near complete model was located in the native dataset using PHASER70, manually adjusted and rebuilt using Coot, and refined using Phenix.Refine. The model was then corrected by ERRASER71 and further refined  data were collected near the Iridium L1 edge 0.9218 Å) at the SER-CAT beamline ID-22 at the advanced photon source (APS). Initial SAD phasing using single datasets did not correctly identify the Ir substructure. A total of 16 Ir-SAD datasets with diffractions extending to 3–5 Å were analyzed by hierarchical agglomerative program BLEND18 Å at t72 and pPET-PKR/PPase73 plasmids. The latter plasmid was used to provide a source of lambda phosphatase while the expressed untagged PKR derived from this plasmid was removed during the first Ni2+ column step. Cells were initially cultured in ampicillin-supplemented and kanamycin-supplemented terrific broth at 37 °C until OD600 reached 1.0. Then the cells were shifted to 30 °C and 0.5 mM IPTG was added to induce protein expression for additional 1.5 h before harvesting. Cells were resuspended in a lysis buffer comprised of 20 mM HEPES–KCl (pH 7.5), 200 mM NaCl, 0.1 mM EDTA, 5% glycerol, 5 mM β-mercaptoethanol, 10 mM Imidazole, supplemented with SIGMAFAST™ Protease Inhibitor Cocktail, lysed using a microfluidizer, clarified by centrifugation, and the supernatant loaded onto a HisTrap HP Ni2+ column on an ÄKTA pure chromatography system. Fractions containing PKR were pooled and further purified on a HiTrap Heparin HP column to remove associated nucleic acids, and was dialyzed into a storage buffer consisting of 20 mM Tris–HCl (pH 7.5), 100 mM KCl, 2 mM MgCl2, 20% glycerol, and 8 mM β-mercaptoethanol and stored at −80 °C. The purity and mass of the purified PKR was verified by SDS–PAGE and liquid chromatography–electrospray ionization–quadrupole-time of flight–mass spectrometry (LC–ESI–Q-TOF–MS), yielding an intact protein mass of 66,240.5 Da, as compared to the calculated mass of 66,239.8 Da (Δmass = 0.7 Da). To further confirm that the purified PKR carries no removable phosphates, PKR was treated with lambda phosphatase (NEB), which led to no change in intact protein mass or phosphate content as assessed by the ratio of Pro-Q Diamond Phosphoprotein Gel Stain (Thermo-Fisher) signal and SYPRO Ruby Protein Gel Stain signal of the same gel.N-terminally his-tagged, dephosphorylated human PKR was heterologously expressed and purified from Rosetta™ 2(DE3)pLysS cells co-transformed with pET28a-HisPKR2, 5% glycerol, and 2 mM DTT for 10 min at room temperature with gentle agitation. A 5× mixture of 250 μM ATP and 0.15 μg/mL poly I:C was then added and incubated at 30 °C for 30 min to challenge the PKR–VA-I complex to induce PKR autophosphorylation. Reactions were quenched by addition of an equal volume of 2.0% SDS in Tris-buffered saline , and subsequently applied to a 48-well Bio-Dot SF (Bio-Rad) microfiltration system containing 400 μL of 0.1% SDS in TBS in each well. PKR was adsorbed onto a nitrocellulose membrane under vacuum. PKR phosphorylation was assessed by western blot analysis using Anti-P-PKR (phospho T446) antibody  and Alexa Fluor 488-conjugated anti-rabbit secondary antibody . Additionally, total PKR was quantified on a separate slot blot using Anti-PKR antibody  and Alexa Fluor 488 conjugated anti-mouse secondary antibody . Band intensities were captured using a GE Typhoon Trio+ Imager and quantified using ImageJ. The ratios of anti-P-PKR (phospho T446) band intensities and anti-PKR intensities were used to assess PKR phosphorylation, and were normalized against the control reaction containing poly I:C but no VA-I RNA. At least triplicate experiments were performed for each RNA. In addition to the slot blot analyses, select reactions are also electrophoretically separated on SDS–PAGE, transferred to PVDF membranes using an iBlot 2 Dry Blotting System (Thermo-Fisher), and quantified by immunoblotting similar to the slot blots , 50 mM NaCl, 4 mM MgCl53 of the RNAstructure suite, using structural constraints derived from the crystal structure of the apical and central domains of VA-I, to produce an updated secondary structure model for VA-II and a consensus secondary structure of both VAs . Data were analyzed using the ASTRA 7.1.4 software (Wyatt Technology Europe).Size-exclusion chromatography with multi-angle static light scattering (SEC–MALS) was used to assess the binding stoichiometry of PKR to WT and ΔTS VA-I RNAs. 30 μM of individual RNA or protein component or their equimolar mixtures were incubated for 15 min in a buffer consisting of 25 mM Tris–HCl pH 7.5, 150 mM NaCl, and 2 mM MgClFurther information on research design is available in the Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Movie 1Reporting Summary


Cryptococcus neoformans. In fungal species, extracellular vesicles (EVs) participate in the export of polysaccharides, proteins and RNA. In the present work, we investigated if EV-mediated RNA export is functionally connected with GRASP in C. neoformans using a graspΔ mutant. Since GRASP-mediated unconventional secretion involves autophagosome formation in yeast, we included the atg7Δ mutant with defective autophagic mechanisms in our analysis. All fungal strains exported EVs but deletion of GRASP or ATG7 profoundly affected vesicular dimensions. The mRNA content of the graspΔ EVs differed substantially from that of the other two strains. The transcripts associated to the endoplasmic reticulum were highly abundant transcripts in graspΔ EVs. Among non-coding RNAs (ncRNAs), tRNA fragments were the most abundant in both mutant EVs but graspΔ EVs alone concentrated 22 exclusive sequences. In general, our results showed that the EV RNA content from atg7Δ and WT were more related than the RNA content of graspΔ, suggesting that GRASP, but not the autophagy regulator Atg7, is involved in the EV export of RNA. This is a previously unknown function for a key regulator of unconventional secretion in eukaryotic cells.Golgi reassembly and stacking protein (GRASP) is required for polysaccharide secretion and virulence in  Extracellular vesicle (EV) formation and release constitute a ubiquitous export mechanism of proteins, DNA and RNA ,2. EVs pDrosophila melanogaster, GRASP participates in the delivery of integrins from the ER directly to the plasma membrane, thus bypassing the Golgi [Dictyostelium discoideum, a GRASP orthologue (GrpA) was necessary for acyl-coenzyme A-binding protein (AcbA) secretion during spore differentiation [Saccharomyces cerevisiae and Pichia pastoris, another GRASP orthologous (Ghr1) was also required for starvation-induced secretion of AcbA [EV biogenesis in fungi is still poorly understood. It has been hypothesized that EV biogenesis in eukaryotes is a complex process that is regulated at multiple levels ,11. EV fhe Golgi . In the ntiation . In the  of AcbA ,19.Cryptococcus neoformans, GRASP was required for polysaccharide export to the extracellular space. Polysaccharide secretion is fundamental for virulence in C. neoformans [graspΔ mutant was hypovirulent in mice [C. neoformans is mediated by EVs but connections between GRASP functions and EV cargo remain uncharacterized.In the yeast-like neuropathogen oformans  and, in  in mice . PolysacToxoplasma gondii Atg8 localizes to the apicoplast and is essential for organelle homeostasis and survival of the tachyzoite stage of the parasite [Autophagy is a self-degradative process conserved in eukaryotes, presenting a housekeeping role by degrading dysfunctional components such as organelles and misfolded proteins . The Atgparasite . Atg7 noparasite , IFNγ-meparasite , adipogeparasite  and cellparasite .C. neoformans [D. discoideum, GRASP-mediated unconventional secretion is mediated by autophagosomes, showing that there is a connection between these processes [Autophagy regulators play key roles in cryptococcal physiology and, in fact, we have recently demonstrated that the putative autophagy regulator Atg7 affects both physiological and pathogenic mechanisms in oformans . In D. drocesses ,29.C. neoformans. Our results suggest that GRASP, but not Atg7, is a key regulator of vesicular export of RNA in C. neoformans.The role of unconventional secretion regulators in vesicular export of RNA is unknown but the functional connections between GRASP and Atg7 led us to evaluate whether these proteins affected extracellular RNA export in C. neoformans strains used in this study included the parental isolate H99 and the mutant strains atg7Δ and graspΔ, which were generated in previous studies by our group [4 (10 mM), KH2PO4 (29.4 mM), glycine (13 mM) and thiamine-HCl (3 µM) for further cultivation for three days at 30 °C, with shaking. All protocols adhered to the biosecurity demands of the Carlos Chagas Institute of Fiocruz .The ur group ,28. Fungg for 15 min at 4 °C and the resulting supernatants were pelleted at 15,000× g for 30 min to remove small debris. The final supernatants were concentrated by a factor of 20 in an Amicon ultrafiltration system . Concentrated supernatants were centrifuged at 15,000× g for 30 min to ensure the removal of aggregates and the resulting supernatant was then ultracentrifuged at 100,000× g for 1 h to precipitate vesicles. Vesicle pellets were washed once in phosphate-buffered saline (PBS) and the final pellets were suspended in PBS. For analysis of EV dimensions, nanoparticle tracking analysis (NTA) was performed on a LM10 Nanoparticle Analysis System, coupled with a 488 nm laser and equipped with a SCMOS camera and a syringe pump . The data was acquired and analyzed using the NTA 3.0 Software . EVs from all samples were diluted 1:30 in filtered PBS (0.22 µM) and measured within the optimal dilution range previously described by Maas and colleagues (9 × 107–2.9 × 109 particles/mL) [EVs were isolated from fungal culture supernatants as previously described . Briefly−1 RNase  for 10 min at 37 °C before RNA extraction, as previously described [Small RNA (sRNA)-enriched fractions were isolated with the miRNeasy mini kit  and then treated with the RNeasy MinElute Cleanup Kit (Qiagen), according to the manufacturer’s protocol, to obtain small RNA-enriched fractions. The success of the sRNA extraction was assessed in representative EV preparations that were treated with 30 U DNase I (Qiagen) and characterized in an Agilent 2100 Bioanalyzer . To confirm that the RNA was confined within the EVs, vesicle samples were treated with 0.4 μg μlescribed .One hundred ng of purified sRNA were used for RNA-seq analysis from two independent biological replicates. The RNA-seq was performed in a SOLiD 3 plus platform using the RNA-Seq kit  according to the manufacturer’s recommendations.g, washed in PBS, suspended in the lysis buffer provided in miRCURY™ RNA Isolation Kit–Cell & Plant  and vortexed 5 times in acid washed glass beads . The lysate was centrifuged for 2 min at 14.000× g and the supernatants were collected for RNA isolation with the mirCURY™ kit, following the manufacturer’s instructions. The RNAs were eluted in ultrapure water and treated with RQ1 RNase-Free Dnase (Promega) following the manufacturer’s instructions. Reverse transcription reactions with the DNAse-treated RNAs were performed with a random primer and the ImProm-II™ Reverse Transcription System (Promega), following the manufacturer’s instructions. Real time PCR reactions were performed using SYBR® Select Master Mix and run and analyzed using the LightCycler® 96 System . The primers corresponded to CNAG_03103 Cullin3 Forward GCCATACGGGAGATACAGAAC, Reverse GAGGTGTTGGACGATGAGAG, CNAG_07590 V_typeH Forward TCATGCTCAACGAAGTCAGG, Reverse GGAAGCAGTGGTTGTGAATG, CNAG_03337 hypothetic Forward CGGTCTTTATCGCTGCTGTAT, Reverse ATTGAAGAGTGGATGTCGTGG and CNAG_00483 Actin Forward CCACACTGTCCCCATTTACGA, Reverse CAGCAAGATCGATACGGAGGAT Each reaction was performed using 10 ng of cDNA. The experiment was performed in triplicates and the expression levels relative to actin were calculated according to Pfaffl’s method using t-test for the statistical analysis [Yeast cells were grown in minimal medium for 72 h, pelleted by 1 min centrifugation at 14.000× C. neoformans-GCA_000149245.3). The alignment was performed as follows: additional 100-base upstream and downstream sequences; 10 minimum number of reads; 2 maximum number of mismatches; −2 nonspecific match limit and minimum fraction length of 0.9 for the genome mapping or 1.0 for the RNA mapping. The minimum reads similarity mapped on the reference genome was 80%. Only uniquely mapped reads were considered in the analysis. The libraries were normalized per million and the expression values for the transcripts were recorded in RPKM (reads per kilobase per million), we also analyzed the other expression values-TPM (transcripts per million) and CPM (counts per million).The sequencing data were analyzed using the version 9.1 of CLC Genomics Workbench©. The reads were trimmed on the basis of quality, with a threshold Phred score of 15. The reference genomes used for mapping were obtained from the NCBI database (The data is deposited to the Sequence Read Archive (SRA) database of NCBI  under study accession number (SRA: SRX2793565 to 67).C. neoformans. WT cells corresponded to strain H99, a standard and widely investigated clinical isolate. Knockout mutant strains (KO) lacked expression of two regulators of cryptococcal pathogenicity, GRASP and ATG7 [Our experimental model included wild type (WT) and two mutant strains of and ATG7 ,28.GRASP (graspΔ) or ATG7 (atg7Δ) expression would affect the EVs composition. The analysis of diameter distribution of wild type EVs by nanoparticle tracking analysis  and the WT strains we first aligned the RNA-seq reads with the C. neoformans H99 genome (GCA_000149245.3) sequences. We used the raw data available for isolate H99 from our previous work [atg7Δ and graspΔ EV RNA  in our previous work [Total RNA was isolated from fungal EVs and two independent biological replicates were subjected to RNA-seq . In ordeΔ EV RNA . For allatg7Δ (r 0.71) sequences was greater than that for WT and graspΔ (r 0.22)  A,B. ThisgraspΔ and WT versus atg7Δ) and applied the statistical negative binomial test [graspΔ analysis, 266 mRNAs were identified as enriched in the EVs from the graspΔ mutant (p ≤ 0.03) such as membrane and endoplasmic reticulum (p ≤ 0.03), the enriched terms included organelle organization, cell cycle and gene expression  in atg7Δ EVs were the nucleus and the mitochondrion (p ≤ 0.03).We next performed paired comparisons  and complemented (graspΔ::GRASP and atg7Δ::ATG7) strains  with the vesicular RNA sequences [The differences in the RNA composition of EVs produced by the carriers . To addrriptome H9 strain equences   that varies depending on the growth conditions. The intron retention (IR) is the prevalent AS mechanism in this fungus [We have previously observed that a great number of e genome , which iproteins . From ths fungus . We alsos fungus . HowevergraspΔ and WT vs. atg7Δ) and applied the statistical negative binomial test [graspΔ we observed 43 ncRNAs enriched in graspΔ . As described for the mRNA analysis, we performed paired comparisons  uses the canonical miRNA machinery to downregulate replication of protein A1 mRNA and other transcripts in B cell lymphoma . Regulatactivity . tRFs aractivity ,43,44,45nfection ,47. In Enfection . Human snfection . It is ugraspΔ EVs had low correlation with WT vesicles. In addition, ncRNA populations were also clearly distinct in EVs from WT and graspΔ cells, where snoRNA predominated in the WT and tRNA/tRFs in the KOs.The mRNA population from C. neoformans and a minor participation of Atg7. Polysaccharides, which lack secretory tags, require GRASP for efficient secretion in C. neoformans [ATG7, however, did not affect polysaccharide export in this fungus [graspΔ mutant. Altogether, these results strongly indicate a novel function for the GRASP family in eukaryotes that could directly affect cell communication, gene expression and host-pathogen interactions.The distinct RNA cargo in the mutants analyzed in this study is in agreement with a key and general role of GRASP in unconventional secretion in oformans . Deletios fungus . Multives fungus ,51. In fs fungus . It has s fungus . This GR


Treatment-emergent depression is a common complication in patients with chronic hepatitis C virus (HCV) infection undergoing antiviral combination therapy with IFN-α and ribavirin. It has recently been shown that changes in A-to-I RNA editing rates are associated with various pathologies such as inflammatory disorders, depression and suicide. Interestingly, IFN-α induces gene expression of the RNA editing enzyme ADAR1-1 (ADAR1a-p150) and alters overall RNA editing activity. In this study, we took advantage of the high prevalence of pharmacologically induced depression in patients treated with IFN-α and ribavirin to test the interest of RNA editing–related biomarkers in white blood cells of patients. In this 16-week longitudinal study, a small cohort of patients was clinically evaluated using standard assessment methods prior to and during antiviral therapy and blood samples were collected to analyse RNA editing modifications. A-I RNA editing activity on the phosphodiesterase 8A (PDE8A) gene, a previously identified RNA editing hotspot in the context of lupus erythematosus, was quantified by using an ultra-deep next-generation sequencing approach. We also monitored gene expression levels of the ADAR enzymes and the PDE8A gene during treatment by qPCR. As expected, psychiatric evaluation could track treatment-emergent depression, which occurred in 30% of HCV patients. We show that PDE8A RNA editing is increased in all patients following interferon treatment, but differently in 30% of patients. This effect was mimicked in a cellular model using SHSY-5Y neuroblastoma cells. By combining the data of A-I RNA editing and gene expression, we generated an algorithm that allowed discrimination between the group of patients who developed a treatment-emergent depression and those who did not. The current model of drug-induced depression identified A-I RNA editing biomarkers as useful tools for the identification of individuals at risk of developing depression in an objective, quantifiable biological blood test.The online version of this article (10.1007/s13365-019-00772-9) contains supplementary material, which is available to authorized users. Catalytic deamination of adenosine (A) to inosine (I) on RNA molecules is a fundamental cellular mechanism that directly impacts expression and function of a large set of proteins. Deamination is brought about by the action of specific editing enzymes, termed ADAR (adenosine deaminase acting on RNA). In vertebrates, the ADAR family is composed of three different genes, namely ADAR1, ADAR2 and ADAR3. All ADARs share common modular organisation of domain structures that comprise a double-stranded RNA-binding domain (dsRBD) necessary for target binding and a catalytic domain conveying deaminase activity  transcript, which is a target of ADARs, is expressed both in brain and in blood tissues and edited in humans and pre-human species. Different studies have indicated the involvement of the 15q chromosomic locus containing PDE8A gene in major depressive disorder and recurrent depression , ADAR1-5 (ADAR1b-p110), ADAR2 and PDE8A mRNA editing profiles in blood samples. We show that a combination of ADAR expression level and specific PDE8A RNA editing profile gene allows discrimination between the group of patients who developed a treatment-emergent depression and those who did not, suggesting our model as a useful tool for the identification of individuals at risk of developing depression during antiviral combination therapy.This study enrolled 10 patients infected with hepatitis C virus (HCV) from three different hospitals in France . Eligible participants were adults due to commence combination antiviral therapy with pegylated interferon alpha-2a (IFN-α-2a) and ribavirin for at least 12 weeks; this comprised weekly IFN-α-2a (180 μg) injection and daily ribavirin tablets . Exclusion criteria included the following: age below 18 years, current diagnosis (at baseline) of major depressive disorder. Written informed consent was obtained from all subjects after a detailed discussion on the study aims and requirements. All subjects were able to understand informed consent detailing the research goals and procedure. The demographic data on age and gender were recorded . The Mini International Neuropsychiatric Interview (MINI) was administered at baseline, to assess current depression or a previous history of depression, and at follow-up assessments for the detection of new-onset cases of depression. The MINI is a structured diagnostic interview for psychiatric disorders according to the DSM-IV  and the 5 cells/well. The next day, culture medium was removed and cells were incubated for 48 h with increasing concentrations of recombinant hIFN-α . Untreated cells were taken as the control group . After incubation, cells were lysed in 600 μl of RLT lysis buffer . RNA isolation and purification were carried out as described by the manufacturer . For homogenisation, cell lysates were passed through QIAshredder spin columns (Qiagen) and the flow-throughs were then transferred to gDNA eliminator spin columns. RNAs were purified on RNeasy spin columns and eluted with 40 μl of RNase-free water. Total RNAs were quantified with the Quant-IT RNA BR assay-500 and a Qubit Fluorometer (Invitrogen). The quality of RNAs (500 ng) was checked on a 1.5% agarose gel.The SH-SY5Y human neuroblastoma cells were purchased from ECACC (ref 94030304). They were cultured in high-glucose D-MEM with stable glutamine (PAA Laboratories) supplemented with 10% dialysed FCS (PAA Laboratories) and a solution of stabilised Antibiotic-Antimycotic (Sigma-Aldrich). SH-SY5Y cells were plated in 6-well plates at a density of 7.10A volume of 2.5 ml of whole blood sample from each patient was retrieved in PAXgene blood RNA tubes 4 weeks prior to antiviral therapy and every 2 weeks during the 12 weeks after onset of treatment, stored at − 20 °C and then transferred to − 80 °C. PAXgene™ Blood RNA tubes  contain a reagent that protects RNA molecules from degradation by RNAses and ex vivo changes in gene expression. Samples were distributed randomly in the different sets of extractions. Total RNA from the 2.5 ml of whole blood was extracted using the PAXgeneTM Blood RNA Kit . Extracted RNA was isolated using a Qiagen QIAcube system, following the manufacturer’s protocol for PAXgene Blood RNA part 1, automated protocol. During sample preparation and RNA extraction, standard precautions were taken to avoid RNA degradation by RNAses. Total RNA concentrations were determined with a Qubit Fluorometer (Invitrogen) and the Quant-IT RNA BR assay (Invitrogen).Reverse transcription was carried out on 12.5 ng/μl of RNA per sample using the Takara Kit . The resulting cDNA was combined with TaqMan universal PCR Master Mix  and with the following specific gene probes: ADAR1-1 (ADAR1a-p150) (Hs01020780), ADAR1-5 (ADAR1-b-p110) (Hs01017596), ADAR2 (Hs00210562), PDE8A (Hs00400174), GAPDH (Hs02758991), HPRT1 (Hs02800695), TBP (HS00427620) and PGK1 (Hs99999906)  in 20 μl final volume. Quantitative PCR was performed in 384-well plates on LightCycler 480 Real Time PCR instrument (Roche). The analysis was performed using a second derivative absolute quantification, normalised by the geometric mean of four housekeeping genes .For next-generation sequencing (NGS) library preparation, a targeted approach was employed in order to selectively sequence the region of interest within intron 9 of the PDE8A gene  as FASTQ file. To evaluate sequencing quality, an initial quality of each raw FASTQ file was performed using FastQC software (version 0.11.3). A minimal sequencing depth of 50,000 reads for each sample was considered for further analysis. A pre-treatment step was performed consisting of removing adapter sequences and filtering of the sequences according to their size and quality score. All short reads (< 100 nts) and reads with average QC < 30 were removed. To improve sequence alignment quality of the sequences, flexible read trimming and filtering tools for Illumina NGS data were used (fastx_toolkit v0.0.14 and PRINSEQ version 0.20.4). After pre-processing steps were performed, an additional quality control of each cleaned FASTQ file was carried out prior to further sequence processing.Alignment of the processed reads was performed using bowtie2 (version 2.2.5) with end-to-end sensitive mode. The alignment was done to the latest annotation of the human genome sequence (GRCh38) and reads multiple alignment regions, reads with poor alignment quality (Q < 30) or reads containing insertion/deletion (INDEL) were taken out of the further analysis. Filtering of file alignment was carried out with SAMtools software (version 1.3.1) that provide various utilities for manipulating alignments in the SAM format, including sorting, merging, indexing and generating alignments in a per-position format.Next, SAMtools mpileup was used to pileup obtained alignment results data from multiple samples simultaneously. An in-house script was run to count the number of different ATGC nucleotides in each genomic location (‘base count’). So, for each genomic location, the home-made script computes the percentage of reads that have a ‘G’ [Number of ‘G’ reads/(Number of ‘G’ reads + Number of ‘A’ reads) × 100]. The genomic location ‘A’ reference with percentage in ‘G’ reads > 0.1 is automatically detected by the script and is considered as ‘A-to-I edition site’. The last stage was to compute the percentage of all possible isoforms of PDE8A transcripts. By definition, the relative proportion of RNA editing at a given editing ‘site’ represents the sum of editing modifications measured at this unique genomic coordinate. Conversely, an mRNA isoform is a unique molecule that may or may not contain multiple editing modifications on the same transcript. For example, the PDE8A mRNA isoform BC contains a modification on both site B and site C within the same transcript.p value below 0.05 was considered as statistically significant. The accuracy of each biomarker and its discriminatory power was evaluated using a receiver operating characteristic (ROC) analysis. ROC curves are the graphical visualisation of the reciprocal relation between the sensitivity (Se) and the specificity (Sp) of a test for various values.All statistics and figures were computed with the ‘R/Bioconductor’ statistical open-source software and GraphPad Prism software (version 7.0)  1, 2 and 3 are the levels of the biomarker.In addition, all biomarkers were combined with each other to evaluate the potential increase in sensibility and specificity using an mROC multivariate approaches  was performed using scatterplot3d (v0.3-40) R package. PCA involves a mathematical procedure that represents the maximum of the data information by reducing the space dimension.In this study, we took advantage of the well-documented and well-characterised mood alterations observed in hepatitis C–infected patients undergoing antiviral therapy with IFN and ribavirin. A small cohort of ten individuals with hepatitis C virus never treated with interferon and ribavirin and without prior records for psychiatric disorders was recruited over different medical hospitals in France. At inclusion, patients underwent harmonised psychiatric assessment and at repeated interval during the course of treatment. In this particular setting, every patient acts as its own control and evolution of the patient can be monitored over time. The general descriptive characteristics of the cohort are given in Table R2 = 0.9827)  induction and RNA editing events on the 5-HT2cR in the neuroblastoma cell line SH-SY5Y by capillary electrophoresis single-strand conformation polymorphism (CE-SSCP)  Fig. . More im27) Fig. , suggestNext, we compared the RNA editing activity in the SH-SY5Y human cell line with that in the RNA extracted from patients’ blood. While most sites were found to be common to both cell types, such as the B site, small variations could be detected Fig. . Among tTranscript levels of the family of ADAR and PDE8A enzymes were analysed at different time points before and during antiviral treatment. While significant changes in gene expression were not observed for PDE8A and ADAR2 transcripts, a significant upregulation of ADAR1-1  and ADAR1-5  transcript level was observed as early as 2 weeks after onset of therapy Fig. . Indeed,Different clinical evaluations have been performed in IFN/ribavirin treated patients, including MADRS, YMRS, MATHys and emotional reactivity during treatment. By dividing the population into two groups composed of patients having a psychiatric event  or not, we analysed the combination of ADAR gene expression. We obtained a marked difference in ADAR1-1  and ADAR1-5  gene expression levels between the groups with and without psychiatric events Fig. . InteresIn this study, we have identified RNA editing blood biomarkers to detect HCV patients at risk to develop treatment-emergent depression during the course of IFN-α and ribavirin therapy. Clinical evaluation of HCV patients using MADRS score was used prior to onset and during the course of IFN-α and ribavirin treatment to track depressive episodes. In parallel, 80 blood samples were collected for analysis of ADAR and PDE8A gene expression and targeted quantification of RNA editing events in WBC. Next, based on clinical assessments, the study population was divided into a group that showed evidence for treatment-emergent depression and a second group that did not. Interestingly, we observed that IFN-α treatment led to an increase in ADAR1-1 (ADAR1a-p150) and ADAR1-5  gene expression as well as a significant increase of RNA editing on the B site of the intron 9 of the PDE8A mRNA. By combining the biomarkers, we generated an algorithm for detecting patients at risk to develop a treatment-emergent psychiatric event. Since depression is one of the most important complications during antiviral treatment with interferon and ribavirin, the assay may be valuable to support patient monitoring.IFN-α-induced depression is one of the most common psychiatric complications in HCV patients who receive IFN-α and ribavirin treatment. Up to 30% of HCV patients develop IFN-induced depression within the first 3 months  expression as well as alterations in the RNA editing activity on the PDE8A gene. Unexpectedly, both ADAR1-1 (ADAR1a-p150) and ADAR1-5 (ADAR1b-p110) displayed an increase in gene expression while ADAR2 did not show alteration in transcript level. While an increase in ADAR1-1 (ADAR1a-p150) expression upon IFN-α treatment has been well documented, this is unexpected and has not yet been clearly described for ADAR1-5  (George and Samuel n = 10) and could be confirmed in a larger population. However, despite the small size of patients, the results are representative of the prevalence of depression observed in the general population; we followed up patients during 16 weeks and then ran analysis on 80 blood samples. Second, this study shows high predictive performance using RNA editing activity of PDE8A. The identification of novel RNA editing targets is currently ongoing and could significantly improve the performance of the test.Our study has a number of limitations. First, the current study was performed in a relatively small number of enrolled patients  and type I interferon gene expression. Interestingly, ADAR gene expression was significantly induced in white blood cells of MDD patients when compared with healthy controls . The forward and reverse primers used for preparation of the sequencing library are shown. (PNG 282 kb)High Resolution (TIF 3323 kb)


Kip1CDKN1B/p27) mRNAs in vivo. P27Kip1 plays an important role in mediating a cell’s response to cisplatin (CP), a widely used chemotherapeutic cancer drug that induces DNA damage and cell cycle arrest. We found that Kip1p27 mRNA is stabilized upon CP treatment of HEK293 cells through elements in its 3ʹUTR. Applying tobTRIP, we further compared the associated proteins in CP and non-treated cells, and identified more than 50 interacting RBPs, many functionally related and evoking a coordinated response. Knock-downs of several of the identified RBPs in HEK293 cells confirmed their involvement in CP-induced p27 mRNA regulation; while knock-down of the KH-type splicing regulatory protein (KHSRP) further enhanced the sensitivity of MCF7 adenocarcinoma cancer cells to CP treatment. Our results highlight the benefit of specific in vivo mRNA-protein interactome capture to reveal post-transcriptional regulatory networks implicated in cellular drug response and adaptation.Post-transcriptional control of gene expression is mediated via RNA-binding proteins (RBPs) that interact with mRNAs in a combinatorial fashion. While recent global RNA interactome capture experiments expanded the repertoire of cellular RBPs quiet dramatically, little is known about the assembly of RBPs on particular mRNAs; and how these associations change and control the fate of the mRNA in drug-treatment conditions. Here we introduce a novel biochemical approach, termed tobramycin-based tandem RNA isolation procedure (tobTRIP), to quantify proteins associated with the 3ʹUTRs of cyclin-dependent kinase inhibitor 1B ( We thus developed a novel two-step tobramycin aptamer-based purification strategy, termed tobTRIP, to identify proteins bound to the 3ʹ untranslated regions (UTR) of specific mRNAs. The approach complements our previous tandem RNA affinity isolation procedure (TRIP) by using ASOs to capture endogenous mRNAs . The melt-curve was included at the end of the run to ensure specificity of the primers. The comparative Ct method was used to measure the amplification of mRNAs relative to β–actin [The RNA from affinity isolates was extracted with the ZR RNA MiniPrep Kit (Zymo #R1064). Ten microliters of RNA obtained from tobTRIP eluates or 500 ng of total RNA isolated from extracts (input) were combined with 2.5 μM of oligo(dT) β–actin .GFP, p27 and c-myc mRNAs relative to β–actin mRNA using respective primers for the p27 ORF, GFP, c-myc, and β–actin. The comparative Ct method was used to calculate the mRNA levels; and curves were fitted, and half-life determined with GraphPad Prism using ‘one phase decay equation’.HEK293 cells were treated for 15 h with CP, while GFP-T and GFP-T-p27(3ʹUTR) cells were induced with 1 µg/ml of tet for 33 h prior to the addition of CP for 15 h. Then, 2 µg/ml of ActD was added for the indicated times prior to RNA isolation. Five hundred nanograms of total RNA was used for RT-qPCR to measure the level of p27  and a fragment (nts 85–947) of the LDRL 3ʹUTR encompassing three AU-rich elements (AREs) [RASM was amplified as described previously [2, 5% glycerol, 1 mM DTT, 0.1% Triton X-100, 0.1 mg/ml heparin, 0.1 mg/ml tRNA, 1 mM phenylmethylsulfonyl chloride (PMSF), 1 µl 20 U/µl RNase OUT  and Complete™ mini EDTA-free protease-inhibitor tablets ) by mechanical disruption of transfected (pEGFPC1-6XHis-FLKSRP) HEK293 cells with glass beads in a Tissue Lyser . Biotin RNA pull-down experiments were performed essentially as described [DNA templates for biotin-RNA synthesis were prepared by PCR from either 100 ng of genomic DNA  or 80 ng of pGL3-p27-3ʹUTR plasmid with oligonucleotides bearing a T7 RNA polymerase promoter sequence. In particular, the entire 3ʹUTR of s (AREs)  were ampeviously . Biotinyescribed . Three hP-values [The human reference proteome (UP000005640) was downloaded from Uniprot considering Swiss-Prot reviewed entries . GO enrichment analysis was performed with the Generic Gene Ontology (GO) Term Finder using the human reference proteome as a background to calculate Benjamini-Hochberg-corrected P-values . The proP-values ) with evP-values  was quer


Flavivirus genus comprises many viruses  which constitute important public health concerns worldwide. For several of these pathogens, neither antivirals nor vaccines are currently available. In addition to this unmet medical need, flaviviruses are of particular interest since they constitute an excellent model for the study of spatiotemporal regulation of RNA metabolism. Indeed, with no DNA intermediate or nuclear step, the flaviviral life cycle entirely relies on the cytoplasmic fate of a single RNA species, namely the genomic viral RNA (vRNA) which contains all the genetic information necessary for optimal viral replication. From a single open reading frame, the vRNA encodes a polyprotein which is processed to generate the mature viral proteins. In addition to coding for the viral polyprotein, the vRNA serves as a template for RNA synthesis and is also selectively packaged into newly assembled viral particles. Notably, vRNA translation, replication and encapsidation must be tightly coordinated in time and space via a fine-tuned equilibrium as these processes cannot occur simultaneously and hence, are mutually exclusive. As such, these dynamic processes involve several vRNA secondary and tertiary structures as well as RNA modifications. Finally, the vRNA can be detected as a foreign molecule by cytosolic sensors which trigger upon activation antiviral signaling pathways and the production of antiviral factors such as interferons and interferon-stimulated genes. However, to create an environment favorable to infection, flaviviruses have evolved mechanisms to dampen these antiviral processes, notably through the production of a specific vRNA degradation product termed subgenomic flavivirus RNA (sfRNA). In this review, we discuss the current understanding of the fates of flavivirus vRNA and how this is regulated at the molecular level to achieve an optimal replication within infected cells.The  Flavivirus within the Flaviviridae virus family comprises almost 70 reported species including the most studied yellow fever virus (YFV), dengue virus (DENV), Zika virus (ZIKV), West Nile virus (WNV), Japanese encephalitis virus (JEV), and tick-borne encephalitis virus (TBEV). The vast majority of flaviviral infections in humans occur through the biting by arthropods such as Aedes-type mosquitoes  in the case of YFV, DENV, and ZIKV or Culex pipiens mosquitoes in the case of WNV. Vaccines do exist for YFV, DENV and TBEV. However, in the case of DENV, the cause of the most prevalent arthropod-borne viral disease, the only available vaccine shows limited efficacy against all DENV serotypes and safety concerns have recently arisen in the Philippines in vaccinated children  whose replication entirely occurs in the cytoplasm and does not generate any DNA intermediates. Most notably, vRNA contains all the genetic information necessary for optimal virus replication. Hence, targeting vRNA or viral processes involved in its metabolism constitutes an attractive strategy for the development of novel antivirals. Moreover, fundamental virology often provides crucial insight into cellular machinery and processes at the molecular level. In this respect, flavivirus vRNA constitutes an exciting and excellent model for investigating the spatiotemporal regulation of RNA metabolism. With that in mind, we focus this review on our current understanding of the multiple fates of vRNA and how it orchestrates the viral life cycle and creates a cellular environment favorable to infection.Flaviviruses are enveloped positive-strand RNA viruses that presumably contain a single copy of the genome RNA. Following receptor-mediated endocytosis of the virion and fusion with the endosomal membrane , Envelop (E) and prM assemble new viral particles while non-structural (NS) proteins NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5 are responsible for vRNA replication Figure . vRNA syin situ hybridization (FISH)-based sub-cellular distribution analyses of flaviviral vRNA and negative strand intermediate RNA have been reported to date in contrast with those of hepatitis C virus, a non-flavivirus member of the Flaviviridae family  of positive-strand RNA viruses, induce massive rearrangements of ER membranes to create a replication-favorable microenvironment that is generically called “replication factories (RF)” . These oe family .The 10–11 kb-long flavivirus vRNA genome is composed of one open reading frame (ORF) flanked by highly structured 5′ and 3′ untranslated regions (UTR) . The vir7GpppAm2)  or a non-functional cap, it was proposed that the 3′SL independently influences translation after cap binding by the small 40S ribosomal subunit , which are absent from the infectious virus particle. Hence, vRNA translation is a critical step for the initiation of vRNA synthesis and subsequent amplification. The flaviviral genome, like cellular messenger RNAs (mRNA), contains a cap structure at the 5′ end which enables translation through canonical cap-dependent translation initiation . The addGpppAm2) . Remarkain vitro . This inin vitro . Moreove subunit .Another stem loop structure in 5′UTR named “capsid-coding region hairpin” (cHP) is also implicated in flaviviral translation . MutatioFlaviviridae such as hepatitis C virus (HCV)  pathway. Moreover, expression knockdown of eIF4E (a component of the eiF4F cap-binding complex) in hamster BHK-21 or monkey Vero cells led to a 60% decrease in total cellular protein synthesis, whereas DENV NS5 protein levels decreased by just 10% . This daus (HCV) .Met ternary complex, which is absolutely required for translation initiation. Surprisingly, it appears that DENV-mediated repression of translation initiation is not functionally linked to PKR, infection-associated eIF2α phosphorylation or SG induction. These observations are consistent with several studies that have reported that DENV, ZIKV and WNV infection inhibits the formation of SGs, especially when cells are under oxidative stress following treatment with the SG inducer sodium arsenite  induce a general shut-off of host cell translation early following infection in human hepatocarcinoma Huh7 cells . This DEarsenite . In sucharsenite . Interesarsenite  are divearsenite . Indeed,arsenite . Overallde novo RNA synthesis . Within RFs (see below), it generates a pool of neosynthesized vRNA molecules that are subsequently used for the formation of new replication complexes, for translation-driven production of viral proteins and for packaging into assembling virus particles. vRNA replication relies on the RNA-dependent RNA polymerase (RdRp) activity of flaviviral NS5 protein, through an asymmetric process. vRNA synthesis is initiated by binding of NS5 to a secondary structure located at the 5′ terminus of the genome called “stem loop A” (SLA) which is critical for the initiation of vRNA synthesis , the 5′ψ and 3′ψ tertiary structures in the DENV vRNA regulate RNA synthesis . Indeed,in vitro analyses, several models have been proposed. NS3 helicase activity is most likely involved in vRNA synthesis from the negative strand. According to the model described above in which vRNA synthesis is initiated from dsRNA, NS3 would be required to unwind this molecule and displace the original vRNA molecule in favor of the nascent genome. Moreover, it cannot be excluded that NS3 also contributes to the synthesis of the antigenome by unwinding secondary and tertiary structures in vRNA. Finally, NS3 might unwind the RNA duplex so that neosynthesized positive-strands can be translated or packaged into assembling viral particles.It is believed that NS3 helicase assists vRNA synthesis presumably through direct interactions with NS5 . Althougin vitro using atomic force microscopy  is important for flavivirus replication and this sequence impacts genome circularization by modulating the topology of the 5′ end. In line with this, changes in the dCS sequence composition affects the formation of the 5′-3′ long range RNA-RNA interactions . Moreovein vitro and consequently of replication. In contrast, increasing the stability of the UFS does impede vRNA circularization. If UFS is too stable, this could result in a “locked-up” conformation of the UAR sequence which is known to be implicated in long range RNA-RNA interactions. Consistently, the circularization of vRNA induced the melting of the UFS structure resulting in a decrease in affinity of NS5 for the 5′ end of the vRNA. These data support a model in which the UFS functions as a riboswitch during RNA replication, which dictates vRNA circularization and NS5 recruitment. Following the binding of NS5 to SLA, the circularization of the vRNA would induce a disruption of the UFS structure, leading to a decrease in the affinity between NS5 and the 5′UTR. This would favor NS5 transfer to the 3′UTR, hence properly positioning the polymerase for negative-strand RNA synthesis.A structure present downstream SLA in the 5′UTR has been recently identified as an important riboswitch, which controls the equilibrium between NS5 recruitment to SLA and the circularization of the vRNA. This motif named “5′ UAR-flanking stem” (UFS) is a U-rich region located in SLB that promotes the formation of a conserved duplex RNA . The conA striking feature of flaviviral infections is the appearance of organelle-like membranous replication factories (RF) resulting from severe alterations of ER membranes. The detailed tridimensional architecture of RFs from several flaviviruses has been reconstructed using electron tomography . RFs areVesicle packets are spherical vesicles which are induced by invaginations of the ER Figure . They shper se  assembly intermediates. Moreover, comprehensive studies about the intracellular distribution of the different vRNA populations are lacking.bona fide RNA packaging signal has been identified for members of the Flavivirus genus. If packaging signal do exist, they are most likely to be located within the highly structured untranslated regions. Indeed, in the case of the related Hepacivirus HCV, the 3′UTR was shown to be important for RNA trans-encapsidation while mechanistic details are still being characterized  influences the production of infectious particles in both insect C6/36 and mammalian BHK-21 cells without affecting vRNA stability, translation and synthesis , the structural dynamics of the vRNA itself allow it to orchestrate the different steps of vRNA replication including vRNA circularization, NS5 binding and RNA synthesis. Considering that some vRNA domains can function as riboswitches, it is tantalizing to speculate that conformational changes in vRNA secondary and tertiary structures drive vRNA transfer from replication complexes in VPs to assembling virions. Moreover, the methylation status of the vRNA might contribute determining its fate. Indeed, it was recently shown that DENV, WNV, YFV, ZIKV and HCV vRNAs are N6-methylated on adenosines by the host methyltransferases METTL3 and METTL14 in infected cells  is important for optimal infectivity . The higcterized . Identifynthesis . Importaed cells . Very inoduction . In the assembly . Based otrans-encapsidation is possible for flaviviruses. Indeed, when structural proteins are expressed in trans, they form virus-like particles that can encapsidate sub-genomic replicons, i.e., replication-competent genomes that express only NS proteins  and interferon-stimulated genes (ISG). ISGs are antiviral effectors that in some cases, specifically target vRNA, may be secreted as proinflammatory cytokines or generate an overall antiviral state to impede virus replication . PatternTLR3 is a member of the Toll-like receptor family and plays a crucial role in activation of the immune response by recognition of dsRNA in endosomes, presumably during viral entry . TLR3 reRIG-I belongs to the RIG-I-like receptor (RLR) family and possesses a dsRNA helicase activity. It is a cytosolic PRR that targets specifically dsRNA and the 5′ tri/diphosphate moiety of short structured uncapped RNAs . It alsoMore recently, it has been shown that the cyclic GMP-AMP synthase (cGAS)/stimulator of IFN genes (STING) pathway, which normally senses DNA virus infection and mitochondrial DNA damage, is also activated upon RNA virus infection (including DENV and WNV) and induces type I IFN production . The vRNFrom a virus-host co-evolution viewpoint, the mammalian innate immune response has evolved to counteract viral infection. Of course, this also implies an adaptation from the pathogens in order to evade innate immunity to the benefit of replication. To this end, several interference mechanisms involving flaviviral proteins have been described over the last decade. Indeed, these viruses can dampen the antiviral signaling pathways by inhibiting for instance, cGAS, STING, RIG-I, MAVS, TBK1 and STAT1/2 functions through interactions with NS5, NS3, NS2B or NS4B . In addiO-methyltransferase activity (O-methylation is an RNA modification on the first and second nucleotides of the mRNA cap structures in which the ribose is methylated at the 2′-OH position by cellular nucleoside 2′-O-methyltransferases (MTase) contributing to form cap 1 (m7GpppNm) or cap 2 (m7GpppNmNm) structures . More specifically, replication and pathogenicity of WNV E218A mutant virus was rescued and comparable to wild-type virus in Ifit1 knockout mice demonstrating the key role of this ISG in antiviral immunity  . In the immunity . As compimmunity . Neverths system . Consists system . This hiO-methylation as a countermeasure against innate immunity has also been recognized in mouse and human coronaviruses. In this case, 2′-O MTase-deficient viruses induced a stronger type I IFN response resulting in attenuation of viral replication  , defectiFlavivirus genus, sfRNA is a highly structured 0.3–0.7 kb-long non-coding RNA and apparently is the most abundant viral RNA species in the infected cell  and the participation of viral proteins as well as host RNA-binding proteins. This intricate network is most likely hosted within viral RFs. Future studies will be needed to explore how all these regulated processes are interconnected to generate a precise integrated model of vRNA metabolism. For instance, does vRNA methylation on specific nucleotides impact vRNA tertiary structure formation, cyclization and/or affinity for host RNA-binding proteins, and vice-versa? Do these structural changes impact the efficiency of vRNA packaging into assembling virions? Importantly, when compared with HCV RFs, little is known about how flaviviruses regulate the morphogenesis of VPs that are very homogenous in size and shape. The same applies to the formation and maintenance of the VP pore that is believed to play a pivotal role in the transfer of vRNA from replication complexes to assembling particles. How is it functionally coordinated with budding viruses? Is this a dynamic structure oscillating between open and closed states? What is its viral and cellular protein composition? Finally, these considerations should ideally always take into account that flaviviruses infect both insects and mammals. Indeed, subtle differences between hosts in the life cycle  may be observed and of great interest.The tremendous work on flaviviruses during the last decade highlights the complexity of the molecular mechanisms governing the fate and functions of the vRNA. This includes dynamic RNA secondary and tertiary structures, RNA modifications such as 2′-Overall, this review highlights how flaviviruses have evolved to confer upon a single RNA species and one viral polyprotein product all the information required for optimal infection in both insect and mammalian hosts. More generally, all of these open questions regarding the vRNA perfectly illustrate the importance of flaviviruses as an exquisite model to study spatio-temporal control of RNA metabolism. Finally, a precise understanding of the dynamic control of vRNA in the flavivirus life cycle will hopefully identify potential therapeutic targets for the development of antivirals, ideally with a broad pan-flaviviral spectrum.CM and WF contributed equally to this work. CM and WF wrote the manuscript and made the figures. LC-C edited the final version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Meanwhile, the observed rapid-changing heteroplasmy frequency can potentially disturb cell functions, which could be an overlooked confounding factor in cell line related studies.Mitochondrial DNA (mtDNA) heteroplasmies are associated with various diseases but the transmission of heteroplasmy from mtDNA to mitochondrial RNA (mtRNA) remains unclear. We compared heteroplasmies in mtRNA from 446 human B-lymphoblastoid cell lines to their corresponding mtDNA using deep sequencing data from two independent studies. We observed 2786 heteroplasmies presenting in both DNA and RNA at 1% frequency cutoff. Among them, the frequencies of 2427 (87.1%) heteroplasmies were highly consistent (less than 5% frequency difference) between DNA and RNA. To validate these frequency consistencies, we isolated DNA and RNA simultaneously from GM12282 cell line used in those two sequencing studies, and resequenced its heteroplasmy sites. Interestingly, we also observed the rapid changes of heteroplasmy frequencies during 4 weeks of the cell culture: the frequencies at Day 14 increased by >25% than those at Day 0. However, the heteroplasmy frequencies from the same time point were highly consistent. In summary, our analysis on public data together with  Studies has shown that there are widespread RNA and DNA differences (RDD) in human nuclear genome3, however, the knowledge of mitochondrial RDD is still limited.The genetic information from DNA needs to be transcribed to RNA for protein synthesis to achieve biological functions. The fidelity of the sequences information from DNA to RNA is critical since the inconsistency introduced during the transcription process may contribute to genetic variations, and further affect protein synthesis and/or the gene expression level4. Recently, there are many studies indicating that mitochondrial heteroplasmic mutations are associated with broad spectrum of human diseases such as muscular dystrophy, diabetes, Alzheimer’s diseases and cancer12. In addition, the allele frequency of a deleterious mutation would be critical for its pathogenicity13. Nevertheless, there are few studies to investigate the transmission of mtDNA heteroplasmies to mitochondrial RNA (mtRNA). It remains unclear whether mutated and wild type mtDNA are transcribed proportionally.In contrast to only two copies of nuclear DNA (nDNA), there can be hundreds to thousands copies of mitochondrial DNA (mtDNA) existing within a single eukaryotic cell. The nature of multiple copies of mtDNA and their highly variable sequences make it possible that mutated mtDNA can co-exist with wild type mtDNA at any ratio, which is termed as heteroplasmy15. We investigated whether the extensively existing heteroplasmic variations in mtDNA were also observable in mtRNA, moreover, whether the heteroplasmy frequencies presented in RNA would keep consistent with that in DNA. Our results demonstrated that the majority of heteroplasmies in mitochondrial DNA (mtDNA) could be transcribed proportionally to RNA regardless of their pathogenic degrees. We further experimentally validate this observation by sequencing DNA and RNA extracted simultaneously from the same cells and confirmed that there was minimal difference in heteroplasmies between DNA and RNA. Interestingly, during the cell culture process, we observed that the heteroplasmies frequencies were changing dramatically with time which may disturb the cellular functions according to the mtDNA heteroplasmy threshold effects.Advances in sequencing technologies  enable us to study heteroplasmy at single nucleotide resolution. By comparing the sequence of mitochondrial RNA and DNA from the same individual, we are able assess the RDDs in mitochondrial genome. In this study, we comprehensively analyzed 446 pairs of RNA and DNA sequences from human lymphoblastoid cell lines, which were a subset of individuals from 1000 genome project15. These individuals were a subset of 1000 Genome Project, thus the DNA-seq data from the same individual were available from 1000 Genome Project14. To investigate mitochondrial heteroplasmy, the sequencing reads were first aligned to human reference genome hg19, and after a series of quality control steps, we retained high quality mtDNA sequencing reads for subsequent mitochondrial heteroplasmy analysis (Methods). We examined two statistics of the mitochondrial genome alignment results: median sequencing coverage and average cover rate of the mitochondrial genome . All identified heteroplasmies were categorized into “Observed in both DNA and RNA” (BDR), ‘Observed Only in DNA” (OD) and “Observed Only in RNA” (OR) groups. We identified 2786 heteroplasmies presenting in both DNA and RNA in total, 219 only found in DNA and 682 only in RNA, respectively. Consistent with previous studies12, heteroplasmy frequencies could be a determinant of the deleterious mutations. We first investigated whether the heteroplasmic mutations in DNA can be transcribed into RNA proportionally. Using these datasets, we compared the heteroplasmy frequency differences between DNA and RNA. For each heteroplasmy in BDR group, we computed its MAF in DNA, then computed the frequency of the same allele in RNA. The frequency differences were calculated as RNA-frequency minus DNA-frequency (RNA-DNA). The heteroplasmy frequencies were highly consistent between DNA and RNA   to the rest of heteroplasmies. However, the CADD scores of the HDLR heteroplasmies were not significantly higher than those of non-HDLR heteroplasmies . In addition, we also identified 219 heteroplasmies in OD group. There was no significant difference between the CADD scores of the heteroplasmies observed in OD group and those of BDR group . The average heteroplasmy frequency of OD group was 2.6%, and more than half of these heteroplasmies had frequency <1.5%, it’s possible that these heteroplasmies were dropouts in the RNA-seq data. Thus, we didn’t find the evidence for negative selections of heteroplasmy transmission from DNA to RNA.According to the heteroplasmy threshold effects theory). For example, the A allele frequency difference of site 929 was 63.1%  in the public dataset. In contrast, from our experiment data, the difference was only 2.2%  at day 0 and 1.4%  at Day 14. Meanwhile, we also observed dramatic frequency changes in all the three heteroplasmy sites during the 28 days , suggesting that the cell cultures for DNA and RNA extraction were performed independently. Assuming that the heteroplasmy frequencies could change during the cell culture period, and that DNA and RNA were extracted at different time points, it is possible that heteroplasmy frequencies between DNA-seq and RNA-seq data are different. To test this hypothesis, we sought to analyze three heteroplasmy sites with |DNA-RNA| frequency differences >45% from the above analysis Table . For exaays Fig. . For exaays Fig. . This obin vitro study suggests that heteroplasmy changes rapidly within 2 weeks of cell culture, which can be transmitted to RNA with high fidelity.Finally, we independently repeated 28-days cell culture using the same batch of cell stocks to address the reproducibility of our results. At Day 28, we observed the heteroplasmy frequency changes comparing to Day 0 in all three experiment repeats  for mtDNA translation. mtDNA is highly susceptible to mutations, since its DNA replication and repair mechanism was not as robust as nuclear DNA20. Recent studies reported that mtDNA heteroplasmic mutations have high prevalence even in general healthy population, ∼90% of the individuals carry at least one heteroplasmy21. The deleterious heteroplasmies among them, once reached the threshold frequency, could be a potential source of mitochondrial dysfunction22 if mutations from mtDNA are transcribed into mtRNA for the further protein synthesis. However, the knowledge of how faithfully the mtDNA heteroplasmic mutations could be transcribed into RNA remains still limited and unclear.Mitochondria are indispensable organelles in eukaryotic cells. Its primary function is converting nutrients and oxygen to ATP by oxidative phosphorylation (OXPHOS) to support various cellular functions. Besides, mitochondria also participate in diverse functions such as calcium signaling, autophagy and apoptosis24. In this project we confirmed that most of the individuals has modification/editing at these three sites . Low frequency heteroplasmies within those regions might be missed due to the fluctuated coverage. In the future studies, RNA heteroplasmy studies could emphasize on protein coding regions. We also investigated RNA editing/modification events in mitochondrial genome by inspecting the heteroplasmies only observed in RNA. There are three well-characterized mtRNA editing/modification sites  from previous studieses Table , and the12. Therefore, the frequency of a heteroplasmy is a critical factor for its functional impacts. The dynamics of heteroplasmy frequencies were studied in different scenarios in previous studies28, Lehtinen et al. showed that pathogenic heteroplasmy 3243 A > G can change during 4 months if growing in selective medium28, and Stewart et al. reviewed the possible mechanisms of how a single mtDNA mutation can eventually reach high frequency after multiple rounds of cell divisions4. Furthermore, Rahman et al. compared the heteroplasmy 3243 A > G frequency in blood samples separated by 9–19 years apart of six Mitochondrial encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) syndrome patients, and found a decline of the frequency in all the patients25. In a recent study, Burgstaller et al. compared oocytes as well as somatic heteroplasmy changes in a mouse model, and reported difference in mean heteroplasmy frequencies between mice generations27. These studies all suggested that heteroplasmy frequency is not a constant value over time. In a long-term context, the heteroplasmy frequency gradually changes. In this study, we tracked heteroplasmy frequencies in cell line cultures in 28 days. Remarkably, we found that the heteroplasmy frequency had dramatic changes in a short time frame: as much as ~30% within seven days. We also noticed that the frequency changes are not monotonous, for a specific allele, the frequency can move both up and down  and Geuvadis RNA Sequencing Project (http://www.geuvadis.org/web/geuvadis/rnaseq-project), respectively. Totally, there were 446 pairs of DNA and RNA sequences. To retrieve mtDNA candidates reads, raw sequencing was first mapped to the mitochondrial genome. The mapped reads were then re-mapped to the combined human genome, hg19 for the nuclear genome and the revised Cambridge Reference Sequence (rCRS) for the mitochondrial genome with bowtie230. To remove possible contaminations from nuclear mitochondrial sequence (NUMTs), we only retained reads which could be uniquely mapped to mitochondrial genome. Retained reads were further processed with the GATK best practice workflow, including Mark duplicates, Indel realignment, and Base quality score recalibration steps31. The sequencing information for each mtDNA sites were compiled with Samtools mpileup function32.DNA and RNA raw sequencing data was retrieved from 1000 Genome Project  Sequencing coverage >200. 2) Minor allele frequency > = 1%. 3) Minor allele must be observed at least twice from each strand. We then integrate heteroplasmy information from both sides to evaluate heteroplasmy concordance between DNA and RNA. To avoid possible artifacts caused sequencing coverage, we only consider sites with depth >200 in both DNA and RNA data. The heteroplasmies were grouped into the following categories: A heteroplasmy was grouped into “Observed in both DNA and RNA” (BDR) if it met any of the followings: 1. Identified as a heteroplasmy at 1% frequency cutoff in both DNA and RNA-seq data of the same individual. 2. If identified as a heteroplasmy only in DNA-seq data, the minor allele of this heteroplasmy needs to have at least 3 sequencing reads to support in the RNA-seq data from the same individual. 3. If identified as a heteroplasmy only in RNA-seq data, the minor allele of this heteroplasmy needs to have at least 3 sequencing reads to support in the DNA-seq data from the same individual. Otherwise, a heteroplasmy would be grouped into “Observed Only in DNA” (OD) and “Observed Only in RNA” (OR) groups if it was identified in either DNA or RNA data.17. The disease associated mtDNA mutations were obtained from the MITOMAP database33.Heteroplasmies were annotated by customized scripts. Pathogenic potential of variants was predicted using Combined Annotation-Dependent Depletion (CADD) score (version 1.3)https://www.coriell.org/). Cells were grown at 37 °C, 5% CO2, in Roswell Park Memorial Institute Medium 1640 with 2 mM L-glutamine and 15% fetal bovine serum with penicillin and streptomycin. Cells used for all experiments in this study were from the same cell aliquots that were prepared in advance and stored in liquid nitrogen. After thawing, cells were grown for 2 days to recover, followed by sub-culture (Day 0). Cell passage was performed every 2–3 days and fresh medium were replaced to maintain cell density with less than 2 × 106 cell/ml. (https://www.atcc.org/~/media/pdfs/culture%20guides/animcellculture_guide.ashx). Cells were then collected and stored at −80C after snap freezing at Day 0, 7, 14, 21 and 28. DNA and RNA were extracted from the collected cells (1 × 106 cells) simultaneously with AllPrep® DNA/RNA Mini Kit . RNA was reverse transcribed to cDNA with SuperScript™ III First-Strand Synthesis System .GM12282 B-lymphocyte cell line was purchased from Coriell Institute . For assessing mtRNA mutation, extracted total RNA was first treated with ezDNase™ Enzyme (ThermoFisher) to remove DNA contamination. Treated total RNA was converted to single strand cDNA by using High-Capacity cDNA Reverse Transcription Kit (ThermoFischer Scientific) after the incubation with ezDNase, followed by PCR amplification. The following primer sets were used for the amplification of region including mtDNA and mtRNA mutation.Location 929 forward: GATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTCACGGGAAACAGCAGTGATT, reverse: GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTATTGGGGAGGGGGTGATCTA;5794 forward: GATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTTTCAATCTACTTCTCCCGCCG, reverse: GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGGGGGTGAGGTAAAATGGCT;15153 forward: GATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTCAGAAACCTGAAACATCGGCA, reverse: GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTCCAATGTATGGGATGGCGGA.10 ng of PCR products were subject to sequencing using the following primers: Location 929, CACGGGAAACAGCAGTGATT; 5794, TTCAATCTACTTCTCCCGCCG; 15153, CAGAAACCTGAAACATCGGCA. Sanger sequencing data were visualized and allele frequencies were quantified by MutationSurveyor (V5.1.1).The 1000 Genomes Project developed guidelines on ethical considerations for investigators doing sampling. All collections included in the Project followed these ethical guidelines and model informed consent language. The 1000 Genomes Project Steering Committee, with input from the Project’s Samples and ELSI Group, made final decisions about which populations and sample sets to include in the Project.Heteroplasmy concordance between mitochondrial DNA and RNA Supplement


Chen  TARDBP account for 1–4% of all ALS cases and almost all arise in the low complexity C-terminal domain that does not affect RNA binding and processing. Here we report an ALS/FTD kindred with a novel K181E TDP-43 mutation that is located in close proximity to the RRM1 domain. To offer predictive gene testing to at-risk family members, we undertook a series of functional studies to characterize the properties of the mutation. Spectroscopy studies of the K181E protein revealed no evidence of significant misfolding. Although it is unable to bind to or splice RNA, it forms abundant aggregates in transfected cells. We extended our study to include other ALS-linked mutations adjacent to the RRM domains that also disrupt RNA binding and greatly enhance TDP-43 aggregation, forming detergent-resistant and hyperphosphorylated inclusions. Lastly, we demonstrate that K181E binds to, and sequesters, wild-type TDP-43 within nuclear and cytoplasmic inclusions. Thus, we demonstrate that TDP-43 mutations that disrupt RNA binding greatly enhance aggregation and are likely to be pathogenic as they promote wild-type TDP-43 to mislocalize and aggregate acting in a dominant-negative manner. This study highlights the importance of RNA binding to maintain TDP-43 solubility and the role of TDP-43 aggregation in disease pathogenesis.Amyotrophic lateral sclerosis (ALS) presents with focal muscle weakness due to motor neuron degeneration that becomes generalized, leading to death from respiratory failure within 3–5 years from symptom onset. Despite the heterogeneity of aetiology, TDP-43 proteinopathy is a common pathological feature that is observed in >95% of ALS and tau-negative frontotemporal dementia (FTD) cases. TDP-43 is a DNA/RNA-binding protein that in ALS and FTD translocates from being predominantly nuclear to form detergent-resistant, hyperphosphorylated aggregates in the cytoplasm of affected neurons and glia. Mutations in  Amyotrophic lateral sclerosis (ALS), also known as motor neuron disease (MND), is the most common adult-onset degenerative disease of motor neurons. The disease is characterized by progressive motor neuron loss from the spinal cord, brainstem and motor cortex, which subsequently leads to muscle weakness and eventual respiratory failure. The average age of disease onset is 55 years. Due to its rapid progression, patients typically die within 3 to 5 years of diagnosis. Riluzole remains the only licensed treatment of ALS in the UK, but only marginally extends life expectancy by a few months . The cauTDP-43 is a DNA and RNA binding protein predominantly localizing in the nucleus but also shuttling between the nucleus and cytoplasm. The architecture of TDP-43 comprises an N-terminal domain, two RRM RNA-binding motifs and a C-terminal region often referred to as the ‘prion-like’ domain, which has low complexity and is enriched for glycine and serine residues. TDP-43 plays a key role in regulating RNA transcription, editing, transport and translation, and is involved in the formation of cellular stress-induced stress granules in the cytoplasm . Both patients had a diagnosis of definite ALS based on revised El Escorial criteria  for 10 min. After blocking in normal serum, the sections were stained and imaged as described in the ‘Immunofluorescence’ section.Sections of 7-μm thickness of human spinal cord tissue samples in 10% formalin-fixed, paraffin-embedded tissue blocks were obtained from the London Neurodegenerative Diseases Brain Bank. The paraffin-embedded tissue blocks were deparaffinized in two changes of xylene and sequential percentage of ethanol solutions for 3 min each . Antigen retrieval was performed by soaking the slides in 10 mM citrate buffer pH 6.0 and microwaved for 15 min. For diagnosis, paraffin sections were stained for haematoxylin and eosin and sections were immunohistochemically stained with the rabbit polyclonal antibody to phosphorylated TDP-43 ; mouse monoclonal antibody to phosphorylated tau , α-synuclein , and amyloid-β , using the Leica BONDMAX™ (Leica Biosystems). Heat induced epitope retrieval was used for all antibodies except for α-synuclein and amyloid-β. For α-synuclein, and amyloid-β, 80% formic acid pretreatment was used. Nuclei were counterstained with Harris’ alum haematoxylin. For immunofluorescence staining, autofluorescence was blocked by the treatment of Sudan black , rabbit anti-mouse TDP-43  , DyLight™ 800 goat anti-mouse IgG , and DyLight™ 488/550/650 anti-rabbit or mouse IgG .Escherichia coli cell strain as proteins fused with a SUMO solubilization tag carrying a 6×His tag. Cells were grown in Luria-Bertani (LB) medium containing 50 µg/ml kanamycin at 37°C until an optical density of ∼0.7 at 600 nm was reached. Protein expression was induced at 18°C by addition of 0.5 mM IPTG. Cells were collected after overnight growth and resuspended in lysis buffer . Cells were lysed by probe sonication and the soluble proteins recovered by centrifugation at 70 000 rcf for 45 min at 4°C. Protein purification included a first nickel affinity chromatography step followed by overnight dialysis in the presence of the Tobacco Etch Virus (TEV) protease at a 1:20 protein:TEV molar ratio to remove the 6×His-SUMO tag. A second nickel-affinity chromatography followed and the flow-through was loaded onto a HiTrap® Heparin column to remove nucleic acids. The protein constructs were finally submitted to size-exclusion chromatography with a HiLoad® 16/60 Superdex 75 prep grade in phosphate buffer pH 7.2. Protein purity was checked by SDS-PAGE.The wild-type tandem RRM domains of TDP-43  and the corresponding K181E mutant variant were encoded in a pET-Sumo expression vector containing the kanamycin antibiotic resistance gene. The plasmids were expressed in a Rosetta2(DE3) 2 flush at 4.0 l/min. CD datasets were an average of 15 scans. Far-UV spectrum was recorded at 25°C in phosphate buffer, pH 7.2. Spectra were corrected for buffer signal and expressed as mean residue molar ellipticity θ (deg × cm2/dmol).Circular dichroism (CD) spectra were recorded on a JASCO-1100 spectropolarimeter with a constant N15N-labelled versions of wild-type RRM1-2 and the K181E mutant were produced by standard methods (15N-HSQC (heteronuclear single quantum coherence) spectra were recorded at 700 MHz frequency at 25°C on Varian spectrometers.Uniformly and selectively Protein aggregation was monitored by following the increment in emission fluorescence of the aggregate-specific dye ProteoStat® on a FLUOstar Omega plate reader. Proteins were diluted in phosphate buffer pH 7.2 to a concentration of 10 μM, with and without equal amount of the AUG-RNA (5′-GUGUGAAUGAAU-3′). The plate was sealed with an optic seal and the assay plates were incubated at 37°C under, shaking for 2 s (≈200 rpm) before each read (every 15 min). The experiments were performed at least in triplicate and the results referred to the blank, normalized and reported as percentage average.2. Cells were plated a day before transfection and media was refreshed before plasmid DNA transfection using Lipofectamine™ 2000 (Invitrogen). Cells were left for 48 h after transfection to be harvested for analysis unless otherwise stated.HEK293T and SH-SY5Y cells were cultured using Dulbecco’s modified Eagle medium (DMEM) and DMEM/F12 (Invitrogen) supplemented with 10% foetal bovine serum (Invitrogen), and maintained at 37°C, 5% COPOLDIP3 forward 5′-TGCTCTGAAGCTCACCAAAA-3′ and reverse 5′-GGAACGGAAGCTATACCATCAT-3′ .RNA was extracted using RNeasy® Mini Kit (Qiagen). cDNA was generated using 1 μg of RNA, oligo d(T) primer and SuperScript™ III First-Strand Synthesis Kit (Invitrogen). Fifty nanograms of cDNA was used for RT-PCR with forward primers labelled with IRDye 700 at the 5′ end. Primers used in this study included: g for 20 min at 4°C. After centrifugation, the supernatant was collected as the RIPA solubility fraction. The pellet, after being washed once with RIPA buffer, was then suspended in 20% of the original lysis volume with urea buffer  and collected as the insoluble, detergent-resistant fraction.The fractionation for protein solubility was performed using a protocol described by Cells were harvested in IP buffer . After a short centrifuge (14 000 rpm for 30 s at 4°C), the supernatant was collected and pre-cleaned with Dynabead® protein G (Invitrogen) 2 h at 4°C. The pre-cleaned lysate was then incubated with immunopurification antibody and fresh Dynabead® protein G (Invitrogen) overnight at 4°C. The Dynabead® protein G-antibody-protein complex was purified using magnetic separation and washed with IP buffer before elution in loading buffer.http://imagej.nih.gov/ij/). Integrated band intensities were normalized to that of loading control or the RIPA fraction.Protein quantification and western blotting were performed as described before  for 20 min and washed with phosphate-buffered saline (PBS) three times for 5 min. Cells were permeabilized by incubation in PBS containing 0.5% Triton™ X-100 (Sigma) for 15 min at room temperature, followed by blocking in PBS containing 1% donkey serum for 1 h at room temperature. Cells were incubated with primary antibody diluted in blocking solution overnight at 4°C. After washing in PBS, cells were subsequently incubated with fluorescent secondary antibodies diluted in blocking solution for 1 h at room temperature. DAPI (Sigma) was then used to stain for nuclei before being mounted on coverslips using FluroSave .2, 30 min) to Hybond-N+ nylon membrane  and UV cross linking (120 mJ/cm2). Biotin-labelled RNA was detected by chemiluminescence with stabilized streptavidin-horseradish peroxidase conjugate provided by the RNA EMSA kit.Using the LightShift Chemiluminescent RNA EMSA Kit  (Thermo Fisher), 5 μg of total cell lysate in RIPA containing RNase inhibitor (NEB) was mixed with 2 μg of tRNA, 1 μM of unlabelled (GU)×6 RNA and 2.5 nM of biotin-labelled (GU)×6 RNA (CGUGUGUGUGUGUGGU) (6) per well were plated on the 15μ-Slide 8 well chamber slide (Ibidi) the day before transfection. Cells were transfected with GFP-TDP-43 for 48 h and Hoechst staining  was applied 20 min before imaging.HEK293T cells  was performed using a Nikon A1R laser scanning confocal microscope fitted with an environmental chamber maintained at 37°C. Confocal images of 512 × 512 pixels were acquired using a ×60 oil immersion objective. Five frames were acquired before bleaching  a 3 × 3 μmThe data supporting the findings of this study are available from the corresponding author on request. A 38-year-old male patient presented with predominantly lower motor neuron signs that progressed over 48 months to a flaccid tetraparesis, but he was still able to speak and had normal cognitive function. His father developed lower motor neuron signs in his upper limbs at the age of 76 and 6 months later developed major personality and behavioural changes consistent with a clinical diagnosis of FTD that eventually led to profound cognitive deficits. Brain MRI was consistent with a diagnosis of FTD. The index patient survived 80 months from symptom onset and his father survived 36 months A. The brThe formalin fixed left half of the brain weighed 782 g. There was some slight cerebral swelling noted but no cerebral atrophy. No other macroscopical abnormality was seen in the brain. The spinal cord did show some evidence of thinning of anterior nerve roots. Histologically the spinal cord showed loss of anterior horn neurons and some surviving neurons contained Bunina bodies [TARDBP: NM_007375:c.541A>G:p.Lys181Glu) (K181E), predicted to be damaging by 15 of 20 prediction tools used to assess pathogenicity. The heterozygous mutation of TDP-43 K181E was validated with Sanger DNA sequencing  of the interaction with AUG RNA by means of biolayer interferometry. We found that wild-type RRM1-2 binds the UG-rich AUG RNA with a Kd of 3.2 ± 0.9 nM, while the K181E mutant displayed a Kd of 2.5 ± 0.4 µM  to the end of RRM2 (Ala260), which are completely devoid of any nonsense or missense variants in the ExAC database . This hiTDP-43 is an aggregation-prone protein that forms oligomers under physiological conditions and detergent-resistant aggregates in disease and overexpression cellular and transgenic models A. OverexIn summary, we have shown that although K181E and K263E ALS and FTD-associated mutants of TDP-43 do not directly bind RNA, they are able to disrupt RNA processing by sequestering endogenous wild-type TDP-43 protein within hyperphosphorylated aggregates, which could have significant functional consequences.TDP-43 is predominantly a nuclear protein; however, it is known to shuttle between the nucleus and cytoplasm, playing a crucial role in regulating RNA transport, metabolism and local cytoplasmic translation . To inveThe formation of hyperphosphorylated TDP-43 cytoplasmic and, less commonly, nuclear aggregates, are a hallmark feature of ALS and FTD, but the factors that contribute to their development are not fully understood. We have confirmed that some disease-associated mutations adjacent to RRM1 and RRM2 disrupt RNA binding and form nuclear and cytoplasmic aggregates. This may impair RNA processing through the sequestration of wild-type protein, acting in a dominant-negative fashion. Furthermore, the binding to its target RNA plays an important role in maintaining the solubility of TDP-43 protein. Disruption of RNA binding promotes TDP-43 to aggregate in the cytoplasm as well as in the nucleus.TARDBP linked to ALS and FTD and are associated with phosphorylated cytoplasmic TDP-43 aggregates in degenerating neurons  mRNA, would be predicted to affect a multitude of important cellular pathways that could lead to neurodegeneration. As more wild-type TDP-43 becomes trapped within aggregates, less soluble TDP-43 is available to the cell. Eventually the RNA binding deficient mutant TDP-43 can deprive the cell of its functional RNA processing counterpart, resulting in a dominant-negative loss-of-function. Furthermore, our studies imply that RNA binding to TDP-43 can inhibit its aggregation, which may open new avenues for therapeutic intervention.In conclusion, we have identified a novel mutation adjacent to the RRM1 domain in a father and son with ALS and ALS/FTD. RRM domains show marked evolutionary conservation and variation largely absent from human genome databases. We have shown that two TDP-43 mutations, adjacent to either RRM domain, will abolish RNA binding and promote the formation of hyperphosphorylated, insoluble TDP-43 aggregates that are the hallmark of FTD and ALS, providing functional evidence that these mutations are likely to be pathogenic. Mutant TDP-43 aggregates may be directly toxic but they can also sequester wild-type TDP-43 protein and the loss of RNA processing function, including the autoregulation of awz313_Supplementary_MaterialsClick here for additional data file.