The method combines two components: injection of a small volume of a hyperosmolar NaCl solution containing the Pal-eGFP-Sindbis RNA construct  in a specific location with enough sparseness and intensity to allow complete and unambiguous reconstructions of their entire axonal tree. The method is based on the “onstruct , followe Long-range projection neurons constitute a broad category defined by their axon leaving the zone where the cell soma is located to target distant regions within the brain or spinal cord. By monosynaptically linking separate local circuits into large-scale networks, LRPN cells confer the brain a functionally robust and efficient small-world network architecture . Moreovein vivo  it is possible to limit the infection to a few or just one cell per brain while consistently labeling its entire axonal tree in exquisite detail  of both sexes raised in our University’s animal facilities were used in this study. Procedures were carried out in accordance with European Community Council Directive 86/609/EEC and approved by our University’s Bioethics Committee. All surgical procedures were conducted under isoflurane anesthesia (0.5–2% in oxygen) following induction with a combination of ketamine  and xylazine . At the time of sacrifice, animals were overdosed with sodium pentobarbital .Aqueoria victoria (eGFP) fusioned with a palmytoilation motif from the growth associated protein 43 (GAP43) under the Sindbis viral subgenomic promoter . To this end, 50 μg of plasmid were linearized by digestion with Not I restriction enzyme. After checking digestion by electrophoresis in 0.7% agarose gel, DNA templates were purified and precipitated. Finally, DNA template in vitro transcription was carried out using 1.5 μg of plasmidic DNA in a 50 μl distilled DEPC water solution containing 10× SP6 buffer (5 μl), rNTP mix (5 μl) , CAP analog (m7G(5′)ppp(5′)G) (5 μl) , Rnasin (1.5 μl)  and SP6 RNA polymerase . After checking the synthesized Sindbis-Pal-eGFP RNA integrity on an electrophoresis gel , this stock RNA solution (1.8–2 μg/μl) was stored at -80°C.An RNA construct engineered to drive the expression of an enhanced variety of the green fluorescent protein from promoter  was usedTable 1) were made using a horizontal puller . To eliminate RNAse activity, micropipettes were then kept in a stove overnight at 240°C. Micropipettes were backfilled with the RNA stock solution and mounted on a holder  that has both a pressure port and electrode connection .Using a Vertical Pipette puller , micropipettes were pulled from Kwick-Fill borosilicate capillaries (1 mm of outer diameter) with an internal glass filament . Inner tip diameter was adjusted to 10–15 μm. In some experiments, narrower tips , the sagittal midline of the scalp was sectioned and retracted, and a small craniotomy was drilled over the intended target region, usually the thalamus. The micropipette tip was positioned into the brain following the coordinates of n = 21) with the complete Sindbis pseudoviral vector. For these experiments, the replication-defective particles were prepared by co-transfecting the same Sindbis-Pal-eGFP RNA construct (25 μl) along with a helper RNA coding for the Sindbis viral envelope and capsid proteins (25 μl), that was kindly provided to us as cDNA by Dr. Sondra Schlesinger , into cultured baby hamster kidney (BHK) cells (5 × 106). Particles were concentrated from the culture supernatant, titrated, and then diluted to 103 particles/μl into a 0.1 M saline phosphate buffer containing 0,5% Bovine Serum Albumin. In each experiment, ~50 nl of this solution were then pressure-injected though a glass micropipette (outer tip diameter 20–40 μm).For control we compared our results with a number of transduction experiments . However, fluorescent labeling of the axon decreased rapidly with distance, and was usually not detectable in terminal regions, probably because of the very thin caliber of the axonal tree branches and the limited sensitivity of the epifluorescence technique.The sections containing the area targeted in each experiment were water-mounted on glass slides and screened for the presence of GFP-expressing cells under an epifluorescence microscope (Nikon Eclipse 600) through 10–20X objectives and BV2A Nikon filter. Transfected cell somata and dendrites were brightly fluorescent . To this end, all sections were incubated, free-floating, in a rabbit anti-GFP serum  followed by incubation with a biotinylated goat anti-rabbit serum  and an avidin-biotin-peroxidase kit . The immunostaining not only revealed the somatodendritic morphology of the transfected cells in exquisite, Golgi like detail but also extensive distal axonal tree domains not visible with standard epifluorescence or confocal microscopy. All sections were serially mounted onto gelatin-coated glass slides and air-dried. As histological reference for the precise delineation of thalamic nuclei and cortical layers, sections were lightly counterstained with Thionin to obtain a pale Nissl-like background labeling. Finally, sections were dehydrated and coverslipped with DePeX .To allow intensive high-magnification microscope analysis, in those cases where transfection was successful, we made the labeling stable and opaque by using immunohistochemistry against eGFP and glucose oxidase-nickel enhacement  .To estimate the efficacy of a given transfection protocol, we calculated the percentage of cases in which any neuronal or glial cell was transfected over the total of injections carried out with that protocol. The efficacy of the various protocols was then compared using two-tailed paired Fischer’s Exact Test . All data are expressed as mean ± SD.in vivo through a micropipette loaded with the Sindbis-Pal-eGFP RNA solution. Besides, as a standard control for efficacy, we also carried out a number of transduction with the complete Sindbis vector following a standard delivery protocol . Specifically, we tested the following parameter combinations: (a) low current intensity in long pulses, as routinely used for electroporating large molecules such as biotinylated dextrans into well-localized brain tissue domains  1–3 ( domains  (Pr1); a domains . DespiteTable 1). In some of the experiments with the longer current pulses (Pr5 and Pr6) occasional cells were transfected (Table 1); however, in these experiments, an extensive electrolytic tissue lesion was evident around the position micropipette tip.Reasoning that failure by the current pulses alone to eject RNA from the micropipette could be a confounding factor for the above negative results, in the remaining protocols we systematically first ejected 50–100 nl of the RNA solution at a rate of 10 nl/s using a precision pressure injection system . We then tested volume injections combined with delivery of different combinations of electric parameters . We then tested the effect of pressure-injecting this high-NaCl RNA solution alone (Pr10), or of doing it followed by short electric current pulses (Pr11). Remarkably, the combination of high-NaCl with electric current pulses (Pr11) produced cell transfection in a large majority of the trials (85.7%). Statistical analysis confirmed the efficacy of Pr11 to be significantly superior to the rest . Note that injection of high-NaCl solution alone (Pr10), seemingly caused per se a significant increase in transfection efficacy . In addition to the concentrations listed in Table 1, higher NaCl concentrations were also tested ; however, at these concentrations the RNA tends to precipitate and clog the pipette tip.Based on reports that presence of salts such as NaCl in the plasmid solution can increase electroporation efficacy in mammalian cells in vivo , we deciFinally, to explore the possible contribution of the pressure injection procedure, we tried simply injecting the base RNA solution (without added NaCl) and no electric current at all (Pr12). This procedure yielded occasional labeling in 9.1% of the trials.Figure 3B). Most labeled cells were neurons , while the rest were glial cells. This proportion is roughly similar to that observed with the Sindbis pseudoviral vector  one or two labeled cells were the most frequent outcome . Subsequent enhancement and stabilization of the labeling with anti-GFP immunohistochemistry revealed in Golgi-like detail the complete axonal morphology up to their terminal branches. The complete cell morphology could be subsequently reconstructed from the serial sections using a Nikon Eclipse microscope fitted with a camera lúcida system under 20–40X objectives .Free RNA-transfected neurons showed somatodendritic morphologies indistinguishable from those labeled by Sindbis infection. Fluorescent eGFP labeling was present both in the somatodendritic domains and proximal axonal tree . In fact, in most of the free RNA transfection cases measured (6/9), the tip position was located within the radius of the labeled neurons dendritic tree , suggesting that electroporation can occur also through dendrites. This was in marked contrast with cells labeled by injections of the encapsidated vector, which were usually located at several hundreds of microns away from the position of the micropipette tip . Only in 1 of 10 cases, the position of the tip was inside de dendritic domain of the transfected neuron. Moreover, transfection mediated by infective particles was often observed occur along the micropipette track when using the encapsidated vector probably due to solution leakage form the tip. In contrast, we never observed cells labeled along the descent of the micropipette in the free RNA experiments .In what concerns to the spatial precision of the transfection experiments, the distance between the tip position and the dendritic tree of the transfected neuron was found to be significantly lower in free RNA transfection experiments (average distance = 16.1 ± 29.6 μm) than after infective particles injections . From these tests, we conclude that the best method is the one that combines a small volume injection of RNA suspended in a 0.5 M NaCl vehicle with the application of continuous square 1 ms current pulses at high frequency and voltage . Using this protocol in a sizable number of in vivo adult mice experiments (n = 28), cell transfection  was achieved in 85.7% of cases. For comparison, this efficacy is as good or higher than that reported with the best published single-cell electroporation protocols of plasmidic DNA in vivo  of the solution immediately before the current pulses reliably ensures the presence of an adequate concentration of RNA around the cells exposed to the current pulses.Finally, an additional factor contributing to labeling efficacy may probably be the fact that we used a RNA construct that can self-replicate in transfected cells, leading to very high levels of a subgenomic RNA that will translate the reporter GFP gene in the cytoplasm .In recent years, several studies have described techniques for transfecting and labeling single neurons in rodent brains by DNA electroporation, but these methods require complex and expensive setups such as two-photon microscope to visualize the target cell  or patchFigure 4A). In contrast, by using the encapsidated vector, cells are very frequently labeled far away from the intended injection site, making it difficult to reliably target a given brain structure . This is a major problem in species whose brain is small in absolute terms, such as mice. We suspect that the small (60 nm) and spherical Sindbis particles (The GFP labeling obtained using the free RNA construct as described here is as rapid, intense and as complete as that obtained with the standard encapsidated Sindbis vector at very low titer  Moreoverarticles  drift awarticles .in vivo with the highly sensitive tracer vector Pal-Sind-eGFP. Thus, it may become a valuable tool for single-cell connectomic studies of long-range projection neurons (Overall, the method described here makes it simpler and more reliable the precise labeling of one or a few isolated neurons per brain  neurons .CP, JR-M, and FC designed the study, carried out the experiments, analyzed the data, and wrote the paper. JQ and CS produced the RNA constructs and revised the paper; TF designed the vector, provided the cDNA plasmid templates and revised the paper.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Post-transcriptional control of mitochondrial gene expression, including theprocessing and generation of mature transcripts as well as their degradation, is akey regulatory step in gene expression in human mitochondria. Consequently,identification of the proteins responsible for RNA processing and degradation in thisorganelle is of great importance. The metallo-β-lactamase (MBL) is a candidateprotein family that includes ribo- and deoxyribonucleases. In this study, wediscovered a function for LACTB2, an orphan MBL protein found in mammalianmitochondria. Solving its crystal structure revealed almost perfect alignment of theMBL domain with CPSF73, as well as to other ribonucleases of the MBL superfamily.Recombinant human LACTB2 displayed robust endoribonuclease activity on ssRNA with apreference for cleavage after purine-pyrimidine sequences. Mutational analysisidentified an extended RNA-binding site. Knockdown of LACTB2 in cultured cells causeda moderate but significant accumulation of many mitochondrial transcripts, and itsoverexpression led to the opposite effect. Furthermore, manipulation of LACTB2expression resulted in cellular morphological deformation and cell death. Together,this study discovered that LACTB2 is an endoribonuclease that is involved in theturnover of mitochondrial RNA, and is essential for mitochondrial function in humancells. Mitochondria are important for many metabolic pathways, including the production ofadenosine triphosphate (ATP) in the process of oxidative phosphorylation. Mitochondriaare an evolutionary remnant of an endosymbiotic event that occurred 1.5 billion yearsago, originating from the α-proteobacterium, where most of the bacterium geneswere transferred from the organelle to the nuclear genome of the ancient host . Themamin vitro and in cultured cells  superfamily. The MBL superfamily includes a variety of proteins that areresponsible for RNA processing, DNA repair and small-molecule metabolism –31. FuncCPSF, Artemis,SNM1/PSO2), whose members are keyplayers in RNA metabolism (CPSF-73 and RNase J) and DNA crosslink repair (Artemis andits homologs mouse SNM1 and yeast PSO2), has been studied over the past decade  and exonucleolytic (exo-) activities and are catalyzed in the same catalyticvicinity (cleavage and polyadenylationspecificity factor) shares similarcharacteristics, as it is responsible for the endo-cleavage of pre-mRNA 3΄processing and the dual endo-/exo- 5΄ to 3΄ activity on pre-histone mRNA were identified as nucleic acid hydrolases ,33. The t decade ,35. RNAvicinity . The provicinity . The mamone mRNA,40,41. Tnnaschii .Prompted by the above findings, we searched for mitochondrial MBL proteins featuring thetraditional characteristics of MBL ribonucleases. β-lactamase-like-protein 2(LACTB2), has been suggested to be located in the mitochondria . Other thttp://www.mitoproteome.org/)(https://www.cgl.ucsf.edu/chimera/) (Human LACTB2 was first identified in (me.org/),45 and lhimera/) .Escherichia coli DE3 pnp- cells (DEHO: C-5691); the column was washed with lysisbuffer containing 25 mM imidazole, then eluted with lysis buffer containing 250 mMimidazole. The eluted fraction was combined with TEV protease (1:20 w/w) and dialyzedovernight at 4°C against buffer A , and reloaded onto a Ni column. The TEVprotease and other impurities bound the Ni column, while LACTB2 was collected in theflow-through fraction. The protein was concentrated using Vivaspin Centricon(Satorius) and loaded on a gel filtration Superdex 200 HR 10/300 column using GFbuffer . The fraction containing LACTB2, which was ∼30–50 kDa, was eitherconcentrated and used directly for crystallization, or (for activity assays) dialyzedagainst low-salt Q buffer , passed through a MonoQ column  andcollected in the flow-through fraction. The purified protein was dialyzed against its2× activity buffer , fast-frozen in liquid nitrogen and stored at−80°C. LACTB2 preparations used for activity assays were assayed bothenzymatically and by immunoblot assay to detect any traces of contaminatingE. coli ribonucleases: RNase E, PNPase, RNase II and RNase R.Human LACTB2 cDNA was polymerase chain reaction (PCR) amplified using the appropriateoligonucleotide primers  and inse C-5691) were tra0 of 1.0 . Protein0 of 1.0 . Cells wPoorly diffracting crystals (7–11Å) were grown by vapor diffusion at20°C in sitting drops , and improved crystals diffracting to 2.6Å were obtained by seeding.For heavy metal derivatization, crystals were formed by seeding in drops of 75 nl ofprotein solution (18 mg/ml) and 75 nl of precipitant consisting of 0.2 Na Formate,0.1M Bis-Tris-Propane, pH 7.5, 20% PEG 3350 and 10% Ethylene Glycol.The crystals were soaked in solution consisting of the mother liquor, supplementedwith 1 mM Thiomersal for 60 min. In all cases, the crystals were cryo-protected usingthe well solution supplemented with 15% ethylene glycol and flash-frozen inliquid nitrogen.Native 2.6 Å dataset—diffraction data were collected from a singlecrystal at the Diamond synchrotron beamline I02 at a wavelength of 0.9795Derivatized dataset—diffraction data were collected from a single crystal atthe Diamond synchrotron beamline I03 at a wavelength of 0.9763The protein crystallized in space group P1 21 1, with six monomers in the asymmetricunit.Repeated attempts to solve the 2.6 A dataset by molecular replacement failed,presumably due to low homology between LACTB2 and the search models. A derivatizedcrystal dataset was collected and scaled to 3.2 Å. Eight mercury sites wereunambiguously identified using SHELXD ,with an32P] ATP,obtaining mono-phosphorylated substrates   wasperfbstrates .The 3΄ e 3΄ end . Full-le32P]-RNA, the annealed dsDNA product was transcribed using T7 RNApolymerase and [α-32P]UTP  . Thefir-32P]UTP . RNAs weIn vitro RNA degradation assays were performed using the recombinantprotein and either 5΄ [32P]-labeled, body-labeled, or 3΄[32P]Cp-labeled RNA substrates. In a common reaction, a protein (7.5μM) was incubated at 37°C with 0.08 μM RNA in a buffercontaining 12.5 mM Tris–HCl pH = 7.4, 125 mM NaCl, 2.5% glyceroland 0.25 mM DTT for the times indicated in the figure legends. When relevant, 10μM yeast tRNA (Sigma) was added to the reaction. Following incubation, the RNAwas analyzed by denaturing PAGE and autoradiography.32P]-labeled RNA and cross-linked at aUV cross-linker that was set at 1.8 joules (Hoefer), followed by RNA digestion with10 μg RNase A and 30 units RNase T1, at 37°C, for 1 h. The proteinswere then fractionated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis(SDS-PAGE) and analyzed by autoradiography.The ultraviolet (UV) light cross-linking of proteins to radio-labeled RNA wasperformed as previously described .Protein12 oligonucleotide degradation assay, the substrate was5΄ end-labeled using [γ-32P]ATP, as described above. Then, 1μg yeast tRNA (Sigma) and either 0.001 U RNase A (sigma) and 0.1 U RNase T1(sigma) were incubated with the radiolabeled (GU)12 RNA on ice for 3 min.Following incubation, the RNA was analyzed by denaturing PAGE and autoradiography.For the PNPase assay, incubation with nuclease P1 was for 30 min at 50°C in abuffer containing: 1 u/μl Nuclease P1, 0.2 mg/ml yeast tRNA, 20 mM sodiumcitrate (pH 9.0), 6.5 M urea and 1 mM ZnCl2. Incubation with RNase A (0.1μg/ml) was on ice for 1 min with the addition of 0.4 mg/ml of yeast tRNA.Incubation with PNPase (15 μg/ml) was at 37°C for 30 min in buffer Econtaining 5 mM Pi (For the (GU) 5 mM Pi . Followi2 indulbecco's modified eagle's medium medium (Sigma) supplemented with10% fetal calf serum, 2 mM L-glutamine, and 1% penicillin-streptomycin. A 27-mer LACTB2-specific siRNA duplex (IDT) (6) were transfected with 0.1 μMsiRNA duplex on 60 mm plates using jetPRIME reagent (Polyplus) and harvested 48 hpost transfection.HEK-293E cells were grown as a monolayer at 37°C, 5% COex (IDT)  or25-me32P]-labeled oligonucleotides that complemented humanmitochondrial transcripts . The quantification of the RNA wereperformed using a NanoDrop spectrophotometer and gel electrophoresis. For qRT-PCRanalysis, 2 μg total RNA were treated with DNase I (Invitrogen) and thenreverse transcribed using random priming (Applied Biosystems). Then, 10 ng cDNA, 0.1μM real-time primer set and SYBR green mix (Quanta Biosciences) were used inthe qRT-PCR reaction for gene expression analysis (Bio-Rad CFX96). RNA blot analysiswas performed, as described . Brieflynscripts . Hybridi9 cells were harvested andhomogenized in swelling buffer . The sucrose concentration was adjusted to 250 mM, and thenucleus fraction was removed by low-speed centrifugation. The mitochondria werewashed twice to remove possible nuclear contaminants and were then pelleted at 15 000× g for 15 min. The washing step was repeated to yield thefinal pellet of the mitochondria fraction. Fractions of the intermembrane space andthe matrix of the mitochondria were analyzed by the proteinase K accessibly test asdescribed (2CO3 (pH 11.5) on ice for 30 min prior to a 30 mincentrifugation at 100 000 × g, to yield the pellet fraction(intrinsic membrane proteins) and soluble fraction .Mitochondria were purified as previously described . Brieflyescribed . AlkalinProteins were analyzed on a 12% SDS-PAGE, blotted to a nitrocellulose membraneand decorated with appropriate antibodies that were detected by chemo-luminescenceusing the ImageQuant (LAS4000) system. The following antibodies were used: LACTB2(Sigma HPA044391 or Thermo Scientific PA5–32043), CPSF3L (Sigma HPA029025),GAPDH (Santa Cruz Biotechnology FL-335), Tom 20 (Thermo Scientific PA5 39247), HSP60(Thermo Scientific PA5–12484), PNPase (Thermo Scientific PA5–22396),CYT C (Santa Cruz Biotechnology H-104) and subunit V of the ATP-synthase , and was then used to solve the native dataset by molecularreplacement. There are six molecules of LACTB2 in the asymmetric unit, arranged astwo trimers. Although PISA analysis  of LACTB2 is a typical MBL fold, with a central doubleβ sheet sandwich flanked by helices and loops Figure . The put2+ ions, denoted as red (LACTB2) or blue (CPSF-73)spheres at the bottom of the structure, are at nearly identical positions (within 0.3Å). A closeup of the catalytic regions . An additionalZn2+ coordinating residue in β-CASP region of CPSF-73, H418,defined as motif C/V, is substituted by a non-homologous his residue (H199) inLACTB2. The presence of a histidine residue at the C/V motif is thought to be adistinguishing feature of MBL ribonucleases, and is absent in MBL deoxyribonucleasessuch as SNM1A and SNM1B  with theMBL domain of CPSF-73   Figure . The strs Figure  shows a nd SNM1B  reveal a32P]-RNA as a substrate, and degradation products were analyzed bydenaturing gel electrophoresis and autoradiography. Several degradation productsaccumulated during the incubation period, indicating that LACTB2 is a ribonuclease20 or poly(GU)12 RNA substrates. While very lowendoribonucleolytic activity was observed with poly(U) and even less with poly(A), adistinct cleavage pattern was observed with poly(GU)12 substrates  or by RNase T1,that cleaves following G. Figure 12 RNAsubstrate, the cleavage pattern of LACTB2 is similar to that of RNase A, cleavingthis substrate following C s Figure. Becauselowing C . Combini2+ at the active site, typically leave a5΄-phosphate and 3΄-OH on the cleavage products. To verify that thecleavage products of LACTB2 are characterized with 3΄-OH, we used the PNPaseassay. PNPase is a 3΄ to 5΄ processive exoribonuclease that digest3΄-OH, but not 3΄-phosphate RNAs . Th. Th32). dessDNA ,42,68. LdessDNA  and 3C. dessDNA ;increasdessDNA : a 30-ntdessDNA  bottom. dessDNA . We inte32P]-labeled RNA severely inhibited the degradation activity, we hypothesized that these residues mayfunction in either RNA binding or protein structure curvature. Unlike thewell-described ribonucleolytic active site of MBL ribonucleases, it remains unknownif additional RNA-binding domains exist. Several of the archaeal CPSF proteinsinclude a KH RNA binding motif recognizing specific nucleic acid sites –80. Howeg pocket ,72,81. Teled RNA . Thedegeled RNA .Additioactivity. Mutatinactivity revealedTaken together, these results suggest an extended RNA-binding site that plays anessential role in the RNA-degrading activity of LACTB2. Deletion of motif II(Δmotif II), the mutation on motif IV (D164A) and two mutations on the surfaceof the protein, H216A::R217T and R220T, lead to reduced RNA binding, resulting in apoor RNA degradation activity of the protein. The impaired RNase activity of themutants N116A and E118A does not correlate with a reduction in RNA binding in thecross-linking assay, so the role of these residues is less clear. Furtherinvestigations and perhaps the crystallization of this protein with an RNA moleculeare required to fully understand the nature of LACTB2 RNA binding. The involvement ofresidues outside the catalytic site in nuclease activity has been recentlydemonstrated in the MBL DNA repair nucleases SNM1A and SNM1B , indicatGiven the localization of LACTB2 in the mitochondria and its activity as anendoribonuclease, we hypothesized that LACTB2 plays a role in mitochondrial RNAmetabolism. Therefore, we next asked if decreased expression of this protein canresult in changes in the accumulation of all or a subset of mitochondrialtranscripts. Decreased expression of LACTB2 was obtained by synthetic siRNAtransfection to HEK-293E cells and then confirmed by qRT-PCR performed 48 h posttransfection Figure . ThisreA modest accumulation of mitochondrial transcripts was observed in LACTB2-depletedcells compared to control cells transfected with scrambled RNA Figure . MitochoUpon overexpression of LACTB2 in HeLa cells , only a In a search for ribonucleases that are responsible for RNA degradation in humanmitochondria, an uncharacterized protein from the MBL superfamily was identified. Inthis study, we report that the previously unannotated protein, LACTB2, displaysendoribonuclease activity. Endogenous LACTB2 localized in the human mitochondrial matrixand existed as a soluble monomeric protein. Unlike other mitochondrial proteins, such asGRSF1 and RNase P  or PNPasBoth strands of the mitochondrial 16K-base circular DNA are transcribed aspolycistronic transcripts. The mRNAs are then derived from the polycistronic RNAs bycleavage of the ‘punctuating’ tRNAs via the action of the enzymes RNaseP and RNase Z (ELAC2). Therefore, it appears that for the major mitochondrialendoribonucleolytic RNA processing events, an additional endoribonuclease is notnecessary. However, there are three additional cleavages sites that are not coupledwith tRNA processing. These include the generation of the 5΄ end of CoxI, thegeneration of the 3΄ end of ATP6 and the 5΄ end of CoxIII by cleavageof the di-cistronic transcript of these genes and the generation of the 5΄ endof Cyt B. An additional previously detected cleavage site on the 3΄ end of theND6 transcript, the unique mRNA transcribed from the light strand, is questionable,since this transcript harbors a long 3΄ UTR . AlthougIn addition to the stable decoration of the 3΄ ends of mitochondrial mRNAs andrRNAs with poly(A)-tails, poly(A)-assisted degradation pathway takes place in thisorganelle as well . In bactIn our experiments aimed to characterize its biological function, increased ordecreased LACTB2 expression resulted in a moderate but noticeable alterations inmitochondrial transcript levels. As expected from an RNase, the decreased expressionof LACTB2 resulted in the accumulation of most tested RNA transcripts by1.5–2.0-fold, and its overexpression caused a reduction to approximately halfof that accumulated in control cells. These two mirrored observations generallyobserved with most of the tested transcripts, are compatible but were too moderate toenable identification of specific initial cleavage site(s). Similar non-specific,moderate and limited effects on the accumulation of mitochondrial transcripts werepreviously obtained with other RNA-mediated degradation proteins in mammalianmitochondria. For example, hPNPase-Suv3, PED12 and REXO2 demonstrated similarbehavior and presented a challenge to identify their specific targets whendownregulated in cell culture ,22,23.OB.subtilis as afunctional replacement of RNase E, an mRNA endoribonuclease that is a major player inRNA degradation in E. coli -tail . Indeed, E. coli ,37,84. R E. coli ,42,80,84T. thermophiles , LACTB2 containsthe MBL domain but lacks the β-CASP and RMM domains; instead, LACTB2 has aC-terminal domain with no homology to other MBL proteins (Figure , LACTB2 s Figure . This shOur results show that human LACTB2 is an endoribonuclease, similar to other MBLmembers, such as the human CPSF73 and the archaeal mjRNase J2 ,42. The 2+ ions, confirmed thatthe histidine and aspartic acid motifs are essential for the catalytic and theRNA-binding activities, as seen with other MBL ribonucleases (Mapping the ribonucleolytic active and the RNA-binding sites of LACTB2 was achievedby mutating key residues of the protein. MBL mutations in motifs II and IV, which areinvolved in the coordination or bridging of the Znucleases ,42,68,722+ ions within its catalytic site. Manipulations of LACTB2 proteinexpression hindered the mitochondrial functioning, resulting in changes in cellmorphology and cell death. Unraveling the specific function of LACTB2 inmitochondrial RNA metabolism still requires further studies that will examine whetherLACTB2 co-localizes with RNA-granules, identify its primary and perhaps specific,substrate in vivo and reveal the mechanism for the mitochondrialdysfunction when this protein is downregulated.In conclusion, we identified a novel human mitochondrial protein, LACTB2, whichfunctions as an endoribonuclease with a preference for cleavage 3΄ topurine-pyrimidine dinucleotide sequences of ssRNA substrates, while usingZnSupplementary DataClick here for additional data file.


The RNA World Hypothesis posits that the first self-replicating molecules were RNAs. RNA self-replicases are, in general, assumed to have employed nucleotide 5ʹ-polyphosphates  as substrates for RNA polymerization. The mechanism by which these substrates might be synthesized with sufficient abundance to supply a growing and evolving population of RNAs is problematic for evolutionary hypotheses because non-enzymatic synthesis and assembly of nucleotide 5ʹ-triphosphates  is inherently difficult. However, nucleotide 2ʹ,3ʹ-cyclic phosphates are also phosphodiesters, and are the natural and abundant products of RNA degradation. These have previously been dismissed as viable substrates for prebiotic RNA synthesis. We propose that the arguments for their dismissal are based on a flawed assumption, and that nucleotide 2ʹ,3ʹ-cyclic phosphates in fact possess several significant, advantageous properties that indeed make them particularly viable substrates for prebiotic RNA synthesis. An RNA World hypothesis based upon the polymerization of nucleotide 2ʹ,3ʹ-cyclic phosphates possesses additional explanatory power in that it accounts for the observed ribozyme “fossil record”, suggests a viable mechanism for substrate transport across lipid vesicle boundaries of primordial proto-cells, circumvents the problems of substrate scarcity and implausible synthetic pathways, provides for a primitive but effective RNA replicase editing mechanism, and definitively explains why RNA, rather than DNA, must have been the original catalyst. Finally, our analysis compels us to propose that a fundamental and universal property that drives the evolution of living systems, as well as pre-biotic replicating molecules (be they composed of RNA or protein), is that they exploit chemical reactions that already possess competing kinetically-preferred and thermodynamically-preferred pathways in a manner that optimizes the balance between the two types of pathways. Life evolved, according to The RNA World Hypothesis, from prebiotic self-replicating catalytic RNA molecules. This hypothesis was initially formulated by Woese , Crick  and OrgeVarious mechanisms of pre-biotic RNA polymerization have been proposed; those currently considered to be the most plausible involve condensation of nucleoside 5ʹ-polyphosphates (such as compound B in rxn (and therefore the ΔGrxn) for the forward reaction in rxn for Nucleoside 2ʹ,3ʹ-cyclic phosphates  has been assumed for rxn due to adduct formation. . If i.e., a catalyst capable of selectively activating a specific reaction pathway), an RNA dimer (or polymer) synthesized via the forward reaction of We propose that the above conclusion cannot be correct. In the absence of an enzyme  geometry (When this third state (Cʹ) is also taken into account, the basis for dismissing geometry . The netWe further hypothesize that a fundamental property of living systems as well as pre-biotic replicating molecules is that they are able to optimize the balance between kinetically-favored and thermodynamically-favored biochemical pathways in such a manner that the probability of their growth and replication becomes maximized. Enzymes, whether self-replicating RNAs, other RNA catalysts, or protein enzymes, have thus evolved as the means for doing this most efficiently. Optimization of the balance between kinetically-favored and thermodynamically-favored biochemical pathways via replication coupled to natural selection is thus likely to be a major driving force behind the evolution of enzyme catalysis and life. We will return to this point below.We have argued that the assumed equal partition between reactants and products in Ribozymes are thought to be, in some sense, molecular fossils. Peptidyl-transferase, for example, likely evolved quite early in biological history, and its preservation in the form of a ribozyme catalyst, as well as its fragility with respect to evolutionary tinkering, is clear and compelling evidence for its ancient origins . At leasIf these modern-day ribozymes are also molecular fossils, it would seem reasonable that the The efficiency of prebiotic RNA self-replication would likely have been limited by the availability of nucleotide substrates. Because nucleoside 2ʹ,3ʹ-cyclic phosphates are comparatively simple to synthesize, and because proto-cellular membranes would likely be much more permeable to the singly-charged nucleotide 2ʹ,3ʹ-cyclic phosphates than they would be to nucleotide polyphosphates having multiple charges, it is likely that nucleoside 2ʹ,3ʹ-cyclic phosphate substrates would be relatively more plentiful and available in higher concentrations for prebiotic RNA synthesis. In addition, since the natural decomposition products of RNA are nucleoside 2ʹ,3ʹ-cyclic phosphates, the starting reagents for pre-biotic RNA synthesis are continually recycled and would likely remain in abundance in a prebiotic environment, limited only by the comparatively slower hydrolysis of the 2ʹ,3ʹ-cyclic phosphate. Nucleoside 5ʹ-polyphosphates cannot be recycled in this way. Instead, nucleoside 5ʹ-phosphates must be regenerated each time they are to be incorporated in a new strand of RNA, in an energetically costly  manner.cf. Figure 1 in [Although purine and pyrimidine nucleotide bases are fairly straightforward to synthesize chemically under plausible pre-biotic conditions, the problem of how these might condense upon ribose to form ribonucleotide adducts is much more challenging. Purine ribonucleotides form inefficiently , and pyrure 1 in ). If 2ʹ,ad hoc heating and cooling cycles, or heterogeneous surfaces are required for catalysis; this is well-known to be the case in RNase A catalyzed reactions as well as the non-catalyzed reaction. Recently, we have demonstrated that three of the four small self-cleaving ribozymes listed above that utilize this pathway also do not require divalent metal ions for catalysis [Nucleoside 2ʹ,3ʹ-cyclic phosphate isomerization  is arguaatalysis . Only thThe product (dimerized or polymerized RNA) may be thought of as a two-state system, in which the initial state that develops instantaneously (compound C) as a result of dimerization is in an approximate 1:1 equilibrium with reactants. Upon nucleotide base-pairing, the initial state then adopts the helical form of the final state (compound Cʹ) that becomes kinetically trapped and therefore inaccessible to the back reaction. Promotion of the initial product state (C) to the final helical product state (Cʹ) depends upon correct base-pairing; an improper fit impedes helical formation and thus increases the propensity for the back-reaction to occur. This increased propensity for the back reaction facilitates elimination of incorrectly-paired nucleotides. Helical editing and fidelity of replication thus arise in a completely natural manner. Hence, a primitive editing mechanism based upon in vitro selection techniques, DNA has been shown to possess the ability to catalyze phosphodiester isomerization reactions as well [in vitro selected deoxyribozymes refutes the absolute necessity of having 2ʹ-OH in a nucleic acid enzyme structure. The remaining advantage of the 2ʹ-OH is that it alone allows formation of 2ʹ,3ʹ-cyclic phosphates. RNA thus possesses an advantage over DNA as a candidate for the original pre-biotic self-replicating polymer in that its constituents can be either nucleotide 5ʹ-polyphosphates, that can be assembled according to The idea that the first self-replicating nucleic acid must have been RNA rather than DNA was based originally on the assumption that DNA lacked the ability to be catalytic; no naturally-occurring deoxyribozymes have yet been discovered. However, using  as well . Nature ad hoc introduction of a new, biologically emergent, thermodynamic quantity that has never been measured. Although this idea has never been reconciled with the second law of thermodynamics, the idea that living organisms are unique in their ability to generate and replicate ordered systems from disordered components certainly appears to be sound.Erwin Schrödinger, in his seminal book “What is Life?”  proposedad hoc invention of a new thermodynamic potential. Instead, we can reformulate Schrödinger’s claim as follows: A fundamental property of living systems as well as evolving pre-biotic replicating molecules is that they exploit chemical reactions that already possess both a kinetically-preferred pathway and a thermodynamically-preferred pathway, and they do this in such a way that optimizes the balance between the two. The optimization process is an evolutionary one, and optimization of the balance can be defined loosely but operationally as changing the propensities of the two pathways relative to one another in successive rounds of replication and natural selection in such a way that maximizes the ability of the system to grow and to replicate itself with reasonable fidelity.Returning to our claim that enzymes have evolved to optimize the balance between kinetically-favored and thermodynamically-favored biochemical pathways, we can begin to understand Schrödinger’s problem without resorting to his It has always been recognized that enzymes have evolved to accelerate the rate of biochemical reactions that benefit an organism. We suggest that an additional selective restraint placed upon enzymes by the evolutionary process is the ability to store information and energy in metastable forms that can then be exploited by the organism or pre-biotic replicating molecule. Kinetically-favored but thermodynamically less favored reaction products, such as RNA polymers, are perhaps the original example, and certainly one of the most important examples, of this principle. However, this principle may be generalized to include much, if not all, of metabolic biochemistry, photosynthesis, and bio-energetics. The description of this principle in its most general form is the subject of a previous communication .in vitro evolution experiments is to discover a sequence of RNA capable of RNA polymerization or even self-replication. Although such an experiment, in the absence of time-travel, cannot prove that the RNA World Hypothesis is correct, successful generation of an RNA replicase would constitute a compelling proof of principle.The Holy Grail of RNA In vitro RNA evolution experiments aimed toward evolving RNA ligases and polymerases have focused on substrates having 5ʹ-triphosphates. Although these have met with significant success, it would be of interest to know whether in vitro evolved ribozymes that use 2ʹ,3ʹ-cyclic phosphates, like their naturally occurring counterparts, might arise more readily or otherwise possess additional assets that might facilitate in vitro selection of a polymerizing or replicating ribozyme, due to the comparative simplicity of the


Cauliflower mosaic virus (CaMV) uses alternative splic-ing to generate several isoforms from its polycistronic pregenomic 35S RNA. This pro-cess has been shown to be essential for infectivity. Previous works have identified four splice donor sites and a single splice acceptor site in the 35S RNA 5’ region and sug-gested that the main role of CaMV splicing is to downregulate expression of open read-ing frames (ORFs) I and II. In this study, we show that alternative splicing is a conserved process among CaMV isolates. In Cabb B-JI and Cabb-S isolates, splicing frequently leads to different fusion between ORFs, particularly between ORF I and II. The corresponding P1P2 fusion proteins expressed in E. coli interact with viral proteins P2 and P3 in vitro. However, they are detected neither during infection nor upon transient expression in planta, which suggests rapid degradation after synthesis and no important biological role in the CaMV infectious cycle. To gain a better understanding of the functional relevance of 35S RNA alternative splicing in CaMV infectivity, we inactivated the previously described splice sites. All the splicing mutants were as pathogenic as the corresponding wild-type isolate. Through RT-PCR-based analysis we demonstrate that CaMV 35S RNA exhibits a complex splicing pattern, as we identify new splice donor and acceptor sites whose selection leads to more than thirteen 35S RNA isoforms in infected turnip plants. Inactivating splice donor or acceptor sites is not lethal for the virus, since disrupted sites are systematically rescued by the activation of cryptic and/or seldom used splice sites. Taken together, our data depict a conserved, complex and flexible process, involving multiple sites, that ensures splicing of 35S RNA.The plant pararetrovirus  ATP in excess was eliminated using an illustra MicroSpin G-25 Column .Recombinant viral proteins P1, P2, P3, P6/TAV and GST-P1P2(D2) were expressed in 2HPO4, 2 mM KH2PO4), Tween 20 1%, milk 5% at room temperature for 12 h. The membrane was incubated either with 32P-radiolabelled viral proteins or with purified virus particles at 4°C for 12 h and washed in phosphate-buffered saline 1x-Tween 20 1% for 10 min. The interaction between the fusion protein and virus particles was detected with anti-P4 polyclonal antibodies  and alkaline phosphatase-labeled secondary antibodies .The GST-P1P2(D2)-containing bacterial fraction was separated by SDS-PAGE, electro-transferred to a PVDF membrane  and saturated in phosphate-buffered saline 1x  and gently suspended in 0.5 M mannitol. This washing step was repeated three times. Fixation and immunolabeling of isolated protoplasts were performed as described by Haas and colleagues [Protoplasts were isolated at 21 dpi from healthy or infected turnip plants as follows. Leaves were chopped and incubated in an enzyme solution ethanesulfonic acid, 1.5% cellulase R10 and 0.4% macerozyme R10 ) at 25°C for 4 h. Leaf debris were removed by filtrating the digestion medium through two layers of Miracloth (Merck Millipore). To eliminate the enzymes, the protoplasts were collected by centrifugation . Moreover, these three sites are totally conserved (99–100% identity) in all the 67 genome sequences that have recently been released [GU sequence that is totally conserved among all CaMV isolates. The corresponding exon-intron junction anneals well with the U1 small nuclear RNA , and threleased   targeting a sequence immediately upstream of the splice acceptor site (ex) that is complementary to a sequence immediately downstream of the splice acceptor site . Amplification products were also obtained in the minus-DNase/minus-RT lane but not in the plus-DNase/minus-RT lane  interacted with purified viral particles, P2 and P3, but neither with P1 nor with P6/TAV  proteins were not immunodetected in crude protein extracts . Mutations were not introduced in ORF I to study the biological relevance of P1P2 fusion proteins because they impair CaMV systemic movement. The P1P2stop mutant was as infectious as wt Cabb B-JI in turnip plants and the capsid protein P4 accumulated at similar level in systemic leaves (The selection of splice donor sites D1 and D2 (and D3 in Cabb-S isolate) and acceptor site A creates fusion ORFs between the 5’ part of ORF I (whose size varies depending on the donor site) and the 3’ part of ORF II, which remains unchanged in the fusion ORFs. The resulting P1P2(D1) and P1P2(D2) fusion proteins have a theoretical molecular mass of about 31 kDa. It has been shown that P2 self-interacts , interach P6/TAV  whereas h P6/TAV  as well . The main pool of spliced RNAs migrated slightly faster than the spliced 35S RNAs present in plants infected with the wt virus. A densitometry analysis indicated that the spliced 35 RNA represented 83% of the 35S RNA global population in D1D2D4Da mutant-infected turnip plants  similar to those found in the CaMV P4 protein [Multiple spliced RNA isoforms arising from single-, double- or triple-splicing events were characterized in CaMV-infected plants. Almost all spliced RNAs contained a new ORF coming from a fusion involving the sORF E, the ORFs I and II or even other reading frames located within ORFs I and II. A complex population of spliced viral transcripts comprising at least 15 spliced isoforms was also characterized in patients chronically infected by hepatitis B virus (HBV) ,49. Singarticles . These iarticles ,38 of tharticles ,54, and articles . The ver protein . In thisCaulimoviruses, FMV and Carnation Etched Ring virus [Mastrevirus Maize Streak Virus [Another aim of our study was to understand the functional relevance of 35S RNA alternative splicing in CaMV pathogenesis. We noticed that the splice donor sites D1 and D2 in ORF I are situated within a GGT triplet coding for glycine, even if this amino acid can be specified by four GGN codons. This could hint toward a selective pressure to conserve both the splice sites and the glycine codons . Glycineng virus . They evng virus  did not ng virus  and in Mak Virus  infectioak Virus ,21, the ak Virus , except ak Virus . This deak Virus  which isak Virus . Thus, tak Virus ,21 mightak Virus ,21 and tak Virus . In the Retroviridae HIV-1 [Alternative splicing could have other roles besides downregulating the expression of ORFs I and II. Recently, it was shown in Arabidopsis that spliced RNAs are less effective substrates for RNA silencing mediated by the RNA-dependent RNA polymerase RDR6, compared to intron-less transcripts ,59. Duriae HIV-1 ,64 and fae HIV-1 . The U1 ae HIV-1 . This reae HIV-1 . In HIV-ae HIV-1  or downsae HIV-1 , thus avcis-elements (splicing enhancers and silencers) is another point which must be elucidated in the future, to gain insight on the temporal regulation and the biological relevance of the various 35S RNA spliced isoforms.The splice donor and acceptor sites described in the present study allow CaMV to produce multiple spliced 35S RNAs, which do not encode for P1 and P2 proteins while still being appropriate mRNAs for P6/TAV-mediated expression of viral proteins via translation reinitiation. Any inactivation of a splice site would be ineffective in impairing CaMV infectivity. The synthesis of intron-deleted DNA by reverse transcription could be a strategy to increase the population of spliced RNAs in a spliceosome-independent fashion. Whether CaMV alternative splicing is regulated by S1 FigE. coli. Recombinant proteins were submitted to SDS-PAGE and transferred to PVDF membranes. 32P-labeled recombinant P1, P2, P3 and TAV proteins or purified virions were used as overlays.Far-western assays on GST or GST-P1P2(D2) expressed in (TIF)Click here for additional data file.S2 FigE. coli crude extract were separated by SDS-PAGE and transferred to PVDF membranes. Polyclonal anti-P1 and anti-P2 antibodies were used as primary antibodies.Recombinant GST, GST-P1P2(D2), P1P2(D2) fusion protein and non-transformed (TIF)Click here for additional data file.S3 FigProteins were extracted from systemic leaves at 21 dpi and analyzed by western-blot. Polyclonal anti-P4 and anti-P2 antibodies were used as primary antibodies.(TIF)Click here for additional data file.S4 FigA: from positions 164 to 2404, B: from positions 844 to 3234, C: from positions 1704 to 4204, D: from positions 2544 to 4974, E: from positions 3404 to 5384, F: from positions 4204 to 6404, G: from positions 4904 to 7434, H: from positions 6404 to 794. (B) RT-PCR products were separated on agarose gel. The control lanes correspond to PCRs performed on viral vectors, using the same primers. Primers targeting region H are designed to specifically amplify contaminating viral DNA.(A) Schematic representation indicating the regions amplified by RT-PCR. (TIF)Click here for additional data file.S1 TableThe 5’ part of the 35S RNA from CaMV Cabb B-JI- or Cabb-S-infected turnip plants was amplified by RT-PCR. Amplicons were fractionated on agarose gel, extracted and cloned into pGEM-T Easy vector. In order to maximize the diversity of the sequenced spliced isoforms, inserted 35S RNA fragments were selected prior to sequencing see “”.(TIF)Click here for additional data file.S1 TextNucleotides corresponding to donor and acceptor sites of Cabb-S isolate are in bold. Donor sites D1 and D2 are in page 17, the donor site D3 is in page 23 and the acceptor site A is in page 27.(DOCX)Click here for additional data file.


The regeneration of axons relies on a previously unknown mechanism that involves the regulation of alternative splicing by CELF proteins. Related research article Chen L, Liu Z, Zhou B, Wei C, Zhou Y, Rosenfeld MG, Fu X-D, Chisholm AD, Jin Y. 2016. CELF RNA binding proteins promote axon regeneration in C. elegans and mammals through alternative splicing of Syntaxins. eLife5:e16072. doi: 10.7554/eLife.16072Image Some mutant worms have problems regenerating axonsInjuries to neural circuits can mean that nerve fibers called axons need to be regrown. However, some nerve cells are better at regenerating than others, and understanding why this is so may open the door to new treatments for traumatic nerve injuries .Researchers have studied the suites of genes that are activated in injured nerve cells for decades . HoweverOne family of RNA binding proteins called CELF proteins regulates alternative splicing in the brain and affects both the development and activity of neurons . Now, inChen et al. reasoned that if, they could identify which RNAs binds to, it might give them some clues as to this protein’s function. They turned to a high-throughput genomics approach called CLIP-seq and generated a list of target RNAs from worm neurons . The majunc-75 mutants have problems regenerating their axons largely because they produce less UNC-64A. This result is quite remarkable. Of the hundreds of RNAs identified as targets of UNC-75, it appears that one – that is to say, UNC-64A – is the key to promoting nerve regrowth following an injury.The mRNA for UNC-64/Syntaxin is spliced differently in neurons and non-neuronal tissues , and CheChen et al. extended their analyses from worms to mammals, and found that two CELF proteins from mice, including CELF2, could partially complement the function of UNC-75 during axonal regeneration in worms. CELF2 is the mammalian version of UNC-75, and mice that did not produce CELF2 in their nervous system had severe defects in axon regeneration. Finally, another round of CLIP-seq in mouse cells revealed that, similar to UNC-75 in worms, CELF2 binds to many Syntaxin mRNAs and can regulate the alternative splicing of two mammalian Syntaxin genes.These experiments with worms and mice suggest that the CELF proteins are important players in axon regeneration, possibly via the post-transcriptional regulation of isoforms of Syntaxin. These new data pose a further question: do these Sytnaxin proteins directly promote axon regeneration, and what are the roles for the distinct isoforms during this process? Chen et al. present data demonstrating that this newly discovered CELF/Syntaxin pathway might control the ability of an axon to extend . NotablyFinally, the CELF2 protein is not new to the spotlight. In fact, CELF2 has already been investigated as a therapeutic target in a neuromuscular disease called spinal and bulbar muscular atrophy, and lowering CELF2 levels can improve symptoms in a mouse model of this disorder . What is


Japanese encephalitis virus (JEV) is one of approximately 70 flaviviruses, frequently causing symptoms involving the central nervous system. Mutations of its genomic RNA frequently occur during viral replication, which is believed to be a force contributing to viral evolution. Nevertheless, accumulating evidences show that some JEV strains may have actually arisen from RNA recombination between genetically different populations of the virus. We have demonstrated that RNA recombination in JEV occurs unequally in different cell types. In the present study, viral RNA fragments transfected into as well as viral RNAs synthesized in mosquito cells were shown not to be stable, especially in the early phase of infection possibly via cleavage by exoribonuclease. Such cleaved small RNA fragments may be further degraded through an RNA interference pathway triggered by viral double-stranded RNA during replication in mosquito cells, resulting in a lower frequency of RNA recombination in mosquito cells compared to that which occurs in mammalian cells. In fact, adjustment of viral RNA to an appropriately lower level in mosquito cells prevents overgrowth of the virus and is beneficial for cells to survive the infection. Our findings may also account for the slower evolution of arboviruses as reported previously. Flavivirus belonging to the family Flaviviridae [Japanese encephalitis (JE) is an important mosquito-borne viral disease, occasionally causing encephalitic symptoms . Nowadayiviridae , the geniviridae . Due to iviridae .Maintenance of genetic diversity theoretically reduces the rapid loss of fitness via Muller's ratcheting during viral passage from one host to another , which pThe viral RNA recombination was first reported in the poliovirus, a picornavirus , and subFlaviviruses naturally comprise multiple genotypes or strains , 24, makThree strains of the JEV, including Nakayama (the vaccine strain), T1P1-S1  , and CJNVirus titers were determined by means of a plaque assay of BHK-21 cells following descriptions in our previous report . CalculaTo detect viral infection in cells, extracted RNA was applied to perform RT with the reverse primer at 42°C for 30 min to generate complementary (c) DNA. PCR cycling was then carried out using the forward primer which was subsequently run to amplify a gene fragment with a size of 529 bp under the following conditions: 25 cycles of 95°C for 30 s, 60°C for 30 s, and 72°C for 1 min. The primers used to amplify specific regions are presented in individual sections below. All procedures in this portion of the study followed our previous description [Coinfection of JEV strains was verified by a method described in our previous report . In brieIn order to evaluate RNA recombination between genomic RNA and a transfected RNA sequence, the p(+)T1P1-5′3′-UTR-1 plasmid was constructed as described here. Viral RNA derived from the T1P1 strain of the JEV was used as a template to generate DNA fragments corresponding to the 5′- or 3′-end of genomic-sense RNA. To prepare the 5′-end sequence, a primer  was used for RT to synthesize the first-strand cDNA. Subsequently, another primer (5′-TAATACGACTCACTATAGAGAAGTTTATCTGTGTG-3′) containing a partial sequence of the T7 polymerase promoter used as a tag  at the 5′-end (nt 1~18) of the T1P1 5′-end sequence was used in the PCR to amplify a 934 bp DNA fragment. In the meantime, the primer 5′-GTGTTCTTCCTCACCACCAGCTAC-3′  was used for RT to generate cDNA. Another primer  was subsequently used for the PCR under conditions described above to amplify a 650 bp DNA fragment. Both types of PCR products were separately digested with the restriction enzyme, AatII; the resultant DNA fragments were ligated to form subgenomic DNA which contained both 5′-end (nt 1~599) and 3′-end  sequences. Subsequently, the subgenomic DNAs were cloned into pGEM-T  to form a plasmid designated p(+)T1P1-5′3′-UTR-I which contained an insert of a 1202 bp fragment.In order to see the stability of viral fragments in host cells, the p(+)5′3′-UTR-II plasmid was constructed. To construct the plasmid, the pT1P1-5′3′-UTR was used as a template, and the PCR was performed under conditions described above with the primers 5′-TAATACGACTCACTATAGAGAAGTTTATCTGTGTG-3′  and 5′-AAGATATCGTGTTCTTCCTCAC CACC-3′ . The PCR products were digested with SpeI and AatII to delete a fragment from nt 178~599; the resultant DNA fragments were then treated with Klenow Fragment enzyme  and ligated to form subgenomic DNA which only contained the 5′- and 3′-UTRs of the T1P1 genome. Subsequently, the subgenomic DNAs were cloned into pGEM-T  to form plasmids designated p(+)5′3′-UTR-II.μL RNase was added to cleave single-stranded RNA that failed to anneal in the mixture. The product was used to evaluate degradation of dsRNA fragments in host cells.Both (+) and (−) sense 5′-end RNA sequences were prepared from the pT1P1-5′3′-UTR-II plasmid. In preparation of the (+) sense 5′-end RNA sequence, the plasmid was linearized by NdeI and transcribed with T7 RNA polymerase using an in vitro transcription system (Fermentas). The RNA products (599 bp) were extracted with phenol-chloroform, precipitated in ethanol, and then stored in a deep freezer until used for transfection. To prepare the (−) sense 5′-end RNA sequence, the plasmid was first linearized, and the 3′-end sequence of the subgenomic DNA was deleted with NdeI. The resultant linear forms of the plasmid were religated and then redigested with SacII. The products were transcribed with T7 RNA polymerase using an in vitro transcription system (Fermentas) to generate the (−) sense 5′-end RNA sequence which was harvested as done for the (+) sense 5′-end RNA sequence. To prepare dsRNA, positive- and negative-stranded RNA described from pT1P1-5′3′-UTR-II were mixed together, incubated at 95°C for 5 min and then 4°C for 10 min. Ultimately, 2 Transfection of dsRNA or (+) sense 5′-end RNA-I prepared from the plasmids (+) pT1P1-5′3′-UTR-II was carried out in BHK-21 and C6/36 cells. At 5 h posttransfection (hpt), cells were infected with the Nakayama strain of the JEV, at an MOI of 5. The detailed procedure followed a previous description, from which efficacy of transfection was demonstrated .μg RNA, 1 μL 100 mM random hexamer primer, and 1 μL 10 mM dNTP, and double-distilled (dd) H2O water was added to bring the volume up to 12 μL. This was heated at 65°C for 5 min, allowed to stand at 4°C for 2 min, and then 4 μL 5x first-strand buffer, 2 μL dithiothreitol (DTT), 1 μL of an RNase inhibitor  were added. After incubation at room temperature for 5 min, 1 μL of reverse transcriptase M-MLV (Invitrogen) was added and allowed to react for 1 h at 37°C, followed by 15 min at 75°C. The cDNA produced was then used for the subsequent PCR under the conditions described above. The primers used for the PCR included the 5′-UTR (5′-AGAAGTTTATCTGTGTGAAC-3′) and 3′-UTR (5′-AGATCCTGTGTTCTTCC-3), which generated PCR products predicted to be 907 bp. To assess integration of dsRNA, the same cDNA and primer pair described above were used to amplify a fragment 807 bp long.Sequences derived from (+)5′3′-UTR-II RNA were transfected into cells either treated or untreated with an exoribonuclease inhibitor   and incubated for 5 h. RNA was extracted with the TRIzol reagent , and then DNAse (Promega) was added to delete interference of genomic DNA. RT was subsequently run in a mixture containing 4 BHK-21 and C6/36 cells transfected with transcribed RNA fragments were then infected by the Nakayama strain of the JEV. Total RNA extracted from cells that had been transfected with (+) sense 5′-end RNA-1 was run for RT using a primer (850-877R: 5′-TCAGTTTTCATGAGATATCGTGTGTGGC-3′) complementary to the sequence of nt 850~877. Amplification using the forward primer (RVF1: 5′-GCGGGATTTAATACGACTCACTATAG-3′) which is a partial sequence of the plasmid that serves as a tag and the reverse primer (RVR1/nt 516~538: 5′-CTGCAATATCCGTATTGTTGAC-3′) produced a specific region comprised of 564 nt. The reverse primer used here was specific for the Nakayama strain. As a result, the fragments amplified by this primer pair must represent a strain of genetic recombination.Viral replication was validated by RNA accumulation through a real-time RT-PCR with cDNAs reverse-transcribed from extracted RNA of infected (at an MOI of 1) or uninfected C6/36 and BHK-21 cells. The primer pairs TS1-F/TS1R (5′-TGTGGCTTGCGAGCTTGGCAG-3′/5′-ACATGTAGCCGACGTCGATT-3′) and CJN1-F/CJN1R (5′-TGTGGCTTGCGAGCTTGGCTA-3′/5′-ACATGTAGCCGACGTCTATC-3′) were used to amplify specific regions of the T1P1-S1 and CJN-S1 strains, respectively. Levels of 18S rRNA designed from the genome of C6/36 or BHK-21 cells were also amplified as an internal control as our previous report . ResultsYates' chi-square test was used to assess the frequency of RNA recombination in cells coinfected by two virus strains or transfected by viral RNA fragments. RsaI RFLP assay as described in our previous report. The result reveals that different strains of the JEV can coinfect a single BHK-21 or C6/36 cell. The C/preM junction comprising 868 nucleotides (nt 10~877) of viral RNA extracted from BHK-21 or C6/36 cells coinfected with the TaP1-S1 and CJN-S1 strains was cloned and used for the SmaI-Alw44I RFLP analysis  which were randomly selected and picked out from infected BHK-21 or C6/36 cells was subjected to ananalysis . The recanalysis . Probabianalysis . In otheA 564 bp fragment was significantly amplified in BHK-21 and C6/36 cells which were infected by the JEV (Nakayama strain) following transfection with the (+)5′3′-UTR-I RNA plasmid, although a light band was also shown in the control group that contained a mixture of RNAs extracted from transfected cells.A specific fragment of viral RNA (529 bp) was amplified as an internal control in all groups with viral infection. In addition, no fragment presenting an artifact of RNA recombination was shown in the control groups of mock treatment (neither infection nor transfection), transfection with only the (+)5′3′-UTR RNA-I plasmid, or infection with only a single strain. An image-density analysis revealed recombination in BHK-21 cells to be 10.7-fold higher than that of the control group, while it was 7.73-fold higher in C6/36 cells, suggesting that RNA recombination may occur in both mammalian and mosquito cells. However, a slightly lower frequency of RNA recombination was eventually shown in mosquito cells .The RNA recombination rate was shown to have increased to a higher level in BHK-21 cells treated with PAP, the inhibitor of exoribonuclease, compared to untreated cells. In contrast, no effect of PAP on increasing RNA recombination was seen in C6/36 cells; only a low level of RNA recombination was found in this test . LookingP value = 0.370; Yates' chi-square test) in coinfected C6/36 cells and even their function of exoribonuclease was inhibited and thus unable to dissolve viral RNA . The RNA recombination rate did not change significantly  was detected in C6/36 cells immediately after transfection (0 hpt) with a fragment of dsRNA derived from (+) or (−) 5′3′-UTR RNA; however, it had faded by 3 and 6 hpt . This suAppropriate accumulation of viral RNA in host cells is essential for prosperous production of progeny virions. According to the results, RNAs of both the T1P1-S1 and CJN-S1 strains accumulated more slowly in C6/36 cells than BHK-21 cells . SpecifiRNA viruses generate new genetic strains with approximately 6 orders of magnitude higher rates of nucleotide substitutions compared to DNA viruses . Thus, tCurrently, 2 possible mechanisms are reported to lead to the occurrence of recombination : a copy-In this study, we infected host cells with Nakayama strains of the JEV, followed by transfection of the (+)5′3′-UTR-I RNA fragment. The result was parallel to our previous observation , showingGenerally double-stranded replicative-form RNA (dsRF-RNA) accumulates to provide an immediate signal which activates specific transcription factors such as type-I interferon (IFN)  and faciRNA recombination creates advantageous genotypes by evolutionary jumps , which p


Indeed, 2AU-2 (50 nM) ameliorates various aspects of SCA10 pathology including improvement of mitochondrial dysfunction, reduced activation of caspase 3, and reduction of nuclear foci. These studies provide a first-in-class chemical probe to study SCA10 RNA toxicity and potentially define broadly applicable compounds targeting RNA AU base pairs in cells.RNA is an important target for chemical probes of function and lead therapeutics; however, it is difficult to target with small molecules. One approach to tackle this problem is to identify compounds that target RNA structures and utilize them to multivalently target RNA. Here we show that small molecules can be identified to selectively bind RNA base pairs by probing a library of RNA-focused small molecules. A small molecule that selectively binds AU base pairs informed design of a dimeric compound ( Expanded RNA repeats in non-coding region of a gene represent a hallmark of several diseases. Here, the authors identify two small molecules that selectively bind AU repeats and use them to design a compound that targets the pathogenic RNA associated with spinocerebellar ataxia type 10. RNA has diverse cellular functions. For example, messenger RNAs (mRNAs) encode protein, microRNAs regulate the lifetime of mRNAs, and the ribosome translates mRNAs into proteins13Because of the important cellular functions of RNA under normal conditions, it is not surprising that mutations in RNA can cause disease. Single-nucleotide polymorphisms can give rise to cryptic alternative pre-mRNA splicing sites, leading to production of aberrant, defective proteins, as is the case with β-thalassaemia6The bacterial ribosome is the most widely exploited RNA target133exp and its dimeric compound (2AU-2) displaces sequestered proteins and improves defects in patient-derived cells. The observation that base pair-binding modules can provide bioactive compounds suggests that many other RNAs can be exploited as targets of small molecules.To aid RNA-targeting endeavours, our group has developed two-dimensional combinatorial screening (2DCS) to identify optimal (high affinity and selective) RNA motif–small molecule interactions. These interactions are deposited into a database and can be used to design small molecules to target RNAs by comparing motifs in the desired target to the database. This approach has been used to design small molecules targeting the RNAs that cause DM1819By using chemical similarity searching, small molecules with features that should pre-dispose them for binding RNA23G°37 for AUAU and AAUU is −8.5 and −6.7 kcal mol−1, respectively, while for GCGC and GGCC it is −17.5 and −19.1 kcal mol−1, respectively. If only eight AU base pairs were present, then the free energy drops considerably to −3.7 and −2.5 kcal mol−1, respectively.Compounds were screened for binding to different base pairs using four model constructs with stretches of AU and GC base pairs embedded in the stem of a common hairpin loop . The fouIn initial compound screens, each small molecule was incubated with the four RNAs and the change in emission was measured . ChangesOn the basis of the substructures within compounds that bind, a Venn diagram was constructed to correlate chemotypes and binding to AU and GC base pairs . For exa1 and 2 provided the most robust and saturable change in emission and were found to bind selectively to AU base pairs ·poly(rC) RNA duplexes and that the compound interacts with approximately 4 bp, or a similar number of interacting sites that are proposed for 2  RNA (r(AUUCU)exp) that causes spinocerebellar ataxia type 10 (SCA10) contains stretches of AU base pairsexp, located within spliced intron 9 of the ataxin 10 (ATX10) mRNA, binds to and sequesters proteins involved in RNA biogenesis such as heterogeneous nuclear ribonucleoprotein K  repeats over other disease-associated RNA repeats including r(CUG)12 (DM1)12 (FXTAS and FXS)812 (DM2)1 has a similar EC50 for all RNA repeats (36–87 nM), indicating that 1 binds to RNA internal loops as well as to AU base pairs with similar binding affinity (2 selectively recognizes r(AUUCU)11 with 5–15-fold selectivity over the other repeats (2 bound r(AUUCU)11 with a Kd of 300 nM and a stoichiometry of ca. 11:1 (2:r(AUUCU)11). Because r(AUUCU)11 contains 10 AU base pairs, it indicates that 2 is binding to each AU base pair in the target. Indeed, a nuclease protection assay revealed that 2 protects each AU base pair in r(AUUCU)11 from cleavage (2 binds to r(AUUCU)11 with positive cooperativity exp simultaneously.We assessed the selectivities of compounds affinity . Compoun repeats . Compouncleavage . Interesy n=1.7; , suggestPreviously, our group has developed modularly assembled small molecules that target various repeating RNAs18193536372 and r(AUUCU)exp, a derivative of 2, AU-azide (3) was synthesized from the furan via two reaction steps as reported previously39AU-azide 11 over other RNA repeats compared with 2 was also improved  were then conjugated by using a Cu-catalysed click reaction  to gain insight into selectivity. Only a modest decrease (∼20%) of 2AU-2 binding was observed, suggesting the compound is selective.The library of dimeric compounds was then tested for binding to r(AUUCU)ng assay . After i11. It is known that DiGeorge syndrome critical region 8 (DGCR8), a protein that is involved in microRNA biogenesis, binds a wide variety of RNAs11, a gel mobility shift assay was completed, affording a dissociation constant of 1.6 μM for the RNA–protein interaction (exp-MBNL1 (muscleblind-like 1), r(CAG)exp-MBNL1, and r(CGG)exp-DGCR8 eraction . Such afCR8 refs , 21, 22.11 and DGCR8Δ analogously. Screening results showed that dimers are better inhibitors than the monomer and that 2AU-2 is the most potent inhibitor amongst them (AU-azide only inhibits ∼25% of r(AUUCU)11-DGCR8Δ complex formation while 2AU-2 inhibits ∼70%. Further, the IC50 of 2AU-2 was 2.7-fold less than that of AU-azide (2AU-2 is the most potent binder of r(AUUCU)11 and that there is considerable enhancement for the dimer relative to the monomer.Time-resolved fluorescence resonance energy transfer (TR-FRET) assays have been developed to screen for inhibitors of each RNA–protein complexes mentioned above2021gst them . At 1.5 AU-azide . Thus, b2AU-2 to r(AUUCU)11 over other RNA repeats was observed (Kd of dimer 2AU-2 to r(AUUCU)11 was measured. The number of 2AU-2 per r(AUUCU)11 was approximately half that of AU-azide , suggesting that each RNA-binding module interacts with each AU pair. The affinity of 2AU-2 for r(AUUCU)11 is twofold greater than the monomer and AU-azide and 2AU-2 maintained positively cooperative binding to r(AUUCU)11. Similar increases in affinity when increasing valency from n=1 to n=2 have been observed with other repeats192AU-2 and r(AUUCU)11 under molecular crowding conditions that mimic a cellular environment by adding 20% (w/v) PEG 8000. A ninefold enhancement in binding was observed (EC50 of 16±4 nM) relative to non-molecular crowding conditions  with similar affinity as the AU-rich RNAs 500 (ref. 500 bound to 2AU-2-Biotin was quantified by using quantitative PCR with reverse transcription (qRT–PCR). Results show that there is a significant enrichment of the r(AUUCU)500 in the pulled-down material, as compared to 18S rRNA, showing that the compound indeed recognizes that target in the presence of cellular RNAs 11 in excess of r(AUUCU)500 were added to the lysate and enrichment of r(AUUCU)500 in the pulled-down fraction was quantified. Results from this competition experiment showed that 100-fold excess of r(AUUCU)11 was required to decrease enrichment of r(AUUCU)500 in the pulled-down fraction  repeats as being a manner in which longer repeats are preferred over shorter ones. Positive cooperativity for binding to 2AU-2 was also observed in vitro thesized . After i500 ref. , bound Rlar RNAs . To furtfraction . Thus, 2We further profiled target selectivity amongst 93 highly abundant transcripts in the pulled-down fraction via qRT–PCR. The RNAs span the diverse biology in the transcriptome, including rRNAs, mRNAs, small RNAs (sRNAs) and tRNAs. The rRNAs included all four rRNA subunits  and 45S rRNA. The 50 mRNAs were chosen from the most abundant mRNAs in HeLa cells for which there are established qRT–PCR primers500, showing 2AU-2 it possesses reasonable selectivity for the desired target  by transfecting a plasmid that encodes green fluorescent protein (GFP); and (ii) co-transfecting plasmids that encode GFP and r(AUUCU)500. GFP is a commonly used gene reporter in translational studies because of its visually identifiable characteristics2AU-2, as compared with an untreated control , these data suggest that binding to tRNAs in a pull-down is not sufficient to elicit a biological effect.Only a fraction of these RNAs were enriched comparably to r(AUUCU)d target . Of the  control . Thus, 22AU-2 was assessed by measuring the ability of the compound to improve three downstream disease-associated defects in SCA10 patient-derived fibroblasts2AU-2 on the caspase-3 activity in control and SCA10 fibroblasts was measured. When SCA10-affected cells were treated with 50 and 100 nM of 2AU-2 for 48 h, caspase-3 activity was reduced to levels observed in healthy cells  diminished ca. 70% of nuclear foci in SCA10 cells 2AU-2 treatment.To confirm that improvement of SCA10-associated defects was due to binding of the compound to r(AAUCU)2AU-2 is not due to reduced abundance of the mutant ATXN10 transcript carrying expanded AUUCU repeats as determined by qRT–PCR analysis exp in SCA10 patient-derived cells, suggesting that the compound binds to the cellular target, r(AUUCU)exp, and frees sequestered proteins. 2AU-2 is the most potent inhibitor known for a traditional non-covalent binder to repeat expansions.Taken together, bis-benzamidine compound 2 is a selective AU base pair binder, as it binds AU base pairs 40-fold more strongly than GC base pairs. This compound was applied to target the pentanucleotide r(AUUCU) expansion that causes SCA10. The repeat periodically displays 5′AU/UA3′ base pair steps in its secondary structure. To improve affinity and selectivity, we modularly assembled 2 and determined that the optimal distance between RNA-binding modules was afforded by two propylamine spacing modules, or 2AU-2. 2AU-2 significantly improves SCA10-associated defects to wild-type levels when patient-derived fibroblasts are treated with 50 nM compound. It is the first bioactive small molecule targeting r(AUUCU)exp. The potent bioactivity of 2AU-2 suggests that base pair-targeting RNA modules could have broad utility to provide bioactive compounds targeting other RNAs in the transcriptome.To identify selective RNA base pair binders, we screened small molecules that have RNA-binding scaffolds. The All pH measurements were performed at room temperature using a Mettler Toledo SG2 pH metre that was standardized at pH 4.0, 7.0, and 10.0. Absorption and emission spectra were measured using SpectraMax M5 plate reader . Sigma Plot (version 11.0) was used for all curve fitting.2HPO4, pH 7.0, 185 mM NaCl, 0.1 mM EDTA)).All small modules were procured from the National Cancer Institute (NCI) or The Scripps Research Institute. Emission spectra  of all compounds were measured in a 384-well plate (Greiner Low-Volume 784076) to select fluorescent compounds for screening (50 μM compound in 1 × Screening Buffer (8 mM Na2O with 0.1% trifluoroacetic acid (TFA) over 60 min was used for compound purification. Analytical HPLC was performed using a Waters Symmetry C18 5 μm 4.6 × 150 mm column. Compounds were analysed using a gradient of 20–60% MeOH in H2O with 0.1% TFA over 30 min. All compounds evaluated had ≥95% purity as determined by analytical HPLC. Mass spectrometry was performed with an Applied Biosystems MALDI ToF/ToF Analyzer 4800 Plus and Microflex (Bruker) using an α-hydroxycinnamic acid matrix. See the Preparative HPLC was performed using a Waters 1525 Binary HPLC pump equipped with a Waters 2487 dual-absorbance detector system and a Waters Sunfire C18 OBD 5-μm 19 × 150 mm column. Absorbance was monitored at 220 and 345 nm. A gradient of 20–100% methanol in HThe RNAs used in fluorescence binding assays, nuclease mapping, and filtre-binding assays were purchased from Dharmacon. The ACE protecting groups were cleaved by using Dharmacon's deprotection buffer by incubating at 60 °C for 30 min. The samples were lyophilized, resuspended in water and gel purified. Concentrations were determined by absorbance using a Beckman Coulter DU800 ultraviolet–visible spectrophotometer at 85 °C. Extinction coefficients (at 260 nm) were calculated using the HyTher server, which uses nearest-neighbour parameters4748F/F0 where F is the fluorescence intensity in the presence of RNA and F0 is the fluorescence intensity in the absence of RNA.RNAs were folded in 1 × Screening Buffer at 95 °C for 2 min followed by slowly cooling to room temperature on the bench top. A 10-μl aliquot of a 1 μM RNA solution was dispensed into each well of a black 384-well plate (Greiner Low-Volume 784076) using an Aurora Discovery FRD-1B liquid dispenser. A 10-nl aliquot of a 2.5 mM stock of small molecule was pinned into each well using Biomek NXP Laboratory Automation Workstation that was equipped with a 384-pin head. The solution was incubated at room temperature for 30 min. Fluorescence intensity was measured using the maximum excitation/emission wavelength for each compound and the change in fluorescence was calculated by the ratio of P value of <0.05 (95% confidence) were chosen as selective binders.Compounds were scored as hits if a >20% change in emission (either enhancement or quenching) was observed upon incubation with RNA. The selectivity of a small molecule was computed by comparing the relative change in emission when incubated different RNAs. Statistically significant differences were calculated by using one-way analysis of variance function in Sigma Plot (version 11.0); compounds that had a http://tripod.nih.gov/?p=46). The functional groups that provided recognition for each type of RNA were then compared.To identify the chemical substructures that facilitate binding, hit compounds were tested by an automated R-group analysis  and serial dilutions of RNA or DNA in 1 × Screening Buffer. For molecular crowding experiments, PEG 8000 was added to a final concentration of 20% (w/v) to the folded RNA and to the solution used for serial dilutions. After a 20 min incubation, fluorescence intensity was measured. The resulting curves were fit to the following equation to determine ECy is fluorescence intensity, x is the concentration of RNA, B is the minimum fluorescence; A is the maximum fluorescence; and the EC50 is the concentration of RNA where half of the compound is bound.where Kd's: fluorescence versus [nucleic acid]/[ligand] to determine stoichiometry and fraction-bound/[nucleic acid] versus fraction bound to determine Kd's. Stoichiometries were determined from the former plots by fitting each of the two slopes (pre-saturated and saturated portions of the curves) to a lineKd's were determined by fitting fraction bound/[nucleic acid] versus fraction bound to Two types of plots were constructed to determine stoichiometries and v is the moles of RNA lattice bound per moles of ligand, [L] is the concentration of ligand, N is the number of repeating units on the RNA, l is the number of consecutive lattice units occupied by the ligand, and k is the microscopic dissociation constant.where 11 was radioactively labelled at the 5′ end with [γ-32P] ATP (Perkin Elmer) and T4 polynucleotide kinase (New England Biolabs) using standard methods and gel purified to homogeneity−1 and the samples were incubated at room temperature for 60 min. RNase V1 was then inactivated by heating at 95 °C for 1 min, and cleavage products were separated on a denaturing 20% polyacrylamide gel. A hydrolysis ladder was prepared by using Alkaline Hydrolysis Buffer (Ambion) and the manufacturer's protocol.r(AUUCU)Details of compound syntheses and characterization are provided in 11 (100 nM) was folded in 1 × PBS buffer (pH 7.4) containing 1 mM MgCl2 by incubation at 60 °C for 5 min followed by slow cooling to room temperature. BSA was added to a final concentration of 50 μg ml−1 followed by addition of 1 μM compound. The samples were incubated at room temperature for 15 min. Nitrocellulose and nylon membranes were incubated in 1 × filtre binding assay buffer (1 × PBS buffer (pH 7.4) containing 1 mM MgCl2 and 50 μg ml−1 BSA). Bound and unbound RNA were separated using a Dot-Blot apparatus followed by washing with 1 × filtre binding assay buffer. The membranes were exposed to a phosphorimager screen and imaged using a Molecular Dynamics Typhoon phosphorimager. The amount of r(AUUCU)11 bound to each membrane was quantified using QuantityOne software (BioRad).To determine the optimal distance between RNA-binding modules, a library of dimers was synthesized and screened by using a filtre-binding assay. Radioactively labelled r(AUUCU)11 was 5′-end labelled as described above and folded in 1 × Folding Buffer  by incubation at 60 °C for 5 min followed by slow cooling to room temperature on the bench top. The buffer was adjusted to 1 × TR-FRET Assay Buffer  and various concentrations of DGCR8Δ were added. The samples were incubated at room temperature for 15 min and loaded onto a pre-chilled native 5% polyacrylamide gel. The gel was imaged and quantified as described above. The resulting curves were fit to r(AUUCU)y is percentage of bounded DGCR8Δ, x is the concentration of protein, Bmax is maximum percentage of protein bound  and kd is dissociation constant.where 11 and DGCR8Δ were 60 and 40 nM, respectively. TR-FRET was measured after an additional 30-min incubation at room temperature. IC50 values were calculated by curve fitting using TR-FRET assays were completed as previously described500 using Lipofectamine 2,000 (Invitrogen) per the manufacturer's recommended protocol. Cells were collected 18–24 h post-transfection and total RNA was extracted by using Trizol reagent (Ambion) according to the manufacturer's protocol. After RQ1 DNase (Promega) treatment, the DNase was removed by phenol:chloroform extraction and total RNA was ethanol precipitated. Next, 100 μg of total RNA was folded in 1 × Screening Buffer by heating at 75 °C for 5 min and cooling to room temperature slowly. The folded RNAs were incubated with 2AU-2-Biotin for 30 min at room temperature. The solution was then incubated with streptavidin beads  for 30 min at room temperature with gentle shaking. The supernatant (containing unbound RNAs) was removed, and the beads were washed with 250 μl 1 × Screening Buffer after gentle shaking for 5 min at room temperature. Bound RNA was released from the beads by heating the beads in 300 μl H2O at 80 °C for 3 min twice. The solution containing bound RNAs was concentrated to 5–50 μl by vacuum concentration. Complementary DNA (cDNA) was generated from 40 ng of RNA using a qScript cDNA Synthesis Kit (Quanta Biosciences) per the manufacturer's protocol. qPCR was performed on an ABI 7900 HT Real-Time PCR System using the following primers to detect the r(AUUCU)500-containing RNA: 5′-AGTCTCTCTATGTTGCCCAGG-3′ and 5′-ACTTCCCGAAACACCGTCTC-3′. The relative fold enrichment of the RNA pulled by the compound was calculated by normalization to 18S rRNA.HeLa cells were maintained as monolayers in growth medium ). Cells were plated in 10-cm dishes and grown to ∼90% confluency and then transfected with a plasmid encoding (AUUCU)www.rtprimerdb.org) and qPrimerDepot (http://primerdepot.nci.nih.gov). The DNA sequences of tRNAs were obtained from tRNAdbhttp://trna.bioinf.uni-leipzig.de/) and primer sets were designed by using Primer 3 software (http://frodo.wi.mit.edu/primer3/). All sequences of primers are listed in Profiling was completed by qRT-PCR as described above. The 93 highly abundant RNAs, including rRNAs, sRNA, tRNAs and mRNAs, were selected based on a previous reportL-glutamine containing 15% fetal bovine serum and antibiotic in 5% CO2 at 37 °C in 75-cm2 flasks. Compounds were dissolved in 50% DMSO and added to the SCA10 cells at the indicated concentrations. Fresh medium with drug was added to the cells after every 12 h and cells were collected after 48 h for the caspase-3 assay, detection of AUUCU RNA foci, or for analysing the subcellular translocation of protein kinase C δ (PKCδ).SCA10 fibroblasts4 cells) were seeded in chamber slides. When cells were 80–90% confluent, fresh cell culture medium containing compound was added to the cells and incubated for 48 h. The drug-treated and control cells were then incubated with mitotracker deep red 633  at a concentration of 250 nM in cell culture medium and incubated at 37 °C for 30 min. The cells were then washed three times with ice-cold 1 × PBS, fixed with 4% paraformaldehyde for 30 min at room temperature, washed three times with 1 × PBS and stored in 70% ethanol for up to 24 h. Cells were blocked with DAKO antibody blocking solution (serum free) and later double stained with anti-PKCδ 1:500 in DAKO antibody diluent. Goat anti-mouse 488 was used to identify PKCδ. Fluorescent photomicrographs were taken using an Hamamatsu Camera Controller using DP controller software.SCA10 fibroblasts (2 × 104 cells) were seeded in chamber slides. When the cells were 60–70% confluent, cell culture medium containing compound was added to the cells and incubated for 48 h. After incubating with compound, the cells were fixed with 4% paraformaldehyde for 30 min at room temperature, and washed three to four times with ice-cold 1 × PBS. The AUUCU RNA foci were detected using a Cy3-labelled (AGAAU)10 RNA oligonucleotide probe as described previously10 Cy3-labelled RNA oligo at 45 °C. Slides were rinsed with 1 × PBS three times and extensively washed 4 × 5 min to remove all non-specific binding of Cy3-labelled RNA probes. Slides were then mounted with DAPI mounting medium and fluorescent images were taken using a confocal microscope.SCA10 and control fibroblasts  or vehicle for 24 h. Later, the culture medium containing LDH was collected and the amount of LDH in the medium was quantified using TOX7 cell toxicity assay kit (Sigma-Aldrich).SCA10 and healthy fibroblasts were treated either with Data supporting the findings of this study are available within the article and its supplementary information files and from the corresponding author upon request.How to cite this article: Yang, W.-Y. et al. Design of a bioactive small molecule that targets r(AUUCU) repeats in spinocerebellar ataxia 10. Nat. Commun. 7:11647 doi: 10.1038/ncomms11647 (2016).Supplementary Figures 1-24, Supplementary Tables 1-4 and Supplementary References


As sequencing costs are being lowered continuously, RNA-seq has gradually been adopted as the first choice for comparative transcriptome studies with bacteria. Unlike microarrays, RNA-seq can directly detect cDNA derived from mRNA transcripts at a single nucleotide resolution. Not only does this allow researchers to determine the absolute expression level of genes, but it also conveys information about transcript structure. Few automatic software tools have yet been established to investigate large-scale RNA-seq data for bacterial transcript structure analysis.Salmonella serovar Typhimurium (S. Typhimurium) 14028s were examined for potential relationships between read mapping patterns and transcript structure. We developed an empirical method, combined with statistical tests, to automatically detect key transcript features, including transcriptional start sites (TSSs), transcriptional termination sites (TTSs) and operon organization. Using our method, we obtained 2,764 TSSs and 1,467 TTSs for 1331 and 844 different genes, respectively. Identification of TSSs facilitated further discrimination of 215 putative sigma 38 regulons and 863 potential sigma 70 regulons. Combining the TSSs and TTSs with intergenic distance and co-expression information, we comprehensively annotated the operon organization in S. Typhimurium 14028s.In this study, 54 directional RNA-seq libraries from S. Typhimurium 14028s may constitute valuable resources that can be used for comparative analyses with other Salmonella serotypes.Our results show that directional RNA-seq can be used to detect transcriptional borders at an acceptable resolution of ±10-20 nucleotides. Technical limitations of the RNA-seq procedure may prevent single nucleotide resolution. The automatic transcript border detection methods, statistical models and operon organization pipeline that we have described could be widely applied to RNA-seq studies in other bacteria. Furthermore, the TSSs, TTSs, operons, promoters and unstranslated regions that we have defined for The online version of this article (doi:10.1186/s12864-015-1555-8) contains supplementary material, which is available to authorized users. Lowered sequencing costs combined with dramatic increases in data output have greatly accelerated bacterial RNA-seq based transcriptome studies. RNA-seq can not only determine the absolute gene expression levels with lower variation compared to microarray technology, but can also be used to find new genes and resolve the structure of transcripts ,2. ThereSalmonella enterica, the transcript organization has been resolved for only select groups of genes [ATG][CTA]T’ was searched in the 35 bp sequences for putative sigma 38 binding sites, and ‘[ATG]TA[AGCT][ACTG][CGTA]T’ was searched for −10 sigma 70 binding sites. A space between 10–25 nucleotides was allowed between two motifs that were used to detect the putative −35 sigma 70 binding motifs. The captured patterns were further shown for the nucleotide composition at each position with WebLogo .RNA-seq data analyzed and used to develop the methods described are available in the SRA database .