The human nuclear poly(A)-binding protein PABPN1 has been implicated in the decay of nuclear noncoding RNAs (ncRNAs). In addition, PABPN1 promotes hyperadenylation by stimulating poly(A)-polymerases (PAPα/γ), but this activity has not previously been linked to the decay of endogenous transcripts. Moreover, the mechanisms underlying target specificity have remained elusive. Here, we inactivated PAP-dependent hyperadenylation in cells by two independent mechanisms and used an RNA-seq approach to identify endogenous targets. We observed the upregulation of various ncRNAs, including snoRNA host genes, primary miRNA transcripts, and promoter upstream antisense RNAs, confirming that hyperadenylation is broadly required for the degradation of PABPN1-targets. In addition, we found that mRNAs with retained introns are susceptible to PABPN1 and PAPα/γ-mediated decay (PPD). Transcripts are targeted for degradation due to inefficient export, which is a consequence of reduced intron number or incomplete splicing. Additional investigation showed that a genetically-encoded poly(A) tail is sufficient to drive decay, suggesting that degradation occurs independently of the canonical cleavage and polyadenylation reaction. Surprisingly, treatment with transcription inhibitors uncouples polyadenylation from decay, leading to runaway hyperadenylation of nuclear decay targets. We conclude that PPD is an important mammalian nuclear RNA decay pathway for the removal of poorly spliced and nuclear-retained transcripts. Cells control gene expression by balancing the rates of RNA synthesis and decay. While the mechanisms of transcription regulation are extensively studied, the parameters that control nuclear RNA stability remain largely unknown. Previously, we and others reported that poly(A) tails may stimulate RNA decay in mammalian nuclei. This function is mediated by the concerted actions of the nuclear poly(A) binding protein PABPN1, poly(A) polymerase (PAP), and the nuclear exosome complex, a pathway we have named PABPN1 and PAP-mediated RNA decay (PPD). Because nearly all mRNAs possess a poly(A) tail, it remains unclear how PPD targets specific transcripts. Here, we inactivated PPD by two distinct mechanisms and examined global gene expression. We identified a number of potential target genes, including snoRNA host genes, promoter antisense RNAs, and mRNAs. Interestingly, target transcripts tend to be incompletely spliced or possess fewer introns than non-target transcripts, suggesting that efficient splicing allows normal mRNAs to escape decay. We suggest that PPD plays an important role in gene expression by limiting the accumulation of inefficiently processed RNAs. In addition, our results highlight the complex relationship between (pre-)mRNA splicing and nuclear RNA decay. Eukaryotic messenger RNAs (mRNAs) undergo a series of maturation events before they are exported to the cytoplasm and translated. The complexity of alternative processing increases the likelihood of mistakes that produce aberrant mRNAs encoding defective proteins. In addition, pervasive transcription occurs across nearly the entire mammalian genome resulting in the generation of nonfunctional RNAs. Consequently, cells have evolved RNA quality control (QC) pathways to eliminate these RNAs ,2.The best-characterized RNA QC pathway is nonsense-mediated mRNA decay (NMD). NMD tarnoncoding snoRNA host genes (ncSNHGs) and transcripts upstream of mRNA gene promoters . Mo. Mo40 pr40 probe . Admitte40 probe  because The two bulk poly(A) pools closely mimicked our observations with RI-RNA and mRNA isoforms. For example, the shorter population was primarily cytoplasmic whereas the hyperadenylated RNAs were nuclear . Moreove35). The processing at the MALAT1 cleavage site is efficient, with ~85% of the RNAs being cleaved by RNase P after a 2-hr transcription pulse  and observed indistinguishable decay kinetics  pathway due to an over-representation of binding sites for the Nrd1p and Nab3p proteins upstream of yeast promoters [Trf4-Air2-Mtr4 polyadenylation (TRAMP) complex then targets the NNS-terminated fragments to the nuclear exosome [The PROMPTs were the most PPD sensitive transcripts based on their fold changes upon PPD inactivation  and theiromoters ,37. The  exosome –40. In c exosome ,42. At l exosome –44. Afte exosome ,43,45,46 exosome . Three s exosome . Visual U1 snRNP is a core component of the spliceosome that recognizes 5´ splice sites, but it also suppresses the use of premature PASs ,50. ThisWe previously demonstrated that an intronless β-globin reporter is rapidly degraded by PPD, but insertion of a single intron into that reporter is sufficient to protect the resulting mRNA from PPD . ConsistWe also found that RI-RNAs are subject to PPD Figs  and 4. RTesting the half-lives of the nuclear RNAs identified herein is complicated by the unusual behavior of nuclear RNAs upon general transcription inhibition Figs  and 5. W35 is unlikely to undergo processive polyadenylation. Nevertheless, PANΔENE-A35 was degraded by PPD  polymerase, marks nuclear RNAs for decay by the exosome. While Trf4 is essential for decay, its polyadenylation activity is not necessary –55. In cd by PPD , suggestd by PPD . Thus, PIn summary, our data show that PPD modulates the levels of functional lncRNAs and mRNAs as well as presumably nonfunctional PROMPTs and the spliced byproducts of snoRNA and pri-miRNA processing. We conclude that PPD is an important nuclear RNA decay pathway that lies at the interface of transcription, splicing, 3´-end formation and mRNA export.http://www.broadinstitute.org/cancer/software/GENE-E/index.html).RNA-seq and sequencing was performed at the McDermott Center Next Generation Sequencing Core and Bioinformatics Core. Libraries were prepared using the TruSeq Stranded mRNA preparation kit and run on an Illumina HiSeq 2500 (paired-end 100 bp reads). The reads were mapped, aligned and assembled using TopHat2 and Cufflinks2.2 ,62. TranWe categorized each of the 353 high-confidence upregulated DEGs by visual assessment of IGV traces . Any DEGAll plasmids were constructed using standard molecular biology techniques. The details of the construction are given in the Expanded View. Transfections and TetRP pulse-chase assays were performed as previously described .Detection of newly made bulk poly(A) tails was performed essentially as previously described ,67.40 probe end-labeled with T4 polynucleotide kinase. Northern blots for specific transcripts were performed using standard techniques with RNA probes. Stripping and re-probing of the membranes were performed as previously described [Bulk poly(A) tails were detected on 1.8% agarose-formaldehyde gel, and detected with a dTescribed . The RNA2, 2 mM MgCl2, 0.1 mM EDTA, 10 mM Tris-HCl (pH 8.0), 1 mM DTT, 0.04 U/ml RNase Inhibitor, 0.5% Triton X-100), incubated on ice for 5 min, centrifuged at 500 x g for 3 min at 4°. RNA in the supernatant was extracted using TriReagent (Molecular Research Center) followed by an additional phenol-chloroform extraction. The pellet was then washed in Buffer I with 150 mM NaCl and once again centrifuged at 500 x g for 3 min at 4°. The resulting supernatant was discarded. The RNA from the remaining pellet was then extracted in TriReagent. We note that in cases in which we analyzed RNA from the wash step, we observed both long and short poly(A) tails; whether this is due to cross contamination of cellular compartments and/or is due to a distinct biological fraction is unclear. This fractionation procedure results in the loss of Triton X100-soluble nuclear material, but it enriches for chromatin and nuclear speckle-associated RNAs [To collect cytoplasmic RNA, cells were resuspended in Buffer I . Random hexamers were used to prime cDNA synthesis with MuLV reverse transcriptase (NEB). Real-time reactions used iTaq Universal SYBR Green Supermix (Biorad).4OAc was added to each tube, and the RNA was precipitated in 70% ethanol.Biotinylation reactions were carried out in a 200μL mixture consisting of 40μg RNA, 20mM NaOAc (pH 5.2), 1mM EDTA, 0.1% SDS, 0.2mg/mL Biotin-HPDP (Pierce), and 50% N,N-dimethylformamide (DMF) for 3 hours at 25°C. Unconjugated biotin-HPDP was removed with three chloroform extractions. After extraction of the aqueous phase, 20μL (10% v/v) of 10M NH4OAc was added to each tube, and the RNA was precipitated in 70% ethanol.Streptavidin selection was carried out using magnetic Streptavidin T1 beads (Invitrogen). Prior to use, the 20 μl bead slurry was washed three times in a 0.1X MPG solution  supplemented with 0.1% igepal. After the final wash, the beads were resuspended in a 1mL solution consisting of 0.1X MPG supplemented with 0.1% igepal, 0.1μg/μL poly(A) (Sigma-Aldrich), 0.1μg/μL ssDNA, 0.1 μg/μL cRNA, and 0.1% SDS, and blocked for one hour. RNA was precipitated, resuspended in a volume of 63μL water, and denatured at 65°C for 5 minutes. Next, RNA was incubated together with beads for one hour while nutating at room temperature. Beads were sequentially washed in: 0.1X MPG, 0.1X MPG at 55°C, 0.1X MPG, 1X MPG, 1X MPG, 0.1X MPG, 1X MPG without NaCl, 0.1X MPG. With the exception of the 55°C wash, each solution included 0.1% igepal. Biotinylated RNAs were eluted twice for 5 minutes each in a 200μL solution of 0.1X MPG containing 5% β-mercaptoethanol. The first elution step was at 25°C and the second was at 65°C. The two eluted fractions were combined and extracted with PCA once and chloroform twice. After extraction, 40μL of 10M NHNuclear run-ons were performed essentially as previously described . The detFollowing knockdown, cells were treated with 2μM of 4SU for one hour. Afterwards, cells were washed twice with phosphate buffered saline (PBS) containing calcium and magnesium (Sigma-Aldrich), and grown in media lacking 4SU for an additional hour. After the one-hour washout step, we collected 0, 30, 60, and 120 min time points. 40μg of RNA was used as input for a biotinylation and streptavidin selection as described above. Selected RNA was reverse transcribed prior to qRT-PCR analysis. β-actin was used as a loading control for qPCR analysis.The cells were given fresh media 4.5 hours prior to the 4SU treatment, which was necessary for consistent results. Cells were treated with 100 μM of 4SU for five minutes and incorporation was quickly stopped by addition of TriReagent. Sixty micrograms of total RNA was used for biotinylation and streptavidin selection as described above except one additional 1X MPG and one additional no salt wash was performed and both elution steps were done at room temperature.S1 Fig(A) Left, Quantitative western blots showing PABPN1 expression levels in the LALA stable cell line as well as knockdowns of PABPN1, UPF1, DIS3 and RRP6. LALA contains a myc-tag. Right, RNA-seq traces of PAPα and PAPγ from “total” samples; ACTB is an unaffected transcript shown for reference. As expected PAPα and PAPγ were listed among the most significant downregulated DEGs in the siPAP datasets ((B) Composite RNA profiles comparing the fold changes from all three datasets of the high-confidence PROMPTs (solid lines) or the entire genome (dotted lines) relative to the TSS. These data are from the second biological replicate whereas those in (C) Scatter plot of DEGs from each of the three datasets using GENCODE lncRNA annotations. Data were analyzed using EdgeR. The log2 fold change (FC) is relative to the matched untreated parental cell line using total RNA or nuclear RNA as appropriate. The relative expression in the control lines is plotted on the x-axis as counts per million (CPM). (D) Venn diagrams of the up-regulated lncRNA DEGs identified in each of the three samples from (C). (E) Venn diagrams of the down-regulated lncRNA DEGs identified in each of the three samples. (F) Scatter plot of the high-confidence up-regulated genes in each of the assigned categories. (G) Box and whisker plots of the expression levels of the high-confidence PPD target categories. The asterisk and ns are statistically significant differences or not significant, respectively, compared with the entire high-confidence group “DEGs (UP)” .datasets . Unfortu(EPS)Click here for additional data file.S2 FigBox and whiskers plot of the fold change in the siPAP total samples compared to the number of exons in the gene. The asterisks denote p-value <0.05 by Mann-Whitney test.(EPS)Click here for additional data file.S3 Fig(A) Cells were fractionated after knockdown with PABPN1 or control siRNAs and RNA was detected by northern blotting (top) and quantitated by Phosphorimager (bottom). The bars are the mean of three biological replicates and the error bars are standard deviation. (B) The mRNA isoforms of each of the indicated genes were quantified after detection by northern blot or qRT-PCR as in (C) NRO analysis as in n = 4). (D) Northern blot of RNAs from total (T), cytoplasmic (C) or nuclear (N) RNA using an exonic probe that recognizes mRNA and RI-RNA isoforms. Lanes 1–3 are duplicated from (EPS)Click here for additional data file.S4 Fig(A) Total cellular poly(A) tails were examined by northern blot at various time points after flavopiridol, triptolide or DRB treatments for the indicated periods of time. Molecular weight marker (kb) are shown on the left. (B) Hela cells and bone marrow derived primary mouse macrophages were treated with ActD and total cellular RNA was used for bulk poly(A) tail analysis.(EPS)Click here for additional data file.S5 Fig(A) Left, cartoon of the PANΔENE-A35 and PANΔENE-AAUAAA RNAs with the positions of the 5´ and 3´ oligonucleotide probes shown (arrows). Right, after a 2-hr pulse, RNA was analyzed by hybridizing first to a 3´ probe, which detects the RNAs not cleaved by RNase P and the same blot was subsequently stripped and re-probed with a 5´ probe that detects both cleaved and uncleaved RNAs. The relative amount of RNA not cleaved by RNase P was determined by comparison of the 3´ signal to the 5´ signal. (B) Transcription pulse chase of PANΔENE-A35 following transfection of pcDNA or LALA, as indicated.(EPS)Click here for additional data file.S6 Fig(A) Inspection of four previously published PROMPTs. In each panel the blue is the minus strand and black is the plus strand sequence. ProRBM39 is essentially undetectable in our cells indicating it is either very efficiently degraded in a PPD-independent fashion or it is not transcribed. ProEIF4ENF1 shows modest up-regulation in the LALA samples but little or no effect upon PAP knockdown. ProCCDC93 shows a weak up-regulation in all three samples. ProFOXP4 is up-regulated under all three datasets, but it was not among the high-confidence target list. (B) Sequence traces from the five mRNA genes that appear to be prematurely terminated. The controls are gray, while the siPAP total, siPAP nuclear, and LALA samples are in black, orange, and purple, respectively. Note that for APOLD1 and FAM120C, full-length RNAs are generated and these are not affected by PPD inactivation as assessed by the signal on the 3´ UTRs of these RNAs. However, the 5´end signal is enriched in all three treated lanes.(EPS)Click here for additional data file.S1 TextDescriptions of plasmid construction and NRO assay.(DOCX)Click here for additional data file.S1 TableThis spreadsheet includes all DEGs from all comparisons in this study. See “notes” sheet for a complete description.(XLSX)Click here for additional data file.S2 TableThese datasets include the upregulated and downregulated genes that were identified in all three datasets.(XLSX)Click here for additional data file.S3 TableThis dataset includes all of the DEGs identified using the lncRNA annotations from GENCODE as described in the Materials and Methods.(XLSX)Click here for additional data file.S4 Table0 refers to the size after RNase H cleavage Click here for additional data file.S5 TableThis table lists names and sequences of all primers used in this study.(XLSX)Click here for additional data file.


These ex vivo experiments identified a single bulged nucleotide (U22) and a putative K-turn (GA motif) structure required for dendritic localization or distal transport, respectively. We generated six transgenic mouse lines (three founders each) containing neuronally expressing BC1 RNA variants on a BC1 RNA knockout mouse background. In contrast to ex vivo data, we did not find indications of reduction or abolition of dendritic BC1 RNA localization in the mutants devoid of the GA motif or the bulged nucleotide. We confirmed the ex vivo data, which showed that the triloop terminal sequence had no consequence on dendritic transport. Interestingly, changing the triloop supporting structure completely abolished dendritic localization of BC1 RNA. We propose a novel RNA motif important for dendritic transport in vivo.BC1 RNA is a small brain specific non-protein coding RNA. It is transported from the cell body into dendrites where it is involved in the fine-tuning translational control. Due to its compactness and established secondary structure, BC1 RNA is an ideal model for investigating the motifs necessary for dendritic localization. Previously, microinjection of  Ala. The central domain features an unstructured Adenosine-rich region as well as a short 3′ stem loop structureshort interspersed repeat element (SINEs) termed ID elements25262728ex vivo and in vivo experiments make it necessary to probe structure-function relationships in intact organisms - in our case, transgenic miceOver the past 15 years, numerous non-protein coding RNAs (RNAs) were identified in all kingdoms of life34567911141516171019in vivo we have generated a series of BC1 RNA variants and expressed them in transgenic mice. Our previously reported BC1−/− knockout mouse line was chosen as recipient backgroundin situ hybridization with emphasis on the dendritic fields of the hippocampi. Once more, the in vivo results deviated to some degree from the previous ex vivo dataIn order to study potential RNA structures and/or motifs responsible for BC1 RNA transport BC1_UCU, TgBC1_AUC, TgBC1_AG, TgBC1_GAU, TgBC1_A and TgBC1), which expressed mutant rat BC1 RNA as well as the rat BC1 wild-type molecule. Rat and mouse BC1 RNA are virtually identical. The genetic background for all transgenic lines was FVB/N lacking the BC1 RNA gene (BC1−/−)in situ hybridization.We generated six transgenic mouse lines  we used wild-type mice and at least three founders for each transgenic mouse line. The probe was in vitro transcribed RNA complementary to the 3′ unique part of BC1 RNA . Control brain slices from BC1−/− animals showed no signal either using sense or antisense RNA probes (data not shown). The ISH on wild-type brain slices with the anti-sense probe showed a clear signal in the CA1–CA3 hippocampal areas including the corresponding dendritic fields 25–27 would not only disturb the GA motif, but also this putative pseudoknot formation. Ex vivo experiments show a significant reduction of BC1 RNA in dendrites suggesting that this mutation might be important for dendritic targetingBC1_AUC mice revealed that mutated BC1 RNA distribution in dendrites was not markedly different from wild-type RNA 1833GA motif . The U ttype RNA .48–51 had been replaced by UCU48–51 in order to remove the GA motif by generating standard Watson Crick base pairing, thus extending the canonical RNA alpha helix  and G35 → A35 (ID4 only) in the terminal stem loop domain at the penultimate base pair abutting the triloop20ex vivo studies tested two BC1 RNA variants where this penultimate base pair is altered to noncanonical G35-G41 and A35-G41, respectively. Both RNA variants were not detected in dendrites under normal conditions but only after potassium polarization35-C41 Watson Crick base pair with A35-G41 juxtaposition  is deleted, RNA transport is completely abolishedin vivo). Previously, there were conflicting ex and in vivo data concerning the ability of the 5′ stem of BC1 RNA to convey transport competence to chimeric mRNAs. Sequences identical or resembling BC1 RNA were fused to a reporter mRNA  appeared to confirm this idea1837Much is known regarding the movement of RNA between cellular compartments; this includes the transport of selected mRNAs and various other RNAs, mostly those involved in translation and its regulation from the cell bodies of neurons to their dendritic processes19in situ hybridization due to the high similarity between the rat and mouse RNA (only two nucleotide substitutions). Hence, a modified minigene was constructed replacing 20 A-residues adjacent to the 5′ stem . A major difference was that the minigene was expressed at significantly lower levels. In general, we observed that transgenes expressed BC1 RNA variants at levels that were comparable to wild-type but some expression was reduced to as low as 25% (see in vivo expression levels sufficient for in situ hybridization could not be achieved in a U22 deletion construct because the RNA polymerase III A-box promoter consensus sequence was affected (53 and C54). While RNA expression was now sufficient, it only reached about 50% of wild-type levels. The two other constructs with lower expression (TgBC1_GAU and TgBC1_AG) featured nucleotide changes that neither affect box A nor box B (for promoter boxes see Pilot experiments showed that a rat BC1 gene fragment including flanking regulatory regions (~1.4 kb) could be specifically expressed in mouse brains. Although on Northern blots we could differentiate between the transgenic and endogenous RNAs with a short specific antisense oligodeoxynucleotide (not shown), we could not hope to achieve discrimination for  5′ stem  with the 25% see . Importaaffected 21. To avBC1_AUC, we changed the sequence of the 5′ section of the loop, thus in all likelihood eliminating the GA motif (BC1_UCU) was to completely replace the apical loop harboring the GA motif by replacing all unpaired as well as noncanonical pairs with bases that form canonical Watson-Crick pairs, thus lengthening the apical stem by three base pairs to altogether fourteen pairs  or slight variants thereof, as represented by some ID repetitive SINE elements (subclass ID1 and ID2) impart dendritic transport behavior on the reporter mRNAin situ hybridization signal in dendritic fields of the hippocampus. This is consistent with ex vivo experiments using the corresponding unfused BC1 RNA (A35⋅G41)The final construct tested, was perhaps the most intriguing. In in vivo study of dendritic transport, it appears that the two structural elements implied in the past, namely the apical GA motif and the single bulge, which have been shown ex vivo to alter the intracellular localization of BC1 RNA18BC1_GAU variant, the actual three-base sequence of the loop is apparently irrelevant. This is further supported by phylogenetic information: in squirrel (Sciurus carolinensis) the unpaired loop sequence is altered from CUA → CUUFrom our in vivo analysis of different BC1 RNA structures influencing dendritic targeting was an important contribution to our understanding of structure/function relationships for RNA transport.Despite the fact that transgenic mouse experiments are time consuming, animal welfare restricted and expensive, we felt that SacI-BamHI 1.4 kb DNA fragment containing the wild-type rat BC1 RNA gene together with 5′ and 3′ flanking regions from the pBC1 plasmidEcoRV-BglII sites of the pT2BH. The resulting plasmid pT2BH_BC1 was used for further mutagenesis. Transgenic mice were generated through regular pronuclei injection in FVB/N BC1−/− embryos as described previously31BC1_AUC and the TgBC1_GAU mutations were generated in the pT2BH_BC1 using pairs of oligonucleotides UAG_AUC_DIR/UAG_AUC_REV and CUA_GAU_DIR/CUA_GAU_REV respectively .For transgenic mouse generation we used the Sleeping Beauty transposase system in vector pT2BHectively . The TgBGC-AGrsp . MutagenEcoRV restriction enzyme (Fermentas) and separated on a 0.8% agarose gel. Following electrophoresis, the DNA was transferred by capillary blotting onto a GeneScreen hybridization membrane (Perkin Elmer). BC1 transgenic probe 800 bp DNA fragment downstream from an EcoRV site located in the 3′ flank of the BC1 RNA gene was labeled with [γ-32P]-dCTP (PerkinElmer) and high prime DNA labeling Kit (Roche).We performed tail biopsies from three-week-old transgenic mice. The DNA was isolated via the phenol/chloroform extraction method. The DNA was digested with 32P]-ATP (PerkinElmer) and T4 polynucleotide kinase (Fermentas) according to the manufacturer’s instructions. Northern blot analysis was performed as previously describedTotal brain RNA was isolated using the Trizol reagent according to the manufacturer’s manual. Ten μg of total RNA was separated on 8% (w/v) polyacrylamide [29:1 acrylamide/bisacrylamide], 7M urea gel and electro-transferred onto a positively charged nylon membrane (Roche). Hybridization probe  into 30 μm coronal slices. Hybridization was performed as previously describedSacI and transcribed by T3 RNA polymerase for the sense probe. The antisense probe was generated by T7 RNA polymerase from pMK1 linearized with Asp718I. In both cases, in vitro transcription was performed in the presence of α-S35UTP. RNA polymerases and restriction enzymes were purchased from Thermo Scientific. pMK1 sense probe: 5′-gggaacaaaagcuggguaccAAAAAAGACAAAAUAACAAAAAGACCAAAAAAAAACAAGGUAACUGGCACACACAACCUUUUg-3′; pMK1 antisense probe: 5′-gggcgaauuggagcucAAAAGGUUGUGUGUGCCAGUUACCUUGUUUUUUUUUGGUCUUUUUGUUAUUUUGUCUUUUUUg-3′. Lower case letters represent transcribed vector sequences.Mice (aged 8 to 24 weeks) were sacrificed with CO6 cpm and incubated in hybridization buffer at 50 °C overnight.For hybridization, we used RNA with 4.5 × 10The slices were washed once with 2x SSC and deionized formamide at a ratio of 1:1 at 50 °C and once with 2x SSC at the same temperature. Subsequently, we treated the slices with 30 μg/ml RNase A at 40 °C and washed again in 5 liters 2x SSC at 50 °C for 3 h and continued washing in the same buffer at room temperature overnight.The slices were mounted onto Superfrost Gold Plus (Menzel) microscopy slides, dried with increasing concentrations of 50%, 70% and 90% ethanol and exposed to autoradiography films (Kodak Biomax MR) overnight.After developing the films the slides were dipped into autoradiography emulsion (Kodak NTB) as previously describedAll mouse procedures were performed in compliance with the guidelines for the welfare of experimental animals issued by the Federal Government of Germany and approved by the State Agency for Nature, Environment and Consumer Protection North Rhine-Westphalia How to cite this article: Robeck, T. et al. BC1 RNA motifs required for dendritic transport in vivo. Sci. Rep.6, 28300; doi: 10.1038/srep28300 (2016).


The RNA world is a very likely interim stage of the evolution after the first replicators and before the advent of the genetic code and translated proteins. Ribozymes are known to be able to catalyze many reaction types, including cofactor-aided metabolic transformations. In a metabolically complex RNA world, early division of labor between genes and enzymes could have evolved, where the ribozymes would have been transcribed from the genes more often than the other way round, benefiting the encapsulating cells through this dosage effect. Here we show, by computer simulations of protocells harboring unlinked RNA replicators, that the origin of replicational asymmetry producing more ribozymes from a gene template than gene strands from a ribozyme template is feasible and robust. Enzymatic activities of the two modeled ribozymes are in trade-off with their replication rates, and the relative replication rates compared to those of complementary strands are evolvable traits of the ribozymes. The degree of trade-off is shown to have the strongest effect in favor of the division of labor. Although some asymmetry between gene and enzymatic strands could have evolved even in earlier, surface-bound systems, the shown mechanism in protocells seems inevitable and under strong positive selection. This could have preadapted the genetic system for transcription after the subsequent origin of chromosomes and DNA. The RNA world refers to the stage of early evolution when RNA macromolecules were responsible both for storing hereditary information and performing enzymatic activities. Conflict arises between these two functions, however, as enzymatic activities of the ribozymes are in tradeoff with their replication rates. Here we address this problem by investigating the evolutionary emergence of a primordial transcription-like system in model protocells inhabited by unlinked replicators. Our numerical analysis demonstrates that division of labor between genes and enzymes could have emerged, given that there was a moderate to strong tradeoff between the enzymatic and template efficiency of one strand of the ribozymes. This division of labor results in a strong asymmetry in the numbers of the enzymatic and genetic strands of the macromolecules, in favor of the former. We offer insight into the emergence of the first transcription-like system, which is today characteristic of all known life forms. It is not inconceivable that complementary strands of RNAs can act as enzymes: Sergei Rodin has convincingly argued that this could have been the case for at least some tRNA As it is likely that some surface-bound metabolic complexity preceded the advent of protocells  one of the tasks while the other do the other task. In our case this means that one strand acts mostly as an enzyme, while the other acts mostly as an information carrier. As only one of the strands in our model has enzymatic activity, that strand can be called an enzyme. Both of the strands need to act as templates, otherwise information is lost, but as one of the strands mainly acts as template and does not have enzymatic activity, we can call this a gene.Ropalidia marginata, even the queen cannot be distinguished from others except for the presence of well-developed ovaries We would like to note here, that division of labor does not require sharp, and full specialization in different tasks: intermediate degree of specialization with the interchangeability of task-performing entities suffices too Chemical difference between the enzymes and the templates is not a requirement for division of labor between genetic and enzymatic functions. The present neat chemical distinction between genes (DNA) and enzymes (proteins) is a rather late invention. Comparative analysis of the genes involved in DNA replication We investigated the evolution of division labor between enzymatic and genetic strands based on the implicit assumption that minus and plus strands can have very different secondary structures. This indeed proves to be the case: on a sample of 10 million sequences, the distances between the secondary structures of minus and plus strands are slightly higher than those between pairs of randomly generated sequences . Furtherβ replicase replicates the X-motif ribozyme The origin of basic genetic operations, including replication and transcription, belongs to the key questions of the origin of life. While there has been considerable progress with template copying An RNA molecule The ribozymes catalyze reactions with metabolic activity ions see  we also Mutation can occur with probability aits see . In the Reactions involving the macromolecules The full replication cycle is completed after two steps of copying: We apply the Gillespie algorithm Degradation is a monomolecular reaction; its probability is proportional to the present amount of the given molecules. The chemical constants We define the sum of all The time At time The population is composed of 7 random RNA sequences of length 50. We have also determined the minimum free energy (MFE) structure of 107 random complementary pairs of RNAs of length 50. Each individual sequence's structure is compared to the structure of the next sequence to obtain the full tree edit distance We have assumed that complementary strands can be quite dissimilar in structure, so that one of them can fold to be a ribozyme while the other has a structure that can be more readily processed by the replicase enzyme. We check if complementary strands can be dissimilar enough to potentially achieve such a state. We have determined the minimum free energy structure of 10We analyzed the set of 305 ribozyme and aptamer sequences mainly from the Aptamer Database


Relative abundance profiles of bacterial populations measured by sequencing DNA or RNA of marker genes can widely differ. These differences, made apparent when calculating ribosomal RNA:DNA ratios, have been interpreted as variable activities of bacterial populations. However, inconsistent correlations between ribosomal RNA:DNA ratios and metabolic activity or growth rates have led to a more conservative interpretation of this metric as the cellular protein synthesis potential (PSP). Little is known, particularly in freshwater systems, about how PSP varies for specific taxa across temporal and spatial environmental gradients and how conserved PSP is across bacterial phylogeny. Here, we generated 16S rRNA gene sequencing data using simultaneously extracted DNA and RNA from fractionated (free-living and particulate) water samples taken seasonally along a eutrophic freshwater estuary to oligotrophic pelagic transect in Lake Michigan. In contrast to previous reports, we observed frequent clustering of DNA and RNA data from the same sample. Analysis of the overlap in taxa detected at the RNA and DNA level indicated that microbial dormancy may be more common in the estuary, the particulate fraction, and during the stratified period. Across spatiotemporal gradients, PSP was often conserved at the phylum and class levels. PSPs for specific taxa were more similar across habitats in spring than in summer and fall. This was most notable for PSPs of the same taxa when located in the free-living or particulate fractions, but also when contrasting surface to deep, and estuary to Lake Michigan communities. Our results show that community composition assessed by RNA and DNA measurements are more similar than previously assumed in freshwater systems. However, the similarity between RNA and DNA measurements and taxa-specific PSPs that drive community-level similarities are conditional on spatiotemporal factors. High-throughput sequencing of conserved marker genes, particularly the small subunit ribosomal RNA gene, allows for rapid and affordable censuses of microbial life , a eutrophic drowned river mouth freshwater estuary  and the SILVA release 119 taxonomy as previously described . Mothur output files, classification files and procedure are available at https://github.com/DenefLab/Frontiers2016Denef and the procedure is also included as an R markdown document   to test if season, filter fraction, lake, station, depth, and day/night could significantly explain variation in the bacterial community composition. We also performed a partial Mantel test  to determine whether environmental factors explained additional variation in RNA level Bray-Curtis dissimilarities after DNA-level variation had been taken into account.To determine differences in bacterial community composition, we merged replicate samples by averaging, rounding down, and scaling each OTU read count to the smallest merged library size   as well as for the top 200 most abundant OTUs. To test for significant differences in OTU overlap based on season, sampling location, filter fraction, or depth, we performed a Kruskall-Wallis test  ordination using the pcoa function (vegan). Similarly, we calculated the Bray-Curtis dissimilarity among RNA samples and among DNA samples and visualized these in a PCoA ordination. A Kruskall-Wallis test was performed as described above to compare whether sample dissimilarity in ribosomal RNA:DNA ratios between PA and FL, Muskegon Lake and Lake Michigan, and surface and deep water was a function of season. We performed a PERMANOVA analysis as described above to determine if season, filter fraction, lake, station, and day/night could significantly explain variation in log2 ribosomal RNA:DNA ratios between samples. Finally, we performed a Procrustes analysis (procrustes and protest functions in vegan) to determine whether the ordination patterns were significantly different between DNA, RNA, and RNA:DNA ratio data.We calculated the Euclidean distance between samples based on the logP > 0.10), but did have significant differences in ribosomal RNA:DNA ratio between summer and fall Lake Michigan PA and FL fractions .To identify the taxa that were drivers of community-level similarities in ribosomal RNA:DNA ratios between FL and PA fraction in spring, and differences in summer and fall, we identified OTUs that did not have differences in ribosomal RNA:DNA ratio between spring Lake Michigan PA and FL fractions . However, the correlation was weaker relative to the correlation between the RNA level Bray-Curtis dissimilarity and the same environmental factors without accounting for the patterns already observed at the DNA level . When repeating the latter analysis one environmental factor at a time, only “Season” and “Lake” resulted in significant partial Mantel test statistics (p < 0.05).Groups of samples clustered primarily by filter fraction [free-living (FL) vs. particulate (PA)], time, and station [particularly Lake Michigan (M15 and M110) vs. Muskegon Lake estuary (MLB) samples; Figure We did not observe a difference in the proportion of OTUs that were identified by both DNA and RNA assays between seasons, filter fractions, stations, or depths Figure . HoweverActinobacteria and to a lesser extent particulate fraction Planctomycetes contributed significantly less to the community RNA pool than to the DNA pool , free-living Betaproteobacteria, and most Cyanobacteria tended to contribute more to the community RNA pool than to the DNA pool. Particulate fraction Betaproteobacteria, and several Bacteroidetes classes showed either limited differential representation between observed RNA and DNA community composition, or displayed a mixed signal . The most over- and/or underrepresented OTUs in both fractions were listed in Table Among abundantly represented phyla, l Figure . Alphaprl Figure . As Actip = 0.001). Most noticeably, while the RNA and DNA spring samples clustered separately from the summer and fall samples and PA and FL communities formed separate clusters, both PA and FL spring RNA:DNA ratio data clustered together with FL Lake Michigan summer and fall communities. In an alternate approach to analyze these patterns, we observed that the distance between community-level RNA:DNA ratios in PA relative to FL fractions, in Lake Michigan relative to Muskegon Lake, and in surface relative to deep water was generally lower in spring relative to summer and fall . In spring, we observed higher between-habitat similarities in RNA:DNA ratios than in summer and fall. Finally, we contribute to the expanding work on analyzing the phylogenetic conservation of traits by revealing how PSP, as measured by ribosomal RNA:DNA ratios, is conserved for some phyla, most likely as a function of cell size.Our observation that sample community composition varies more across spatiotemporal gradients than between RNA and DNA measurements opposes previous analyses by Jones and Lennon . They shIn Muskegon Lake there was less overlap between OTUs observed in the RNA and DNA measurements than between replicate DNA or replicate RNA samples. This result may be explained by higher bacterial richness in urban estuaries due to urban runoff and influx of riverine and soil bacteria . This allowed us to evaluate the impact of stochastic effects associated with sampling and sequencing on OTU overlap. Subsequently, we contrasted DNA and RNA OTU overlap to replicate sample overlap. This allowed us to identify significant impacts of habitat on DNA and RNA sample OTU overlap that we could not have made without the availability of replicate measurements. We also reduced the impacts of stochasticity by averaging OTU counts from replicates and scaling the read depth to the smallest merged library size to remove the impact of many rare and spurious OTUs . Similarly, RNA:DNA ratios in spring for the offshore FL samples resembles all other spring RNA:DNA ratios, despite the higher similarity of DNA and RNA community composition to lake Michigan samples taken at other times of the year Figures , 5. The While we cannot confidently infer bacterial contributions to ecosystem processes from ribosomal RNA:DNA ratios, our analyses identified several intriguing patterns that deserve follow-up. In particular, the seasonal changes in habitat-dependent ribosomal RNA:DNA ratios pose the question: to what extent are bacterially mediated ecosystem processes more similar across all habitats in spring? This is particularly pertinent for communities occupying the particulate and free-living fractions of freshwater lakes, as many studies in recent years have described large differences in community composition across these habitats. Future studies will need to extend our findings beyond the marker gene level, either through activity assays or by characterization of the transcriptome of the populations driving the seasonal shifts in ribosomal RNA:DNA ratios observed here.VD designed the study, analyzed data, and was the primary author of the manuscript; MF analyzed data and contributed to the writing of the paper; MB and MS provided new analytical tools, analyzed data, and contributed to the writing of the manuscript.VD was supported by the Community Sequencing Program  and funds from the Fred A. and Barbara M. Erb Family Foundation and the University of Michigan Water Center. MS was supported by the National Science Foundation Graduate Research Fellowship (GRFP) program.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.


Once administered and internalized by host cells, the mRNA transcripts are translated directly in the cytoplasm and then the resulting antigens are presented to antigen presenting cells to stimulate an immune response. Alternatively, dendritic cells can be loaded with either tumor associated antigen mRNA or total tumor RNA and delivered to the host to elicit a specific immune response. In this review, we will explain why RNA vaccines represent an attractive platform for cancer immunotherapy, discuss modifications to RNA structure that have been developed to optimize mRNA vaccine stability and translational efficiency, and describe strategies for nonviral delivery of mRNA vaccines, highlighting key preclinical and clinical data related to cancer immunotherapy.RNA vaccines traditionally consist of messenger RNA synthesized by Cancer immunotherapy seeks to stimulate a host antitumor immune response, leading to tumor shrinkage and improved clinical outcomes in patients. In recent years, this field has exploded with the development of many different classes of agents aimed at enhancing immune responses against tumors. These include cytokines, immune checkpoint inhibitors, adoptive T cell therapies, and numerous vaccine strategies –3. SeverCompared to some other types of cancer immunotherapy, vaccines can be more cumbersome to produce and, for the most part, have shown more modest clinical responses in patients –12. Howe in vitro transcription (IVT) using a bacteriophage RNA polymerase and template DNA that encodes the antigen(s) of interest. Once administered and internalized by host cells, the mRNA transcripts are translated directly in the cytoplasm and then, like DNA vaccines, the resulting antigens are presented to APC to stimulate an immune response [By definition, nucleic acid vaccines are vaccines containing antigens encoded by either DNA or RNA. More specifically, DNA vaccines consist of antigen-encoding gene(s) inserted into a bacterial plasmid under the control of a eukaryotic promoter. The DNA plasmid is administered to the host and internalized by host cells, where it is transcribed in the nucleus and translated in the cytoplasm by host cellular machinery. The resulting proteins are processed into peptides, which are ultimately presented on the surface of host antigen-presenting cells (APC) in the context of major histocompatibility complex (MHC) molecules. This can occur by APC being directly transfected with the DNA or by cross-presentation from non-APC to APC. The peptide-MHC complex is recognized by antigen-specific T cells, resulting in a cellular host immune response . Alternaresponse . It is iresponse , 16. Addresponse .The nucleic acid vaccine platform is appealing because it allows easy delivery of multiple antigens with one immunization and induces both humoral and cellular immune responses, which makes tumor escape less likely. Additionally, unlike peptide-based vaccines, nucleic acid-based vaccines do not require prior knowledge and are not restricted by the patient's HLA type. Finally, like other vaccine types, nucleic acid-based vaccines have proven to be safe and tolerable , 18–20.Despite the promising features of DNA vaccines, in general, they have been found to elicit less of an immune response than other types of vaccines, including peptide vaccines, cellular vaccines, viral vector vaccines, and RNA vaccines. The reasons for this are not completely clear, but possible explanations include inefficient delivery of DNA into human cells, the need for DNA to cross both cell and nuclear membranes and be transcribed in the nucleus in order to transfect a cell, low expression of DNA-sensing machinery, and differing expression of nucleic acid sensing pattern recognition receptors , 21. The in vitro transcription, RNA can also be isolated from a limited tumor sample and amplified using techniques such as polymerase chain reaction (PCR), yielding large amounts of patient-specific antigens [RNA vaccines are attractive because they retain the same appealing characteristics as DNA vaccines but also offer some additional benefits. Unlike DNA, RNA only needs to gain entry into the cytoplasm, where translation occurs, in order to transfect a cell. Moreover, RNA cannot integrate into the genome and therefore has no oncogenic potential. In addition toantigens , 14. Finantigens . For the7G) cap and a 3′ poly(A) tail. The m7G cap, poly(A) tail, and UTR are all critical for mRNA stability and translation [Eukaryotic mRNA is composed of a coding region flanked by 5′ and 3′ untranslated regions (UTR), as well as a 5′ 7-methylguanosine triphosphate (mnslation –25. A ke5′ m7G Cap. A 7-methylguanosine triphosphate (m7G) cap is added to the 5′ end of almost all eukaryotic mRNA transcripts during transcription. The cap protects the 5′ end of the mRNA transcript from 5′ to 3′ exonucleases [ucleases  and is rucleases , thus plCapping of IVT mRNA transcripts can be achieved using cap analogues. However, it has been shown that cap analogues are often incorporated in the reverse orientation with the methylated G proximal to the RNA , resultiAdding a cap analogue, conventional or ARCA, during IVT is not 100% efficient; thus a portion of the resulting transcripts is not capped at all. These uncapped RNAs are not efficiently translated. A method of adding a cap structure posttranscriptionally is gaining favorability among those developing RNA-based therapies.Poly(A) Tail. A poly(A) tail is added to the 3′ end of the majority of eukaryotic mRNA transcripts during transcription. The poly(A) tail regulates mRNA stability and translation synergistically with the m7G cap by binding poly(A) binding protein (PABP) [7G cap and eIF4E [n (PABP) , which ind eIF4E .There are two ways to add a poly(A) tail to IVT mRNA: (1) encoding the poly(A) tail on the DNA template from which the IVT mRNA is transcribed or (2) using recombinant poly(A) polymerase to extend the IVT mRNA after transcription. In contrast to enzymatic polyadenylation with recombinant poly(A) polymerase, which yields mRNA transcripts with poly(A) tails of varying lengths, mRNA transcribed from a DNA template yields transcripts with a defined poly(A) tail length and is therefore preferred . Studies5′ and 3′ Untranslated Regions. Eukaryotic mRNA transcripts include 5′ and 3′ untranslated regions (UTR), which contain important regulatory elements.IVT mRNA can be optimized by incorporating 5′ and 3′ UTR known to enhance RNA stability and translational efficiency. The most well recognized examples of such UTR in IVT mRNA are the alpha- and beta-globin mRNAs. Beta-globin 5′ and 3′ UTR improve translational efficiency, and alpha-globin 3′ UTR stabilize mRNA –39. ThesChemically Modified Nucleosides. Finally, IVT mRNA can be created by incorporating chemically modified nucleosides, which are known to reduce immunogenicity. Natural nucleosides are added to mammalian RNA during posttranslational RNA processing in eukaryotes [karyotes . This haIVT mRNA containing modified nucleosides such as pseudouridine possesses increased stability and translation , 45. Thi7G cap, poly(A) tail, 5′ and 3′ UTR, and nucleosides are fundamental to optimize the stability and translational efficiency of all IVT mRNA for all RNA vaccines. Once an IVT mRNA transcript has been generated, it must be administered and ultimately must reach the cytoplasm of target cells. In general, nonviral delivery methods are preferred over viral vectors for their low cost, ease of large-scale production, and potential for improved safety [The previously described modifications to the 5′ md safety , 49. We  in vivo expression of those reporter genes in mice [The initial data for the use of mRNA as a vaccine platform emerged 25 years ago when Wolff et al. demonstrated that intramuscular injection of mRNA coding for reporter genes induced in mice . Five ye in mice . Both of in mice , 52–56.  in mice , 58. MorGene Gun. The gene gun is an alternative delivery method, in which IVT mRNA is injected directly into the target cell cytoplasm, thereby limiting exposure to extracellular exonucleases that might degrade it. More specifically, in the gene gun technique, IVT mRNA is coated onto gold particles, which are then accelerated toward a stopping plate by a pressurized helium pulse. The gold particles penetrate into the cytoplasm of target cells, serving as carriers for the mRNA [the mRNA , 60. Thethe mRNA . HoweverProtamine Condensation. Another method to improve the stability of naked IVT mRNA is to condense it in order to provide protection from RNA degradation. This can be accomplished by incubating IVT mRNA with protamine, a small arginine-rich polycationic protein, normally involved in DNA condensation [ in vivo in both mice and humans. For example, the injection of protamine-protected naked mRNA into mice stimulated production of antigen-specific IgG antibodies as well as activation of a specific cytotoxic T lymphocyte response and effective lysis of target cells [ensation . Protamiensation –63. Protet cells . In a phet cells .An adjuvant can be broadly defined as a component that is added to a vaccine to enhance the immunogenicity of the vaccine. Naked IVT mRNA possesses inherent self-adjuvanticity. Additionally, several other molecules, including protamine, as described previously, poly I:C RNA, and CpG containing motifs can be combined with naked IVT mRNA to augment an mRNA-based vaccine's ability to induce an adaptive immune response. However, even with the addition of an adjuvant, a vaccine may not be potent enough to overcome the powerful immunosuppressive effects of tumors. To address this issue, mRNA encoding costimulatory molecules, such as CD40L, CD70, OX40L, GITR, and CD83, can be incorporated into mRNA-based vaccines to further boost their immunogenicity –67. in vivo cytotoxicity. Recent studies have shown that encapsulating mRNA in nanoparticles protects the mRNA from nuclease degradation and enhances cell uptake and delivery efficiency [ in vivo and eliciting an antitumor immune response [Despite the previously described strategies to improve the stability of naked IVT mRNA, RNA degradation remains a significant concern with naked RNA vaccines. Because of this, carriers have been developed to encapsulate IVT mRNA, thereby protecting it from degradation and improving vaccine delivery . To dateficiency . Moreoveficiency . Preclinresponse , 75, 76.mRNA transfected dendritic cell (DC) vaccines represent a distinct type of vaccine strategy involving RNA. DCs are professional antigen presenting cells that play an essential role in bridging innate and adaptive immune responses. When used as a vaccination platform, dendritic cells (DCs) are transfected with mRNA encoding a desired tumor antigen and then delivered to the host in order to elicit an immune response against the antigen of interest. DCs can be transfected with tumor associated antigen (TAA) mRNA or total tumor RNA. Both methodologies have their advantages and drawbacks.Tumor Associated Antigens. Vaccine strategies utilizing DCs transfected with defined TAA mRNA circumvent the need for growth of patient specific tumor cells and/or isolation of patient specific tumor antigen [ antigen –79. MoreSeveral studies to date have utilized TAA mRNA-loaded DCs to stimulated antitumor responses –84. In aTotal Tumor RNA. An alternative to the use of TAA to generate an anticancer immune response is the use of patient derived total tumor RNA [umor RNA –93. Thisumor RNA .Clinical studies utilizing total tumor RNA vaccination strategies have been tested in several tumor models including brain cancer, lung adenocarcinoma, melanoma, renal cell carcinoma, and ovarian cancer –100. In Of note, loading DCs with total tumor RNA can lead to expression of self-proteins and induction of autoimmunity. However, this has not been an issue in the studies conducted thus far.Costimulatory Molecules and Checkpoint Inhibitors as Part of mRNA Transfected DC Vaccine Strategies. The benefit of DC based vaccinations has been limited primarily due to the presence of regulatory T cells as well as upregulation of checkpoint molecules. Recent studies have focused on new strategies to enhance the efficacy of RNA transfected DC vaccines. Cotransfection of DCs with mRNA encoding OX40-ligand in addition to tumor antigen improves mRNA-DC vaccine efficacy in preclinical models [l models . Moreovel models , 103.In addition, studies where expression of checkpoint inhibitors, such as PD-1, is silenced utilizing siRNA have shown improved vaccine efficacy , 105. OtAntigen-specific immune responses have been achieved in animal models and in humans by administration of RNA vaccines via various routes, including intramuscular, intradermal, subcutaneous, intravenous, intrasplenic, intranodal, intratumoral, and intranasal methods. These strategies each have their own advantages and disadvantages. However, they have not all been compared directly, and therefore the optimal route of RNA vaccine delivery is not known. For a more detailed discussion of RNA vaccine routes of administration, please refer to the following review papers: intramuscular , 52; intThe field of cancer immunotherapy has undergone many changes in recent years, and immunotherapy has now been clinically validated as an effective way to treat many types of malignancies –3. RNA vMoving forward, combining RNA vaccines with other therapies that have distinct mechanisms of action could potentially result in better treatment outcomes. For example, the vaccination methods described above could be used simultaneously or sequentially with immune checkpoint inhibitors such as anti-CTLA-4, anti-PD-1, and anti-PD-L1 . In suchMoreover, in this review we discuss the importance of RNA strategies to directly block immune checkpoints and engineer the tumor microenvironment, similarly to antibodies against immune checkpoints. These strategies would perhaps limit the toxicities and side effects associated with systemic delivery of antibodies against immune checkpoints.Further studies are needed to specifically investigate combinations of RNA vaccines with other immunotherapies as well as targeted and cytotoxic agents, with the overall goal of improving clinical outcomes and cancer care.


Bunyaviridae is a family of arboviruses including both plant- and vertebrate-infecting representatives. The Tospovirus genus accommodates plant-infecting bunyaviruses, which not only replicate in their plant host, but also in their insect thrips vector during persistent propagative transmission. For this reason, they are generally assumed to encounter antiviral RNA silencing in plants and insects. Here we present an overview on how tospovirus nonstructural NSs protein counteracts antiviral RNA silencing in plants and what is known so far in insects. Like tospoviruses, members of the related vertebrate-infecting bunyaviruses classified in the genera Orthobunyavirus, Hantavirus and Phlebovirus also code for a NSs protein. However, for none of them RNA silencing suppressor activity has been unambiguously demonstrated in neither vertebrate host nor arthropod vector. The second part of this review will briefly describe the role of these NSs proteins in modulation of innate immune responses in mammals and elaborate on a hypothetical scenario to explain if and how NSs proteins from vertebrate-infecting bunyaviruses affect RNA silencing. If so, why this discovery has been hampered so far.The  Bunyaviridae, with more than 350 identified species, is divided in five genera and contains several important viruses that cause major problems in human/animal health and agriculture production systems. All five genera of this family contain viruses pathogenic to either animals/humans  or plants (Tospovirus). Most bunyaviruses are arthropod-borne viruses (arboviruses), as they replicate in the arthropods by which they are transmitted RNA genome that is encapsidated by a nucleocapsid (N) protein and small amounts of the viral RNA-dependent RNA polymerase . The bunyavirus RNA genome is tripartite and segments have either a negative or ambisense polarity  activity . Du. Du18]. A strategy used by many RSS is the binding and sequestration of dsRNA. Binding to dsRNA can be limited by the size of the molecule and some RSS proteins are able to bind only siRNAs. Size-selective binding to siRNAs has been shown for, e.g., P19 , P21 , γB  and HC-Pro  ,48 Figu, Table 1Crinivirus), which contains endonuclease activity and cleaves siRNAs into inactive smaller products which are unable to activate RISC [Another strategy of interference involves the inhibition of HEN1-dependent methylation of siRNAs (in plants and flies this methylation is necessary for stabilization of siRNA). Interference with methylation has been shown for HC-Pro (TEV) and P19 (CIRV) . A more ate RISC ,46. Seveate RISC . Some RSate RISC . Anotherate RISC . While mate RISC .The amplification phase of RNA silencing is also targeted by several RSS. Interaction of V2 (TYLCV) with SGS3 (a cofactor of RDR6) was demonstrated to be essential for its RSS activity . NucleorCaenorhabditis elegans and plants, one of the first evidence of natural antiviral silencing in insect species came in 2002 during studies on flock house virus (FHV) infection of Drosophila melanogaster cells, in which FHV B2 protein was identified as a RSS [Drosophila Dicer proteins [Drosophila C virus (DCV) binds dsRNA and blocks Dicer-2 processing of dsRNA into siRNAs but does not bind siRNAs [After the discovery of RNA silencing in as a RSS . The preas a RSS ,84. Drosas a RSS ). In theas a RSS ,86,87,88as a RSS ,90,91). as a RSS , but alsproteins . B2 fromproteins ). Anotheproteins , while 1d siRNAs .Flaviviridae, Togaviridae, Reoviridae and Bunyaviridae). During persistent-propagative transmission, both plant- and animal-infecting arboviruses replicate in the arthropod vector [Anopheles gambiae mosquitoes presents evidence that RNA silencing is active however it does not seem to have antiviral impact on the initial midgut infection, but only at later stages of infection [Arthropods are vectors for viruses (arboviruses) from several families . d vector ,101. Hownfection . Supportnfection ,104, whinfection  and the nfection .Bunyaviridae are arboviruses, and as such replicate in both the host (plant or vertebrate) as well as in the arthropod vector. While infecting arthropods, bunyaviruses are targeted by RNA silencing, as illustrated by production of virus-specific siRNAs  isolates and more severe symptoms . About aMore recently it was also shown that NSs of TSWV, as well as those of groundnut ringspot virus (GRSV) and tomato yellow ring virus (TYRV), suppresses long-distance systemic silencing , which iNext to the siRNA-mediated silencing, there is a branch of the RNA silencing pathway involving micro (mi)RNAs, which resemble siRNAs structurally and in size, but differ from those in their biogenesis, being processed from host-encoded transcripts that fold into imperfect stem-loop structures. The miRNA pathway plays a major role in regulation of eukaryotic endogenous gene expression, including expression of RNA silencing components. The best known example is the regulation of AGO1 expression by miR168 . InterfeInterference in the miRNA pathway has been described for TSWV NSs, shown to bind miRNAs in vitro, and to interfere with the miRNA pathway in planta . In lighR) genes codes for the NB-LRR type, which are proteins consisting of three main domains. The N-terminal end is presented by a coiled-coil (CC) or Toll and interleukin-1 receptor (TIR) domain, followed by an internal nucleotide binding site (NBS) domain and a leucine-rich repeat (LRR) at the C-terminal end [Besides RNA silencing, another layer of the innate immunity system is represented by resistance genes and their R protein products. In plants the major class of dominant resistance  viral fitness and virulence, preventing virus clearance from the plant by antiviral RNA silencing. A study on engineered NSs mutants indicated that NSs RSS and Avr functions can be uncoupled. Furthermore, some of the resistance-breaking NSs mutants , mutated in predicted RNA-binding or putative Argonaute-binding domains, still suppress systemic silencing spread while having lost the ability to suppress local silencing [Recently, TSWV NSs protein was identified as the Avr for the single dominant RR genes . In the ilencing ,127. Altilencing ,127.Antiviral RNA silencing has already been demonstrated in several arthropods ,128. AlsFrankliniella occidentalis , especially in midgut epithelial cells (primary infection site), associated muscle tissue and cells in the salivary glands, from where tospoviruses are transmitted to the plant during thrips feeding [In contrast to the situation in plants, less is known about the function of tospovirus NSs in the insect vector (thrips). Evidence shows that TSWV NSs is expressed in cells of thrips  feeding ,132,133.The role of NSs as a transmission determinant in thrips was recently demonstrated in experiments with NSs-defective TSWV isolates which lost RSS activity and became non-transmissible by thrips. A closer look indicated that these viruses were still acquired by thrips larvae upon feeding on infected plants, however virus accumulation in adult thrips was severely reduced, which likely explains why adult thrips do not support transmission of these NSs-defective isolates upon a next probing-feeding attempt . In lighAedes albopictus) [Ixodes scapularis) [Spodoptera frugiperda insect (lepidopteran) cell lines [Although not in thrips, silencing suppression function of tospovirus NSs has been investigated in arthropods including mosquitoes (Diptera), caterpillar (Lepidoptera) and ticks. Two studies showed that TSWV NSs is able to suppress luciferase silencing, when expressed in mosquito cell lines (U4.4— opictus)  and in tpularis) . In anotll lines  and incrll lines . These oThere is indication that TSWV infection activates other thrips antiviral genes and defense responses—such as genes coding for antimicrobial peptides—proteins involved in pathogen recognition, receptors that activate innate immune response—such as Toll3—and members of signal transduction pathways activated by toll-like receptors . WhetherN gene and encoding a NSs protein with weak IFN-antagonistic properties [Vertebrate-infecting viruses must deal with the innate immunity in their mammalian hosts, including the well-known IFN-based defense ,140. NSsoperties ,153. In operties , howeverBesides the IFN-induced innate immune responses, mammals contain additional layers of innate immunity that act against viruses, including antiviral RNA silencing ,21. ProtThus far, only two papers have appeared on the identification of RSS activity in NSs proteins from vertebrate-infecting bunyaviruses in mammals, both with LACV NSs, but with contradictory outcomes. In one paper, RNA silencing was triggered by transfecting cells (human cell line 293T) with siRNAs, showing that in the additional presence of (transiently) over-expressed LACV NSs an apparent decrease of siRNA-triggered silencing was observed, which tempted the authors to suggest that LACV NSs exhibits RSS activity in mammals . In anotBunyaviridae genera.Vertebrate-infecting bunyaviruses, with the exception of hantaviruses, are transmitted by arthropod vectors, including arachnids (ticks) and insects  where, similarly to tospoviruses, they replicate and establish a persistent infection . During Aedes albopictus cell line U4.4), and infection with NSs-deletion virus in mosquito Aedes aegypti revealed lower titers and delayed dissemination to salivary glands when compared to wild-type virus, suggesting that in the absence of NSs the virus has difficulties in overcoming cellular defenses in the midgut [Still, little is known regarding the role of NSs during propagative transmission of vertebrate-infecting bunyaviruses in the arthropod vector. Analysis of BUNV and a recombinant NSs-deletion BUNV indicates that NSs is required for efficient replication in cell lines  of RSS activity with NSs from vertebrate-infecting bunyaviruses in arthropods is still lacking. While bunyaviruses are targeted by antiviral RNA silencing in plants, vertebrates and arthropods, currently only the NSs from plant-infecting tospoviruses has been clearly shown to contain RNA silencing suppressor activity. Considering the close ancestral relation of bunyaviruses, as well as many structural and functional similarities, the lack of clear proof on suppression of RNA silencing by the NSs from vertebrate-infecting bunyaviruses in both arthropods and vertebrates remains a matter for debate.In light of this it is important to highlight that the two reported (contradictory) studies on the effect of orthobunyavirus LACV NSs in mammalian RNA silencing made use of different experimental set ups to induce silencing and to express NSs. The first study by Soldan et al. , in whicAnother reason why studies on this point for the vertebrate-infecting bunyaviruses so far have remained unresolved is that in mammalian cells the IFN-pathway is a major antiviral mechanism, while antiviral silencing is still being debated by some  and mighIn conclusion, NSs from bunyaviruses remains an enigmatic protein, being a virulence factor in different cellular environments. However, as reviewed above, its interference on antiviral RNA silencing is only clearly described for tospovirus infections in plants, while still being debated in arthropods and vertebrates. As new data are gathered on the relevance of RNA silencing during infection in mammals as well as during persistent infections in arthropods, this may eventually also contribute to a deeper understanding of how bunyaviruses possibly affect this antiviral defense mechanism. The advancement in deep-sequencing technologies and the use of undifferentiated cell lines may provide further tools to functionally analyze NSs during bunyavirus infection. Considering the importance of NSs for plant- and vertebrate-infecting bunyaviruses, understanding its role during infection and its modus operandi will remain a continuing challenge for many years to come.


GABA transporters belong to a large family of neurotransmitter:sodium symporters. They are widely expressed throughout the brain, with different levels of expression in different brain regions. GABA transporters are present in neurons and in astrocytes and their activity is crucial to regulate the extracellular concentration of GABA under basal conditions and during ongoing synaptic events. Numerous efforts have been devoted to determine the structural and functional properties of GABA transporters. There is also evidence that the expression of GABA transporters on the cell membrane and their lateral mobility can be modulated by different intracellular signaling cascades. The strength of individual synaptic contacts and the activity of entire neuronal networks may be finely tuned by altering the density, distribution and diffusion rate of GABA transporters within the cell membrane. These findings are intriguing because they suggest the existence of complex regulatory systems that control the plasticity of GABAergic transmission in the brain. Here we review the current knowledge on the structural and functional properties of GABA transporters and highlight the molecular mechanisms that alter the expression and mobility of GABA transporters at central synapses. The brain utilizes GABAergic synaptic transmission to modulate the ongoing activity of neuronal networks. By acting on ionotropic and metabotropic receptors, GABA controls the generation of membrane potential oscillations, the time window over which synaptic inputs are integrated and the temporal structure of the activity patterns produced by entire populations of neurons , neurotransmitter amino acid , monoamine  and nutrient amino acid/orphan transporters . The currently accepted nomenclature that is used to identify members of the SLC6 family represents the nomenclature of GABA transporters in humans (A1-20) and rats . The nomenclature of GABA transporters in mice is different and somewhat confusing when compared to the ones mentioned above. In mice, GAT2 corresponds to A12/BGT1, GAT3 corresponds to A13/GAT2 and GAT4 corresponds to A11/GAT3, while GAT1 carries the same name as in humans and rats (A1)  family. The SLC6 family is composed of 20 members and, based on sequence composition, it is subdivided into four groups including GABA, osmolyte and creatine transporters  are the most extensively characterized GABA transporters and will be the main focus of discussion in this review.First, they showed that GABA transport is an active process, not directly coupled to ATP hydrolysis but requiring the presence of an inward electrochemical gradient for Na+, typically created by the membrane Na+/K+ ATPase. Second, GABA transporters have low micromolar, steady-state affinity for GABA, with a Michaelis-Menten constant of 2.5 μM [later found to be 3.1–10.6 μM for GAT1  . The counter argument, however, is that the functional relevance of an electroneutral exchange of Cl− across the cell membrane is unclear. Recent kinetics experiments indicate that Cl− is only required for the generation of GABA-induced steady-state currents, not for GABA-induced pre-steady-state currents, which reflect the first electrogenic steps in the transport cycle  intracellular dissociation of GABA and Na+ and rapid Cl− exchange , the initial estimates at 22°C suggested a value of 2.5 sXenopus oocytes did not provide an indication that GAT1 could generate any other current than the stoichiometric current described above  an agonist-induced Na+ inward current  and as a high-affinity, non-competitive antagonist for the Na+ inward current [iK = 0.03 μM  and often reproduced during electrophysiological patch-clamp recordings  and was first isolated near underwater volcanic vents in the Aeolic Islands, north of Sicily (hence aeolicus). LeuTAa is an experimentally tractable prokaryotic leucine transporter. It is evolutionary distant from GAT1 and shares 20–25% sequence similarity with GAT1 and other members of the SLC6 family including glycine, dopamine and serotonin transporters  indicates that substrate transport via LeuTAa is not Cl−-dependent, in contrast to GAT1. Despite these differences, the structural conservation between LeuTAa and other members of the SLC6 family is thought to be remarkably high , intracellular N- and C-terminal domains and extracellular, glycosylated regions  and TM11 to be displaced by 2.8Å and 2.2Å, respectively . These tentative estimates are useful to develop quantitative frameworks to determine the function of GABA transporters in the synapse  interneurons evokes post-synaptic currents that last <1 ms. In contrast, the currents evoked by GABA release from neurogliaform cells last >100 ms  with phorbol esters like phorbol-12-myristate-13-acetate (PMA) and 1-oleoyl-2-acetyl-sn-glycerol (OAG), or with N-heptyl-5-chloro-1-naphtalenesulphonamide (SC-10) and (-)-indolactam V (indoV) enhances GABA uptake via GAT1, whereas inhibiting PKC with bisindolyilmaleimide reduces it , not in the Michaelis-Menten constant for GABA uptake via GAT1 (mK), suggesting that they do not alter GAT1 binding affinity for the substrate  or OAG  does not alter GABA uptake in neurons but decreases it in glial cells (Gomeza et al., Xenopus oocytes, cultured neurons and glial cells may be attributed to differences in the concentration of PKC in all these different cell types. This suggests that in vivo the influence of PKC on GAT1 activity may vary significantly depending on the amount and specific PKC isoform expressed in different neurons and, possibly, in different sub-cellular compartments (Takai et al., GABA transporters, like other neurotransmitter transporters, are dynamically regulated at the level of their surface-to-cytoplasm partitioning and anchoring to the cell membrane Figure . In XenoA distinct mechanism through which neurons can regulate the cell-surface expression of GAT1 is via tyrosine kinase phosphorylation of intracellular tyrosine residues (Law et al., Previous works indicate that the localization of individual GAT1 molecules within the plasma membrane is not fixed in time and is not entirely predicted by passive diffusion in the lipid bilayer (Imoukhuede et al., There have been tremendous advances in our understanding of the molecular architecture and function of GABA transporters. A number of works has shown that there are intracellular signaling cascades that control the cell surface expression and mobility of GABA transporters, suggesting that the regulation of GABA transporter activity may be more complex than previously thought. It remains currently unclear how these short- and long-term changes in the expression, mobility and activity of GABA transporters affect the time course and spatial spread of GABAergic signals in the brain. This missing piece of information hold the promise of providing novel insights into the regulation of synaptic transmission by GABA transporters and will shed new light on our current understanding of the synaptic mechanisms underlying the generation and maintenance of neuronal network oscillations in distinct regions of the living brain.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Trypanosoma brucei. In addition to the catalytic 20S editosome, multiple accessory proteins are required for this conversion. Recently, the multiprotein mitochondrial RNA binding complex 1 (MRB1) has emerged as a key player in this process. MRB1 consists of six core proteins but makes dynamic interactions with additional accessory proteins. Here we describe the characterization of one such factor, the 72 kDa MRB1590 protein. In vivo experiments indicate a role for MRB1590 in editing mitochondrial mRNA transcripts, in particular the transcript encoding the ATP synthase subunit 6 (A6). Structural studies show that MRB1590 is dimeric and contains a central ABC-ATPase fold embedded between novel N- and C-terminal regions. The N-terminal domains combine to create a basic pore and biochemical studies indicate residues in this region participate in RNA binding. Structures capturing distinct MRB1590 conformations reveal that the RNA binding pore adopts closed and open states, with the latter able to accommodate RNA. Based on these findings, implications for MRB1590 function are discussed.Kinetoplastid RNA (kRNA) editing is a process that creates translatable mitochondrial mRNA transcripts from cryptogene encoded RNAs and is unique for kinetoplastids, such as  Trypanosoma brucei, Trypanosoma cruzi and Leishmania spp., are the causative agents of life threatening afflictions that include African sleeping sickness, chagas disease and leishmaniasis . These studies suggest that p22 functions as an adaptor by linking the editosome to the kRNA editing protein, TbRGG2  in kRNA editing. However, the current model posits two key functions for this complex. The first is in synchronizing multiple rounds of editing by the editosome by somehow mediating the exchange of the many gRNAs needed to edit pan-edited mRNAs. A second important function of the MRB1 complex is in coordinating kRNA editing with other RNA processing events , was discovered and shown to be essential for kRNA editing –24. MRB1 editing . MRB1187 editing . Subsequg events . Indeed,In vivo experiments suggest that MRB1590 effects editing of several mitochondrial mRNA transcripts, in particular that encoding subunit 6 of ATPase synthase (A6). Structural studies show that MRB1590 consists of three domains. These include novel N-terminal and C-terminal domains, which encompass a central region that contains an ATPase ABC fold. We show that residues in the basic N-terminal domain are involved in RNA binding and crystal structures capturing open and closed states of MRB1590 suggest possible mechanisms for how this protein may participate in RNA editing processes.A key aspect of MRB1 function, however, that has made it difficult to study is its dynamic nature. Specifically, in addition to core proteins, a number of other factors have been shown to associate with MRB1 during editing. One such protein, MRB1590, was originally identified in MRB1 complexes immunoprecipitated or tandem affinity purified with GAP1/GAP2 . MRB1590To generate MRB1590 RNAi cells, a fragment of the MRB1590 open reading frame from nucleotides 1299–2002 was amplified using PCR and cloned into the BamHI and ClaI sites of p2T7–177  that hadT. brucei 29–13 cells. Cells were selected with 1 μg/ml puromycin and cloned by limiting dilution.For integration of the tandem affinity purification PTP tag into the endogenous MRB1590 locus, primers MRB1590 5′ ApaI (5′-GAGGGCCCCCATTACGCCTTTCGTGGAGCG-3′) and MRB1590 3′ NotI (5′-GAGCGGCCGCCTTTTCGCAGTAACGGTCCGGAG-3′), from nucleotides 1299–2002 of the MRB1590 open reading frame, were used to amplify the 3′ end of MRB1590 and introduce it into the ApaI-NotI restriction sites of pC-PTP PURO . pC-PTP-Total RNA was isolated from uninduced and tetracycline-induced MRB1590 RNAi cells on day 4 post-induction, treated with DNase and qRT-PCR was performed as described previously –32, normRNA was extracted from uninduced and tetracycline-induced MRB1590 RNAi cells. Oligo-dT primed cDNA was prepared by reverse transcription with Superscript III. A6-specific primers, 5′-GCGAATTCAAATAAGTATTTTGATATTATTAAAG-3′ and 5′-ATTAACTTATTTGATCTTATTCTATAACTCC-3′, which anneal to the never edited 5′ and 3′ regions that flank the edited part of the A6 mRNA, were used for RT-PCR analysis.10 procyclic form cells containing the endogenous MRB1590-PTP construct. Expression of MRB1590-PTP was verified by western blot using affinity purified rabbit anti-protein C. Tandem affinity purification and nuclease treatment of cellular lysates was performed as described previously  cells. Protein expression was induced when the cells reached an A600 = 0.4–0.6 by the addition of 0.1 mM isopropyl β-d-1-thiogalactopyranoside for 4 h at 37°C. The protein was purified in one step using nickel-nitrilotriacetic acid column chromatography. Structure determination (see below) of full length MRB1590 revealed that its N-terminal region was largely disordered. Indeed, residues ∼1–30 are predicted to encode a mitochondrial targeting sequence, which are rich in basic and hydrophobic residues. Thus, different MRB1590 truncations were generated and cloned into pMCSG7. Studies showed that MRB1590(10–668) expressed optimally and was as active as the full length protein in RNA binding and ATPase activity assays. Hence MRB1590(10–668) was used for all studies unless otherwise stated.To obtain pure MRB1590 for structural and biochemical studies, the sequence encoding the full length  cloning . The vecRwork/Rfree values of 20.4%/26.2% to 2.6 Å resolution. The structure of MRB1590 bound to AMP–PNP was obtained by mixing MRB1590(10–668) (in which ADP had been removed by EDTA treatment) with AMP-PNP and MgCl2 to final concentrations of 5 mM and 1 mM, respectively. This complex was then combined 1:1 with the crystallization reagent composed of 0.1 mM 1:2:2 dl-malic acid:MES [2-(N-morpholino)ethanesulfonic acid]:Tris (MMT) buffer, pH 9.0 and 20–25% (w/v) PEG 1500. The structure, which contains two subunits in the ASU, was solved by molecular replacement using Phaser  bound to ADP in the presence of poly-U15 was obtained by mixing a protein-ADP solution equimolar with polyU15 RNA and using 25% (w/v) PEG 3350, 0.2 M magnesium acetate as a crystallization solution. The structure was solved using Phaser  PEG 3350, and 0.15–0.2 M sodium/potassium tartrate as a crystallization reagent. The structure was solved by single wavelength anomalous diffraction (SAD) using data collected from a crystal that had been soaked overnight with saturating concentrations of thimerosal. The heavy atom substructure was obtained using Phenix  and the g Phaser . The finHKL 2000 . The fin2, 2 mM ADP or AMP–PNP with 1 nM fluoresceinated oligonucleotide. All FP experiments were fit to a bimolecular binding model by nonlinear regression. The MRB1590 concentration used to fit the data corresponds to that of the dimer. MRB1590 mutants, R199A–R200A, R525A–R526A–R527A–R529A, F96A, Y637A and R191A were generated with Quikchange and analyzed for RNA binding using FP as per the wild type protein.FP experiments were performed using a PanVera Beacon 2000 FP System at 25 °C. 3′-Fluoresceinated oligonucleotides were utilized for RNA binding experiments. The oligos were heated to 99°C and slow cooled to room temperature prior to use to remove any secondary structure. RNA substrates that were measured include the GC-rich putative pause site in the A6 transcript, its complement, a double stranded (ds) GC-rich RNA, poly-U, poly-A and a dsRNA harboring the predicted hairpin in the A6 putative pause site. Regions of the RSP12, ND7 and COXII mRNA transcripts were also used in binding studies. For each experiment, MRB1590 was titrated into a 0.995 ml reaction cell containing a buffer of 20 mM Tris HCl, pH 7.5, 150 mM NaCl, 5% (v/v) glycerol, 1 mM MgCl2, 5% (v/v) glycerol. ADP or AMP–PNP was dissolved in the same buffer. 28–30 aliquots of 10 μl of 1 mM nucleotide were titrated into 1.3 ml of MRB1590 (36 μM). The experiments were all done at 25°C.Isothermal titration calorimetry experiments were conducted with a MicroCal VP-ITC. Before ITC experiments, MRB1590 was treated with 0.1 mM EDTA for 1 h at 4°C to remove bound nucleotide. The protein was then dialyzed overnight into an ITC buffer consisting of 20 mM Tris–HCl, pH 7.5, 150 mM NaCl, 5 mM MgCl−1 cm−1) at 340 nM, as measured using a PerkinElmer Lambda 25 UV-VIS Spectrometer. The assay buffer contained 20 mM Tris–HCl, pH 7.5, 150 mM NaCl, 5 mM MgCl2 and the reaction mixture included 0.5 mM phosphoenolpyruvate, 0.3 mM NADH, 20 units/ml pyruvate kinase, 20 units/ml lactate dehydrogenase, and 0.5 mM ATP. Hydrolysis was plotted against MRB1590 concentration and the slope of the line taken as the hydrolysis rate. This assay was carried out in the absence and presence of RNA.The ATPase activity of MRB1590 was measured using a continuous, regenerative assay, as previously described . BrieflyFor SEC, purified MRB1590 was concentrated to 5 mg/ml and applied to a Superdex 200 size exclusion column in a buffer containing 20 mM Tris–HCl, pH 7.5, 300 mM NaCl, 5% (v/v) glycerol. A single peak was obtained from the size exclusion column. The elution volume of MRB1590 was compared to a series of standard proteins to determine the molecular weight and hence oligomeric state of MRB1590.T. brucei cells in which MRB1590 was down-regulated by RNAi in a tetracycline-dependent manner. MRB1590 mRNA was depleted to ∼50% of the level in uninduced cells and its down-regulation caused a modest growth defect that became apparent by day 6 post-induction . The structure was refined to final  solvent . Size ex solvent . The ove solvent -B. MRB15 solvent .http://www.ebi.ac.uk/msd-srv/ssm/). These searches revealed that the central domain in MRB1590     ,44. In fure S4A) . Unlike ure S4A) . The N-dure S4C) .Kds of 885 ± 49 and 1270 ± 210 nM, respectively . These binding affinities are comparable to those obtained for other ATPase ABC proteins  and measured binding of MRB1590 in the presence of ADP to several RNA fragments. Poly-U sequences are found at the 3′-ends of gRNAs, hence we measured binding to a poly-U15 RNA. Also, as MRB1590 depletion correlated with altered editing of the A6 transcript and led to stalling at a certain GC-rich region, we also analyzed the ability of MRB1590 to bind a single stranded (ss) RNA fragment containing that site (5′-AGGCGGGCGGGCGACGGCGGU-3′). The FP experiments showed that MRB1590 bound these RNAs with Kds of 182 nM and 2.2 nM for the poly-U15 RNA and GC-rich sequence, respectively  (Supplementary Table S1). We next measured MRB1590 binding to a ssA15 sequence, the ssRNA complement of the GC-rich sequence, a double stranded version of the GC-rich sequence (dsGC-rich) and the GC-rich hairpin that is predicted to form at the stalled region of the A6 transcript. MRB1590 showed no binding to the GC-rich hairpin and weak binding to the other RNAs  and COXII mRNA transcripts, which encode ribosomal protein subunit 12, subunit 7 of the NADH dehydrogenase complex and cytochrome C oxidase subunit II Table . MRB1590Kd of 1.3 nM, indicating that length does play a role in high affinity RNA interactions by MRB1590  than the other structures, due to its fewer cross dimer contacts.Our data indicate that MRB1590 binds RNA with high affinity in the presence of ADP. To try and gain insight into the molecular basis for the MRB1590–ADP interaction with RNA, we obtained a structure of MRB1590-ADP in the presence of poly-Uon Table , superimon Table . As noteon Table . The MRBThe notable consequence of the structural changes found in the MRB1590 structure obtained in the presence of RNA is that the attached N-domains are significantly rotated relative to the rest of the protein. As a result the central cleft or pore located between the N-domains adopts an ‘open’ state. The average distance between N-domains in the RNA bound or open conformation is ∼28 Å compared to ∼3 Å in the closed state. In fact, there are direct contacts between residues in the N-domains in the closed state Figure . SpecifiWhile MRB1590 contains no RNA interaction domains or motifs, our structure of MRB1590-ADP solved in the presence of RNA revealed spurious electron density near the N-domains as well as a distinct conformation in which the N-domains are splayed open. These data implicate the N-domain pore or cleft as the possible RNA binding region. Consistent with this hypothesis, the electrostatic surface potential of MRB1590 revealed strong basic patches within this cleft Figure . ExaminaTo determine if the MRB1590 arginine patches are involved in RNA binding, we mutated them to alanines and measured the binding of the resultant mutant proteins to the GC-rich sequence by FP. The R525A–R526–R527A–R529A mutant displayed a 14-fold reduction in binding compared to wt, while the R199A–R200A mutant decreased MRB1590 binding by 590-fold Figure -C. Mutat15, the 21mer single stranded GC-rich sequence and dsRNA. In the presence of the dsRNA and the poly-U15 substrate the ATPase rate was unchanged while the addition of the GC-rich sequence resulted in a slight increase in the activity. Studies have shown that the ATPase activities of helicases are significantly affected in the presence of target, usually double stranded, RNA or DNA substrates  of RNA binding by MRB1590 and its roles in kRNA editing.The hypothesis that the N-terminal regions of MRB1590 mediate RNA binding was supported by mutagenesis studies, which revealed that several arginine residues in this channel, including Arg199, are important for high affinity RNA binding. Strikingly, our structures suggest that ATP binding would favor the closed state, in which the RNA binding cleft is largely blocked at the bottom edge by Phe95–Phe95’ and Arg199–Ag199’ stacking interactions Figure . BecauseMRB1590 coordinates and structure factors have been deposited in the Protein Data Bank under the Accession codes 4YJ1, 4YIX and 4YIY.