Bacillus sp. CDB3 possesses a novel eight-gene ars cluster  with some unusual features in regard to expression regulation. This study demonstrated that the cluster is a single operon but can also produce a short three-gene arsRYC transcript. A hairpin structure formed by internal inverted repeats between arsC and arsD was shown to diminish the expression of the full operon, thereby probably acting as a transcription attenuator. A degradation product of the arsRYC transcript was also identified. Electrophoretic mobility shift analysis demonstrated that ArsR interacts with the ars1 promoter forming a protein-DNA complex that could be impaired by arsenite. However, no interaction was detected between ArsD and the ars1 promoter, suggesting that the CDB3 ArsD protein may not play a regulatory role. Compared to other ars gene clusters, regulation of the Bacillus sp. CDB3 ars1 operon is more complex. It represents another example of specific mRNA degradation in the transporter gene region and possibly the first case of attenuator-mediated regulation of ars operons. Assessed in E. coli host, orf8 exhibited a significant resistance to arsenate but it could not complement the function-loss of arsC gene, indicating its role not as an arsenate reductase  differs from the common five-gene ars operon (arsRDABC) in E. coli; (2) two inverted repeats exist between arsC and arsD potentially forming a hairpin structure, which may play a role in transcriptional regulation; and (3) the ArsD protein sequence lacks the C-terminal vicinal cysteine pairs which E. coli ArsD bears, bringing into question its role as a repressor regulator. In this study, we investigated the transcriptional pattern of CDB3 ars1 under varying arsenic stresses and assessed the function of potential regulatory factors.clusters . The arssubtilis , while are novel . The orfeductase . In addiechanism . (1) ArrBacillus sp. CDB3 at 30 °C or E. coli strains at 37 °C, respectively.The bacterial strains, plasmids and primers used in this study are listed in Cell pellets of appropriately treated cultures at mid-exponential growth phase were suspended in NETS buffer  followed by phenol-chloroform extraction. Total RNA in the aqueous phase was collected by ethanol precipitation and then redissolved in RNase-free water. Probes were produced from pAR27 by PCR amplification using a DIG PCR labelling kit  following manufacturer’s protocol. For northern blotting, 10 µg of total RNA was loaded per lane and run in 1.8% (w/v) agarose denaturing formaldehyde gels in MOPS buffer  before being transferred onto nylon membranes . Prehybridization, hybridization, washes and film  image development were conducted as described in the product manuals.arsD cDNA with primer pair RT-DF/RT-DR was carried out using a SensiFAST™ SYBR No-ROX kit  on a LightCycler@480 detection system . The PCR conditions were: one cycle of 95 °C for 5 min; 45 cycles of 95 °C for 10 s, 60 °C for 20 s and 72 °C for 10 s. Each run was followed by a melt analysis comprising a 2.2 °C/s ramp rate from 95 °C to 65 °C. The cDNA was quantified using a Nanodrop® ND-1000 spectrophotometer.Total RNA samples isolated above were treated with RQ1 RNase-free DNase  before cDNA synthesis using a Transcriptor First Strand cDNA Synthesis kit from Roche following manufacturer’s instruction. Real-time qPCR to quantify ® 3130xl Genetic Analyzer  with GeneScan™-500 LIZ® internal size standards  and analysed using GeneMapper® software (Version 4.0).Primer PED-R labelled with 5’-end 5-6-carboxyfluorescein (6FAM) was used in reverse transcription before the resultant cDNA products were examined using an ABIarsC and arsD, a mutant 638-bp DNA was synthesised  to replace the original KpnI-RsrII fragment in pAR27. The resultant plasmid pAR27HPΔ was introduced into E. coli AW3110 for RNA assays.To eliminate the inverted repeats between arsR and arsD were amplified from pAR27 and cloned into pQE30  at BamHI and HindIII sites resulting in His-tag recombinant vectors pQENR and pQEND, respectively. Upon sequence verification, the two constructs were transformed into E. coli strain M15 followed by induction at 37 °C for 4 h using 0.5 mM isopropyl β-D-1-thiogalactopyranoside (IPTG). Cell pellets were lysed by French press  at 20,000 psi. The target recombinant proteins were isolated and purified from the filtered lysates using HisTrap™ HP, HiLoad16/60 Superdex75 and Vivaspin 10 kDa MWCO columns  and then stored in small aliquots at −80 °C before use.The coding regions of ars1 was PCR amplified using primer pair ProR-F/ProR-R from plasmid pAR27 and named proR. Another 132-bp fragment within orf7 was also amplified using ProN-F/ProN-R as a non-specific DNA control named proN. After purification by Sephadex G50 spin column chromatography , but not probes 2 and 3 , detected two other RNA fragments of approximately 1.9 kb and 1.5 kb. A further northern blot assay  can both hybridize to the 1.9-kb RNA band, corresponding to the length of arsRYC but the 1.5-kb RNA band was only detected when probe C was applied. It was also observed that the amount of 1.5-kb RNA increased with treatment time, which is evident in the 4 mM arsenite samples: at 3 min the 1.5-kb RNA was not detected but was remarkably abundant after 30 min . RNA samples isolated from AW3110 strains harbouring pAR27 (ars1) or pAR27HPΔ (hairpin knockout mutant) with or without arsenite treatment were reverse transcribed and analysed using qPCR assay. The results showed that in the mutant strain, expression levels of the long eight-gene transcript increased more than one fold over that in the control strain  displays features of a Rho-independent terminator, where GC is rich in the repeat followed by concentrated U nucleotides . This type of anti-termination usually involves an anti-terminator . They all contain a hairpin sequence in the intergenic region between arsC and D so assumingly have the similar transcription pattern as CDB3 ars1. Such internal terminator-like structures my also exist in other types of ars operons.Transcription attenuation has been recognised as a strategy in bacteria to modulate expression of operons through transcription pausing or termination, often in response to extracellular environmental changes. The region between leotides  which is E. coli . Termina E. coli . In the echanism . Hence, rminator ; howeverobtained . Since Aolerance , it is rarsY coding region. The electropherogram of extension products showed a predominant peak corresponding to a 261-bp cDNA fragment (arsY. This nucleotide (uridine) is immediately downstream of an inverted repeat region in Y RNA sequence  was annealed from 295 bp downstream of the fragment , demonstsequence . This fuE. coli plasmid R773 arsRDABC operon found rapid degradation of ArsB messenger and proposed that two RNA hairpins, one in the intergenic region before arsB and the other in the 5’ end of arsB, are responsible  (E. coli hairpin on ArsB translation (E. coli mRNA), it also assumes a main role in the specific degradation. The 5’ end of a degradation product has been identified as U35 which is straight after the hairpin, not before, suggesting a cleavage point between C34 and U35 (arsYC RNA fragment (1.5 bp) was identified, it is reasonable to assume that this specific cleavage also took place in the full operon transcript, which was not observed due to co-migration of the cleaved (∼5.4 kb) and intact RNA (∼5.8 kb) on our RNA gels (arsR plus 34 bp of Y) was not detectable by northern analysis, indicating that it had been rapidly degraded. Unlike the first hairpin in E. coli R773  , with a nslation . However and U35 . AlthougRNA gels . The upsoli R773 , the 5-bproteins . This rears operon regulator of Bacillus subtilis Skin element with 89% similarity  demonstrated that the ArsR protein retarded the mobility of a 167-bp DNA fragment (proR) containing the promoter region of ars1 by forming a DNA-protein complex (proN) even at 25.5 mM (255-fold concentration as that used for proR)   right, wBacillus CDB3 ArsR possesses three cysteine residues: Cys35, Cys36 and Cys38 in the ELCVCDL binding motif, which vary in positions from other known ArsRs  in the C-terminal region are involved in regulatory function in E. coli R773. Our result supports the proposition. Conservation of the three N-terminal cysteine residues , which were shown to be essential for the metallochaperone function of ArsD in E. coli R773 (arsD in arsenic resistance of Bacillus sp. CDB3 ars1 has been demonstrated (The regulatory function of ArsD in arsenic defence has been demonstrated in at least two species,  AIU 301 . Howeveroli R773 , points nstrated  and the Bacillus sp. CDB3 ars1 has been revealed to express its genes in a more complicated way than other known ars operons. It represents another example of specific mRNA degradation in the transporter gene region and possibly the first case of transcription attenuation in ars operons. The possible coordination of the two transcripts and other factors involved in both the transcriptional and post-transcriptional processes warrant further investigation.The eight-gene operon of 10.7717/peerj.1230/supp-1Data S1Figure S1. Raw data for qPCR results in Figure 2BClick here for additional data file.


Cnot2 is an inherited metastasis susceptibility gene. In this study, using orthotopic metastasis assays and genetically engineered mouse models, we show that one of the enzymatic subunits of the CCR4-NOT complex, Cnot7, is also a metastasis modifying gene. We demonstrate that higher expression of Cnot7 drives tumor cell autonomous metastatic potential, which requires its deadenylase activity. Furthermore, metastasis promotion by CNOT7 is dependent on interaction with CNOT1 and TOB1. CNOT7 ribonucleoprotein-immunoprecipitation (RIP) and integrated transcriptome wide analyses reveal that CNOT7-regulated transcripts are enriched for a tripartite 3’UTR motif bound by RNA-binding proteins known to complex with CNOT7, TOB1, and CNOT1. Collectively, our data support a model of CNOT7, TOB1, CNOT1, and RNA-binding proteins collectively exerting post-transcriptional control on a metastasis suppressive transcriptional program to drive tumor cell metastasis.Accumulating evidence supports the role of an aberrant transcriptome as a driver of metastatic potential. Deadenylation is a general regulatory node for post-transcriptional control by microRNAs and other determinants of RNA stability. Previously, we demonstrated that the CCR4-NOT scaffold component  Cnot7, is also a metastasis-associated gene, and that enzymatic activity of Cnot7 is required for its promotion of metastatic disease. These results suggest that large-scale control of RNA abundance may be modulating specific metastasis-related transcriptional programs, and that inhibition of specific RNA deadenylases may be a viable avenue in the development of anti-metastatic therapeutics.The majority of human cancer related death is due to the effects of metastasis, the process of cancer dissemination to and growth in distant organs. Primarily due to its complexity, the metastatic process remains incompletely understood. This complexity stems from the tumor cell’s dependence on multiple cellular and molecular systems for the successful colonization of distant sites. In this study, we demonstrate that one of the factors that contributes to metastatic progression is the control of tumor cell RNA stability. Previously, we demonstrated that a structural component of the CCR4-NOT transcription regulatory complex was an inherited metastasis susceptibility gene. Here we demonstrate that one of the enzymatic components of the CCR-NOT complex,  Metastasis is a complex process in which tumor cells disseminate from the primary tumor site to form life-threatening lesions at distant sites. To successfully complete the metastatic process tumor cells must activate a series of molecular functions. These include motility and invasion to escape the primary site and penetrate the parenchyma at the secondary organ, anti-apoptotic programs to survive anoikis during transit through the lymphatic or hematic vasculature, and proliferative programs to establish clinically relevant macroscopic lesions . Each ofCnot2, a scaffolding component of the CCR4-NOT RNA deadenylase complex F1 mice –also shosurvival . FurtherCnot1 knockdown cells were unsuccessful and therefore orthotopic assays were not performed. Tob1 knockdown in 6DT1 cells . 3’ untranslated regions (3’UTRs) were then interrogated for known consensus RNA-binding protein (RBP) sequence motifs enriched in the Cnot7 inversely-correlated transcripts  antibody in 4T1 cells overexpressing FLAG-CNOT7. Co-precipitated RNA was subjected to high throughput sequencing (RIP-seq). 149 transcripts showed enrichment relative to input and control (M2 RIP in 4T1 empty vector cells not expressing FLAG-CNOT7) and showed an inverse correlation with pression . These tpression . PUM andpression  suggestiWe next tested if the 71 transcripts that shared the tripartite motif were prognostic in human breast cancer data sets. Forty-six (65%) human orthologs of the CPE/CPSF/NRE containing genes were present in GOBO  S3 Tabl. These 4Cnot7 expression yielded 293 genes  transcripts previously annotated as cancer-associated. The top five represented canonical pathways associated with extravasation signaling, and cancer-associated HER2 signaling, HGF signaling, and MAPK-signaling. The top five upstream regulators in this network included ignaling . Gene seignaling . Consistof ERBB2 , 47.CCR4-NOT is a highly conserved protein complex that has been implicated in diverse functions associated with gene regulation , 13. It Cnot2 that was capable of discriminating breast cancer patient outcome. Furthermore, in vivo modeling demonstrated that suppression or over-expression of CNOT2 within tumor cells resulted in enhanced or reduced pulmonary metastases, respectively, indicating that Cnot2 has metastasis suppressing activities [Cnot2 to metastatic capacity through the CCR4-NOT complex could therefore occur through a variety of CCR4-NOT molecular functions.Previously our laboratory implicated the CCR4-NOT complex as an important determinant for metastatic mammary cancer. Generation of co-expressed gene maps from mouse strains with differing inherited sensitivity for pulmonary metastasis identified a network module centered on the CCR4-NOT component tivities . CNOT2 htivities . CNOT2 ctivities . Despitetivities , cellulativities . The conCnot2 also implicated other genes associated with RNA deadenylation  as potential modulators of metastatic disease, suggesting that this function of CCR4-NOT might be a critical determinant [CNOT8 and TOB1 were associated with distant metastasis free survival in human patients. We therefore selected Cnot8 and its highly conserved paralog Cnot7 to determine whether the deadenylation function of CCR4-NOT plays a critical role in tumor progression.In this study we have begun to dissect the role of CCR4-NOT in mammary tumor metastasis by investigating the role of RNA deadenylation in tumor progression. The integrated genetics and gene expression analysis that initially identified erminant . BioinfoCnot7 and Cnot8 are members of the DEDD superfamily of deadenylases [Cnot7 knockdown had little or no effect on primary tumor growth, indicating that its role in tumor progression is related to the metastatic process. In contrast Cnot8 suppressed both primary tumor growth and metastatic disease, suggesting a more general role in regulating tumor cell proliferation. Further elucidation of the commonalities and differences in molecular pathways controlled by these two deadenylases would likely provide interesting insights into tumor growth and progression.enylases . Both geenylases  and are enylases . Biochemenylases , 51. ThiCnot7 to promote metastatic disease was dependent on deadenylase activity. Point mutations eliminating enzymatic activity or that disrupted interactions mediating recruitment of RNA binding proteins to the CCR4-NOT complex suppressed the pro-metastatic activity of CNOT7. Knockdown of the adaptor protein TOB1, which acts as a bridge between CNOT7 and RNA binding proteins CPEB3 and CPEB4, which recruit RNAs to the complex for deadenylation, had similar effects. Attempts to knockdown CNOT1, which is responsible for recruitment of the NANOS1 and PUM2 RNA binding proteins, was unsuccessful. However, due to the central role of CNOT1 in the CCR4-NOT complex and increased apoptosis in CNOT1-depleted cells [Tob1 depletion results suggest that the majority of the effect on metastasis was likely due to the influence of CCR4-NOT on RNA equilibrium or translational efficiency, rather than on the other many functions ascribed to the complex.Importantly, due to the multifunctional nature of the CCR4-NOT complex, the ability of ed cells –54, thisCnot7 and Cnot8 knockdowns suggest that the different complexes likely target overlapping subsets of RNAs within the cell [Cnot7-containing CCR4-NOT complexes target global gene expression analysis was performed in two independent experiments. We focused specifically on genes that were inversely correlated with Cnot7 levels to enrich for those that were likely direct targets rather than those dysregulated due to secondary effects on the transcriptome. Since transcript recruitment to the CCR4-NOT complex is specified by RNA binding proteins a screen for RNA binding proteins was performed [Cnot7 metastatic suppressive program is mediated by specific RNA binding partners. Further investigation of these RNA binding proteins and their RNA targets will likely provide additional insights into the molecular pathways important for metastatic progression.The CCR4-NOT complex is thought to be one of two general deadenylase complexes in mammalian cells . Howeverthe cell . Alternaerformed , 38, 55[erformed . This scCnot7 RIP-seq experiment, providing more direct evidence of the interaction of the CNOT7-containing CCR4-NOT complex with the Cpsf, Nanos and Cpeb families of RNA binding proteins. Furthermore, almost half (71/149) of the RNAs identified by the RNA-immunoprecipitation contained all three motifs. When used as a weighted gene signature, this set of transcripts was capable of discriminating metastasis outcome in human breast cancer. Global transcriptome analysis in 4T1 mammary tumor cells revealed only 293 expressed genes bearing all three RNA binding protein motifs, consistent with previous findings of only limited numbers of genes changing after Cnot7 knockdown [Tgfb1 signaling as enriched in the 293-gene set.Encouragingly enrichment of three of the four RNA binding protein motifs  was replicated in the nockdown . Like thCnot6 and Cnot6l in metastatic progression may reveal additional important insights into tumor autonomous metastatic mechanisms. Moreover, these results also suggest that targeting Cnot7 deadenylase activity may be useful for anti-metastatic therapy. Cnot7 knockout animals are viable, with limited known phenotypes, indicating that pharmaceutical suppression of Cnot7 deadenylase activity may not be unacceptably toxic in the clinic. If true, this would provide a novel class of therapeutic agents to suppress colonization or the emergence of disseminated but dormant tumor cells, ultimately leading to a reduction in the morbidity and mortality associated with metastatic disease.Overall this study supports the hypothesis that in addition to initiation of specific transcriptional programs, such as epithelial-to-mesenchymal transition, degradation of specific RNAs may play an important role in the establishment of metastatic capacity. Further investigations of the RNA binding proteins that recruit transcripts for deadenylation and studies into possible roles of the other CCR4-NOT deadenylase subunits The research described in this study was performed under the Animal Study Protocol LCBG-004, approved by the NCI Bethesda Animal Use and Care Committee. Animal euthanasia was performed by cervical dislocation after anesthesia by Avertin.The mouse mammary carcinoma cell lines 4T1, 6DT1, Mvt-1   constructs or 10ug/mL (shRNA) puromycin for shRNA constructs.Two milliliter suspensions of 10Co-immunoprecipitation was conducted as described in  using moProliferation and wound healing assays were performed on the Incucyte ZOOM (Essen BioScience) system following the previously described protocols . For sofCnot7-knockdown and CNOT7-overexpressing 4T1 tumor cells was performed on Affymetrix GeneChip Mouse Gene 1.0 ST arrays by the Microarray Core in the NCI Laboratory of Molecular Technology.Array-based transcriptome profiling of Library preparation was performed using the NEBNext Ultra Directional RNA Library Prep Kit for Illumina with NEBNext multiplexing oligos using manufacturer’s protocol. RNA sequencing was conducted on the Illumina HiSeq 2500.RNA was isolated from tumors and cell lines using RNeasy kit (Qiagen) or TriPure (Roche) and reverse transcribed using iScript (Bio-Rad). Real-Time PCR was conducted using VeriQuest SYBR Green qPCR Master Mix (Affymetrix).cDNA sequences of human FLAG-CNOT7, FLAG-CNOT7-D40A, and FLAG-CNOT7-E247A/Y260A were describe previously . LentiviCNOT7 lentivector by restriction digest and ligation.Site directed mutagenesis was employed to generate CNOT7-M141R mutant using the primers listed in 2, 10mM HEPES (pH 7.3), 0.5% NP40, 200 units RNasin (Promega), Protease inhibitor cocktail (Roche)). The resulting 4mL of cell lysate was spun down twice at 21,000*g for 20 minutes at 4°C. 40uL of lysate was saved 1% input to confirm immunoprecipitation. 200uL of lysate was saved for 5% RNA-seq input and was purified by TriPure (Roche) RNA extraction. 25uL beads per 1mL lysate of Protein G Dynabeads were blocked with 1mL 0.5% BSA in PBS at room temperature for 20 minutes then washed twice with 1mL NT2 wash buffer (50mM Tris-HCl (pH 7.4), 150mM NaCl, 1mM MgCl2, 0.05% NP40). 24ug antibody was added to each sample and incubated at 4°C overnight then beads were added to each sample and rotated for 30 minutes at room temperature. Beads were washed four times in 1mL NT2 buffer. Eighty five percent of beads were subjected to RNA extraction with TriPure. Protein from the remaining 15% of beads was eluted with Laemli buffer to confirm immunoprecipitation.4T1 cells expressing 8166-M24-658 control vector or FLAG-CNOT7 constructs were grown to ~85% confluence in ten 15cm culture plates. Cells were then rinsed with 15mL ice cold sterile PBS, scraped off, pelleted (~1mL pellet), and snap frozen in liquid nitrogen. Cells were thawed on ice and lysed in 3.5mL lysis buffer  was used for western blotting. PVDF membrane (Millipore) containing transferred proteins was incubated overnight in solution of 5% milk protein, tris-buffered saline supplemented with 0.05% Tween-20, and primary antibody. The membrane was then incubated with horse-radish peroxidase linked anti-mouse , anti-rat, or anti-rabbit (Santa Cruz Biotechnology) IgG secondary antibodies. Immunoblot was visualized using Amersham ECL Prime Western Blotting Detection System and Amersham Hyperfilm ECL . Rabbit origin anti-CNOT7 was a generous provided by G. Sebastiaan Winkler. Commercial antibodies used in this study include rabbit origin anti-TOB1 (GeneTex), rabbit origin anti-CNOT1 (Protein Tech), rat origin anti-HA (Roche), mouse origin monoclonal anti-FLAG .5 cells were injected orthotopically into the fourth mammary fat pad of age-matched virgin females. After 30 days the mice were euthanized by intraperitoneal injection of 1mL Tribromoethanol with subsequent cervical dislocation. Primary tumors were resected, weighed, and snap frozen in liquid nitrogen. Lungs were resected, surface metastases were counted; lungs were inflated with 10% nitrate-buffered formalin and sent for sectioning and staining. For tail vein injection, 105 were injected into the lateral tail vein, mice were euthanized 22 days post-injection. All procedures were performed under the Animal Safety Proposal (LCBG-004) and approved by the NCI-Bethesda Animal Care and Use Committee.Female FVB/NJ or Balb/cJ mice from Jackson Laboratories were injected at 6–8 weeks of age. Two days prior to orthotopic injections, cells were placed in non-selective media. On the day of injection, 1x10Statistical analysis comparing two samples were conducted using the Mann-Whitney test on Prism Version 5.03 . Multiple-comparison data was analyzed by Kruskal-Wallis test with post-hoc Conover-Inman correction for multiple analyses by R-script. Survival data was conducted with the Mantel-Cox test on Prism.RBP motif sites were mapped on the 3’UTR regions of genome genes (27305 mRNA FASTA format) by using a Perl script. RBP motif site enrichment analysis was performed by the random sampling the genes from genome in the same number  and calculate the numbers of the gene with the RBP motif site and of the RBP motif site and repeat the sampling 1000 times. The p-value was estimated by one tail t-test. Differential gene expression analyses were done by t-test using the software package R.http://www.ncbi.nlm.nih.gov/geo/ under the accession numbers GSE73296 (array) and GSE73366 (RIP-seq).Array-based gene expression and RIP-seq studies from this study have been submitted to the NCBI Gene Expression Omnibus for 6DT1 and Mvt1 cells is shown in panels (A) and (B). Effects of shRNA knockdown are shown for the primary tumor for 6DT1 (C) and Mvt1 (D) are shown at the top of the figure. The macroscopic pulmonary surface metastases counts are shown in panels (E) and (F). Macroscopic pulmonary surface metastases after normalization for primary tumor mass are shown in panels (G) and (H). N.S. = not significantly different. * = p< 0.05; ** = p<0.01; *** = p<0.001.Relative (EPS)Click here for additional data file.S2 FigCnot7 knock down on migration of 6DT1 and Mvt1 cells, as measured by wound healing assays (B). Soft agar colony formation assays for 6DT1 and Mvt1 Cnot7 knock down cell lines (C).Results of (EPS)Click here for additional data file.S3 FigTob1-knockdown cells into BALB/c animals (B). Pulmonary surface metastasis count of 4T1 Tob1-knockdown tumors (C). Tumor normalized metastasis counts of 4T1 Tob1-knockdown cells (D).Primary tumor burden after orthotopic transplantation of 4T1 (EPS)Click here for additional data file.S4 FigCnot7 inversely correlated gene set. The blue curve represents the predicted range based on 1000 permutations.Results of motif search (B). The vertical red line represents the observed number of times a particular motif was found in the (EPS)Click here for additional data file.S5 FigGray lines indicate patients with tumors that expressed the individual gene signatures below the median value. Red lines indicate patients with tumors expressing the gene signatures above the median.(EPS)Click here for additional data file.S1 Table(XLS)Click here for additional data file.S2 Table(XLS)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S4 Table(XLSX)Click here for additional data file.S5 Table(XLS)Click here for additional data file.S6 Table(XLS)Click here for additional data file.


Honeybees inoculated with 109 RNA copies per bee developed paralysis symptoms within 6 days after inoculation. The number of CBPV RNA copies increased significantly throughout the infection. Moreover, the negative strand of CBPV RNA was detected by RT-PCR, and CBPV particles were visualized by electronic microscopy in inoculated honeybees. Taken together, these results show that CBPV RNA 1 and CBPV RNA 2 segments can induce virus replication and produce CBPV virus particles. Therefore, the three minor RNAs described in early studies are not essential for virus replication. These data are crucial for the development of a reverse genetic system for CBPV.Chronic bee paralysis virus (CBPV) causes an infectious and contagious disease of adult honeybees. Its segmented genome is composed of two major positive single-stranded RNAs, RNA 1  and RNA 2 . Three minor RNAs  have been described earlier but they were not detected by sequencing of CBPV genome. In this study, the results of  Apis mellifera) is one of the most important pollinators with a big impact on agriculture and economics  recombinant plasmid containing the pSP sequence) or cDNAs in a total volume of 25 μL. The thermal cycling conditions were 2 min at 50°C (active temperature for UNG to degrade any carryover DNA amplified from previous reactions) and 10 min at 95°C (activation of AmpliTaq Gold DNA Polymerase and degradation of UNG), followed by 40 cycles of denaturation at 95°C for 15 s and annealing/extension at 60°C for 1 min.The number of RNA copies was estimated using 5 Before the inoculation, a multiplex RT-PCR  was perfTo assess the CBPV replication, we used a specific RT-PCR to detect the negative strand form of CBPV RNA as developed by Celle et al. .10 RNAs copies per μL were mixed together. After addition of 2.5 mL carbon tetrachloride (CCl4), homogenates were incubated for 1 h at 4°C and then clarified at 200 ×g for 10 min at 4°C. Aqueous phases were recovered and centrifuged at 1,700 ×g for 1 h at 4°C. Ultracentrifugation of supernatants was carried out at 75,000 ×g for 3.5 h at 4°C. The pellet was resuspended in 50 μL PB. All the samples were diluted with water to 0.1 mg/mL, the negative control was diluted with the same 1/25 ratio, and 3.5 μL drops were applied onto glow-discharged formvar-carbon coated grids (Agar Scientific). After 1 min incubation at room temperature, the liquid excess was blotted off and the grids were rinsed with water before being stained with uranyl acetate 1%. Grids were transferred into a FEI Tecnai Spirit G2 Electron Microscope operated at 120 kV and imaged with an EAGLE 2kX2k CCD camera at a nominal magnification of 18,000x and with an underfocus of approximately 2.5 μm.The formation of viral particles was checked using electron microscopy. All RNA samples of bees inoculated with CBPV major RNAs showing a load of >1010 copies per μL and purified RNA 2 was about 8.3 × 1010 copies per μL. Each RNA was diluted to 1.0 × 109 RNAs copies per μL. Then, RNA 1 and RNA 2 were mixed together (major CBPV RNAs) to be inoculated to the bees.The total RNA extracted from the purified CBPV particles (A79-P isolate) was run on a MOPS/formaldehyde gel to separate RNA 1 and RNA 2 . After t10 DNA concentration of each run (R2 = 0.998). The slope of the DNA standard curve was −3.27 and the average efficiency of RNA 2 RT-qPCR was 102%. The limit of quantification was 100 DNA copies per reaction.In order to evaluate the CBPV RNA 2 RT-qPCR, four independent runs were performed using a 10-fold serial dilution of a plasmid DNA control as standard. The standard curve  showed ap < 0.001). However, no significant difference in symptoms and mortality was found between bees inoculated with CBPV naked RNAs and those inoculated with CBPV major RNA (p = 0.766). This result confirms that the clinical signs observed could be associated with RNA inoculations (naked RNA as well as major RNA).Bees were exposed to five different treatment groups including (i) bees not inoculated, (ii) bees inoculated with PB, (iii) bees inoculated with CBPV particles, (iv) bees inoculated with CBPV naked RNAs, and (v) bees inoculated with CBPV major RNAs . CBPV clIn addition, two other treatments were tested: bees inoculated with CBPV RNA 1 or with CBPV RNA 2. Around 12.5% (5 bees out of 40 bees) of bees inoculated with CBPV RNA 1 and 25% (7 bees out of 40 bees) of those inoculated with CBPV RNA 2 were dead. However, no chronic bee paralysis symptoms were observed (data not shown).10–1011 RNA 1 copies/bee). In contrast, the asymptomatic bees inoculated with naked or major CBPV RNAs presented a lower amount, ranging from 107 to 108 RNA copies/bee. Bees inoculated with CBPV RNA 1 or CBPV RNA 2 only did not show any increase of the viral load throughout the infection (data not shown).The number of copies of RNA 1 and RNA 2 was estimated using RT-qPCR methods. The quantities of RNA 1 and RNA 2 copies per bee were estimated using the two RT-qPCR methods . The resThe replicative RNA strand was tested by a specific RT-PCR on several samples from each condition studied . InteresTransmission electron microscopy was used to confirm the formation of viral particles . Six dayNo other bee viruses were detected by conventional RT-PCR in the bees used for the experiments.This study demonstrates that the RNA 1 and RNA 2 segments of the CBPV genome are the essential genetic elements for CBPV replication and that they are sufficient to induce the chronic bee paralysis disease. alphavirus methyltransferase guanylyltransferase. In addition, they found that ORF3 of CBPV RNA 2 shares significant similarities with a virion membrane protein found in various insect and plant viruses, and they suggested that ORF2 of CBPV RNA 2 may be a virion glycoprotein.CBPV, a worldwide virus, infects the honeybees. The anisometric morphology of its particles is unique among bee viruses as most of them are picorna-like viruses with icosahedral particles. CBPV genome consists of positive single-stranded RNA segments. Overton et al.  reported9 RNAs copies per μL. We also inoculated bees with a lower amount of CBPV major RNAs in an independent experiment (104 major RNAs copies per bee) and found a significant increase of CBPV RNA level and CBPV replication in 16% of those bees (data not shown). However, no clinical signs were observed in this last experiment. Therefore, the efficiency of the infectivity depends on the amount of inoculated major RNAs.Recently, Chevin et al.  showed tIn the current study, five different treatments were tested . In each12 RNAs copies/bee in dead bees. No other bee virus could be detected by conventional RT-PCR indicating that the mortality and the clinical signs were associated with CBPV replication. The amount of CBPV RNAs needed to induce chronic paralysis is much higher when using major RNAs (4.0 × 109 CBPV copies per bee) compared to that needed when using the CBPV particles (4.0 × 104 CBPV copies per bee). This may be due to several reasons: a lower stability of purified RNAs compared to native RNA protected by the viral capsid, partial degradation of RNAs during the purification process, and/or lower efficiency of infection through RNA transfection compared to that obtained with the virus. The infection by the native virus particles probably occurs via interaction with a cell receptor, which may facilitate entry into cells.Bees inoculated with major RNAs showed the signs of chronic paralysis disease within 6 days after inoculation and 87.5% of the bees were dead or symptomatic , suggestCBPV is a positive single-stranded RNA virus, so the synthesis of antigenomic RNA (negative strand) is carried out during viral replication . The neg


Picornaviridae virus family is one of many viral families that disrupt the nucleocytoplasmic trafficking of cells to promote viral replication. Picornaviruses contain positive-sense, single-stranded RNA genomes and replicate in the cytoplasm of infected cells. As a result of the limited coding capacity of these viruses, cellular proteins are required by these intracellular parasites for both translation and genomic RNA replication. Being of messenger RNA polarity, a picornavirus genome can immediately be translated upon entering the cell cytoplasm. However, the replication of viral RNA requires the activity of RNA-binding proteins, many of which function in host gene expression, and are consequently localized to the nucleus. As a result, picornaviruses disrupt nucleocytoplasmic trafficking to exploit protein functions normally localized to a different cellular compartment from which they translate their genome to facilitate efficient replication. Furthermore, picornavirus proteins are also known to enter the nucleus of infected cells to limit host-cell transcription and down-regulate innate antiviral responses. The interactions of picornavirus proteins and host-cell nuclei are extensive, required for a productive infection, and are the focus of this review.The compartmentalization of DNA replication and gene transcription in the nucleus and protein production in the cytoplasm is a defining feature of eukaryotic cells. The nucleus functions to maintain the integrity of the nuclear genome of the cell and to control gene expression based on intracellular and environmental signals received through the cytoplasm. The spatial separation of the major processes that lead to the expression of protein-coding genes establishes the necessity of a transport network to allow biomolecules to translocate between these two regions of the cell. The nucleocytoplasmic transport network is therefore essential for regulating normal cellular functioning. The  In this section, we will first provide a brief review of nucleocytoplasmic trafficking in uninfected eukaryotic cells, followed by an outline of the salient features of picornavirus gene expression and replication. Refer to Table The nucleus is bound by a double membrane of phospholipids termed the nuclear envelope. The inner nuclear membrane is associated with a network of the scleroprotein lamin, comprising the nuclear lamina, and the outer nuclear membrane is an extension of the endoplasmic reticulum  sequences that form intrinsically disordered motifs and act as the major impediment to free diffusion through the main channel of the NPC  of non-enveloped, small (~30 nm in diameter) viruses containing a positive-polarity, single-stranded RNA genome of ~7–9 kb in length with a viral protein (VPg) covalently attached to the 5′-terminus of the genome. These RNA molecules contain both 5′ and 3′ untranslated regions that function, in association with viral and host cell proteins, to facilitate both translation of the single open reading frame flanked by these regions, as well as RNA replication for genomic amplification. The long  tract for some aphthoviruses), highly structured, 5′-noncoding region (NCR) contains an internal ribosome entry site (IRES) that directs the cap-independent translation of a large polyprotein from the viral genome  RNA molecule, producing a double-stranded RNA structure called the replicative form. This opposite polarity molecule is, in turn, used for the production of positive-sense RNA molecules, generating a multiple-stranded RNA complex composed of negative-sense RNA templating multiple genomic RNA molecules simultaneously, called the replicative intermediate. These nascently produced molecules are then recycled back through the translation/replication process or packaged into progeny virions. Importantly, proteins predominantly localized to the nucleus in uninfected cells are utilized by these cytoplasmic viruses from the very primary steps of the replication process.The positive-sense RNA genome of a picornavirus is competent for immediate IRES-driven translation upon uncoating. In addition to picornavirus RNAs, it has been suggested that up to 10% of cellular mRNAs contain an IRES element  was the first host protein shown to interact with, and promote translation from, the IRES regions of encephalomyocarditis virus (EMCV), foot and mouth disease virus (FMDV), poliovirus, and human rhinovirus (HRV) 2  has also been implicated in having a role in the cap-independent translation of type I and II IRESs. La has been shown to bind a portion of the poliovirus IRES as a dimer and enhance the production of poliovirus proteins -binding protein 2 (PCBP2) binds single-stranded nucleic acids through three hnRNP K-homologous domains (KH domains) and is involved in the stabilization of several cellular mRNAs  to promote cap-independent translation. The SR proteins comprise a group of splicing factors with a multitude of functions related to gene expression including: constitutive and alternative splicing, mRNA export and stability, and translation  with the FMDV IRES element. A chimeric Theiler's murine encephalomyelitis virus  containing the FMDV IRES element in place of the TMEV IRES was unable to replicate in mouse neurons, suggesting the absence of a necessary ITAF for minimal FMDV translation. This ITAF was identified as PA2G4 (“ITAF 45”) in assaying for the formation of 48S initiation complexes through biochemical reconstitution and was shown to bind directly to viral RNA corresponding to the FMDV IRES through UV cross-linking  as well as the use of different strains of virus/sequences of reporter constructs. These apparently different results likely point to the fact that there are some minimal ITAFs required for Type I and Type II IRES elements but a multitude of ITAFs that play some stimulatory or translation-enhancing role depending on the specific context of an infection. For example, it is possible that different ITAFs are utilized by viral IRES elements depending on the cell type infected , as the availability of particular proteins that function in this regard likely dictate whether viral protein production and growth are supported, and to what extent , PTBP1 has been shown to be both required or unnecessary  heterodimerizes with nuclear factor of activated T cells, 45 kDa , and inhibits translation initiation from the HRV 2 IRES in neuronal cells. The recombinant oncolytic poliovirus PVS-RIPO exploits the incorporated HRV 2 IRES to permit attenuated neurovirulence in the treatment of malignant glioma, possibly due to the presence of DRBP76:NF45 heterodimers in the cytoplasm of neuronal cells  interacts with, is stabilized by, and is stimulated by PTBP1 , they utilize host cell factors in order to augment the function of this replicase enzyme. As with IRES-dependent translation, most of the factors utilized in the process of RNA synthesis are nuclear-resident proteins with RNA-binding functions that can be used by the virus to facilitate the replication of viral RNA molecules. These host proteins act in the context of RNP complexes they form with picornavirus RNAs to impart replication specificity to the polymerase, as 3D is able to replicate RNA non-specifically when provided with a primed template As discussed previously, a template usage switch from translation to RNA replication occurs as viral proteins accumulate and, in some cases, proteinases cleave host cell factors functioning as ITAFs. Prior to this transition, it has been proposed that optimal translation of poliovirus RNA requires the circularization of the RNA molecule, likely allowing ribosomes to be efficiently reloaded on the template , has been demonstrated to promote the amplification of positive-strand poliovirus RNA from the negative-strand template and, as with the majority of other host proteins involved in the infectious cycles of picornaviruses, is a nuclear-resident protein. hnRNP C1 and C2 are produced by alternative splicing, with the C2 isoform containing 13 additional amino acids , with other cellular proteins that may be species-specific that act to further enhance the efficiency of RNA replication. As with translation, the cohort of proteins utilized for picornavirus RNA replication may also be dependent upon the availability of particular proteins within the context of the cellular microenvironment where the viral RNA molecules are located. The different levels of enhancement provided to picornavirus RNA replication through the activities of different (or available) nuclear-resident host-cell proteins may contribute to the variable ratios of positive- to negative-sense RNA ratios, which have been reported to range from 30:1 to 70:1 for poliovirus  infection alters both the classical import pathway, which relies on a heterodimer consisting of an importin-α (karyopherin α) adaptor protein that binds the arginine-lysine-rich NLS of the cargo protein and transport receptor importin-β1 (karyopherin β1), as well as the transportin-1 (karyopherin β2) pathway in which the import receptor transportin recognizes a glycine-rich motif known as the M9 NLS  have been demonstrated to occur in picornavirus infected cells, these mechanisms are not mutually exclusive. Enterovirus-induced cleavage of Nups 62, 98, 153, 214, and 358 by the 2A and 3CD/3C proteinases, or cardiovirus-induced hyper-phosphorylation of Nups 62, 98, 153, and 214 through actions of the Leader protein, is directly responsible for both the increased “leakiness” of the nuclear envelope and the lack of nuclear import receptor–cargo complex docking at the cytoplasmic face of the NPC. Nucleocytoplasmic trafficking is an intricate and tightly regulated process allowing for precise control of gene expression. As a result, drastic alterations to the components of the NPC gateway between the two major compartments of the eukaryotic cell will have diverse and far-reaching consequences on trafficking pathways. The picornaviruses have evolved to take advantage of this important regulatory node to provide the functions of nuclear-resident proteins to the cytoplasm to promote their replication.The five Nup proteins that are the targets of alteration by picornavirus proteins have critical roles in shuttling macromolecules through the NPC Figure . Nup 214The picornavirus-mediated changes to components of the NPC critical to nucleocytoplasmic trafficking increase the bidirectional transport of proteins within the infected cell, thus causing a massive dysregulation of the transport process as a whole. However, there appears to be some level of specificity to which import and export pathways are affected, as a subset of nuclear-resident proteins relocalize while others do not. For example, nuclear-resident proteins fibrillarin, TATA-box-binding-protein 1 (TBP1), and serine/arginine-rich splicing factor 2 (SRSF2 or SC35) do not relocalize from the nucleus to the cytoplasm during poliovirus infection . This response is activated by the cellular recognition of viral molecules called pathogen associated molecular patterns (PAMPs). One major receptor of intracellular PAMPs that recognize picornavirus family members is interferon-induced helicase C domain-containing protein 1   binds with high affinity to RNA molecules containing AU-rich elements , is a predominantly nuclear-resident protein that shuttles to the cytoplasm, and promotes mRNA turnover  is a functionally significant event that many picornaviruses have evolved to accomplish in order to mount effective cytoplasmic replication strategies.In addition to shutting down most host-cell translation through actions in the cytoplasm, some picornaviruses also target the transcriptional components of cellular gene expression. Picornavirus infection has long been known to greatly reduce the initiation rate of cellular RNA synthesis , a component of SL1. Although UBF is not targeted by 3C in vitro, it does appear to be modified by a 3C-dependent mechanism within poliovirus infected cells  synthesizes ribosomal RNA (except 5S rRNA), which accounts for over half the RNA produced in the cell, and is carried out in the nucleolus  system, which controls the production of tRNA and 5S rRNA within eukaryotic cells, is also altered in poliovirus-infected cells. Studies similar to those discussed above demonstrate that 3C is responsible for cleaving the general transcription factor IIIC polypeptide 1 (TFIIIC box B-binding subunit or TFIIICα), leading to reduced activity of this transcription factor and subsequent inhibition of Pol III transcription , a central component of the spliceosome, causing interference with pre-mRNA splicing and mRNA synthesis  subunit of NF-κB , 3C, and 3D localize to the nucleus, and specifically to the nucleolus, of EMCV and mengovirus infected cells (Aminev et al., in vitro, thus inhibiting euchromatin formation and subsequent transcription (Falk et al., FMDV has distinct interactions with the transcription apparatus of cells. In contrast to other picornaviruses, FMDV modulates cellular transcription by targeting the regulation of transcriptionally active chromatin through the cleavage of the nucleosome component histone H3 (Griger and Tisminetzky, The inhibition of cellular gene expression by picornaviruses is carried out in diverse ways that target translation as well as splicing and transcription. To affect the latter two processes, some picornavirus proteins enter the nucleus of the infected cell. The pro-viral effects of reduced cellular gene expression are two-fold: to liberate cellular proteins with functions advantageous to viral replication and to re-route metabolic energy from cell-specific to viral-centric functions. It is somewhat surprising that the RNA-dependent RNA polymerase, 3D, of many picornaviruses becomes localized to the nucleus of infected cells, because it would seem that maximizing viral RNA production in the cytoplasm would be prioritized during the infectious cycle. The fact that many picornavirus 3D proteins contain an NLS suggests that there are specific functions of 3D and/or the precursor 3CD in the nucleus, some of which remain to be uncovered, which are balanced with the polymerase function of 3D in the cytoplasm. It is a given that further experimentation will continue to reveal the elegant ways in which picornaviruses alter the cellular functions that occur in the nucleus and the way in which viral proteins are able to infiltrate the command center of the cell to appropriate cellular components and functions for their benefit.Picornaviridae family have evolved to orchestrate a well-balanced promotion of viral replication with host-cell attenuation of nucleocytoplasmic trafficking demonstrates that interactions with the nucleus are functional and that these coding-capacity-limited cytoplasmic RNA viruses are master manipulators of even the most complex of cellular processes.Picornaviruses have traditionally been labeled as “cytoplasmic” RNA viruses as a result of the subcellular region in which these viruses produce viral proteins and replicate their genomic RNA molecules. Indeed, picornaviruses are able to complete the replicative cycle and produce infectious progeny in nucleus-free cytoplasts and cytoplasmic extracts (Pollack and Goldman, 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.


Most transporters share specificity for other neutral or cationic amino acids. Na+-dependent co-transporters efficiently accumulate glutamine while antiporters regulate the pools of glutamine and other amino acids. The most acknowledged glutamine transporters belong to the SLC1, 6, 7, and 38 families. The members involved in the homeostasis are the co-transporters B0AT1 and the SNAT members 1, 2, 3, 5, and 7; the antiporters ASCT2, LAT1 and 2. The last two are associated to the ancillary CD98 protein. Some information on regulation of the glutamine transporters exist, which, however, need to be deepened. No information at all is available on structures, besides some homology models obtained using similar bacterial transporters as templates. Some models of rat and human glutamine transporters highlight very similar structures between the orthologs. Moreover the presence of glycosylation and/or phosphorylation sites located at the extracellular or intracellular faces has been predicted. ASCT2 and LAT1 are over-expressed in several cancers, thus representing potential targets for pharmacological intervention.Glutamine together with glucose is essential for body's homeostasis. It is the most abundant amino acid and is involved in many biosynthetic, regulatory and energy production processes. Several membrane transporters which differ in transport modes, ensure glutamine homeostasis by coordinating its absorption, reabsorption and delivery to tissues. These transporters belong to different protein families, are redundant and ubiquitous. Their classification, originally based on functional properties, has recently been associated with the SLC nomenclature. Function of glutamine transporters is studied in cells over-expressing the transporters or, more recently in proteoliposomes harboring the proteins extracted from animal tissues or over-expressed in microorganisms. The role of the glutamine transporters is linked to their transport modes and coupling with Na However, in 2003 Mackenzie et al suggested that Li+ tolerance is not per se a useful criterion to classify amino acid transporters, since in some instance it depends on membrane potential and on the parameters used in experiments  and two neutral amino acid transporters (SLC1A4 and SLC1A5). In humans, the five glutamate transporters possess 44–55% amino acid sequence identity with each other, whereas the two neutral amino acid transporters exhibit 57% identity. The human isoform of SLC1A5 gene, isolated in 1996 from human placenta  is associated with breast cancer  form, in the rat ASCT2, a CXXC metal binding motif missing in the human isoform. Function/structure relationships studied in proteoliposomes highlighted the importance of Cys residues in substrate translocation which is potently inhibited by specific Cys reagents. Interestingly, ASCT2 harbors the two hairpins, HP1 and HP2, which are important for substrate translocation . The half-saturation constant for glutamine in proteoliposomes corresponded to a Km measured in oocytes  K+, transport was stimulated while at higher K+ level it was inhibited. Since the intracellular K+ level depends on ATP concentration, it can be assessed that the nucleotide indirectly modulates the B0AT1 transporter activity. High ATP level, causing increase in K+, would impair B0AT1 activity; lower ATP level will stimulate B0AT1  of the α-carbon, net neutral charge, size and, to a lesser extent, hydrophobicity of the side-chains  has been crystallized and resolved at 2.95 Å  of regulatory sites within the SLC6A19 amino acid sequence predicted five N-glycosylation sites. Three  are reported in the homology model  and the Light subunits of Amino acid Transporters (LATs) of the Heterodimeric Amino acid Transporters. Some general information will be given here to highlight the peculiarity of the glutamine transporters belonging to this family. HATs and CATs originate from a common ancestor with 12 trans-membrane domains (TMD) which duplicated the last two TMD originating a 14 TMD structure  with 12 putative TMD with the N- and C-terminals localized intracellularly and an heavy chain with the C-terminal localized extracellularly , PKC phosphorylation motifs, tyrosine kinase dependent phosphorylation sites  and includes 11 membrane transporters most of which are known to mediate the net uptake of glutamine, alanine, asparagine, histidine, arginine, and some other neutral amino acids in those tissues where they are expressed  correspond to the old systems A. System A was firstly isolated in 1965 . SNAT3 gene was identified in 1999 and annotated on the chromosome 3p21.31  gene is localized on the chromosome 14q23.1 and is expressed mainly in brain and liver  gene is localized on the chromosome 16q23.3 and the protein has a distribution similar to that of SNAT7. The transcripts of the other members SLC38A9, SLC38A10 and SLC38A11 derive from genes annotated on chromosome 5q11.1, 17q25.3 and 2q24.3 respectively  or EAAT2 (Glutamate transporter SLC1A2) . In addition, SNAT7 has unique properties resembling those of both system A and N. Differently by SLC1 and SLC6 transporters, homology models built for human and rat SNAT7 proteins by Modeler do not have the same fidelity of the others due to the low sequence similarity to the available crystallized bacterial homologs (AdiC and ApcT). SNAT2 model was already built on the basis of LeuT crystal structure  and ASCT2 (SLC1A5) are those for which over expression in several tumors has been documented since 2005 , is clearly associated to a glutamine transporter, i.e., Bn Broer, . The draP. pastoris and in the bacterium E. coli, respectively  which may be related with some specific signaling role of the transporter arisen later in evolution. While the function of several transporters has been widely described, no structures are available and the molecular mechanisms of regulation are still not completely solved. Thus, a lot remains to be done for a comprehensive knowledge of glutamine fluxes through tissues. This is very important not only for the biochemical knowledge of the glutamine homeostasis but also, and more importantly, for the involvement of this special amino acid in the hottest topic of human health: cancer biology.Glutamine is a “conditionally essential” amino acid which homeostasis is maintained owing to the different mode of transport performed by several membrane transporters. Thus, on the one hand, concentrative transporters allow glutamine absorption from diet in intestine and reabsorption from urine in kidney. On the other hand, both co-transporters and antiporters allow delivery of glutamine in all the tissues and equilibration with other amino acids. The redundancy of transporters with overlapping functional properties is very probably related to the need of avoiding derangements from homeostasis even in case of malfunctioning of one transporter. The lack of known defects of genes encoding these transporters, with very few exception, is in some instances an evidence that derangements of glutamine homeostasis is incompatible with life. Notwithstanding all the transporters dealt with share specificity for glutamine, they display different tridimensional structures. This is in line with their classification in different protein families; however, within the same family, the orthologs are very conserved from rat to human. The only exception is represented by ASCT2 (Lorena Pochini, Mariafrancesca Scalise contributed in collecting bibliography, preparing figures and writing; Michele Galluccio contributed in drawing structure figures and writing; Cesare Indiveri contributed in writing and supervision of all the activities.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.


Transcription of genetic information from archival DNA into RNA molecule working copies is vital for proper cellular function and is highly accurate. In turn, RNAs serve structural, enzymatic, and regulatory roles, as well as being informational templates for the ribosomal translation of proteins. Following RNA synthesis, maturing of RNA molecules occurs through various RNA processing events. One component of the collection of processes involving RNA species, broadly defined as RNA metabolism, is the RNA-editing pathway and is found in all animals. Acting specifically on RNA substrates with double-stranded character, RNA editing has been shown to regulate a plethora of genomic outputs, including gene recoding, RNA splicing, biogenesis and targeting actions of microRNAs and small interfering RNAs, and global gene expression. Recent evidence suggests that RNA modifications mediated via RNA editing influence the biogenesis of circular RNAs and safeguard against aberrant innate immune responses generated to endogenous RNA sources. These novel roles have the potential to contribute new insights into molecular mechanisms underlying pathogenesis mediated by mishandling of double-stranded RNA. Here, we discuss recent advances in the field, which highlight novel roles associated with the RNA-editing process and emphasize their importance during cellular RNA metabolism. In addition, we highlight the relevance of these newly discovered roles in the context of neurological disorders and the more general concept of innate recognition of self versus non-self. Alu repeats  modification, mediated by a highly conserved protein family known as adenosine deaminases acting on RNA ADAR; . Acting  repeats , the mos repeats .Adar in Drosophila leads to frequent seizures, chronic uncoordination, and age-dependent neurodegeneration (Caenorhabditis elegans (C. elegans) lacking RNA-editing activity through the deletion of both encoded adar genes exhibit defects in chemotaxis (adar null C. elegans is rescued by mutants impaired in RNA interference (RNAi) response  from what the authors named, adar-dependent loci  is interpreted by the ribosome as CGG(R) , and results in the insertion of the charged residue arginine in the polypeptide chain rather than the encoded polar residue glutamine. Genomic recoding is extensively utilized in Drosophila as a means of neuronal proteome diversification (+) channels  or AA-to-AI → AG(acceptor); Alu element within the human nuclear prelamin A recognition factor generates a novel 3′ splicing acceptor site regulating its exonization in a tissue-specific pattern  and subsequent generation of a hypomorphic allele (IG → GG) can prevent its recognition by the spliceosome machinery. The observation that ADAR1 knockdown leads to aberrant exonization of an Alu element in the seryl-tRNA synthetase transcript, and suggests that RNA editing also plays a destructive role in eliminating unwarranted acceptor sites to ensure proper splicing  and 3′ splice acceptor sites (AG), specific RNA editing in introns can generate novel splicing signals . First, as miRNA precursors are edited, pre-miRNA cleavage and other processing steps of the pathway can be inhibited  is achieved by dsRNA molecules, which trigger a highly conserved biological response known as RNAi . In addinhibited . Second,nhibited . Finallynhibited .Alu repetitive sequences are highly associated with exon circularization .Transcriptional profiling studies in metazoans have revealed mysterious new RNA species comprising unbroken circles . Termed rization . Strikinrization . Moreoverization . Similarrization . This an elegans . Second, at 18°C . The acc at 18°C . Clearly at 18°C . NeverthCellular infection by viruses activates a protective mechanism that involves an inflammatory response . The phyAdar1(-/-)) mouse embryonic lethal phenotype was substantially rescued to pup birth in Adar1(-/-)/Mavs(-/-) double-mutant mice. Finally, transfection of dsRNA species containing multiple IU pairs was unable to elicit an immune response in fibroblast cells of ADAR1-deficient mice, which indicates that editing activity is necessary for blocking the response. In a similar study, E861A/E861AAdar1). Through transcriptional profiling of homozygous mutant mice, they observed an atypical upregulation of ISGs, highly similar to the one observed in the ADAR1 protein null mice (E861A/E861AAdar1/MDA5(-/-) double-mutant mice also rescued the embryonic lethality phenotype suggesting that MDA5 is the principal sensor of non-edited endogenous dsRNAs. Clearly, these observations suggest a pivotal role for RNA editing in the regulation of innate immune responses toward self-generated dsRNA molecules. More importantly, ADAR1 acts hierarchically upstream in the immune cascade and through RNA editing it fingerprints dsRNAs as endogenous, thus preventing undesired cellular immune responses and allowing robust measures to be taken against invading non-self dsRNA signaling events .In these studies, it was reasoned that the elevated cellular apoptosis exhibited by ADAR1 null mice is due to induction of immune/inflammatory responses triggered by non-edited, endogenous dsRNA sources. In agreement with this notion, ull mice . FurtherA universal property of RNA, and trait of transcriptomes in general, is the generation of RNA molecules with varying degrees of double-strandedness that orchestrate diverse cellular functions. Despite variation in length, many endogenous molecules with dsRNA character intersect with the A-to-I RNA-editing system, since no sequence specificity is required for ADAR binding and editing .Rather, ADAR modification conceptually generates two radically opposed forces determined by substrate structure. When structures are imperfect dsRNA, only specific editing events occur. Because, in general, these imperfect structures involve conserved secondary and tertiary RNA interactions of exquisite detail, specific editing must be seen as a creative force in the evolutionary tool-kit – a novelty generator. Conversely, less specific hyper-editing of near-perfect duplexes serves a preventative or intervening role, competing with other enzymes that have different designs on the fate of long perfect dsRNA. Deconvolution of these opposing roles of editing in organismal phenotype will remain a challenge into the near future.RNA-editing enzymes have the inherent capacity to regulate many dinstinct pathways that are involved in RNA metabolism and serve to fine-tune and optimize transcriptional outputs . AlthougAdar1 gene were identified in individuals diagnosed with AicardiGoutières syndrome (AGS), an autoimmune disorder (Adar1 mutations exhibited high levels of expression for a number of IFN-stimulated genes, a phenotype observed previously in ADAR1-deficient mice (Adar1 mutations can result in autoimmune diseases. Thus, it is becoming increasingly clear that ADAR-mediated modifications occupy a central role in cellular physiology. Therefore, a more comprehensive understanding on how RNA-editing systems regulate global cellular RNA metabolism can shed light in the development of therapeutic strategies of ADAR-mediated human diseases by providing insights into their pathogenesis.Previously, multiple missense mutations in the disorder . In addient mice . TherefoAll authors listed, have made substantial, direct and intellectual contribution to the work, and approved it for publication.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.


Sin3b exon usage after RBM39 knockdown. SIN3B targets histone deacetylases to chromatin to repress transcription. In mouse, Sin3b produces long and short isoforms, with the short isoform lacking the ability to recruit HDACs. BMP4 induced a shift in SIN3B expression to the long isoform, and this change in isoform ratio was prevented by RBM39 knockdown. Knockdown of long isoform SIN3B enhanced BMP4-dependent transcription, whereas knockdown of the short isoform did not. We propose that BMP4-dependent transcription is negatively autoregulated in part by SIN3B alternative splicing, and that RBM39 plays a role in this process.BMP signalling is negatively autoregulated by several genes including SMAD6, Noggin and Gremlin, and autoregulators are possible targets for enhancing BMP signalling in disorders such as fibrosis and pulmonary hypertension. To identify novel negative regulators of BMP signalling, we used siRNA screening in mouse C2C12 cells with a BMP-responsive luciferase reporter. Knockdown of several splicing factors increased BMP4-dependent transcription and target gene expression. Knockdown of RBM39 produced the greatest enhancement in BMP activity. Transcriptome-wide RNA sequencing identified a change in  ID genes, which are negative regulators of cell differentiation and positive regulators of cell proliferation10Members of the BMP/TGFβ superfamily play critical roles in pluripotency, embryonic stem cell differentiation and developmental patterningBMPR2 are found in 80% of familial and between 15–40% of idiopathic cases of pulmonary arterial hypertension5Reduction in BMP signalling is a causal mechanism in certain disorders. Haploinsufficient mutations in BRE-luc) transcriptional reporter. We screened 9968 genes  produced the strongest increase in 24 hours . While kVegfa mRNA isoform expression was altered in C2C12 cells , encodes an RNA-binding protein of the U2AF65 small nuclear ribonucleoprotein family. RBM39 splices VEGF isoforms in metastatic breast cancer cells and loss of RBM39 in sarcoma cells alters VEGF isoform ratio towards more mitogenic/angiogenic forms of VEGF12 cells , indicat12 cells . Analysi12 cells . Of thess  in human cells, and we observed that it spliced VEGF in mouse cells. To investigate if RBM39 also negatively regulates BMP4-dependent transcription in human cells, we transfected U2OS osteosarcoma cells with siRNAs to human RBM39 and a  to BMP4 . Knockdo to BMP4 . This denscripts . Similarificance . Expressificance . We measis ratio . Knockdois ratio  attenuatRbm39 mRNA expression, RBM39 protein or its nuclear localisation , we used transcriptome-wide sequencing to identify targets of RBM39, which identified SIN3B. The mammalian SIN3/HDAC complex comprises multiple subunits, including HDAC1, HDAC2, SAP18/30, SUDS3, RBBP4/7 and either SIN3A or SIN3B20Sin3b-201 to Sin3b-202. As we observed that knockdown of RBM39 increased BMP4-dependent transcription, and that SIN3B-201 was likely to possess greater repression than SIN3B-202 due to the presence of the HID, we explored this further. To clarify, we used isoform specific qPCR to measure both Sin3b-201 and Sin3b-202 levels in C2C12 cells. This was done with and without BMP4 and in the presence or absence of RBM39 knockdown. While we found an apparent trend for increased expression of Sin3b-201 mRNA after RBM39 knockdown by qPCR (consistent with the results observed from RNA sequencing), the difference did not reach statistical significance. Instead we reproducibly found that BMP4 induced an increase in long SIN3B-201 mRNA and protein. This change was prevented by knockdown of RBM39. The difference in detected significance between sequencing data and qPCR may be a technology specific effect. RNA-sequencing provides a raw count of exon numbers compared to the relative expression by qPCR. As we do not have sequencing data from cells treated with BMP4, it is not possible to say what effect BMP4 treatment has on exon expression by sequencing. We speculate that because levels of Sin3b-201 were not statistically different after RBM39 knockdown when measured by qPCR, data from sequencing may be representative of a (relatively) small change in low basal transcript levels that are amplified in terms of significance. It is, however, important to note that by both methods, RBM39 knockdown trends towards increasing Sin3b-201, at least at the mRNA level. Such an observation may suggest dual-activity of RBM39, where it slightly increases Sin3b-201 in the absence of BMP4, but prevents BMP4 induced splicing in favour of Sin3b-201. In the latter part of this discussion, we refer to the likely importance of post-translational and complex-dependent regulation of RBM39. A future focus would need to investigate any potential for dual-activity arising from such regulation. In the interests of simplicity, our considerations below are limited to the validated splicing change mediated by RBM39 in the presence of BMP4.RNA-sequencing identified a change after knockdown of RBM39, which increased the ratio of Sin3b-201 and Sin3b-202 share a transcriptional start site. Therefore, the most likely explanation for the BMP4 induced regulation of Sin3b by RBM39 is that it occurs via alternative splicing rather than transcription. Knockdown of long SIN3B-201 enhanced transcription, while knockdown of short SIN3B-202 did not. These results indicate that BMP4-dependent transcription is negatively autoregulated in part by SIN3B alternative splicing. RBM39 knockdown prevented not only the increase in long SIN3B-201 induced by BMP4, but also the overall change in long-to-short isoform ratio. Due to limitations of pulling down RBM39, we have been unable to provide a direct physical link between RBM39 and Sin3b transcripts, e.g. using RNA immunoprecipitation.Both Sin3b mRNA isoform expression occurred concurrently with increased expression of Id1 and Id2. For both, increased expression preceded the change in SIN3B protein. This indicates that mechanisms other than altered Sin3b splicing are required for enhanced early induction of Id1/Id2 after RBM39 knockdown. At 24 hours, the return of Id1/Id2 expression towards basal levels coincided with sustained induction of long SIN3B-201 expression. Preventing splicing in favour of SIN3B-201 by knocking down RBM39 may therefore result in sustained induction of Id1/Id2 in response to BMP4. The dynamics of BMP4-dependent Id1/Id2 expression after knockdown of SIN3B or long SIN3B-201 were similar to those observed after knockdown of RBM39. This data supports long SIN3B-201 being the major isoform involved in the suppression of BMP signalling, and thus that the balance between isoforms is functionally important. Taken together, our results suggest that Sin3b splicing is a novel negative autoregulatory feedback mechanism induced by BMP4.We observed that the BMP4 induced change in Sin3b-202 mRNA. This was surprising, as there are only two described transcript isoforms of Sin3b in mouse. It may therefore be expected that a ratio should be maintained based on the observed change in Sin3b mRNA levels in cells treated with BMP4. Based on the actual observed changes, RBM39 is required for active splicing in favour of Sin3b-201, but not for BMP4-induced loss of Sin3b-202. It is important to note that the approach used here may be complicated by other post-transcriptional processes, including mRNA degradation, or post-translational modifications, which are speculated to regulate SIN3B functionSIN3B gene is more elaborately spliced, unknown splice variants in mouse Sin3b may also prevent simple interpretation. Further, our data indicates that in the presence of RBM39, total SIN3B mRNA and protein levels remain relatively static in response to BMP4. Knocking down RBM39 disrupts maintenance of SIN3B isoform ratios and produces a BMP4-dependent overall loss of SIN3B from basal levels. The two SIN3 isoforms, SIN3A and SIN3B, have both unique and shared gene targetsOur observations on the role of RBM39 in mediating BMP4 induced alternative splicing are complicated by finding that knockdown of RBM39 did not restore Rbm39 mRNA levels or RBM39 protein. RBM39 has an arginine/serine (RS) rich domain, similar to the SR family of splicing proteins which are phosphorylated in the RS domainChanges in cellular splicing programs in response to activated cell signalling cascades have been described and can occur through mechanisms including splicing factor synthesis/degradation, different protein-protein interactions and altered nuclear translocation 13BMP/TGFβ superfamily signalling is made more complicated by the diversity of both ligand and receptor (comprehensively recently reviewed elsewhere with respect to cardiovascular development and disease)SIN3B alternate splicing by RBM39 can occur across species. Knockdown of RBM39 in U2OS cells also increased transcription in response to BMP4. The functional link between RBM39 and the BMP signalling pathway therefore appears to be present in both mouse and humans.In human U2OS cells, we observed changes in transcripts that contained exons encoding the HID domain of human SIN3B. This change in transcript ratio of HID transcripts to most other transcripts was both BMP4-responsive and inhibited by siRNA knockdown of human RBM39. This indicates that regulation of BMP4 induced In this study, we have found initial evidence that mRNA splicing factors provide a level of regulation of BMP signalling. As these factors are reported to interact with one another, their role in this regulation is likely complicated, and context specific. We have identified that RBM39 is required for BMP4 induced regulation of a transcriptional repressor of BMP signalling, and that different isoforms of mouse SIN3B have different regulatory effects on transcription induced by BMP4 (summarised in BRE-luciferase C2C12 is previously describedBRE-luciferase (hereafter BRE-luc) consists of two copies of the BMP responsive elements of the mouse Id1 gene cloned into the pGL3 luciferase vectorBRE-luc C2C12 cells were maintained in DMEM supplemented with 10% FBS, 2 mM L-glutamine and 700 μg/ml Geneticin/G418. U2OS cells were transiently transfected with pGL3 BRE-lucBRE-luc assays , alkaline phosphatase expression and all qPCR, cells (stable C2C12 lines and transient U2OS) were transfected with siRNA using RNAiMax (Invitrogen) for 24 hours in DMEM with 10% FBS, L-Glutamine and then cultured with rhBMP4  in DMEM with 0.1% FBS and L-glutamine. For all experiments except timecourse data and expression of alkaline phosphatase, cells were treated with rhBMP4 for 24 hours. For alkaline phosphatase expression, cells were treated with rhBMP4 for 72 hours. For timecourse data, cells were cultured in DMEM with 0.1% FBS and L-glutamine for 24 hours before being treated with BMP4.Mouse myoblast C2C12 and human osteosarcoma U2OS (both from ATCC) were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% Foetal Bovine serum and 2 mM L-glutamine. The BMP reporter cell line http://web-cellhts2.dkfz.de/cellHTS-java/cellHTS2/). The average Z’ over all plates was 0.443.All liquid handling was performed using the Perkin Elmer Janus liquid handling platform unless noted. To create a customised siRNA library, we identified druggable genes in the Therapeutic Targets databaseFor validation and follow up of target genes, cells were transfected with 20 nM of siRNA for 96 well assays, scaled up to maintain this concentration in larger vessels. Initial siRNAs used were ON-TARGETplus SMARTpool from GE Dharmacon; siRNAs used were to PABPN1 (L-045150-01), PHF5A (L-014987-01), RBM39 , DHX15 (L-044844-01), SF3A1 (L-041779-01), SF3A3 (L-064034-01), PRPF19 (L-057330-01), PLRG1 (L-062980-01), SNRNP200 (L-041847-01) and SIN3B . Custom siRNAs were picked to target only SIN3B-201 (sense 5′-UGGGAUAAGCAGAGAAAUUUU-3′) and SIN3B-202 (sense 5′-CUGCACAGCUGAGGAGUGAUU-3′). All changes were controlled by comparison to the effect of a non-target control (NTC) siRNA (D-001810-10). The effect of knockdown of several targets was further independently confirmed with Silencer Select siRNAs from Ambion; siRNAs used were to PABPN1 (s79501), PHF5A (s86709), RBM39 (s172699) and DHX15 (s64901). All changes were controlled against non-target control siRNA (4390843).4, 2 mM CDTA, 1% Triton X-100). Lysates were assayed with luciferase assay buffer . Total luciferase activity was normalised to cellular protein levels by BCA assay of lysate.After knockdown and treatment with BMP4, cells were lysed in luciferase lysis buffer (25 mM Tris-POPabpn1 (Mm00479791_m1), Phf5a (Mm00466078_m1), Rbm39/RBM39 (mouse Mm00504698_m1 and human Hs00863502_g1), Dhx15 (Mm00492109_m1), Id1/ID1 (mouse Mm00775963_g1 and human Hs03676575_s1), Id2/ID2 (mouse Mm00711781_m1 and human Hs04187239_m1), Smad6/SMAD6 (mouse Mm00484738_m1 and human Hs00178579_m1), Sin3b-201 (Mm01249269_m1), Sin3b-202 (Mm01247120_g1), total Sin3b (Mm01247119_m1) and human SIN3B (Hs01006369_m1 and Hs01006375_m1); all qPCR data was normalised to either Hprt (Mm01545399_m1) for mouse or GAPDH (Hs02758991_g1) for human samples and changes analysed using the ddCt method. PCR for Vegfa was carried out using IMMOLASE Taq polymerase (Bioline); primers used to detect alternative Vegfa isoforms were forward 5′-GCTGTAACGATGAAGCCCTG-3′, reverse 5′-CCGCCTTGGCTTGTCACA-3′, altered Vegfa isoform expression was normalised to Gapdh using primers forward 5′-GAGAGTGTTTCCTCGTCCCG-3′ and reverse 5′- ACTGTGCCGTTGAATTTGCC-3′. Vegfa isoform PCR products were gel extracted and Sanger sequenced to confirm their identity. For RNA sequencing, total RNA was extracted from C2C12 cells after knockdown with RBM39 siRNA (or non-target control siRNA) for 24 hours and DNase-I treated. Libraries were prepared with a TruSeq Stranded mRNA Library Prep Kit (Illumina) and 100 bp paired-end sequenced on an Illumina Hi-Seq platform.Total RNA was isolated from cells using an RNeasy Micro kit (Qiagen) and converted to cDNA with a QuantiTect Reverse Transcription Kit (Qiagen) to the manufacturer’s instructions. Quantitative PCR (qPCR) was carried out using 2× Taqman Universal PCR Master Mix no-AmpErase UNG (Life Technologies) on a BioRad CFX96 qPCR system. Assays used (Life Technologies) were Alkaline phosphatase expression was determined with a Phospha-Light kit (Life Technologies). Cells were transfected with siRNA for 24 hours, followed by treatment with BMP4 for 72 hours. Cells were lysed in dilution buffer containing 0.2% Triton X-100 and assayed using the manufacturer’s instructions, with the exception of not adding buffer containing non-placental alkaline phosphatase inhibitors.Total protein was extracted from cells in RIPA buffer  supplemented with a protease and phosphatase inhibitor cocktail; protein lysate was combined with SDS loading buffer  and boiled for 5 minutes. Protein samples were run on Bis-Tris (Novex) gels and transferred to nitrocellulose. Blots were blocked in 50% Seablock blocking buffer  and incubated with primary antibody overnight in blocking buffer. Antibodies used were to HCC1/RBM39 , SIN3B , Phospho-SMAD1/5 (Ser463/465)  and beta tubulin (Sigma T5283). Secondary antibodies used were IRDye 680RD Donkey anti-Rabbit IgG and IRDye 800CW Donkey anti-Mouse IgG (LI-COR), incubated for 1 hour in blocking buffer. Blots were imaged on a LI-COR Odyssey CLx. Nuclear and cytoplasmic fractions were extracted with an NE-PER kit (Thermo Scientific), to the manufacturer’s instructions. Separation of cellular fractions was confirmed by blotting for anti-Histone H3 . Phosphorylated and unphosphorylated fractions from C2C12 cells were extracted with a PhosphoProtein Purification Kit (Qiagen) to the manufacturer’s instructions.Cells were fixed with ice cold methanol, blocked in 5% BSA and stained overnight with Phospho-SMAD1/5, followed by Goat anti-Rabbit IgG (H + L) Alexa Fluor 488 (Life Technologies) for 1 hour. Cells were counterstained with Hoechst 33342 and imaged on a PerkinElmer Operetta.https://ccb.jhu.edu/software/tophat/index.shtml) to mouse genome GRCm38/mm10 and ENSEMBL annotated (ENSEMBL release 75). Potential PCR duplicates were removed with MarkDuplicates command part of Picard tools v1.92 (http://broadinstitute.github.io/picard). Gene expression and exon usage were determined by counting uniquely mapped reads with HTSeq-count and dexseq-count.py respectively (http://www-huber.embl.de/users/anders/HTSeq/doc/count.html), with default parameters for both tools. Differentially expressed genes between RBM39 siRNA and non-target control siRNA samples were identified with edgeR using FDR < 0.05. Differential exon usage between the samples was determined with the DEXSeq package using FDR < 0.05. Graphs are expressed as mean +/− standard error (S.E.M.). Densitometry of PCR and western blot bands was carried out with Image Lab software (BioRad). Statistical analysis of differences between groups was by one way ANOVA with post-hoc Tukey’s test or Student’s t-test. Analysis was carried out with GraphPad Prism software (GraphPad).RNA-seq data was mapped using Tophat2 v2.0.12 .


The detailed investigation reveals a unique innersphere interaction to uracil and extends the knowledge of the influence of metal ions for conformational changes in RNA structure. Furthermore, we could demonstrate that an accurate localization of the metal ions in the X-ray structures require the consideration of several crystallographic and geometrical parameters as well as the anomalous difference map.Due to the polyanionic nature of RNA, the principles of charge neutralization and electrostatic condensation require that cations help to overcome the repulsive forces in order for RNA to adopt a three-dimensional structure. A precise structural knowledge of RNA-metal ion interactions is crucial to understand the mechanism of metal ions in the catalytic or regulatory activity of RNA. We solved the crystal structure of an octameric RNA duplex in the presence of the di- and trivalent metal ions Ca Metal ions play a crucial role in the folding of RNA and its catalytic mechanism, which are central in RNA biology. Folded RNA is involved in almost every aspect of cellular metabolism, including protein synthesis, RNA splicing, catalysis, and gene regulation ,2,3,4, wMetal Ions in Nucleic AcidS) database [Nucleic acids, with their negative charge, are excellent targets for metal ions and metal-containing compounds. Most of the metal ions interact non-covalently with RNA, for example by electrostatic attraction, outersphere binding via hydrogen bonds, π–π interactions between a ligand of the metal complex and the nucleobases, or shape selective binding to the grooves ,8. A secdatabase  allows sThere is no ideal spectroscopic or biochemical method that provides in a single experiment details of metal ion occupation sites, specific ligands environment, and structural response of nucleic acids to cations. Biochemical and chemical methods predict metal ion binding sites and their involvement in catalytic activities of RNA, but this requires either RNA modification or cleavage, or the exchange of the natural cofactor by another metal ion ,12,13. NX-ray crystallography is an excellent method to locate metal ions precisely. Nevertheless, small molecules and metal ions are often misinterpreted in macromolecular structures if only parameters as distances, coordination numbers, B-factors, and valence are verified. A recent study reported that around 10% of the metal ion binding sites in macromolecular structures are problematic and over 30% cannot be identified with sufficient evidence. The CheckMyMetal web server  uses eig2+ binding site and an uncommon interaction of most tested metal ions to O4 of uracil in an octameric RNA duplex. From the crystal structure in the presence of Ca2+ we could collect a high quality and redundant data set to get sufficient anomalous signal to solve the crystal with native-SAD . Furthermore, five octameric RNA crystal structures were solved with metal ions appearing less relevant in the context of nucleic acid binding, although they are often applied as chemical probes in nucleic acid biochemistry, as crystallization agents, or as Mg2+-mimicking atoms. Therefore, it was important to determine the sites of metal ion binding with confidence, which was achieved by collecting sufficient anomalous signals of four metal ions at different wavelengths together with careful inspection of the B-factor, valence bond parameter [In a previous study 13 different metal ions were localized in two 23-nucleotide long subtypes of the RNA dimerization initiation site (DIS) in the human immunodeficiency virus (HIV)-1 RNA genome. Despite similar sequences the two structures showed different metal preferences and binding sites . We extearameter , and met2+, Sr2+, and Tb3+ have a slightly bigger major groove width  and afterwards soaked in solutions containing either Mn2+, Cu2+, or Tb3+. We measured all solved crystal structures at the corresponding X-ray absorption edge to get a strong anomalous signal. We could thereby unambiguously localize the ions by calculating anomalous difference maps in various resolution ranges. Further we verified that the B-factor of the metal and the environment are in the same range and we considered the distance and coordination to surrounding waters  [3+. In other macromolecules this interaction is frequently found for Mn2+ and Co2+, as suggested by the MINAS database [3+, Ca2+, and Sr2+ to phosphate oxygens, as well as Co2+ and Cu2+ coordination to N7 of guanine [In addition to the innersphere binding to the phosphate oxygen of guanine, there is an uncommon innersphere binding to O4 of uracil by Caand Cu2+ . While ink (PDB) . Outerspdatabase . Our stu guanine ,31, alth2+, the unusual elongated bond lengths to the coordinating waters [2+ is located at this position  at this position. This was quantitatively demonstrated by integrating the anomalous difference map and by determining the occupancy of Cu soaking .Chemically synthesized and desalted RNA oligonucleotides with the sequence 5′-UCGUACGA were purchased from Microsynth, Switzerland in a quantity of 2 μmol and purified by denaturing 20% polyacrylamide gel electrophoresis (PAGE) following standard procedures . The exc2+ grew after two days in a solution consisting of 200 mM CaCl2, 28% PEG 400, 2 mM spermine, and 50 mM hepes sodium buffer (pH 7.5). Soaking was done in reservoir solutions with 100 mM MnCl2, 2 mM TbCl3, or 20 mM CuCl2, instead of CaCl2, for 24 h. The conditions for the two co-crystallized RNA were 2 mM CoCl2, 20 mM MgSO4, 25% MPD, 0.5 mM spermine, and 50 mM potassium cacodylate (pH 6.0)  and 200 mM SrCl2, 200 mM ammonium acetate, 10 mM magnesium acetate, 28% polyethylene glycol (PEG) 8000, and 50 mM sodium cacodylate pH 6.5 . Crystals were looped in cryosolution containing reservoir solution with 25% glycerol and flash-frozen in liquid nitrogen.RNA (0.6 mM) was annealed for 1 min at 50 °C in water and cooled to room temperature. Crystals were grown by the hanging drop vapor diffusion method in a 1:1 mixture of RNA and reservoir solution. The colorless needle-shaped crystals in the presence of Ca2+ was solved with native-SAD at a wavelength of 1.61 Å (List 1). The long needle-shaped crystals were well-suited for measuring at different positions to prevent radiation damage and to get highly redundant data of high quality. All the other crystal structures were solved by molecular replacement using MOLREP [Single crystal datasets were collected by beamline X06DA Swiss Light Source  at 100 K with a PILATUS 2M-F detector . Data were processed with XDS  and scalg MOLREP . Refinemg MOLREP  and moleg MOLREP . FurtherList 1. Statistics of sub-structure determination and phasing (Ca2+ data).weak (%)/CCall (%) (for the top solution)SHELXD CC48.8/29.3SHELXD CFOM (for the top solution)78.1SHELXD PATFOM (for the top solution)18.6Number of correct sites (for the top solution)14SHELXE CC (%)69.20SHELXE FOM0.656PHASER EP FOM0.697Map CC (%) 82.4The ideal A-RNA duplex was predicted with the web-accessible tool RNAComposer .List 1.2+, as the proposed M-O distance was 2.62 Å due to the longer ionic radii. For outersphere binding a maximum distance of 3.2 Å from water to RNA was set. The bond valence parameter as described in [i by iv = ∑exp((R0 − Ri)/b), where R0 is a constant describing ideal distance, if the bond valence is 1, and b is an empirical constant. Both values are reported in [The identity of metal ions with sufficient anomalous scattering was confirmed by calculating an anomalous difference map in various resolution ranges with PHENIX . The M–Oribed in  is calcuorted in . The bon2+ the anomalous difference map was integrated with Mapman [2+ and P were used to determine the ratio of their anomalous signals at the measured wavelength of 1.37 Å. The integrated value for phosphorus was then multiplied by this factor. For phosphorus, a 100% occupancy was assumed to calculate the relative occupancy for Cu2+.For quantification of the anomalous signal of Cuh Mapman  to add u2+, Co2+, and Cu2+.Here we describe a detailed study of six different metal ions coordinating to a short RNA duplex. The cations have a rather strong influence on RNA structure, although none of the metal ions employed shows a specific individual structural influence. Different parameters were considered to carefully assign the positions of the individual metal ions, yielding two prevalent positions in the octameric RNA duplex. The first is coordinated to the phosphate backbone, the second cation binds in the major groove of the RNA, interacting by a particular innersphere coordination to O4 of uracil in the presence of CaFinally, this study demonstrates the importance of considering several parameters when assigning metal ion positions in X-ray structures. Even when there is a strong anomalous signal present, we have to inspect other geometrical and crystallographic parameters for an accurate localization. On the other hand, geometrical consideration without examining the anomalous signal or other crystallographic parameters may lead to wrong interpretations as the geometry could be distorted by structural constraints of the RNA due to the metal ions or crystal packaging effects.