Thermotoga maritima in complex with a truncated tRNA. The RNA is mainly bound by the N-terminal ferredoxin-like domain (NFLD) and the THUMP domain of one subunit within the ThiI homo-dimer thereby positioning the U8 close to the catalytic center in the pyrophosphatase domain of the other subunit. The recognition of the 3’-CCA end by the THUMP domain yields a molecular ruler defining the specificity for U8 thiolation. This first structure of a THUMP/NFLD-RNA complex might serve as paradigm for the RNA recognition by THUMP domains of other proteins. The ternary ThiI–RNA–ATP complex shows no significant structural changes due to adenosine triphosphate (ATP) binding, but two different states of active site loops are observed independent of the nucleotide loading state. Thereby conformational changes of the active site are coupled with conformational changes of the bound RNA. The ThiI–RNA complex structures indicate that full-length tRNA has to adopt a non-canonical conformation upon binding to ThiI.In prokaryotes and archaea transfer ribonucleic acid (tRNA) stability as well as cellular UV protection relies on the post-transcriptional modification of uracil at position 8 (U8) of tRNAs by the 4-thiouridine synthetase ThiI. Here, we report three crystal structures of ThiI from  The widthe tRNA , but addthe tRNA .4U8 is catalyzed by 4-thiouridine synthetase (ThiI)  (Bacillus anthracis (ThiIBa) and of a putative ThiI ortholog from the archaeon Pyrococcus horikoshii have been solved  has been proposed (The biosynthesis of se (ThiI) , which cin (RLD) . The cryn solved ,13. Baseproposed ,14. Whilproposed ,15.E. coli (ThiIEc) requiring the cysteine desulfurase IscS . The bulge (U8-C14) connecting both stems of TPHE39A is composed of the target U8, the adjacent A9 and the variable loop sequence G10-C14, which is equivalent to G44-C48 of the full-length tRNAPhe. Surprisingly, even though the 3D structure of TPHE39A differs in the T-stem and T-loop in comparison to full-length tRNA  in complex with the truncated tRNAPhe (TPHE39A) from E. coli. With regard to the crystal structure of ThiIBa, the structure of each domain of ThiITm remains unchanged upon RNA binding, but their relative orientation to each other changes considerably. The tRNA acceptor stem and the crucial single-stranded ACCA 3’-end form multiple interactions with the NFLD and THUMP domain of one monomer, respectively. This binding mode enables correct positioning of U8 close to the active site in the PPase domain of the other subunit within the homo-dimeric ThiI. The complex structure also suggests that the conformation of full-length substrate tRNA has to largely deviate from the canonical L-shape for productive binding.In order to gain insight into the RNA binding mode of ThiI and to understand the structural basis of ThiI's specificity for both tRNA and the target U8, we determined the crystal structure of ThiI from Tm, and the preparation and crystallization of the ThiITm–RNA complex, has previously been described (4)2SO4) was mixed with 3 μl of reservoir . Single crystals were obtained at 20°C after 1 week in sitting drops by vapor diffusion both in the presence of ATP (2 mM) and MgCl2 (2 mM) or in the absence of ATP and MgCl2. For data collection, crystals were transferred into cryo buffer  glycerol).The overexpression and purification of ThiIescribed . BrieflyTm–RNA and ThiITm–RNA–ATP) have been collected at the beamline X11 of the EMBL outstation Hamburg at DESY, Germany, at 100 K and a wavelength of 0.9780 Å and 0.8140 Å, respectively. The diffraction images of the ThiITm–RNA–ATP complex have been processed with the XDS package  and placed into the head of the Free Mounting System  in an adjustable and reproducible stream of humidified gas, which allows a controlled change of relative humidity . At the Tm–RNA complex crystallized in the space group P212121  with two protein and two minimal substrate RNA molecules in the asymmetric unit (solvent content of 68.7%). The data set collected from the crystal treated by controlled dehydration using the FMS was used in order to solve the phase problem by Molecular Replacement (MR) method and build the model. The known crystal structure of ThiIBa  and 30.3% (Rfree), the density for oligonucleotides became visible and allowed manual building of the two TPHE39A molecules. The model was validated against SIGMAA-weighted simulated annealing (SA) (difference) omit maps as calculated with CNS. The last refinement steps were performed using PHENIX (2 prior FMS treatment) was refined at the resolution of 2.85 Å to Rwork and Rfree factors of 18.5% and 22.7%, respectively. Stereochemical quality of the structure was assessed by MolProbity  have been scored according to the log-likelihood gain (LLG) values as calculated by PHASER. The protein model revealing the highest LLG score has been used as a partial solution for the search of tRNA molecules using truncated models of TPHE39A . The structure was manually rebuilt and verified against SIGMAA-weighted SA omit maps, composite omit maps and difference maps using COOT. The structure was refined in CNS using strong NCS restraints between equivalent residues in the two monomers and weaker ones between acceptor and T stems of tRNA molecules. The NCS restraints were later relaxed for regions showing different conformations in each ThiITm monomer . For refinement, 5% of the reflections were randomly chosen and left out for cross-validation as the free R-factor. In order to reduce the risk of over-fitting, refinement was based on slow-cooling SA (torsion angle dynamics) combined with standard minimization and grouped B-factor refinement. Remaining non-interpreted electron density in the active site clearly exhibited the size and shape of a bound nucleotide triphosphate. After adding an ATP molecule to both catalytic centers, in each active site a difference peak of mFo-DFc electron density at a contour level of 3.5 sigma has been observed within 2.5–3.3 Å distances to three oxygens of the triphosphate moiety. Those peaks have been interpreted as Mg2+ ions as they were present in the crystallization buffer. Refinement of individual atomic displacement factors for the Mg2+ ions resulted in B-factors in the same range as those of the ATP molecules (refined as grouped B-factor). Non-ideal Mg2+–O distances could result from low resolution of the diffraction data and consequently larger positional error, however we can not rule out the possibility that the observed difference electron density peaks could correspond to a highly coordinated water molecules or a mixture of the two mentioned individuals.The ThiITm monomers and two copies of TPHE39A (nucleotides 1–39). To model anisotropic displacements of the ThiI domains and tRNA molecules, we performed the final refinement step using PHENIX with eight TLS groups at 3.42 Å resolution resulting in Rwork and Rfree factors of 23.04% and 27.38%, respectively. The ATP-free ThiITm–RNA complex structure is isostructural with the ThiITm–RNA–ATP complex structure and has been refined using the same strategy at the resolution of 3.5 Å to the Rwork and Rfree factors of 23.02% and 26.86%, respectively.The final protein model consists of residues 3–388 for each of the ThiIB-factor sharpened SA omit electron density map of ATP was calculated with CNS  cells were transformed with the pET15b plasmids encoding either wild-type ThiITm, or ThiITm C165S or ThiITm C344S. Overnight cultures (3 ml) were grown in lysogeny broth (LB) medium in the presence of 50 μg/ml ampicillin at 37ºC with shaking at 250 rpm and used to seed 50 ml cultures. When the cultures reached an OD600 = 0.6, expression was induced with isopropyl β-D-1-thiogalactopyranoside (IPTG) (1 mM) and the temperature was raised to 45ºC for the remainder of the overnight incubation period. Unfractionated tRNA was isolated, digested and analyzed using high pressure liquid chromatography (HPLC) at a wavelength of 330 nm as previously described  were then removed and applied to DE81 filter discs (Whatman) and processed for scintillation counting as previously described (Reactions (50 μl) containing 50 mM Tris, pH 7.5, 5 mM Mg(OAc)Aliquots  μl were 2, 50 mM KCl, 0.2 mM ATP, 1 mM DTT, 1 μg IscSTm, 1 mg ThiITm and 20 μM TPHE39A (or tRNAPhe) RNA. After incubation for 10 min at 37°C, mixtures were analyzed by HPLC using a Supelco LC-18S column eluted with a 40 min gradient of 0–20% methanol in 20 mM potassium phosphate, pH 6.0, with UV detection at 260 nm.Complete reaction mixtures (50 μl) contained 50 mM Tris, pH 7.5, 5 mM MgClT. maritima (ThiITm) was crystallized with bound truncated tRNAPhe (TPHE39A) from E. coli , and the truncated tRNAPhe TPHE39A from E. coli is in vitro an excellent substrate for ThiITm, showing similar levels of s4U modification as a full-length tRNA transcript . The crystals usually exhibited poor diffraction properties, but optimization of the crystallization and crystal cryo-protection conditions followed by an extensive crystal screening yielded a few crystals diffracting to a resolution limit of about 3.5 Å. The diffraction quality of one ThiITm-TPHE39A crystal was further improved by controlled dehydration providing data to 2.85 Å resolution (ThiITm–RNAFMS), concomitantly leading to significant unit cell shrinkage , while the other one represents an ATP-bound form (ThiITm–RNA–ATP). These structures were refined at a resolution of 3.50 Å and 3.42 Å, respectively, with reasonable stereochemistry and R-values (Table B-factors) reflecting a high degree of crystal disorder most likely caused by the high flexibility of the ThiI–RNA complexes (see below), which correlates with the limited resolution and the Wilson B-factors of the corresponding diffraction data.ThiI from the hyperthermophilic bacterium  E. coli . ThiITm ge Table . The Thif ThiIBa  as searces Table . All strTm monomer has an elongated overall shape and comprises three structural domains: an N-terminal ferredoxin-like domain (NFLD: Met1-Lys72), followed by a THUMP domain (Gly73-Asp161) that is connected via a short flexible linker region (Arg162-Leu169) with the C-terminal PPase domain (Pro170-Glu388)  Figure . In the f ThiIBa , but theTm protein molecules, each binding one RNA molecule. The size and properties of the contact surface between the two ThiITm molecules in the asymmetric unit indicate the presence of a physiologically relevant homo-dimer . The interactions at the interface include 23 hydrogen bonds and 10 salt bridges, which are mainly formed by residues of the PPase domain and partially extend to the NFLD (three interactions), but exclude the THUMP domain. One hydrogen bond is formed between the backbone amide group of Gly168, which is part of the flexible linker, and the side chain of Asp319 located in the PPase domain of the other monomer. The dimer observed for the ThiITm–RNA complex corresponds to the dimer of ThiIBa defined by crystallographic 2-fold symmetry axis. The superposition of ThiIBa and ThiITm dimers unveiled that only the PPase domains represent the rigid and well superimposable part (r.m.s.d. of 1.4 Å for 301 Cα atoms).The asymmetric unit of the crystals contains two ThiIr Figure . The dimEach of the two RNA molecules present in the crystal's asymmetric unit interacts with both subunits of the ThiI dimer Figure  and D. TBa and ThiITm–RNA structures reveals an asymmetry of the ThiITm homo-dimer due to different relative orientations of the NFLD and THUMP domains with respect to the PPase domains . Interestingly, the observed asymmetry correlates well with the number of crystal and protein–RNA contacts and does not cause significant structural changes within the individual domains as indicated by negligible r.m.s.d. values obtained from domain-wise comparison. The largest differences are observed for the ThiITm–RNAFMS structure, most likely caused by the crystal dehydration and unit cell shrinkage. The ThiITm–RNA and ThiITm–RNA–ATP structures show a similar, moderate degree of asymmetry indicating that ATP binding does not cause large conformational changes in the protein.The comparison of the ThiIs Figure . This flPhe , trans Watson–Crick ‘Levitt’ (G15-C48), trans Watson–Crick sugar-edge (G18-U55) and cis Watson–Crick (G19-C56) base pairs found in unmodified tRNAPhe  and the T-loop (G20-A23), while the acceptor stem and T-stem structures are very similar . In addition to the THUMP domain the NFLD, which closely resembles the RNA recognition motif of ribosomal protein S6, also binds the RNA mainly by residues located in the loops Arg10-Lys19, and Trp44-Arg46, but there are also important contacts with residues of α-helix α1 (e.g. Arg21) and of β-strand β2 (e.g. Arg42).The pattern of interactions between the 3’-ACCA moiety and the THUMP domain is very similar in all ThiIs Figure  and D. Ts Figure . In addiTm–RNA complexes in the ATP-free as well as ATP-bound form. Comparison of both structures depicts that ATP binding does not cause large conformational changes. The adenosine moiety of the ATP is bound in the active site of the PPase domain similarly to the ThiIBa–AMP complex structure (Tm and ThiIBa (except Thr205 which is His208 in ThiIBa) and have identical side chain conformations . The adenine ring is bound deeply in the active site and forms both polar and van-der-Waals interactions with ThiITm . Notably, the active site loop carrying this catalytic cysteine is fully disordered in the ThiIBa–AMP complex structure, whereas it is traceable in the ThiITm–RNA and ThiITm-ATP–RNA structures  and upper (Lys338–Asn351) active site loops that comprise Lys17 and Lys19 or the catalytic Cys 344, respectively.The cysteine residue 344 within the PPase domain of ThiIatalysis . Mutatios Figure . While ny Figure . They coIn case of the RNA molecule bound to the NFLD-B and THUMP-B, the bulge region is positioned further away from the PPase-A domain and makes only two interactions with the active site residue Lys350-A Figure . The NFLin silico as putative RNA-binding domain . Interestingly, with exception of Pus10 all of these enzymes contain also a NFLD that forms together with the THUMP domain a functional entity similar to that of ThiI. This applies especially for the tRNA-modifying enzyme CDAT8, which catalyzes a C-to-U editing of archaeal tRNAs at position 8 . Therefore, full-length substrate tRNAs are expected to undergo large conformational rearrangements upon binding to ThiI. This conclusion is supported by circular dichroism (CD) spectroscopic studies indicating conformational changes to occur in tRNAPhe when it binds to ThiI .RNA-complex structures of other tRNA-modifying enzymes revealed that the substrate tRNA is either bound primarily as a rigid body or it is remodeled significantly thereby increasing the affinity and/or enabling access to an otherwise buried target base. Based on these tRNA conformations required for catalysis tRNA-modifying enzymes have been divided into two families . Interes to ThiI . The non to ThiI  can be rTm and ThiIBa orthologs is quite similar, significant rearrangements of the individual domains relative to each other can occur. They seem to represent an essential inherent feature of ThiI to accommodate productive substrate binding and target U8 recognition. Structural changes of loops around the active site in the PPase domain result in two main shapes of its rim. The ATP binding pocket is either rather shallow—probably preventing U8 to flip into it—or larger and wider making it accessible for ATP and U8 binding . Interestingly, the information about the conformation of the bound RNA seems to be transferred to the ATP binding pocket. This signaling cascade employs a set of loops flanking the active site that originate on one side from the NFLD of one monomer or on the opposite side from the catalytic PPase domain. These loops undergo coinciding rearrangements and place the catalytically essential Cys344 of the active site loop close to the bound ATP when the RNA has a proper position and conformation for U8 flipping, thus facilitating specific s4U8 formation. However, the crystal structures do not show a flipped out U8 bound in the catalytic center or even the adenylated U8 in case of the ATP complex. Probably the flip of U8 and the adenylation reaction only occur in the presence of IscS bound to the ThiI–RNA complex, which would be consistent with biochemical studies (unpublished). Such an IscS-induced mechanism appears reasonable as the adenylated U8 could be attacked by the sulfide immediately after the flip and the activating adenylation reaction, thereby preventing a wasteful hydrolysis of the U8-AMP intermediate. To understand the underlying mechanism to greater extent, structural information on the ThiI–RNA–IscS complex and the U8-adenylated ThiI-bound RNA coordinated at the active site is needed.Although the overall structure of the three domains of the tripartite ThiIEc the Cys456 of the RLD forms an essential persulfide which was suggested to either directly attack the adenylated U8 or to serve as a source for the generation of hydrogensulfide anion (HS−) which then performs the nucleophilic attack  strictly depends on the sulfurtransferase NifZ, which transfers the sulfhydryl group onto a not yet identified cysteine of ThiIBs , which exclusively modifies U34 in the anticodon loop of some tRNAs. In the crystal structure of MnmAEc a disulfide bond between two active site cysteines  was observed (The reaction mechanism of 4-thiouridine synthetase appears to show striking variability depending on the organism. In ThiIc attack . In contf ThiIBs . In vitrobserved .Tm the mechanism has to be unique as it contains only one cysteine in the active site (Cys344). There is only one additional cysteine residue (Cys165) present in ThiITm located in the loop linking NFLD and THUMP domain. However, Cys165 plays no role, as the C165S mutant of ThiITm is as active as wild-type protein both in vivo and in vitro , which is consistent with the structure as Cys165 is much too distant from the active site . Since the reaction requires the formation of a disulfide bond, it is tempting to speculate that it is formed between Cys344 and the persulfide carrying Cys of the IscS sulfurtransferase. The formation of AMP during in vitro thiolation of TPHE39A by ThiITm indicates the adenylation of the substrate , however, the covalent bond between the α-phosphate and U8 was not formed prior to crystallization of the ThiI-ATP–RNA complex as it was observed e.g. in one of the crystal structures of the 2-thiouridine synthetase MnmA .The atomic coordinates and structure factors have been deposited in the Protein Data Bank, Supplementary Data are available at NAR Online.


Thousands of long non-coding RNAs (lncRNAs) have been identified in mammalian cells. Some have important functions and their dysregulation can contribute to a variety of disease states. However, most lncRNAs have not been functionally characterized. Complicating their study, lncRNAs have widely varying subcellular distributions: some reside predominantly in the nucleus, the cytoplasm or in both compartments. One method to query function is to suppress expression and examine the resulting phenotype. Methods to suppress expression of mRNAs include antisense oligonucleotides (ASOs) and RNA interference (RNAi). Antisense and RNAi-based gene-knockdown methods vary in efficacy between different cellular compartments. It is not known if this affects their ability to suppress lncRNAs. To address whether localization of the lncRNA influences susceptibility to degradation by either ASOs or RNAi, nuclear lncRNAs (MALAT1 and NEAT1), cytoplasmic lncRNAs (DANCR and OIP5-AS1) and dual-localized lncRNAs  were compared for knockdown efficiency. We found that nuclear lncRNAs were more effectively suppressed using ASOs, cytoplasmic lncRNAs were more effectively suppressed using RNAi and dual-localized lncRNAs were suppressed using both methods. A mixed-modality approach combining ASOs and RNAi reagents improved knockdown efficacy, particularly for those lncRNAs that localize to both nuclear and cytoplasmic compartments. Advancements in genomics technologies over the last decade have led to the identification of tens of thousands of previously unsuspected long non-coding RNAs (lncRNAs), revolutionizing the way we view the complex eukaryotic transcriptome ,2. WhileLncRNAs comprise a remarkably diverse population of cellular RNAs. By definition, these are non-protein coding RNAs longer than 200 nt. Length varies from the small 7Sk species (∼330 nt) to the very large Airn (∼118 kb). LncRNAs can be transcribed from intergenic, intragenic or intronic regions. Further, these species can be spliced or remain unspliced and are often 5′-capped and 3′-polyadenylated (reviewed in ). ThrougMethods to degrade cellular RNAs using synthetic oligonucleotides have been used for many years as an approach to study gene function. The present study employs both ‘antisense’ and RNA interference (RNAi) approaches. ‘Antisense methods’ were first used to suppress gene expression over 35 years ago –19 and eTm) of the oligonucleotide, which can lower potency. A chimeric ASO design was developed that employs Tm-enhancing modifications at the ends to increase binding affinity to the target, retaining a central DNA core to form a substrate for RNase H when duplexed with the RNA target (termed a ‘gapmer’)  RNA and locked nucleic acids (LNA)  ,31. The ds (LNA) . ASOs cods (LNA) ,33,34.In recent years, RNA interference (RNAi) has gained favor in the research community as a primary tool to reduce mRNA levels. RNAi is a natural process to regulate gene expression that exists in most eukaryotic cells. RNAi employs small, double-stranded RNA molecules that associate with multiple protein factors to form the RNA-Induced Silencing Complex (RISC), leading to either suppression of translation or degradation of a target mRNA. Natural RNAi effector molecules either arise from processing of endogenous hairpin transcripts by the nucleases Drosha and Dicer (microRNAs or miRNAs) or from long double-stranded RNAs (dsRNAs) processed by Dicer alone  . SynthetAlthough both RNAi and antisense methods have already been successfully used to knockdown specific lncRNAs –45, the We investigated if the intracellular localization of individual lncRNAs influences which knockdown method is optimal to employ. We also compared different ASO and RNAi reagent designs and chemical modification strategies to determine if a specific reagent showed any particular advantage in this application. A set of seven lncRNAs with various cellular distribution patterns was selected from the literature to perform a comparative survey Table . MALAT1 ® Select siRNAs) were designed using the Ambion site selection algorithm .Three design variants were tested for both the ASO and RNAi arms of the study Table . Reagent® Select siRNAs were purchased from Thermo Fisher Scientific  and LNA™ longRNA GapmeRs were purchased from Exiqon . All oligonucleotide sequences are shown in Supplementary Table S1 in the online Supplemental Data.Antisense oligonucleotides (DNA-PS and 2′ OMe-PS gapmers), primers and probes were chemically synthesized using standard phosphoramidite chemistry. RNA oligonucleotides (21mer unmodified siRNAs and 27mer DsiRNAs) were chemically synthesized using t-Butyl-dimethylsilyl (TBDMS) chemistry (Integrated DNA Technologies). Probes for qPCR were purified using reversed phase high performance liquid chromatography (RP-HPLC) (Integrated DNA Technologies) while all other oligonucleotides were prepared as sodium salts. All oligonucleotides were analyzed by electrospray-ionization mass spectrometry (ESI-MS) and were within ±0.02% predicted mass. Oligonucleotide concentrations were calculated using modification-specific extinction coefficients based on measured ultraviolet (UV) absorbance at 260 nm. RNA duplexes were annealed in IDT duplex buffer  (Integrated DNA Technologies). Silencer® 2000 at 10, 3 and 1 nM doses. HeLa cells were cultured in ATCC recommended media. For transfections (optimized for this system), oligonucleotides were reverse transfected in 96-well plates by complexing the various oligonucleotide doses with 0.5 μl Lipofectamine® 2000 (Thermo Fisher Scientific) in OptiMEM® I (Thermo Fisher Scientific) for a total volume of 50 μl in each well. HeLa cells (20 000) were suspended in 100 μl Dulbecco's Modified Essential Medium (DMEM) containing 10% fetal bovine serum (Thermo Fisher Scientific), added to the lipid-oligonucleotide complexes, then incubated for 24 h at 37°C and 5% CO2. For HOTAIR, transfections were performed in 48-well plates with 40 000 HeLa cells per well, with doubling of all reagent volumes. Each 96-well transfection plate included a positive transfection control  and two or three compound-specific negative transfection controls. All transfections were performed in biological triplicate and each experiment was performed at least twice. The results of all transfections performed were averaged.To compare the knockdown efficiency between reagents, oligonucleotides were transfected in biological triplicate into HeLa cells with Lipofectamine® II Reverse Transcriptase (Thermo Fisher Scientific) per the manufacturer's instructions. qPCR reactions were performed using ∼10 ng cDNA with Immolase DNA polymerase , 500 nM of each primer and 250 nM probe in 10 μl reactions in 384-well plate format. Amplification reactions were run on an Applied Biosystems 7900HT (Thermo Fisher Scientific) with cycling conditions consisting of enzyme activation at 95°C for 10 min, followed by 40 cycles of 2-step PCR . All qPCR reactions were performed in triplicate and averaged. Linearized cloned amplicons were used as copy number standards to establish absolute quantitative measurements for each assay. LncRNA expression levels were quantified by multiplexing two 5′-nuclease assays per target (one assay located toward the 5′ end and one located toward the 3′ end of the transcript) and normalized against both HPRT (NM_000194) and SFRS9 (NM_003769) as internal reference controls. LncRNA knockdown levels were calculated by comparing cells transfected with ASO or RNAi reagents to cells transfected with appropriate chemical modification-specific negative control oligonucleotides. Data are plotted graphically with error bars representing standard error of the mean (SEM).LncRNA knockdown was measured by reverse transcription quantitative PCR (RT-qPCR), comparing each knockdown reagent with the appropriate chemically modified negative control oligonucleotide at the same dose and calculated from an average of two or three separate experiments. RNA was isolated 24 h after transfection with the SV 96 Total RNA Isolation Kit  with DNase1 treatment. cDNA was synthesized from ∼200 ng total RNA with anchored oligonucleotide dT and random hexamer primers (Integrated DNA Technologies) using SuperScript® 6.0 software using the non-linear regression formula: log (inhibitor) versus normalized response—variable slope .HeLa and HCT116 cells were transfected in biological triplicates with ASOs or siRNAs as previously described. A range of nine doses for each knockdown reagent was selected based on empirically determined oligonucleotide activity and tested in both cell lines. RNA extraction, cDNA synthesis and qPCR reactions were performed as previously described. IC50 values were calculated with GraphPad PrismO-methoxyethyl phosphorothioate gapmer ASO (ISIS395254) (MALAT1 (metastasis associated in lung adenocarcinoma transcript 1) is a highly abundant ∼8.7 kb lncRNA that localizes to nuclear speckles and plays a role in both regulating alternative splicing and cellular proliferation –67. MALAS395254) .Results are shown for MALAT1 and NEAT1 Figure  and B wiFor the two nuclear lncRNAs studied, the ASOs were consistently more effective at suppressing RNA levels than were the RNAi reagents. For MALAT1 Figure , severalTo verify that the RNAi reagents and ASOs did not cause non-specific inhibition of nuclear lncRNAs, all of the MALAT1 or NEAT reagents were individually transfected into HeLa cells as previously described and RNA levels for both MALAT1 and NEAT1 RNA were measured. None of the MALAT1 knockdown reagents affected NEAT1 levels , and none of the NEAT1 knockdown reagents affected MALAT1 levels , demonstrating target specificity.DANCR (Differentiation Antagonizing Non-Protein Coding RNA) is a small ∼0.8 kb lncRNA involved in maintaining the undifferentiated state of certain cell types in the epidermis and regulating osteoblast differentiation; earlier reports indicated that this target can be suppressed using either shRNAs or siRNAs ,80–82. RThe cytoplasmic lncRNA DANCR was effectively suppressed by all 28 RNAi reagents tested, of which 17 out of 28 showed 80% or higher reduction in DANCR RNA levels at the 1 nM dose Figure . While sin vitro and in vivo, and Bhan et al. inhibited HOTAIR with a DNA-PS ASO , a ∼7.5 kb lncRNA necessary for retinal development and involved in the upregulation of growth-control genes ,16,84–86A-PS ASO ,91,93–98Both ASO and RNAi reagents were able to reduce TUG1 expression Figure , althougA summary compilation of the knockdown studies performed on all seven lncRNAs  at the 10 nM dose is shown in Figure Many lncRNAs are not restricted to either the nuclear or cytoplasmic compartment but exist in variable concentrations between the different subcellular locations . SimultaIt is desirable to employ knockdown reagents which are potent and have minimal toxicity. SiRNAs have been reported to be ≥100-fold more potent than ‘first generation’ DNA-PS ASOs for RNA knockdown, as evidenced by IC50 values in comparative studies at the same target sites –103. ‘Se® Select siRNAs). Oftentimes, potent siRNA sites do not correlate with potent DsiRNA sites and viceversa . However, general knockdown trends were the same between the two classes of ASOs, with good DNA-PS ASO sites also serving as good 2′ OMe-PS ASO sites, which may allow for the less expensive DNA-PS ASOs to be employed for screening ‘walks’ to identify good sites in high value targets with later conversion to a more active 2′ OMe-PS gapmer design.Antisense and RNAi are both widely used techniques for sequence-specific reduction of cellular RNA expression. They employ distinct mechanisms of action with different protein co-factors and/or effector molecules that have different intracellular distribution patterns. While neither method is restricted to a single subcellular compartment, relative activity can vary between compartments and can affect the ease with which that method can be used to knockdown an RNA target. RNase H-mediated ‘degradative’ antisense relies upon hybridization of an ASO to the RNA target to form an RNA:DNA heteroduplex substrate for RNase H1, which is largely (but not exclusively) found in the nucleus –24. The Targeting mRNAs is somewhat simplified by the single purpose of protein coding transcripts, which are made in the nucleus and translated to protein in the cytoplasm. Hence, RNase H active-ASOs can be used to trigger degradation of nascent pre-mRNA transcripts in the nucleus and siRNAs can be used to trigger degradation of mature mRNAs in the cytoplasm; use of either method serves to prevent translation of the targeted mRNA. Although antisense methods were the first gene knockdown technology developed, RNAi has become the dominant technology employed in research applications, largely due to its ease of use. Efforts to develop algorithms to predict effective ASO sites have not had the same success as for RNAi, probably due to their different mechanisms of action. ASO activity relies upon hybridization of the single-stranded oligonucleotide to the target RNA, which is highly dependent upon both the folded structure of the RNA and the presence of RNA-binding proteins that may limit access. Tertiary structure and protein binding are difficult to predict, particularly for longer RNAs of the type studied herein. ASO site selection was done by combining RNA folding prediction, analysis of oligonucleotide sequence properties and a cross-reactivity search to help ensure specificity (for the DNA-PS and 2′ OMe-PS sites) or by Exiqon using proprietary methods (for the LNA-PS sites). RNAi, on the other hand, relies upon a protein effector complex (RISC) that evolved to mediate gene regulation by endogenous dsRNAs and seems to facilitate interaction between the siRNA and the RNA target. Algorithm development for siRNA site selection has been very successful , reducinTargeting lncRNAs is complicated by their wide range of functions and subcellular distribution patterns. LncRNAs play many different roles in the cell, including, for example, association with chromatin or with mRNA, or binding miRNAs or proteins (‘sponge’ effects). Some lncRNAs reside mainly in the nucleus, some reside primarily in the cytoplasm, others are present in both compartments, while yet others concentrate in specific subcellular organelles. It is, therefore, not surprising that lncRNAs might be more difficult to suppress than mRNAs or that greater challenges would be encountered when trying to use the different gene knockdown techniques available.The present study observed that achieving high levels of knockdown of nuclear lncRNAs was easier using antisense methods while knockdown of cytoplasmic lncRNAs was easier using RNAi methods. Consistent with this observation, antisense methods are thought to be particularly effective in suppressing nuclear-retained RNAs, including lncRNAs such as MALAT1 ,114. IntIt is interesting that the cytoplasmic targets DANCR and OIP5-AS1 both associate with 40S/60S ribosomal subunits, placing them in proximity to known sites for high RNAi activity ,51. ThisThe results reported and discussed herein employed measurements of RNA levels taken 24 h post-transfection and, therefore, assess the immediate effects seen on degradation of target molecules present in accessible compartments. Similar results were observed if measurements were taken 48 h post-transfection (data not shown); however, longer incubation periods were not studied. Since transcription occurs in the nucleus, antisense methods, which utilize nuclear RNase H1, should eventually be effective at suppressing all lncRNAs, regardless of where the mature lncRNA product later accumulates in the cell. The time course of this knockdown will vary with the turnover rates for each lncRNA species.The present study employed two RT-qPCR assays to measure levels of each lncRNA. One assay was located toward the 5′-end and the other toward the 3′-end of the target. Using two (or more) assays is needed to help rule out assay-related artifacts, such as false positive results due to interference of the oligonucleotide-based knockdown reagents with reverse transcription or qPCR . Also, tDifferent methods can be used to suppress expression of lncRNAs. Due to their varied function and subcellular distribution, antisense methods (for nuclear species) or RNAi methods (for cytoplasmic species) may show better performance. When the distribution of a lncRNA is unknown, antisense methods may be more reliable for achieving knockdown if only one approach is employed. Combined use of antisense with RNAi methods can lead to better knockdown than use of either method alone, especially for lncRNA targets that are distributed throughout the cell.


Aedes mosquitoes, infections with arthropod-borne viruses (arboviruses) trigger or modulate the expression of various classes of viral and host-derived small RNAs, including small interfering RNAs (siRNAs), PIWI interacting RNAs (piRNAs), and microRNAs (miRNAs). Viral siRNAs are at the core of the antiviral RNA interference machinery, one of the key pathways that limit virus replication in invertebrates. Besides siRNAs, Aedes mosquitoes and cells derived from these insects produce arbovirus-derived piRNAs, the best studied examples being viruses from the Togaviridae or Bunyaviridae families. Host miRNAs modulate the expression of a large number of genes and their levels may change in response to viral infections. In addition, some viruses, mostly with a DNA genome, express their own miRNAs to regulate host and viral gene expression. Here, we perform a comprehensive analysis of both viral and host-derived small RNAs in Aedes aegypti Aag2 cells infected with dengue virus 2 (DENV), a member of the Flaviviridae family. Aag2 cells are competent in producing all three types of small RNAs and provide a powerful tool to explore the crosstalk between arboviral infection and the distinct RNA silencing pathways. Interestingly, besides the well-characterized DENV-derived siRNAs, a specific population of viral piRNAs was identified in infected Aag2 cells. Knockdown of Piwi5, Ago3 and, to a lesser extent, Piwi6 results in reduction of vpiRNA levels, providing the first genetic evidence that Aedes PIWI proteins produce DENV-derived small RNAs. In contrast, we do not find convincing evidence for the production of virus-derived miRNAs. Neither do we find that host miRNA expression is strongly changed upon DENV2 infection. Finally, our deep-sequencing analyses detect 30 novel Aedes miRNAs, complementing the repertoire of regulatory small RNAs in this important vector species.In  Aedes family transmit many important viruses, including dengue virus, between their vertebrate hosts. In the mosquito, the growth of these viruses is limited by the antiviral RNA interference pathway. Key to this pathway is a class of small non-coding RNAs known as small interfering RNAs (siRNAs). In addition, two related but distinct small RNA pathways known as the microRNA (miRNA) and the PIWI-interacting RNA (piRNA) pathway are implicated in regulating virus replication in mosquitoes. Thus, since small RNAs may critically influence the transmission of dengue virus, we set out to analyze the populations of viral and mosquito small RNAs that are produced in infected Aedes mosquito cells. We found that besides the well-known viral siRNAs, dengue virus-derived piRNAs were produced in these cells and we identified the PIWI proteins that these small RNAs rely on. In addition, we found that viral miRNAs were not expressed from the dengue virus genome and that the levels of mosquito miRNAs were barely changed upon infection. Finally, our data allowed for the identification of novel Aedes miRNAs, complementing the repertoire of these important regulatory RNAs in vector mosquitoes.Mosquitoes of the  Aedes mosquitoes are essential vectors for the transmission of important arthropod-borne viruses (arboviruses), including dengue virus (DENV), yellow fever virus, and chikungunya virus [ya virus . While sya virus .After ingestion in a mosquito’s blood meal, arboviruses need to overcome a number of anatomical and immunological barriers to reach sufficiently high titres in the saliva. Only then can transmission to a naive vertebrate host efficiently occur. One of the most important immune responses to arboviral infection is antiviral RNA interference (RNAi) –5. This Aedes mosquitoes, miRNA levels or modifications have been reported to be changed upon infections with DENV, West Nile virus, and chikungunya virus [MicroRNAs (miRNAs) are a distinct class of small RNAs that are produced from genome-encoded stem loop-containing transcripts known as primary miRNA (pri-miRNAs). During the canonical miRNA biogenesis pathway, the stem loop structures, known as precursor miRNA (pre-miRNA), are released from the pri-miRNA by the microprocessor complex with at its core the RNase-III enzyme Drosha. After translocation into the cytoplasm, pre-miRNAs are cleaved by Dicer-1 (Dcr1) to produce a small RNA duplex comprised of the two mature miRNA strands. Usually, one of these strands is then preferentially incorporated into the Argonaute-1 (Ago1) containing miRNA-induced silencing complex (miRISC), whereas the other strand (the passenger or miRNA* strand) is usually discarded . Loaded ya virus –20. For Besides modulation of host miRNAs, some DNA and retroviruses encode their own miRNAs to regulate viral and host mRNAs . The expDrosophila, piRNA maturation involves endonucleolytic cleavage of precursor transcripts by the Zucchini nuclease and the three PIWI proteins Piwi, Aubergine (Aub) and Argonaute-3 (Ago3) [Aedes mosquitoes, suggesting that they contribute to the regulation of virus replication [The third, most enigmatic class of small RNAs are PIWI interacting RNAs (piRNAs). These are processed from long RNA precursors that are transcribed from genomic loci known as piRNA clusters. In sharp contrast to siRNAs and miRNAs, their biogenesis into mature piRNAs is Dicer-independent. In 3 (Ago3) –30. The lication . At preslication –39. Howelication .Aedes aegypti Aag2 cell line to investigate the production of small RNAs during DENV infection. We find that in addition to the well-characterized vsiRNAs, specific vpiRNAs are produced from DENV, which for their biogenesis in Aag2 cells rely on Piwi5 and Ago3 and, to a lesser extent, on Piwi6. We do not detect DENV-derived miRNAs, or prominent changes in host miRNA levels upon infection. Finally, we identify novel host miRNAs in our small RNA deep-sequencing libraries, complementing the currently annotated miRNA repertoire in Aedes aegypti vector mosquitoes.Here, we make use of small RNA deep-sequencing in the siRNA, miRNA, and piRNA competent 2 in Dulbecco’s modified Eagles medium (DMEM) supplemented with 10% FCS and pen/strep. Stocks of DENV serotype 2 (DENV2), New Guinea C (NGC) and 16681 strains were grown on C6/36 cells and titred on BHK-15 cells as detailed in [Aag2 cells were cultured at 25°C in Leibovitz L-15 medium (Gibco) supplemented with 10% heat inactivated fetal calf serum , 2% tryptose phosphate broth solution (Sigma), 1x MEM non-essential amino acids (Gibco), and 50 U/ml penicillin and 50 μg/ml streptomycin . U4.4 and C6/36 were kept in the same culture medium at 28°C. BHK-21 cells were cultured at 37°C, 5% COailed in .Aag2 cells were seeded one day prior to infection and infected with DENV2 at a multiplicity of infection (MOI) of 0.5 by directly adding the virus to the culture medium. Three days post infection the culture medium was removed and cells were harvested for RNA and protein isolation as detailed below.For the detection of the DENV NS1 protein in samples used for small RNA deep-sequencing, 5% of the cells were harvested in 50 μl lysis buffer ; 1 mM DTT). 12.5 μl of 5x Laemmli buffer was added to each sample, incubated at 95°C for 5 min, and 30μl of each sample was loaded on a 12.5% polyacrylamide gel. After gel electrophoresis, proteins were transferred to a nitrocellulose membrane (Bio-Rad) using a semi-dry blotting system (Bio-Rad). The membrane was blocked in 5% non-fat dry milk (Bio-Rad) in 0.1% Tween20 in PBS (PBS-T) for 30 min at room temperature. Mouse anti DENV NS1 antibody was kindly provided by Dr. Peter Mason . The antin vitro transcription from T7-promoter flanked PCR products as detailed in [dsRNAs targeting PIWI/AGO transcripts or luciferase as a negative control were produced by ailed in . Primers5 Aag2 cells were seeded in one well of a 24-well plate. For each condition, three wells were plated. The following day, transfection mixes containing 300 μl non-supplemented L-15 medium, 450 ng dsRNA and 1.8 μl X-tremeGENE HP (Roche) were prepared according to the manufacturer’s recommendations. 100 μl of the mix was added dropwise to one well. After 2–3 hours the medium was replaced with fully supplemented L-15 medium. 48 hours later, the transfection was repeated to enhance knockdown efficiencies. Where indicated, the cells were infected with DENV2 which was added to the L-15 medium used to replace the transfection medium as specified above.For dsRNA transfection, 7.5x10Aag2 cells were lysed in Isol-RNA Lysis reagent (5 PRIME) as described in the manufacturer’s instructions. Briefly, 200 μl of chloroform was added to 1 ml of Lysis reagent and mixed well. After centrifugation, the aqueous phase was collected and total RNA was purified using isopropanol precipitation. RNA was quantified on a Nanodrop spectrophotometer and RNA integrity was checked by ethidium bromide staining of ribosomal RNA bands after agarose gel electrophoresis.For RT-(q)PCR, 1 μg of total RNA was DNaseI (Ambion) treated according to the manufacturer’s instructions. The RNA was subsequently reverse transcribed in a 20 μl reaction using the Taqman reverse transcription kit (Applied Biosystems). Complementary DNA (cDNA) was diluted 5–10 times before PCR amplification. Endpoint PCR was performed using Thermoperfect DNA Polymerase. Quantitative PCR (qPCR) analysis was performed using the GoTaq qPCR SYBR mastermix (Promega) on a LightCycler 480 instrument (Roche). The relative changes in gene expression were calculated using the ΔΔCt method  using ly4) oxidation and β-elimination of total RNA was performed as described previously [4 and 40 μl 5x borate buffer. As a control, RNA was treated with water instead of NaIO4. The reaction was incubated at room temperature for 30 min and 10 μl glycerol was added to the reaction. The reaction was incubated for another 10 min before 10 μl of 500 mM sodium hydroxide (NaOH) was added to induce β-elimination. The reaction was incubated at 45°C for 90 min. After these treatments, total RNA was purified by ethanol precipitation in the presence of 300 mM NaCl and 5 μg of glycogen. Electrophoretic mobility of Aedes aegypti miR-2940-3p and DENV2 piRNAs was then analyzed by small RNA northern blotting as detailed below.Sodium periodate  using T4 Polynucleotide kinase (Roche). Hybridization to the oligo-probes was performed overnight at 42°C in Ultrahyb Oligo hybridization buffer (Ambion). Membranes were then washed three times at 42°C in 0.1% SDS with decreasing concentrations of SSC . Membranes were exposed to X-ray films (Carestream) or Phosphorimager screens (BioRad). Sequences of DNA oligonucleotide probes are indicated in Small RNA northern blot was performed as described in . Briefly2 flasks of Aag2 cells were infected in parallel with DENV2 NGC. Three additional flasks were left uninfected. Total RNA was then isolated from these six flasks as specified above and 30 μg of total RNA was size separated on a 15% Polyacrylamide, 7 M urea, 0.5x TBE gel. Subsequently, the small RNAs in the size range from 18 nt to 33 nt were excised from gel using radioactively-labelled RNA oligos, loaded in the adjacent lanes of the gel, as rulers. The gel was crushed and the small RNAs were eluted in 300 mM sodium acetate overnight at 4°C under constant rotation. The RNA was recovered from the elution buffer using ethanol precipitation and eluted in 10 μl of nuclease-free water. 5 μl of the sample was directly used as input for small RNA deep-sequencing library preparation using the TruSeq small RNA library preparation kit (Illumina) following the manufacturer’s recommendations. The RNA was ligated to 3’ and 5’ adapters, reverse transcribed, and PCR amplified. The small RNA libraries were then size-purified from 1x TBE, 6% polyacrylamide gel using overnight elution in 300 mM sodium acetate followed by ethanol precipitation. The individual small RNA libraries were pooled and sequenced on a single sequencing lane on a HighSeq2500 by Baseclear .Small RNA libraries were prepared as described previously ,45. BrieFASTQ sequence reads were generated using the Casava pipeline (v.1.8.3) and initial quality analysis was performed using the Illumina Chastity filter and an in-house filtering protocol by Baseclear. Subsequent quality assessment was based on the FASTQC quality control tool (v.1.10.0). The individual small RNA sequencing libraries were separated based on the TruSeq indices (no. 1 to 6) that were introduced during PCR amplification. The individual libraries were subsequently analyzed using the Galaxy bioinformatics tool shed ,47. For Re-analysis of the data published by Hess et al.  was perfhttp://dx.doi.org/10.14806/ej.17.1.200) with parameters -O 6 -m 17 -n 5 -q 20. Within each library, the resulting reads were collapsed to generate a non-redundant set of FASTA sequences, subsequently processed to the format required for miRNA prediction with miRDeep2 software [Aedes genome  and the DENV2 NGC genome using the mapper.pl component of miRDeep2 (parameters: -o 20 -l 19 -r 100 -c). The resulting outputs were parsed to remove alignments that were not full length and perfect match (FLPM). miRDeep2 predictions were generated from the FLPM aligned sequences, with miRBase v21 Arthropoda mature miRNA and pre-miRNA sequences as templates [Aedes-specific genome reads within each sample group, using the lowest number of reads aligning as the baseline. Subsequently, the counts were converted to abundances within each sample, converted to log2 equivalent counts, and all samples quantile normalized prior to linear model fitting with the limma package [Analysis of miRNA expression levels was performed using the miRDeep2 tool. Raw data were assessed for quality using FASTQC. Subsequently, adapters were removed from the raw reads, and the reads were quality trimmed using cutadapt software  infected cells, each. The efficiency of the three infections was comparable as assessed by western blot for the DENV2 NS1 protein  strand RNA virus belonging to the proteins . Since v protein . As expe protein . Interes protein . In cont protein . In facts et al. . We analAedes albopictus mosquitoes, which we have previously shown to be competent in producing SINV-derived vpiRNAs [To exclude the possibility that the piRNA-like molecules are sequencing artefacts and to characterize this small RNA population in more detail, we performed small RNA northern blotting for the highly-abundant small RNAs starting at DENV2 genome positions 9180 or 9985–9990. Indeed, small RNAs in the expected size range could readily be detected specifically in DENV2 infected Aag2 cells . In addi vpiRNAs ,37 . In another publication, several ‘miRNA-like’ RNAs (termed vsRNA-1 to 6) were proposed to be produced from the 5’ and 3’ UTRs of the DENV2 RNA, based on the analysis of small RNA sequencing data [bona fide Dicer products , Apis mellifera  or Bombyx mori . We therefore suspected that the repertoire of published mosquito miRNAs is not yet complete and we set out to identify novel Aedes aegypti miRNAs in our small RNA sequencing data using miRDeep2. We obtained a list of 399 miRNA predictions  contains 101 dictions , 73 of w miRBase ,58  have been detected in small RNA deep-sequencing data from invertebrates, simple chordates, vertebrates and even viruses –68. In tosophila  5’ moRs Aedes mosquitoes, viral siRNAs were detected from several virus families, including Togaviridae, Flaviviridae, Bunyaviridae, and Reoviridae [Aedes aegypti cells [Aedes mosquitoes, DENV2 titres and transmission are enhanced, underlining the pivotal role of RNA interference in limiting DENV2 replication [Small RNA pathways critically influence the outcome of virus infections in many host organisms, including plants, fungi, invertebrates, and vertebrates –5,15,69.ti cells ,36. Theslication .Drosophila [Besides siRNAs, our DENV2 infected small RNA libraries contained a substantial number of virus-mapping reads in the size range of piRNAs. Their expression was confirmed using small RNA northern blotting, validating that these small RNA reads were no sequencing artefacts. Only very few DENV2 genomic locations near the 3’ end of the DENV genome give rise to these vpiRNAs but the origin of this spiky pattern remains obscure. We hypothesized that perhaps endogenous, transposon-derived piRNAs would loosely bind the DENV2 genome at these positions triggering the production of secondary vpiRNAs. A similar mechanism has been suggested to initiate piRNA production from specific mRNAs in osophila . HoweverAedes aegypti mosquitoes or Aag2 cells [Aedes mosquitoes. Although in this study the exact location of vpiRNA spikes differs from the positions we found in Aag2 cells, these data suggest that similar mechanisms may be responsible for piRNA biogenesis in Aag2 cells and adult mosquitoes. Yet, genetic evidence for the PIWI protein dependency of vpiRNA production in vivo is lacking. Of note, piRNAs are far less abundant in the small RNA libraries reported by Scott et al. [et al. [Previous analyses of DENV2-derived small RNAs identified vpiRNAs in g2 cells ,36. Alsog2 cells , a majort et al.  and HessAedes PIWI proteins are specialized in producing piRNAs from various sources. Whereas in mosquito cells piRNA biogenesis from transposons directly and indirectly depends on Piwi4-6 and Ago3, piRNA biogenesis from SINV predominantly requires Piwi5 and Ago3 only [Aedes PIWI proteins are even further specialized towards RNA substrates from different viruses. This may be caused by virus-specific sequence elements or structures that are preferentially recognized by certain PIWI proteins. Alternatively, but not mutually exclusive, differences in replication strategies or replication sites might favour recognition of viral RNA by distinct sets of PIWI proteins.Using knockdown of PIWI proteins, we identified Piwi5, Ago3 and, to a lesser extent, Piwi6 as responsible for the production of vpiRNAs in Aag2 cells. Therefore, DENV2 vpiRNA biogenesis in Aag2 cells relies on a similar set of PIWI proteins as SINV vpiRNAs, which also depend on Piwi5 and Ago3 . We havego3 only . The addDrosophila [Aedes or Aubergine in Drosophila) slices a complementary target RNA and transfers the 3’ slicer products as the new piRNA precursor to a second PIWI protein (Ago3 in Aedes and Drosophila). From this precursor, an Ago3-bound secondary piRNA is produced that in turn is able to slice a target RNA, giving rise to a new piRNA precursor. This precursor will be matured to generate the same primary piRNA sequence that initiated the amplification. Therefore, this model predicts the presence of piRNAs derived from both strands. Although also during SINV infection (-) strand derived piRNAs are only a minor fraction, they can be identified as the primary piRNAs by a nucleotide bias that is characteristic for Piwi5/Aub bound piRNAs (uridine at position one). During DENV2 infection, 25–30 nt reads from the (-) strand are extremely scarce and they do not have the nucleotide bias that would classify them as primary piRNAs. Therefore, exactly how the production of the secondary, (+) strand vpiRNAs is triggered or whether a different, amplification-independent mechanism is responsible for their production, remains unclear.The almost complete loss of DENV2 piRNAs upon knockdown of Piwi5 and Ago3 indicates that both proteins are equally important for vpiRNA biogenesis in Aag2 cells, similar to piRNA biogenesis during SINV infection . SINV piosophila ,29. DuriThe expression of miRNAs from DENV2 genomic RNA is still debated. Based on small RNA sequencing data, Hussain and Asgari have described a set of six viral small RNAs that have miRNA-like properties . InhibitAedes aegypti mosquitoes, a total of 31 miRNAs were recently shown to be differentially regulated following infection at three different time points [per se, but could reflect an indirect effect of homeostatic or metabolic responses during the infection. It should be remembered that miRNA expression can be highly cell-type specific; if miRNA levels are responsive to DENV2-infection only in selected cell types within the entire mosquito, we may miss those in our Aag2 cell-based assays.Modulation of host miRNAs after virus infection may be a mechanism that coordinates gene expression during the course of the immune response. Alternatively, it may be a consequence of a viral strategy to manipulate host gene expression. Comparing uninfected with DENV2-infected Aag2 cells showed that the expression of almost all miRNAs was unchanged upon infection. Only a handful of miRNAs were up or down-regulated after exposure to DENV2. The fold changes ranged from approximately 4 fold up to 4 fold down. In whole e points . The setAedes aegypti miRNAs to the small RNA repertoire in this medically important virus-host interaction.In summary, here we provide an in-depth analysis of small RNAs in DENV2 infected Aag2 cells in comparison to uninfected cells. Aag2 cells provide a powerful model system for studying biochemical details of small RNA biogenesis pathways, as they are fully competent in producing siRNAs, miRNAs and piRNAs originating from both virus and host. Our analyses add both DENV-derived piRNAs and novel S1 FigAedes mosquitoes (9 days post infection) published by Hess et al. [(A) Size profile of small RNA reads mapping to the sense strand (black) or antisense strand (grey) of the DENV2 JAM1409 genome. Inlay shows reads of 23 to 32 nt in size, with a different scale for the y-axis. (B) Peaks indicate the number of 5’ends of small RNAs of 21 nt (left panel) or 25–30 nt (right panel) across the sense (red) or antisense (blue) strand of the viral genome. Read counts have been normalized to the depth of the library. Red numbers in D and E indicate the genome position of the 5’ end of the small RNA peaks.Re-analysis of small RNA sequencing data from DENV2-JAM1409 infected s et al. . (A) Siz(TIF)Click here for additional data file.S2 Fig(A) Quantification of DENV2 RNA levels by RT-qPCR in the samples used for (B) Knockdown (KD) efficiency of Piwi5 and Ago2 (upper panel) and the corresponding levels of DENV2 RNA (lower panel) as assessed by RT-qPCR. Bars indicate mean +/- SEM of three independent experiments. Statistical significance was determined using two tailed, unpaired student t-test. * p<0.05; **p<0.01.(TIF)Click here for additional data file.S3 Fig(A) miRNA predictions using miRDeep2 on the DENV2-NGC genome. The read counts of the individual sequences, combined from all the six sequencing libraries, that support the predictions are shown to the left. The predicted mature miRNA and the expected miRNA* sequences are indicated in red and blue, respectively. (B) Genome coverage of small RNAs mapping to the DENV2-NGC 3’ UTR. Bars represent the mean of the normalized coverage at each nucleotide position (n = 3). Green bars show all small RNA reads mapping to the region. Red bars show the 22–24 nt reads derived from the viral (+) strand only. (C) Size distribution of (+) strand-derived small RNA reads that end at the 3’ terminus of the DENV2-NGC genome. Bars represent the average read count and SEM of the three deep-sequencing libraries normalized to their corresponding size (displayed as % of library).(TIF)Click here for additional data file.S4 Figet al. or Akbari et al. [Aedes genome. Also aae-miR-11894a-1 and aae-miR-11894a-2 are identical sequences that map to two very similar hairpins. Aae-miR-11895-1 and aae-miR-11895-2 are two identical hairpins that map to two locations in the Aedes genome. Similarly, the mature aae-miR-11899 sequence maps to two identical hairpins in the genome.Folding of miRNA hairpins that have been published by Hu i et al. ,58, and (TIF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S2 TableSheet 1 provides an overview of the selection procedure to reach the final miRNA predictions. Sheets 2 to 6 show the unfiltered miRNA predictions and the filtered miRNA predictions after the individual rounds of selection.(XLSX)Click here for additional data file.S1 Dataset(XLSX)Click here for additional data file.


Drosophila disease models, as well as to patient-derived fibroblasts, P3 inhibited expanded-CAG-RNA-induced nucleolar stress and suppressed neurotoxicity. We further examined the combined therapeutic effect of P3 and polyQ-binding peptide 1 (QBP1), a well-characterized polyQ protein toxicity inhibitor, on neurodegeneration. When P3 and QBP1 were co-administered to disease models, both RNA and protein toxicities were effectively mitigated, resulting in a notable improvement of neurotoxicity suppression compared with the P3 and QBP1 single-treatment controls. Our findings indicate that targeting toxic RNAs and/or simultaneous targeting of toxic RNAs and their corresponding proteins could open up a new therapeutic strategy for treating polyQ degeneration.Polyglutamine (polyQ) diseases represent a group of progressive neurodegenerative disorders that are caused by abnormal expansion of CAG triplet nucleotides in disease genes. Recent evidence indicates that not only mutant polyQ proteins, but also their corresponding mutant RNAs, contribute to the pathogenesis of polyQ diseases. Here, we describe the identification of a 13-amino-acid peptide, P3, which binds directly and preferentially to long-CAG RNA within the pathogenic range. When administered to cell and  Drosophila Collection: We identified a novel peptidylic inhibitor against expanded CAG RNA toxicity. Our study highlights the importance of simultaneously targeting both toxic RNA and protein species in treating polyQ degeneration. Polyglutamine (polyQ) diseases represent a group of dominantly inherited progressive neurodegenerative diseases . These dRibosome biogenesis is essential for cellular protein synthesis. The ribosome is a ribonucleoprotein complex composed of ribosomal RNAs (rRNAs) and ribosomal proteins. Failure in rRNA transcription induces nucleolar stress, and cells undergo apoptosis. Thus, nucleolar stress is a cellular response designed to eliminate cells that fail to carry out efficient protein synthesis due to ribosome biogenesis defects . A reducTsoi and Chan, 2013KD=52.70±2.21 µM). Next, we further demonstrated that P3 disrupted the interaction between endogenous NCL protein and the expanded-CAG RNA. The introduction of the P3 peptide to cells expressing expanded-CAG RNA resulted in the restoration of the interaction between NCL and UCE, and the level of pre-45s rRNA expression. We further showed that P3 suppressed cell death in both cell and Drosophila disease models, and in MJD patient-derived fibroblasts. Our findings indicate that expanded-CAG-RNA toxicity can be targeted by peptidylic inhibitors. Finally, various peptidylic inhibitors , and the purified GST-NCL protein was determined by a glutathione S-transferase pull-down assay . Furthermore, P3 expression had no effect on the level of pre-45s rRNA in cells expressing the control construct CAG27EGFP (P3 expression would affect the expression levels of genes that are transcribed by RNA polymerases II (GAPDH) and III (U6 and mettRNA), and observed no change in RNA-polymerase-II- or III-mediated transcription in either CAG27EGFP or CAG78EGFP RNA-expressing cells . This indicates that P3 expression does not affect cellular gene transcription in general.We next investigated whether P3 could mitigate expanded-CAG RNA toxicity in cells. Following overexpression of CAG RNA – GFPCAG27 D. HoweveGFPCAG27  -PCR analysis, and their expression did not alter the levels of CAG78EGFP RNA . Next, we examined the expression level of pre-45s rRNA in HEK293 cells co-transfected with both the CAGEGFP and individual P3MT constructs. In contrast to the P3WT positive control, rRNA transcription could not be restored via the expression of any of the P3MT constructs in CAG78EGFP RNA-expressing cells  interactions , including cells D. This iP3 prevents sequestration of NCL by expanded-CAG RNA, allowing it to resume its normal cellular role in rRNA transcription regulation. To test this, HEK293 cells were co-transfected with CAG78EGFP and P3 constructs, and chromatin immunoprecipitation was performed to determine whether P3 restores the binding of endogenous NCL to UCE. Indeed, co-expression of P3 restored the interaction between endogenous NCL and UCE in cells expressing CAG78EGFP RNA . When cells were co-transfected with P3 and CAG78EGFP, caspase 9 activity was significantly suppressed when compared with the CAG78EGFP-transfected cells  value was 4.369±1.140 µM.Cell-penetrating peptides (CPPs) have been widely used as a vehicle to enhance delivery of therapeutics across the cell membrane , includiPopiel et al., 2007NT . The TATNT . We therNT  and testNT A, as detNT . The calCAG78EGFP RNA . When compared with CAG78EGFP RNA-expressing HEK293 cells, CAG78pEGFP-transfected cells that were co-treated with the synthetic TAT-P3 peptide (12 µM) showed an increased level of pre-45s rRNA  CAG mRNAs of different repeat lengths . We observed no effect of TAT-P3 on the levels of ATXN2 proteins . Next, we found that the adult eclosion rate of wild-type Drosophila  and the viability of primary rat cortical neurons  were not compromised when these models were treated with up to 1 mM and 25 µM of the TAT-P3 peptide, respectively. Finally, we showed that TAT-P3 had no effect on staurosporine (STS)-induced cell death in vitro , nor in vivo on toxicity induced by the expression of expanded-CUG . Taken together, our findings demonstrate that the P3 peptide displays specificity for expanded-CAG-RNA-induced toxicity.Next, we tested whether the effect of the TAT-P3 peptide on cytotoxicity is due to the suppression of NCL-mediated nucleolar stress . We firs45s rRNA B, 18S rR45s rRNA C and UCE45s rRNA D, as wel45s rRNA E, p53 pr45s rRNA F and cas45s rRNA G. The abnded-CUG  and -CGGnded-CUG  RNAs in CAG78-EGFPRNA-toxicity-only cell model . We further showed that DX-assisted intracellular delivery of P3 did not alter RNA-polymerase-II- and III-mediated gene expression , indicating that the suppressive effect of the P3 peptide is specific for pre-45s rRNA transcription mediated by RNA polymerase I . After validating the DX-assisted peptide-delivery protocol, we utilized the CAG78MJD cell model  to generate the active spliced XBP1S mRNA for subsequent production of the functional XBP1 protein . This indicates that the QBP1 peptide suppresses polyQ-protein toxicity and that the suppression is not affected by the co-delivery of the P3 peptide. An effective suppression effect of QBP1 was also detected in MJD patient-derived fibroblasts (The binding immunoglobulin protein (BiP), also known as GRP-78 , is a mo protein . To monicription D and XBPoduction E in our roblasts F.BiP induction and XBP1S production  or QBP1 (P3MT5+QBP1) peptide yielded only a partial inhibition of cell death . Intriguingly, the P3WT+QBP1 co-treatment group suppressed CAG78MJD RNA/protein-induced cell death more effectively when compared with the single-treatment groups  or QBP1  peptidylic inhibitor . This indicates that the in vivo suppression effect of P3 is not simply confined to the photoreceptor neurons in the eye, but can further be extended to other nervous tissues.toxicity . Peptide, a marked preservation of retinal integrity was observed as evidenced by a significant increase in the pseudopupil score (4.93±0.14) when compared with the single-treatment groups  and animal lethality  investigations, in which P3 and QBP1 peptidylic inhibitors did not elicit any dominant toxicity effect under our experimental conditions. We further found that treating flies with both non-functional peptidylic inhibitors, TAT-P3MT5 and TAT-QBP1 SCR, did not cause any suppression of neurodegeneration . Upon TAT-QBP1 administration, we also observed that the stacking gel-residing SDS-insoluble expanded polyQ MJD protein was partially diminished . Furthermore, our findings illustrate that the mitigating effects of P3WT  on RNA and protein toxicity, respectively, were not influenced by the other co-administered peptide. Taken together, our data suggest that the simultaneous targeting of RNA and protein cellular toxicities using peptide agents is a viable approach for developing effective treatments for polyQ diseases.To further confirm that P3+QBP1 co-treatment mitigates both expanded-CAG-RNA and -polyQ-protein toxicities, we examined the expression levels of rRNA  . An increasing number of peptide therapeutics have entered clinical-trial phases in recent years ; one of CAG27pcDNA3.1-MJD, CAG78pcDNA3.1-MJD, CAA/G78pcDNA3.1-MJD, CAG27pEGFP and CAG78pEGFP constructs were reported previously myc-His A vector using EcoRV enzyme. To generate peptide expression constructs, oligonucleotide linkers were employed. All DNA oligos were ordered from Life Technologies. The P3WT linker was generated by annealing the following oligos: P3WTF 5′-AATTCATGGATGGTAAGTCAAAGGGTATCGCTTACATCGAGTTCAAGTAAC-3′ and P3WTR 5′-CGAGTTACTTGAACTCGATGTAAGCGATACCCTTTGACTTACCATCCATG-3′. The P3MT1 linker was generated by annealing the following oligos: P3MT1F 5′-AATTCATGGATGGTGCTTCAAAGGGTATCGCTTACATCGAGTTCAAGTAAC-3′ and P3MT1R 5′-TCGAGTTACTTGAACTCGATGTAAGCGATACCCTTTGAAGCACCATCCATG-3′. The P3MT2 linker was generated by annealing the following oligos: P3MT2F 5′-AATTCATGGATGGTAAGTCAGCTGGTATCGCTTACATCGAGTTCAAGTAAC-3′ and P3MT2R 5′-TCGAGTTACTTGAACTCGATGTAAGCGATACCAGCTGACTTACCATCCATG-3′. The P3MT3 linker was generated by annealing the following oligos: P3MT3F 5′-AATTCATGGATGGTAAGTCAAAGGGTATCGCTGCTATCGAGTTCAAGTAAC-3′ and P3MT3R 5′-TCGAGTTACTTGAACTCGATAGCAGCGATACCCTTTGACTTACCATCCATG-3′. The P3MT4 linker was generated by annealing the following oligos: P3MT4F 5′-AATTCATGGATGGTAAGTCAAAGGGTATCGCTTACATCGAGGCTAAGTAAC-3′ and P3MT4R 5′-TCGAGTTACTTAGCCTCGATGTAAGCGATACCCTTTGACTTACCATCCATG-3′. The P3MT5 linker was generated by annealing the following oligos: P3MT5F 5′-AATTCATGGATGGTAAGTCAAAGGGTATCGCTGCTATCGAGGCTAAGTAAC-3′ and P3MT5R 5′-TCGAGTTACTTAGCCTCGATAGCAGCGATACCCTTTGACTTACCATCCATG-3′. The annealed linkers were ligated to pcDNA3.1 vector digested with EcoRI and XhoI.The eviously . To genein vitro binding was over 95%. Desalted peptides were used in Drosophila feeding assays. All RNAs were synthesized using the MEGAscript® kit (Ambion) as previously described  and MJD-patient-derived fibroblasts (GM06153) were obtained from the Coriell Institute for Medical Research . Both HEK293 cells and fibroblasts were cultured at 37°C with 5% COescribed . Transie78CAG RNA/GST-NCL mixture. The reaction mixture was incubated at 4°C with end-to-end rotation for 2 h. The beads were then washed three times with 1 ml of binding buffer. Each wash was conducted for 10 min at 4°C. After the washing steps, 100 µl of GST elution buffer  was used to elute the protein-RNA complex. RNA extraction was performed. Reverse-transcription PCR was performed to amplify the CAG amplicon with primers CAGF 5′-AAAAACAGCAGCAAAAGC-3′ and CAGR 5′-TCTGTCCTGATAGGTCC-3′. Band intensity was measured using ImageJ (Purified nucleolin protein (GST-NCL) was purchased from Abnova (Taiwan), and the control GST protein was expressed and purified as mentioned in g ImageJ . Each exactin was amplified by primers actinF 5′-TGTGCAAGGCCGGTTTCGC-3′ and actinR 5′-CGACACGCAGCTCATTGTAG-3′; the amplicons of P3WT and P3MT1-5 were amplified by primers P3F 5′-TAATACGACTCACTATAGGG-3′ and P3R 5′-TAGAAGGCACAGTCGAGG-3′; the amplicon of 78CAG RNA was amplified by primers CAGF 5′-AAAAACAGCAGCAAAAGC-3′ and CAGR 5′-TCTGTCCTGATAGGTCC-3′; the amplicon of XBP1S for human was amplified by primers XBP1SF 5′-GGAGTTAAGACAGCGCTTGG-3′ and XBP1SR 5′-ACTGGGTCCAAGTTGTCCAG-3′; and the amplicon of Xbp1S for Drosophila was amplified by primers Xbp1SF 5′-CAACAGCAGCACAACACCAG-3′ and Xbp1SR 5′-AGACTTTCGGCCAGCTCTTC-3′. Taqman gene expression assays were performed on an ABI 7500 Real-time PCR system and data were analyzed as previously described (pre-45s rRNA (Assay ID: AILJIZM), pre-rRNA (Assay ID: AIMSG5U), 18S rRNA (Assay ID: Hs03928985_g1), human GAPDH (Assay ID: Hs99999905_m1), Drosophila GAPDH (Assay ID: Dm01841186), U6 (Assay ID: AII1MM6), mettRNA (Assay ID: AIN1FB2), UCE (Assay ID: AIHSOGY) and actin (Assay ID: Hs99999903_m1), human BiP/GRP78 (Assay ID: Hs99999174_m1), Drosophila BiP/GRP78 (Assay ID: Dm01813415-g1), EGFP (Assay ID: Mr04097229_mr) and ATXN3 (Assay ID: Hs01026440_g1). Each experiment was repeated at least three times.RNA was extracted from cells or ten 6-day-old adult fly heads by Trizol reagent (Life Technologies), and 1 µg of purified RNA was then used for reverse-transcription using the ImPromII™ Reverse Transcription System (Promega). Random hexamer (Roche) was used as primers in reverse transcription. The amplicon of escribed . The folAll protein samples were resolved on 12% SDS-PAGE, and detected using the following antibodies: 7F5  for p53, 9B11  for myc-tagged proteins. Tubulin was detected by E7 . Each experiment was repeated at least three times, and comparable results were obtained.HpaII methylation assay, genomic DNA was extracted from cells, followed by digestion with 2 units of HpaII or MspI (New England Biolabs) for 4 h at 37°C. The DNA products were incubated at 85°C for 15 min to heat-inactivate the restriction enzymes. The resulting DNA products were amplified by PCR. Amplicon of the human UCE was amplified by UCEF, 5′-CGTGTGTCCTTGGGTTGACC-3′ and UCER, 5′-CGCGTCACCGACCACGCC-3′. Each experiment was repeated at least three times, with consistent results obtained.Chromatin immunoprecipitation was performed according to Tsoi et al., 2011®8 and Caspase-Glo®9 assay systems (Promega) following the manufacturer's instructions. TNF-related apoptosis-inducing ligand (TRAIL) served as a positive control for the caspase 8 activity assay, whereas staurosporine (STS) served as a positive control for the caspase activity 9 assay. EnVision® Multilabel Reader (PerkinElmer) was used to measure the luminescence. Each sample was measured in duplicates, and each experiment was repeated at least three times.Caspase activity was measured using the Caspase-Glo® scientific plotting software version 7 . All RNAs and peptides were dissolved in binding buffer . The concentration of RNA was estimated with appropriate extinction coefficients at 260 nm on a Nanodrop 2000 (Thermo Scientific). A reference power of 8 μcal/s was used with an initial 0.5 µl of injection of peptide followed by 2.5 µl for all subsequent titration points, with a 60 s initial equilibrium delay and 150 s pause between injections. The samples were stirred at a speed of 1000 rpm throughout the experiment. The thermal titration data were fitted to the ‘one binding site model’ to determine the dissociation constant (KD). At least two batches of independently synthesized peptides were used in the experiments. Each experiment was repeated at least three times with consistent results obtained.Experiments were carried out using a MicroCal iTC200 isothermal titration calorimeter  at 25°C. Data were analyzed using the Origin5, and CAG27/78pcDNA3.1-MJD or CAG78pEGFP DNA construct was used to transfect the cells. Four hours after DNA transfection, peptide transfection was performed as follows: P3WT-QBP1, P3WT-QBP1 SCR, P3MT5-QBP1 and P3MT5-QBP1 SCR (2 μM each). For STS (CAG78pEGFP transfection experiments) post-treatments using the Cytotox 96 non-radioactive cytotoxicity assay (Promega). Each experiment was repeated at least three times, with consistent results obtained.Human embryonic kidney 293 (HEK293) cells were seeded on a 24-well plate at a density of 0.5×10 For STS  treatmenCAG78EGFP RNA-expressing HEK293 cells, the LDH assay was employed. A density of 0.5×105 HEK293 cells were transfected with CAG78pEGFP and various amounts of the TAT-P3WT peptide – 0.1, 0.5, 1, 2, 4, 5, 10 and 25 μM – were then added to individual culture wells. Seventy-two hours after treatment, LDH enzyme activity in the cell culture medium was measured as described before. Experimental groups were normalized to the untransfected control. After normalization, data were analyzed using the dose response-inhibition curve (nonlinear regression-variable slope) to determine the IC50 value .To detect the effect of P3WT on inhibiting cell death in CAG27UAS-flMJD and CAG84UAS-flMJD (90UAS-EGFP-CGG (480UAS-(CTG) , and consistent results were obtained. For lifespan analysis, third instar larvae were fed with 200 µM of TAT-P3WT or TAT-P3MT5 (dissolved in 2% sucrose solution) for 2 h and then continued to culture in standard fly food at 25°C. Two days after eclosion, 10-15 adult flies were allocated to individual fresh non-drug-containing food vials. At least 120 flies were analyzed per treatment group. The flies were transferred to fresh vials every 3 days during the whole course of the experiment, and the number of surviving flies was counted every 3 days. Survival rate was calculated as area under survival curve followed by one-way ANOVA analysis. For the wild-type adult eclosion test, Oregon R third instar larvae were fed with 500 µM or 1 mM of TAT-P3WT peptide dissolved in 2% sucrose solution for 2 h, and then continued to culture in standard fly food at 25°C. Adult eclosion rate was calculated as the number of adult flies divided by the number of larvae examined. Each experiment was repeated three times (n=60 larvae). Two batches of independently synthesized peptides were used in the experiments.Flies were raised at 21.5°C or 25°C on cornmeal medium supplemented with dry yeast. Fly lines bearing MJDCAG84  were gifFP-CGG90  and UAS-(CTG)480  fly linet 21.5°C . The pset 21.5°C . Images P<0.05, P<0.01 and P<0.001, respectively, which are considered statistically significant.Data were analyzed by one-way ANOVA followed by post-hoc Tukey test. *, ** and *** represent


Scientific Reports5: Article number: 13750; 10.1038/srep13750 published online: 09022015; updated: 10202015.In the original version of this Article, affiliations 1–6 were incomplete. The correct affiliations are listed below.Affiliation 1:Shanghai Ruijin Hospital, Shanghai Institute of Digestive Surgery and Biobank of Gastrointestinal Carcinoma, Ruijin er Road, No. 197, Shanghai, ChinaAffiliation 2:Collaborative Innovation Center of Systems Biomedicine; Ruijin er Road, No. 197, Shanghai, ChinaAffiliation 3:State Key Laboratory of Medical Genomics; Ruijin er Road, No. 197, Shanghai, ChinaAffiliation 4:Department of Pathology of Shanghai Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Ruijin er Road, No. 197, Shanghai, ChinaAffiliation 5:Department of Radiology of Shanghai Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Ruijin er Road, No. 197, Shanghai, ChinaAffiliation 6:Genome Center of WuXiAppTec (Shanghai) Co., Ltd. Middle Fute Road, ShanghaiThese errors have been corrected in both the HTML and PDF versions of the Article.


ADAR proteins are among the most extensively studied RNA binding proteins. They bind to their target and deaminate specific adenosines to inosines. ADAR activity is essential, and the editing of a subset of their targets is critical for viability. Recently, a huge number of novel ADAR targets were detected by analyzing next generation sequencing data. Most of these novel editing sites are located in lineage-specific genomic repeats, probably a result of overactivity of editing enzymes, thus masking the functional sites. In this study we aim to identify the set of mammalian conserved ADAR targets.We used RNA sequencing data from human, mouse, rat, cow, opossum, and platypus to define the conserved mammalian set of ADAR targets. We found that the conserved mammalian editing sites are surprisingly small in number and have unique characteristics that distinguish them from non-conserved ones. The sites that constitute the set have a distinct genomic distribution, tend to be located in genes encoding neurotransmitter receptors or other synapse related proteins, and have higher editing and expression levels. We also found a high consistency of editing levels of this set within mice strains and between human and mouse. Tight regulation of editing in these sites across strains and species implies their functional importance.Despite the discovery of numerous editing targets, only a small number of them are conserved within mammalian evolution. These sites are extremely highly conserved and exhibit unique features, such as tight regulation, and probably play a pivotal role in mammalian biology. The canonical post-transcriptional modification of adenosine to inosine (A-to-I) deamination is catalyzed by enzymes of the ADAR family, which bind to double-stranded RNA (dsRNA) -3. This Drosophila[Alu pairs [Vast amounts of sequencing data have become available over the last few years. Consecutive computational approaches were developed to identify novel RNA editing sites, taking advantage of the available large RNA sequencing (RNA-seq) datasets. Such analysis is generally performed by comparing DNA to RNA sequences ,20-24. Erosophila,30. Mostlu pairs ,31-33 inlu pairs ). Yet, i−/− and ADAR2−/− mice exhibit embryonic and postnatal lethal phenotypes, respectively [2CR which regulates mood, appetite, and behavior [Although most of the sites are located in genomic repeats, and seem to have no functional importance, RNA editing is an essential process, as ADAR1ectively ,35, and ectively . Indeed,ectively . Other ebehavior -43, and behavior .In this study, we wished to identify from within the large number of novel editing sites, those sites that became important over the course of mammalian evolution. In order to achieve this goal, we used evolution as the key selection tool, looking for sites that are common in several lineages, which suggest that they were functionally selected. Creating such a catalog of selected editing sites will have a remarkable effect on functional editing research, since it illuminates the few ‘meaningful’ sites, by not only defining the functional sites, but also by suggesting a method to define them.Surprisingly, we found that the number of such conserved editing sites is extremely small, and probably only a limited set of such functionally important editing sites exist. We found that the plethora of sequencing data did not contribute much to the discovery of novel conserved sites, as most of the functionally important sites were known before the next generation sequencing revolution. Nevertheless, we were able to discover, based on their extreme conservation, the few editing sites that probably play a pivotal role in mammalian biology. In addition, our results demonstrate that editing, in parallel to the established mutational processes that shape genomes, add another layer of variability and complexity that can be spatiotemporally regulated.Drosophila fly [Most of the known editing sites seem to be located in lineage-specific regions, mainly in inverted repeats as is the case in the human ,28, moushila fly ,46  and specificity rates seem to be strikingly high. The high recall rate was confirmed as the set contains all the conserved functional sites known so far. To measure the specificity of the ESS, we estimated the effect of accumulating a species-specific RNA editing dataset on the ESS size. In order to demonstrate that the size of the ESS does not dramatically change as the data accumulate, we found that even a small fraction of the available data is sufficient to retrieve most of the ESS. We used data from 15 mouse strains  to buildThere are two reasons for explaining the few cases in which the editing signal was not detected in a specific mouse strain. The first is the low read depth (low expression), which makes measurement of editing levels difficult. The second explanation is editing levels under the detection threshold . There is clear distinction between the conserved and the non-conserved sites: while there were only a few cases of sufficient coverage with no signal for editing in the conserved sites, there were a higher proportion of sites with sufficient coverage but no editing signal for the non-conserved ones. Many of the latter are not reproduced across different samples . VisualiWe analyzed RNA-seq data to find editing levels for the matched position of the ESS in four additional mammals: rat, cow, the non-placental marsupial opossum, and the monotrem platypus ,55. Theyversus the non-conserved sites. As expected, most of the sites in the ESS are located in coding regions , . In contrast to exons that have a clear potential for evolutionary benefit, such as amino acid changes, introns are considered as fitness-neutral in nature, and the reason they are evolutionarily conserved might look enigmatic. One probable explanation for the editing sites found in introns, is that the intronic sites are located in exon complementary sequence (ECS), a genomic region needed for dsRNA structure formation, which is required for ADAR binding . Indeed,Three additional sites were found in non-coding regions. Two sites are located in miRNAs . The first one, previously published , is locaAlu sites). Although both sets adhered to the previously published neighbor preferences [Previous studies indicated that ADARs have a sequence preference for G depletion on the upstream nucleotide to the editing site and have excess of G at its 3′ base. We compared the nucleotide frequency for both the ESS and a control set . As a control, we applied the same procedure to adjacent but non-edited As. Here, no significant trend was found, and a similar percent (19%) of the human and mouse sites have a G at the corresponding position, suggesting this result is specific for edited adenosine. These results suggest that in the majority of cases, editing serves as a mechanism to compensate for a loss of phenotype caused by G-to-A evolution. This versatile reversion may occur in only part of the transcripts in parallel to the non-edited version and in a tissue-specific manner. Thus, editing allows the functional co-existence of two independently evolved genome versions. Furthermore, our results suggest that in addition to the ESS, there are additional functional editing sites in humans that have a G in the mouse genome  harbor a G in the mouse genome, only 18%  of mouse editing sites harbor G in the human genome , we found at least one editing site which seems to be involved in exonization of a retroelement Figure A. In thiWe also analyzed RNA-seq data to identify RNA editing candidates in zebrafish (see Methods). We used the same method to find conserved sites between human and mouse and zebrafish, which is one of the most evolutionarily distant vertebrate relatives of human for which genome and transcriptome data are available. We found 17 ESS sites that are also conserved in zebrafish  identity in 45 of the sites , at very low evolutionary cost . InteresOur studies revealed a comprehensive set of mammalian conserved sites. Yet, it is still possible that additional sites do exist and can be found if more samples from additional tissues (as most of the mouse data are derived from brain and liver) or genomes with higher coverage are used, or if less stringent criteria are used for conservation. However, our results suggest that adding more data or using relaxed parameters will not add many additional sites that are as highly edited and highly expressed. Therefore, we believe that this set is close to being inclusive, and if such additional sites do exist they are probably few in number.We carried out the first systematic screening for conserved mammalian RNA editing. Although we explored comprehensive editing sets in human and mouse, we found that there are only a few RNA editing sites that are conserved between these species. Our results demonstrate that although additional RNA-seq data can lead to the identification of novel editing targets, they are unlikely to add many additional conserved sites. We found that the mammalian conserved set of editing sites is limited and has distinguishing characteristics that set these sites apart from others. The conserved sites have a unique genomic distribution and tend to be located in regions with evolutionary impact. Most of the conserved edited genes are related to neural-specific functions; yet, we found an editing signal in a wide variety of tissues. In addition, we found editing to be involved in the creation of a new functional unit by exonization of a repetitive element in the SLC9A6 gene. Finally, we showed that editing tends to preserve ancient genome versions and allows the co-existence of two evolved phenotypes.The human RNA editing dataset was created by merging published datasets. These datasets were created by seeking RNA-DNA differences using RNA-seq analysis. The first dataset that we used contained all editing candidates identified by Ramaswami et al. , who appThe mouse RNA editing dataset was created by merging five datasets; the main dataset was created by Danecek et al., who screened for RNA-DNA differences, using whole brain RNA-seq analysis in 15 different mouse strains .The second dataset published by Lagarrigue et al., investigated for RNA-DNA differences in liver and adipose tissues revealing 63 and 188 editing candidates, respectively . The thiThis dataset was created by analysis of RNA-seqs . Fastq files were aligned to the ZF genome (Zv9/DanRer7) using tophat, command: tophat -r 530 index fastq1,fastq1_replication fastq2,fastq2_replication fastq3,fastq3_replication. We then realigned the fastq files to the zebrafish reference genome, and added the splice junction file, achieved from the first run, as input.Command: tophat -r 530 -j splice_junctions_file index fastq1,fastq1_replication fastq2,fastq2_replication fastq3,fastq3_replication.Mpileup was then used to find RNA to reference-genome differences. Only sites with more than five edited reads and editing levels higher than 0.01 were taken into account.Human cDNA SNPs were downloaded from dbSNP137 (All SNPs table), using the UCSC table browser . We madeorganism1_dadaset.fa -strand ‘both’ -db organism2_blastdb -out out_file -task ‘dc-megablast’.The sequences surrounding each editing site were downloaded using the UCSC table browser . We used several sequence sizes and compared them by calculating signal-to-noise ratio, using SNP conservation as a control .Mouse editing and expression levels were obtained from a previously published study . For eacWe aligned the Human-body-map dataset to the human genome (hg19), using Bowtie aligner  with libEach RNA-seq was aligned to the matched reference genome  using STAR aligner with default parameters. Mismatches were screened using the same procedure as with human body map. Accession numbers for RNAseq: cow- SRR594491, rat- SRR594419, opossum- SRR306744, SRR306746, platypus- SRR306727, SRR306729. Human body map- GSE7905.Sequence logos were generated for 10 nt upstream and downstream to the editing sites using WebLogo. .RNA secondary structures were predicted using mfold .Codon changes were calculates using ANNOVAR .ADAR: Adeonsine deaminase acting on RNA; BLAST: Basic local alignment tool; DARNED: Database of RNA editing; ECS: Exon Complementary Sequence; ESS: Evolutionary selected set; GLUR2: Glutamate receptor2; GO: Gene ontology; LINE: Long interspersed nuclear element; miRNA: microRNA; SNP: Single nucleotide polymorphism; UTR: Untranslated region.The authors declare that they have no competing interests.YP performed the research, analyzed the data, and wrote the manuscript. HYC participated in the design of the study and writing. EYL conceived the study, helped to analyze the data, and to write the manuscript. All authors read and approved the final version of the manuscript.Table S2. Additional sites that were found to be conserved by UCSC liftOver. Table S3. Editing profiles for the ESS in rat, cow, opossum, and platypus. For each organism we provide the matched nucleotide, number of A + G reads, and editing levels. Table S4. Editing levels among 16 Human body map tissues. Table S5. Enriched GO terms in the ESS. Table S6. Human editing sites which harbor ‘G’ in mouse genome. Table S7. ESS sites that were found to be edited in zebrafish; coordinates are in Zv9/DanRer7 genome version. Table S8. Sequence conservation among mouse, dog, opossum, chicken, and zebrafish. Table S9. Editing levels among 16 Human body map tissues for human specific sites (deleted regions in mouse). Table S10. List of all non-conserved non-synonymous sites. Table S11. List of predicted complementary region for each site in the ESS.Additional information on the ESS, including human and mouse coordinates, year of publication, if they were found using next generation sequencing data analysis and molecular type of SNPs found in this position. Click here for fileP value = 7.24 × 10-10, Student’s t-test). Figure S2. Spatial proximity of conserved sites. The secondary structure shows spatial proximity of the conserved sites of (A) gria3, and (B) five intronic sites in the gria4 gene. Editing sites are depicted in orange and marked by an arrow. Figure S3. Conserved editing sites in microRNAs. Editing sites in pre-mir (A). The editing site is located in the seed region of mir376c. (B) Editing site within mir27b. Editing sites are highlighted in orange and marked by an arrow. Figure S4. Editing levels are conserved between human and mouse. RNA editing levels were measured in both human and mouse brains. We found positive correlation between editing levels in both species by calculating Pearson’s correlation coefficient (R = 0.55). Figure S5. Signal-to-noise ratios. Signal-to-noise was measured by the ratio of editing hits to normalized SNPs hits. Both were calculated using the pipeline as described in the paper. We used 40 nt, 80 nt, and 100 nt blast alignment length and the UCSC liftover.Incidence of editing sites per strain. The prevalence of editing sites was measured for the ESS (conserved sites) and all other sites (Click here for file