RNA–protein interactions play important roles in gene expression control. These interactions are mediated by several recurring RNA-binding motifs including a well-known and characterized ribonucleoprotein motif or so-called RNA recognition motif (RRM).nrb, SART3, or p110, using a RNA-based yeast three-hybrid cloning strategy. Six putative RNA targets were isolated and found to contain a consensus sequence that was identical to nucleotides 34–46 of U6 small nuclear RNA. Tip110 binding to U6 was confirmed to be specific and RRM-dependent in an electrophoretic mobility shift assay. Both in vitro pre-mRNA splicing assay and in vivo splicing-dependent reporter gene assay showed that the pre-mRNA splicing was correlated with Tip110 expression. Moreover, Tip110 was found in the spliceosomes containing pre-spliced pre-mRNA and spliced mRNA products. Nonetheless, the RRM-deleted mutant (ΔRRM) that did not bind to U6 showed promotion in vitro pre-mRNA splicing, whereas the nuclear localization signal (NLS)-deleted mutant ΔNLS that bound to U6 promoted the pre-mRNA splicing both in vitro and in vivo. Lastly, RNA-Seq analysis confirmed that Tip110 regulated a number of gene pre-mRNA splicing including several splicing factors.In the current study, we set out to identify the RNA ligand(s) of a RRM-containing protein Tip110, also known as p110Taken together, these results demonstrate that Tip110 is directly involved in constitutive eukaryotic pre-mRNA splicing, likely through its binding to U6 and regulation of other splicing factors, and provide further evidence to support the global roles of Tip110 in regulation of host gene expression. In eukaryotic cells, gene expression begins in nucleus with transcription of protein-coding genes to primary RNA transcripts, or so-called pre-messenger RNA (pre-mRNA) or heterogeneous nuclear RNA (hnRNA) . The presnRNP are important components of the spliceosomes in which the pre-mRNA splicing occurs . There anrb, SART3, or p110, has been proposed to serve several important biological functions. These include tumor antigen function [Tip110, also known as p110function –18, contfunction –21, regufunction , 23, andfunction –26. In afunction , U4/U6 sfunction , 28, YB-function , and C-Mfunction  and has function , 30, 31.2 incubator. Cells were transfected using the standard calcium phosphate precipitation method.293T and Hela cells were purchased from American Tissue Culture Collection  and maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, in a 37°C, 5% COGA ATT CAC CAT GGC GAC TGC GGC CGA A-3′ and 5′-CCG CTC GAG TCA ATG ATG ATG ATG ATG ATG GGC AAC TGC AGG AGC CG-3′ (the restriction enzyme sites underlined). The Tip110 siRNA expression plasmid was constructed in the backbone of pSHAG-1 by annealing oligonucleotides 5′-CGGGAT CCG ACT CAG CCT CGG GTT CTG AA-3′ and 5′-CGG GAT CCA AAA AAT TGG ACT CAG CCT CA-3′, and inserting the annealed DNA at the Bam HI site of pSHAG-1. All recombinant plasmids and deletion mutants were verified by sequencing. The sources of the other plasmids used in the study are: pGEX-4T-3 from Amersham , pSHAG-1 from Dr. Gregory Hannon, pIIIA.MS2 from Dr. Marvin Wickens, pT7.U6 from Dr. Iain Mattaji, pDM138 from Dr. Thomas Hope, pcRev from Dr. Bryan Cullen, and pSP64-Hβδ6 from Dr. Adrian Krainer.Plasmids pTip110.His, pTip110s.His, and deletion mutants ∆NLS and ∆RRM, and pGEX-Tip110, pGEX-Tip110s, and deletion mutants ∆NLS and ∆RRM, and pAD.Tip110 were described elsewhere . The staA hybrid RNA library and yeast three-hybrid screening were performed as previously described , with soE. coli BL21, as previously described [d-thiogalactopyranoside for 2 h at 37°C. GST fusion proteins were purified using a Pierce GST fusion protein purification kit . When necessary, GST was removed by treating the eluted protein with thrombin protease (10 units)  at room temperature for 18 h. The digested protein solution was dialyzed overnight in 4 l of phosphate-buffered saline and cleared of GST protein by additional incubations with fresh glutathione beads. The purified proteins were electrophoresed on a 8% SDS-polyacrylamide gel and stained with Gold–Blue (Pierce) to ensure the complete removal of GST protein as well as undigested fusion protein and other contaminated proteins.GST-Tip110, GST-Tip110s, Tip110 deletion mutants ∆NLS and ∆RRM, and GST proteins were expressed in and purified from escribed . Briefly32P-labeled UTP in the presence of T7 RNA polymerase at 30°C for 1 h, followed by removal of free nucleotides by an CentriSpin column . 32P-labeled-U6 RNA was then mixed with purified GST-Tip110 or its mutant proteins in a total volume of 20 μl containing 1 mM HEPES, pH 7.6, 1 μM MgCl2, 16 mM KCl, 2.5% glycerol (v/v), and 10 nM DTT. The mixture was incubated on ice for 15 min and then subjected to 5% nondenaturing polyacrylamide gel electrophoresis  in the 0.5× TBE buffer. The gel was dried and exposed for autoradiography.U6 RNA was synthesized using an Ambion in vitro transcription kit . Briefly, pT7-U6 plasmid was linearized with Dra I and incubated with ATP, CTP, GTP and 32P-labeled UTP at 37°C for 2 h. 32P-labeled pre-mRNA was then purified on a 5% denaturing polyacrylamide gel and suspended in 10 mM Tris.HCl, pH 7.9. The 32P-labeled pre-mRNA was then aliquoted and stored at −80°C. Splicing reactions were carried out in a 25 μl volume containing 12.8 μM MgCl2, 500 μM ATP, 20 mM creatine phosphate, 2.7% (w/v) polyvinyl alcohol, 1,000 U/ml RNasin, 12.8 mM HEPES, pH 8.0, 14% (v/v) glycerol, 62 mM KCl, 0.12 mM EDTA, and 0.7 mM DTT at 30°C for 2 h. The reactions contained 6 μl Hela nuclear extract  and 12.5 ng 32P-labeled pre-mRNA prepared above.Human β-globin minigene pre-mRNA was synthesized using an Ambion in vitro capped RNA transcription kit (Ambion). Briefly, pSP64-Hßδ6 plasmid was linearized with BamH I and incubated with ATP, CTP, GTP, cap anolog and 2 with a purity of higher than 99%.Anti-Tip110 monoclonal antibodies were produced by Promab Biotechnologies, Inc. . Briefly, purified GST-Tip110 protein was used to immunize BALB/c mice. The mice were given an additional boost immunization after 3 weeks. Three days after the boost, the spleen cells were isolated from the immunized mice and fused with SP20 cells (ATCC) using polyethylene glycol . Resulting hybridoma were screened to be specific anti-Tip110 antibody using ELISA. To purify the monoclonal antibody, the hybridoma cells were cultured in a production module of the miniPERM device , and the antibody was further purified from the culture supernatant using a Protein A column and a AKTA prime-automated liquid chromatography system . The antibody protein purity was determined to be IgGPurified Tip110 monoclonal antibody (20 μg) was incubated with 50 μl of 50% slurry protein A-Sepharose (Amersham) in a buffer containing 20 mM HEPES, pH 8.0, 100 mM KCl, 0.2 mM EDTA, 20% (v/v) glycerol, 0.5 mM PMSF, 1 mM DTT at 4°C for 4 h with continuous mixing. The protein A beads were then recovered by centrifugation and repeated washes with the same incubation buffer to remove excess anti-Tip110 antibody. An aliquot of the beads were then incubated with Hela nuclear extract at 4°C for 2 h with continuous mixing, and the supernatant was used as Tip110-depleted Hela nuclear extract. Tip110 protein on the immunoabsorbed beads was eluted by adding 20 μl of 4× SDS sample buffer, followed by boiling 5 min. The eluent and aliquot of the Tip110-depleted Hela nuclear extract were subjected to 10% SDS-PAGE gel and Western blot analysis to ensure efficient Tip110 depletion.6 cells/well and 1 day later transfected with 1 μg pDM138, 1 μg pcRev, 1–5 μg pTip110.His or pTip110.siRNA. In all transfections, a CMVβGal plasmid was included to normalize transfection variations, and pcDNA3 was used to equalize the amount of transfected DNA. Forty-eight hours after transfection, the cells were harvested and subjected to 3 rounds of freeze–thaw in 0.25 M Tris.HCl, pH 8.0, and the lysates were collected for β-galactosidase and CAT activities. β-galactosidase was determined using a colorimtric assay [293T cells were plated in a 6-well plate at a density of 0.72 × 10ic assay , while Cic assay .4, 150 mM NaCl, 1% Triton X-100, 0.1% SDS, 0.2% sodium azide, 0.5% sodium deoxycholate, 0.004% sodium fluoride, and 1 mM sodium orthovanadate and incubated on ice for 10 min. Cell lysates were obtained by centrifugation, removal of the cell debris, and electrophoretically separated on 10% SDS-PAGE and analyzed by immunoblotting. Anti-His antibody was from Qiagen , while β-actin was from Santa Cruz Biotech. . The blots were first probed with primary antibodies, followed by appropriate horseradish peroxidase-conjugated secondary antibodies, and then visualized with the ECL system (Amersham).Cells were washed twice with ice-cold phosphate-buffered saline (PBS), and cell pellets were suspended in two volumes of whole cell lysis buffer containing10 mM NaHPOHela were transfected with human Tip110 siRNA or the universal control siRNA. Total RNA was purified from transfected cells using Trizol according to the manufacturer’s instructions  and used for RNA-Seq. The RNA-Seq was performed at the Genomic Core of UT Southwestern Medical Center, Dallas, TX, USA. The RNA-Seq reads were aligned to the human genome sequences for further differential and spliceosome pathway analysis using the Panther Pathway Analysis software.®Green PCR kit (Life Technologies) according to the manufacturer’s instructions. The qPCR program consisted of one cycle of 95°C for 10 min, 40 cycles of 95°C for 15 s and 60°C for 1 min.Total RNA was extracted using Trizol and used to synthesize cDNA using a ScriptII RT reagent kit (Promega). cDNAs were used for qPCR using a Power SybrTo determine if Tip110 bound to specific RNAs, we took advantage of a RNA–protein interaction-based yeast three-hybrid cloning strategy . This ge32P-UTP and used the 32P-labeled U6 RNA as a probe. We then incubated 1, 5, 10 and 50 ng purified recombinant GST-Tip110 protein with 32P-labeled U6 RNA and determined Tip110-U6 complex formation using a gel mobility shift assay. The same amounts of purified GST protein were included as a control in the experiments. Complex formation between Tip110 and U6 became evident when 5 ng Tip110 was added and increased significantly when more Tip110 protein was present  and determined Tip110-U6 complex formation using the same assay. Increasing amounts of unlabeled U6 effectively competed out 32P-labeled U6 for Tip110 binding, as Tip110 and 32P-labeled U6 no longer formed a complex in the presence of unlabeled U6 RNA  at aa601–650 [To ascertain Tip110 binding to U6 RNA, we synthesized U6 RNA in vitro in the presence of α-a601–650 . To detea601–650 . CompareSmall nuclear U6 RNA directly participates in pre-mRNA splicing . Our earTo determine the significance of Tip110 function in pre-mRNA splicing, we decided to immunodeplete Tip110 protein from the Hela nuclear extract that was used in the in vitro splicing assay. Thus, we first generated anti-human Tip110 monoclonal antibodies using recombinant GST-Tip110. We then incubated Hela nuclear extract with anti-human Tip110 antibodies and protein-A agarose beads. Tip110-depleted Hela nuclear extract was recovered by brief centrifugation, followed by removal of the beads. An isotype-matched IgG was included as a control. Western blot analysis showed that immunodepletion efficiently removed Tip110 protein from Hela nuclear extract Figure a. We usein trans. We reasoned that if Tip110 enhanced pre-mRNA splicing, Tip110 expression would negate HIV-1 Rev-dependent expression of intron-containing pre-mRNA. Thus, we transfected 293T cells with DM138 alone, or in combination with HIV-1 Rev plasmid and increasing amount of Tip110 expression plasmid. We then determined CAT activity. As expected, Rev expression significantly improved CAT gene expression . Using the Panther pathways analysis, those genes were mainly grouped as the following categories: protein-binding transcription factors (37%), catalytic molecules (35%), nucleic acid-binding transcription factors (9%), structural molecules (6%), and receptors (4%) . The cells transfected with Tip110 siRNA and control siRNA were compared for the alternative gene spliced transcripts. Bioinformatics analysis of the RNA transcripts indicated that Tip110 knockdown led to changes of alternative spliced RNA transcripts of 17,498 genes with statistical significant difference (In this study we aimed to identify the RNA targets for Tip110 binding. We demonstrated that Tip110 specifically interacted with small nuclear U6 RNA through the yeast 3-hybrid screen and RNA EMSA Figures . Tip110 Biogenesis of the snRNP U1, U2, U4, U5, and U6 is a complex process. Newly synthesized snRNP undergo various modification and assembly, and then export from nuclear to cytoplasm where they mature and transport back into the Cajal bodies in the nucleus. snRNA/snRNP then recruit other splicing factors, undergo further assembly and maturation in the Cajal bodies –44. Our After exiting Cajal bodies, mature snRNP enter nuclear speckles. Nuclear speckles are sites for pre-mRNA splicing that serve as storage and/or modification sites for splicing factors . Our preTo further determine the significance of Tip110 involvement in pre-mRNA splicing, we performed RNA-Seq analysis of the alternate pre-mRNA splicing transcripts. As expected, Tip110 knockdown led to alterations in a large number of the gene transcripts including four known genes in pre-mRNA splicing Figure . Tip110


Gene expression profiling is being widely applied in cancer research to identify biomarkers for clinical endpoint prediction. Since RNA-seq provides a powerful tool for transcriptome-based applications beyond the limitations of microarrays, we sought to systematically evaluate the performance of RNA-seq-based and microarray-based classifiers in this MAQC-III/SEQC study for clinical endpoint prediction using neuroblastoma as a model.We generate gene expression profiles from 498 primary neuroblastomas using both RNA-seq and 44 k microarrays. Characterization of the neuroblastoma transcriptome by RNA-seq reveals that more than 48,000 genes and 200,000 transcripts are being expressed in this malignancy. We also find that RNA-seq provides much more detailed information on specific transcript expression patterns in clinico-genetic neuroblastoma subgroups than microarrays. To systematically compare the power of RNA-seq and microarray-based models in predicting clinical endpoints, we divide the cohort randomly into training and validation sets and develop 360 predictive models on six clinical endpoints of varying predictability. Evaluation of factors potentially affecting model performances reveals that prediction accuracies are most strongly influenced by the nature of the clinical endpoint, whereas technological platforms (RNA-seq vs. microarrays), RNA-seq data analysis pipelines, and feature levels (gene vs. transcript vs. exon-junction level) do not significantly affect performances of the models.We demonstrate that RNA-seq outperforms microarrays in determining the transcriptomic characteristics of cancer, while RNA-seq and microarray-based models perform similarly in clinical endpoint prediction. Our findings may be valuable to guide future studies on the development of gene expression-based predictive models and their implementation in clinical practice.The online version of this article (doi:10.1186/s13059-015-0694-1) contains supplementary material, which is available to authorized users. Microarray-based gene expression profiling is being widely applied in cancer research to identify biomarkers for clinical endpoint prediction, such as diagnosis, prognosis, or prediction of treatment response –5. The cThe advent of next-generation sequencing technologies has revolutionized eukaryotic transcriptome analysis. RNA deep-sequencing (RNA-seq) provides a powerful tool to decipher global gene expression patterns far beyond the limitations of microarrays, including an unprecedented capability to discover novel genes, alternative transcript variants, chimeric transcripts, and expressed sequence variants as well as allele-specific expression –12. RNA-MYCN proto-oncogene. In addition, a number of microarray-based gene expression models have been proposed to predict neuroblastoma patient outcome [In this study of the Sequencing Quality Control (SEQC) consortium, we therefore aimed to systematically investigate the potential of RNA-seq-based classifiers in predicting clinical endpoints in comparison to microarrays. To this end, we selected neuroblastoma as a model, a pediatric malignancy arising from the developing sympathetic nervous system . The cli outcome , 17. How outcome , the pre outcome –20.Here, we determined global gene expression profiles from 498 primary neuroblastoma samples using both RNA-seq and Agilent’s 44 k oligonucleotide-microarrays to compare the performance of RNA-seq and microarray-based models in predicting clinical endpoints. We generated 360 gene expression-based models using a broad range of algorithms to predict six different endpoints with a varying degree of predictability, and analyzed the effects of a range of variables on the prediction performances. We found that prediction accuracies were most strongly influenced by the nature of the clinical endpoint, whereas neither the expression profiling technology nor the RNA-seq data analysis pipeline affected prediction accuracy systematically. To our knowledge, we present the first study on the evaluation of predictive models using RNA-seq in comparison to microarrays, which may provide valuable information for designing future experiments on gene expression-based classifiers using high-throughput technologies.To comprehensively characterize the neuroblastoma transcriptome, we sequenced 30,753,066,000 reads from 498 primary neuroblastoma samples covering the entire spectrum of the disease Table . DiscontIn total, 88.75 % of the aligned reads mapped to genes annotated in either of the reference databases RefSeq , AceView , or Gencode n = 56,071), while 4.52 % of the reads mapped to newly discovered exons or exon-junctions, 5.91 % to intronic and 0.65 % to intergenic sequences Fig. . In the , while 4P value cutoff (MAQC-I) [U test  , 24, 25;NF1 and MDM4 ; the belonging to a patient subgroup with extreme disease outcome ; the occurrence of events, that is, progression, relapse, or death (EFS ALL); the occurrence of death from disease (OS ALL); and the occurrence of events (EFS HR) and death from disease (OS HR) in the subset of current high-risk patients  mapP = 0.043), while models based on the two platforms performed similarly well in predicting the remaining five endpoints , n = 3), stage 2 , stage 3 , stage 4 , stage 4S . Events were defined according to a revised version of the International Neuroblastoma Response Criteria [This project comprised tumor samples of 498 neuroblastoma patients from seven countries: Belgium (n = 1), Germany (n = 420), Israel (n = 11), Italy (n = 5), Spain (n = 14), United Kingdom (n = 5), and United States (n = 42). All patients were registered in respective clinical trials with informed consent. The patients’ age at diagnosis varied from 0 to 295.5 months . Tumor stage was classified according to the International Neuroblastoma Staging System (INSS): stage 1 . Magic includes quality control, alignment, annotation, quantification, and normalization of RNA-seq data. Magic maps the read pairs in parallel on all targets, including the genome (NCBI 37), the RefSeq 37.104, the Gencode v15, and the AceView 2011 gene models. Gene expression is measured in sFPKM, adding to the standard FPKM (fragments per kilobase of transcript per million mapped reads) a gene-specific and experiment-specific threshold of significance (sFPKM) for low counts, together with a number of batch effect corrections. The Magic-AceView pipeline is described in detail as a Supplementary Note 1  is based on the Magic analysis tool  (ftp://fFor the TopHat-AceView (TAV) pipeline, raw reads of RNA-seq were filtered using an in-house pipeline . Clean RNA reads were aligned to the human genome (hg19) using TopHat . CufflinFor the TopHat-UCSC pipeline (TUC), RNA-seq reads were mapped by TopHat  to a refIn addition to the microarray expression profiles, three different RNA-seq processing pipelines were applied to generate expression profiles from the sequencing raw data.Six data analysis teams received 10 expression profiles (nine profiles based on RNA-seq data and one profile based on microarray data) to predict six different endpoints as extensively described in the Results. Data analysts were asked to include a five-fold cross validation for 10 iterations to assess model performances in the training datasets, but were otherwise free in their choice of modeling algorithms and statistical tests to generate and select suitable prediction models. Models with highest average performance metrics were selected and submitted for testing on adequate blinded validation sets for each endpoint. More details and an overview of the applied classification algorithms are given in Additional file P <0.05 as significant. All analyses are regarded as explorative.Matthew’s correlation coefficient (MCC) was used to evaluate the prediction performance of classification models as described . Differewww.ncbi.nlm.nih.gov/geo/) with accession numbers GSE49710 and GSE49711, respectively, which are included in SEQC Project SuperSeries GSE47792.Microarray and RNA-seq data can be accessed from the GEO database (


In the past decade, an increasing number of studies have begun to focus on the molecular pathogenesis of FMDV NSPs and NCEs. This review collected recent research progress on the biological functions of these NSPs and NCEs on the replication and host cellular regulation of FMDV to understand the molecular mechanism of host–FMDV interactions and provide perspectives for antiviral strategy and development of novel vaccines.Foot-and-mouth disease virus (FMDV) represses host translation machinery, blocks protein secretion, and cleaves cellular proteins associated with signal transduction and the innate immune response to infection. Non-structural proteins (NSPs) and non-coding elements (NCEs) of FMDV play a critical role in these biological processes. The FMDV virion consists of capsid and nucleic acid. The virus genome is a positive single stranded RNA and encodes a single long open reading frame (ORF) flanked by a long structured 5ʹ-untranslated region (5ʹ-UTR) and a short 3ʹ-UTR. The ORF is translated into a polypeptide chain and processed into four structural proteins , 10 NSPs (L Aphthovirus genus, Picornaviridae family. FMDV is one of the most contagious viruses in cloven-hoofed animals and can cause both acute and prolonged, asymptomatic but persistent infection [Foot-and-mouth disease (FMD), an acute highly contagious viral disease in susceptible cloven-hoofed animals, was described 100 years ago. The etiologic agent, FMD virus (FMDV), is a positive-sense, single-stranded RNA virus that belongs to the The RNA virus genome of FMDV displays a very high mutation rate because the virus-encoded RNA polymerase lacks a proofreading mechanism , 3. The pro) and 3Cpro) to yield structural and NSPs [FMDV virion has a symmetric protein shell (or capsid) enclosing the genomic RNA. Genome RNA contains a positive single-strand chain approximately 8.3 kb long and encodes a single long open reading frame (ORF) of about 7 kb with two alternative initiation sites. The ORF is flanked by a long 5ʹ-untranslated region (5ʹ-UTR) and a short 3ʹ-UTR, and ends with a genetically encoded poly-(A) tail . A genomand NSPs  Fig. 1)pro) and pro. P1 region, encoding a precursor for capsid polypeptide, which can generate four mature capsid proteins  upon cleavage by viral protease. P2 region encodes three viral proteins  in the middle region of the genome. And P3 region, which encodes four viral proteins: 3A, 3B, 3Cpro and 3Dpol, in which, 3C is a viral protease and 3D an RNA-dependent RNA polymerase [pro, P1-2A, 2BC and P3 by Lpro, 2A and 3Cpro. The precursors P1-2A, 2BC and P3 are further processed into mature viral proteins and some cleavage intermediates with relative stability, such as VP0 or 1AB, 3ABC, 3BCD, 3AB, and 3CD by 3Cpro , which are self-assembled into an icosahedral structure with a diameter of 30 nm , 13  and NSPs play essential roles in FMDV-induced viral evasion. This review focus on the molecular biology of FMDV, along with the functional roles of FMDV NSPs and NCEs in viral replication and virulence, to elucidate how FMDV evades the host immune response and evolves into such an aggressive pathogen.The 5ʹ-UTR is a central element that initiates replication and translation of the picornavirus genome. Similar to other picornaviruses, the FMDV RNA genome does not contain the 5ʹ-terminal cap structure (m7GpppN…), which can be recognized by the translation initiation machinery in all eukaryotic cells . InsteadFMDV RNA, which consists of a long 5ʹ-UTR containing over 1300 nucleotides (nt), exhibits extensive secondary structure and is commonly divided into five regions  tract exhibiting a variable length of 150–200 nt in typical field strains. Investigations on genetically engineered viral genome revealed that a certain threshold length of poly(C) is required to rescue live virus, although no evidence has shown that poly(C) tract length is directly associated with virulence . On the bus), also known as cis-acting replication element (cre), which is a highly conserved stem-loop of 55 nt and is essential for viral genome RNA replication [bus elements contain a conserved “AAACA” motif in the loop region. This motif acts as the template for uridylylation of VPg (3B) to produce VPgpU and/or VPgpUpU, collectively termed as VPgpU(pU), by the viral RNA polymerase 3D [bus structures are embedded in protein-coding regions of the viral genome, except for that of FMDV, which has a bus structure that lies in the 5ʹ-UTR region [bus sequence [cre” also functions in trans.Downstream of the PK region lies a 3B-uridylylation site  element, a type II IRES element . Consistcis-acting RNA sequence that adopts diverse three-dimensional structures to recruit the translation machinery using a mechanism that is independent on the 5′end [pro removes its N-terminal portion, the binding sites for eIF4E, impairing cap-dependent protein synthesis in host cells. By contrast, the C-terminal portion of eIF4G retains the binding sites for eIF4A and eIF3, which is sufficient for FMDV IRES activity.The featured IRES element is an essential structural region for initiation of protein synthesis in picornavirus genome RNA , 35, 36.he 5′end . Nearly he 5′end , 38, conHowever, viral IRES are characterized by the presence of ignored AUGs upstream of the functional start codon, heavy RNA structure and high GC content , 40. DesIn addition to the eIFs described above, many other cellular proteins are also involved in the modulation (stimulate or repress) of IRES activity. They are all termed IRES-transacting factors (ITAFs), including PTB, PCBP2, the SR splicing factor (SRp20), the far upstream element binding protein 2 (FBP2), the lupus antigen (La), unr (upstream of N-ras), nucleolin, or Gemin5, etc .The polypyrimidine PTB was the first protein identified as an ITAF , 47, 48.As a consequence of in depth RNA–protein interaction studies performed with picornavirus IRES, the list of ITAFs is still growing incessantly . The GlyFMDV RNA 3ʹ-UTR consists of two components, a structural sequence of 90 nt folding into two separate stem-loops and a poly(A) tail with variable length , 57 Fig. These eIn addition to direct RNA-RNA interaction, 5′–3′ end bridges could also involve protein-protein and protein–RNA interaction. Studies found that cellular proteins PCBPs and p47 can both interact with the S region and 3ʹ-UTR by directly binding to them . As mentpro is a region in the polyprotein preceding the capsid precursor protein [pro, the first protein in FMDV to be translated, is initiated at two different start codon AUGs separated by 84 nt. Lpro has two alternative forms, namely, Labpro and Lbpro. Both forms have been detected in vitro and in vivo [pro protein (synthesized from the second AUG) is the major protein type in vivo. Viable viruses can be recovered from synthetic genomes containing mutations in the first AUG but not in the second [pro is a well-characterized papain-like proteinase [The viral protein L protein . Lpro, t in vivo , 61. Lbpe second , 63. Thee second , 64. FMDoteinase –67 that oteinase , 68.pro is an important determinant of virulence. Previous studies showed that the Lpro-deleted virus shows only a slightly slower replication rate than the wild-type (WT) virus [pro is not required for viral replication but is indispensable for the pathogenesis of FMDV.LT) virus  but exerT) virus  and failT) virus , 71. Thupro also represses host cell translation by cleaving the translation initiation factor eIF4G [pro recognizes and cleaves the crucial host translation initiation factor eIF4G at the site between Gly479 and Arg480 [pro does not impair viral RNA translation initiation. Thus, FMDV freely uses the host protein synthesis machinery to synthesize viral protein  activity directly and indirectly by inhibiting the central upstream regulatory factor [pro coding region, induces higher levels of type I IFN (IFN-α and IFN-β) mRNA level than WT virus, and type I IFN downstream signaling can only be detected in cultures with the leaderless virus infection [pro. Lpro also inhibits dsRNA-induced IFN-λ1 expression, which is a type III IFN demonstrating significant antiviral activity against FMDV [Ly factor –77. IFN y factor . Secretey factor , includinfection , 76, 80.pro. Translational repression of IFNs is probably resulted from the blocking of FMDV Lpro-induced cap-dependent mRNA translation by Lpro-mediated cleavage of eIF4G [pro virulence. Promoter activity and protein studies have shown that FMDV Lpro down-regulates interferon regulatory factor 3/7 (IRF-3/7) expression both at the transcription and translation levels [No evidence demonstrated the direct cleavage of IFNs by Lof eIF4G , 81. Furn levels , 83. IRFn levels . Their in levels , 83.pro is a novel viral deubiquitylation (DUB) enzyme [pro, the shorter form of Lpro, in all seven serotypes. The topology of FMDV Lbpro is remarkably similar to that of some known DUBs, such as ubiquitin-specific protease 14, a cellular DUB [pro), a coronaviral DUB [pro can remove ubiquitin (Ub) moieties from cellular substrates, function on both lysine-48- and lysine-63-linked polyubiquitin chains, a feature shared by other known viral DUBs, such as human cytomegalovirus UL48, herpes simplex virus type 1 UL36, and SARS-CoV PLpro [pro exhibits deubiquitinating activity. A new mechanism of Lpro blocking IFN antiviral response is revealed by the evidence that Lpro antagonizes type I IFN induction by deubiquitinating the critical signaling components RIG-I, TBK1, TRAF3, and TRAF6 [A recent study found that FMDV Lb) enzyme . Ubiquit) enzyme . Viruses) enzyme , 88. Seqular DUB , as welliral DUB . In addioV PLpro , 92, indnd TRAF6 .pro generally functions as a multifunctional protein that blocks IFN-mediated antiviral response via multiple distinct mechanisms  Lprpression ; (2) Lpr IRF-3/7 ; and (3)ignaling .Fig. 4Knpro or 3CDpro [The P2 portion in the picornavirus genome encodes three mature viral proteins, namely 2A, 2B, and 2C Fig. . FMDV 2Br 3CDpro .The FMDV 2A peptide, along with the first aa of the 2B protein, can mediate cleavage in artificial polyprotein systems , 95. HowPicornaviruses 2B are viroporins, a class of low-molecular-weight hydrophobic transmembrane proteins encoded by a wide range of animal viruses. The transmembrane hydrophobic domains interact with the phospholipid bilayer to induce dispersion, increasing membrane permeability and promoting the release of viral particles. Thus, viroporins are crucial for viral pathogenicity , 102.2+ abnormalities, activating autophagy [Knowledge on FMDV 2B is limited. FMDV 2B encodes a 154 aa peptide, which is slightly longer than other viroporins and contains two predicted putative transmembrane domains located in 83–104 aa and 119–137 aa, respectively . Topologutophagy . HoweverSome reports have indicated that 2B functions synergistically with other viral proteins, such as 2C, in FMDV . PreviouViroporins participate in multiple stages of the viral life cycle, such as cell entry and genome replication. Viruses with viroporin deletion cannot properly assemble and release from cells , 106. ThProtein 2C is one of the highly conserved molecules among the viral proteins encoded by FMDV, and 2C-like (2CL) proteins generally exist in many other RNA viruses across animals and plants . The FMDMany other functions of 2C in other picornavirus members have been reported, including viral RNA binding activity , 132, NTA recent report discovered that FMDV 2C is involved in apoptosis induction and type 1 IFN response. Yeast two-hybrid system and immunoprecipitation approaches revealed that FMDV 2C interacts with N-myc and STAT interacting (Nmi) protein , a factoet al. further investigated the interaction between FMDV 2C and cellular Beclin1 using a yeast two-hybrid model, immunoprecipitation and confocal microscopy. They found that FMDV 2C binds to Beclin1, a factor that plays dual roles in the autophagy pathway. Beclin1 is involved in initiation of autophagosome formation and fusion of autophagosome to lysosome [Some other research reported that FMDV 2C is involved in FMDV-induced autophagy. FMDV triggers cellular autophagy and enhances viral replication . Gladue lysosome , 149. Solysosome . 2C-Becllysosome .Viral 2C protein is speculated to act as an important regulator integrating multiple cell signaling during FMDV infection, including apoptosis, immune response, and autophagy. This protein reduces the cellular killing effect against viruses and promotes virus survival and proliferation, thereby facilitating viral proliferation and release of virus particles Fig. .FMDV 3A protein is a 153 aa peptide, which is larger than other picornaviral 3A protein, like the 87 aa-long poliovirus 3A . By contet al. [Based on its hydrophobic motifs, FMDV 3A exerts membrane binding activity . Fluoreset al.  generateet al. . In addiet al. .Reports demonstrated that 3A plays a role in virulence and host range. Some amino acids substitutions or deletions in 3A protein are associated with change in host range and tropism of FMDV , 155 andIn addition to 2C protein, 3ABC, the 3A precursor, is also used to differentiate potential carrier from vaccinated animals both in cattle and swine . Actuall1, 3B2, and 3B3), which are 23–24 aa long [Protein 3B, which is also known as VPg, is covalently bound to the 5ʹ terminus of the genome and antigenome and primes picornavirus RNA synthesis . In cont aa long . No natu aa long , althoug aa long , suggestpol). In this process, the viral 3Dpol catalyzes the binding of two uridine monophosphate (UMP) molecules to the hydroxyl group of this Tyr3 using as template a cis-replicating element (bus/cre) in FMDV genome [1, 3B2, and 3B3) can all be uridylylated in vitro, although 3B3 is likely the most efficient substrate for 3Dpol activity [in vitro using purified components, including VPg (3B) with 3Dpol, 3CD precursor, UTP, and an RNA template containing a stem-loop structure (bus) [Uridylylation of the VPg peptide primer is the first stage in the replication of the picornavirus genome Fig. . The picV genome . Three iactivity . VPg pepre (bus) . VPgpU(pre (bus) , 168.3 coding sequence exerts a deleterious effect on FMDV RNA replication, resulting in production of a non-infectious RNA transcript [1 and 3B2 also reduces viral RNA synthesis levels and infective particle formation in vitro, attenuats disease in pigs, but not drastically [3 is more important than 3B1 and 3B2 to maintain viral RNA replication, but co-existence of all three 3B copies exerts the best RNA replication efficiency. Whereas the underlying mechanisms of their respective roles on viral RNA replication and how the three copies are integrated during viral replication still require further investigation.A reverse genetics study demonstrated that deletion of the 3Banscript , 169. Lastically . These spro from P1, 2A cleavage between P1-2A and 2BC, and maturation cleavage of VP0 to VP2 and VP4, 3Cpro can efficiently process all other 10 cleavage sites in FMDV polyprotein although the rate of cleavage varies at different junctions [pro cleavage sites show great heterogeneity, with cleavage occurring between multiple dipeptides, including Gln-Gly, Glu-Gly, Gln-Leu, and Glu-Ser [pro cleavage site located exclusively between Gln-Gly and 3CDpro is implicated as the major viral proteinase in structural protein cleavage [FMDV 3C proteinase, responsible for most cleavages of viral polyprotein, was recently identified as a chymotrypsin-like cysteine protease , 171, alunctions . FMDV 3C Glu-Ser . By contcleavage , 177.pro is also associated with inhibition of host cell transcription and translation. As mentioned above, FMDV Lpro is involved in eIF4G cleavage, which shuts off host cell gene translation. FMDV 3Cpro can also cleave eIF4A, a portion of the cap-binding complex with the function of an RNA helicase [pro, FMDV 3Cpro cleaves eIF4G late in the infection cycle at an alternative site, although it may not be favorable for the translation of viral proteins at this stage [pro is the agent that cleaves histone H3 following FMDV infection. Histone H3 is an important component of nucleosome and is crucial in maintaining the conformation of nucleosomes, thereby affecting cellular transcription [pro also removes 20 N-terminal aa residues from histone H3, resulting in inhibition of host cell transcription [FMDV 3Chelicase . Compareis stage . Moreovecription . FMDV 3Ccription .pro can enter nuclei through its precursor 3CD, which contains a nuclear localization sequence (NLS) in the N-terminus of the 3Dpol protein [pro also cleaves multiple factors and regulators. Recent studies found that FMDV 3Cpro directly cleaves the 68 kDa Src-associated substrate during mitosis (Sam68), one of the nuclear RNA-binding proteins that participate in viral replication in cells. Immunofluorescent and immunoblot assays revealed that 3Cpro removes the NLS-containing C-terminus of the Sam68 protein (~18 kDa). The truncated Sam68 (~50 kDa) was subsequently redistributed into the cytoplasm [Similar to other picornaviruses, FMDV 3C protein . 3Cpro aytoplasm . Moreoveytoplasm .pro with innate immune regulation  essential modulator (NEMO), a bridging adaptor protein essential in activating both the NF-kB and IFN-regulatory factor signaling pathways [pro specifically targets the NEMO Gln383 residue, which lies between the C-terminal leucine zipper motif and a zinc finger (ZF) domain [Other reports associated FMDV 3Cion Fig. . Wang etpathways , to redu) domain . The ZF ) domain , 184.pro cleaves many other factors and regulators associated with cellular DNA-dependent RNA polymerases I, II, and III, such as TATA-box binding protein, octamer-binding protein, transcription activator p53, cyclic AMP-responsive element binding protein, and DNA polymerase III [pro may widely perturb gene transcription and translation in host cells.In addition, the picornavirus 3Crase III . Althougpol sequences are highly conserved among the different sero- and subtypes of FMDV [pol remains inactive until protein processing is complete [FMDV 3D protein, the virus-encoded RNA-dependent RNA polymerase (RdRP) , is the  of FMDV . Picornacomplete .pol shares similar structure and catalytic mechanism to all other RNA virus-encoded RdRPs of several other families [pol imaginatively resembles a cupped “right hand” consisting of “palm,” “fingers,” and “thumb” subdomains, which determine the correct geometrical arrangement of substrate molecules and metal ions at the active catalytic site [Crystal structure analysis revealed that FMDV 3Dfamilies . The ovetic site . The cattic site .pol or 3CD, termed VPg uridylylation complex, to initiate RNA replication in picornavirus [pol are present among different members of this family [Picornavirus replication is initiated in a primer-dependent manner. The protein primer VPg provides the hydroxyl nucleophile and forms a complex with 3Drnavirus , 190. Bis family . In FMDVs family , and in s family . The cons family ).The structure and biochemical activity of RdRP offer the opportunity to develop very selective anti-drugs against this viral enzyme in FMDV. Reports have indicated that some base and nucleotide analogues, particularly 5-fluorouracil and ribavirin. 5-Fluorouracil, a pyrimidine analogue, is mutagenic for a number of RNA viruses, including FMDV . RibavirFMDV remains a severe pathogen that hampers modern agriculture worldwide, despite the extensive work has been conducted in the past several decades to control it. Traditional vaccine development is limited by the potential risk of virus transmission. Synthetic capsid vaccine, an important novel vaccine against virus infection is limited because of the high potential for genetic and antigenic variation , 5. SpecThe NSPs and NCEs of FMDV play a critical role in viral proliferation and virus–host interaction. Studies on the involvement of these proteins and elements provided important insights into the molecular mechanism of virus-induced diseases. Nearly all NSPs and NCEs of FMDV are under investigation for their involvement in either virus detection or potential anti-viral strategy development –205, alt


PAN RNA has been implicated in KSHV gene expression, replication, and immune modulation. PAN RNA expression is required for optimal expression of the entire KSHV lytic gene expression program. Latent KSHV episomes are coated with viral latency-associated nuclear antigen (LANA). LANA rapidly dissociates from episomes during reactivation. Here we review recent studies suggesting that PAN RNA may function as a viral lncRNA, including a role in the facilitation of LANA-episomal dissociation during lytic replication.Kaposi’s sarcoma-associated herpesvirus (KSHV/human herpesvirus 8) is a γ-herpesvirus linked to Kaposi’s sarcoma (KS) and two lymphoproliferative disorders, primary effusion lymphoma (PEL or body-cavity B-lymphoma [BCBL]) and a subset of Multicentric Castleman’s Disease. During lytic growth, pervasive viral transcription generating a variety of transcripts with uncertain protein-coding potential has been described on a genome-wide scale in β- and γ-herpesviruses. One class of such RNAs is called long non-coding RNAs (lncRNAs). KSHV encodes a viral lncRNA known as  Recent new developments in epigenetic control of transcriptional programs by chromatin modifiers are highlighted by findings that long non-coding RNA (lncRNA), previously thought to be “transcription noise”, interact with chromatin-associated proteins to modulate their functions ,2. lncRNCurrently, the most dominant function explored in lncRNA studies relates to epigenetic regulation of target genes. This role is typically associated with gene repression, which has been studied in many cellular lncRNAs including ANRIL, HOTAIR, H19, and XIST ,11,12. Tde novo infection [de novo infection there is a rapid association of KSHV genomic DNA with histones, followed by a biphasic period of chromatinization [lysine methyltransferases (KMT), protein arginine methyltransferases (PRMT), and lysine demethylases (KDM) are targets of viral proteins and important for the viral life cycle [per se, act as epigenetic marks for inheritance [et al. showed that methylated histones were not detected during S-phase of the cell cycle, however Trithorax and Ezh2, which are H3K4 and H3K27 methylases were continuously associated with their response elements on the newly replicated DNA which thus re‑establishes the histone marks on newly assembled unmethylated histones. This finding suggests that dislodging PRC1/PRC2 polycomb complexes from the KSHV genome may be important for effective viral replication, because the KSHV genome has been shown to be heavily loaded by polycomb complexes [While the KSHV genome is histone-free in virions, the viral genome adopts a highly organized chromatin structure in infected cells that have established latency ,21 and fnfection . During nization . These enization . During nization ,21. Uponnization ,21. In anization ,21,22, tnization  (see belfe cycle ,26,27,28eritance . In the omplexes ,21,22. Tomplexes , it is tomplexes  and contpolyadenylated nuclear RNA or PAN RNA, an abundant early gene product. High level expression of PAN RNA is directly regulated by a master switch gene K-Rta (replication and transcription activator [KSHV encodes a 1.1 kb viral lncRNA known as ctivator ,32,33. Wctivator ,34,35 asctivator ,39,40,41ctivator ,42,43,44ctivator  supportictivator  and virictivator . Moreovectivator ,46 suggeOur discovery of PAN RNA involvement in the regulation of LANA began with the observation that LANA is released from the KSHV genome immediately after triggering reactivation by K-Rta expression . For thein vivo [in vitro and in vivo. The results clearly demonstrated that LANA has a property to bind PAN RNA [What molecular mechanisms would underlie LANA dissociation from KSHV episomes? As our lab and another group independently found; LANA associates with multiple RNA-binding proteins in vivo ,51. We hin vivo . Could P PAN RNA . LANA ma PAN RNA . Subsequ PAN RNA . By usin PAN RNA . The RNA PAN RNA ; this ma PAN RNA ,53. It iin vitro [in vivo [To study if PAN RNA/LANA association is responsible for dissociation of LANA from KSHV genomes, we adapted the strategy reported by the Steitz lab  which utin vitro  and in v[in vivo . Togetheet al. [bona fide mRNA with the potential to produce several small viral peptides [PAN RNA has been reported to interact with both viral and cellular proteins suggestive of a multifunctional role for PAN RNA in the KSHV lifecycle. This interaction list includes KSHV ORFs 26, 57, and 59 ,43,54,55et al.  have shopeptides .Pathogen-encoded lncRNAs have been reported for several virus groups, including herpesviruses (see  for a re


Nucleic Acids Res. (2014) 42 (16): 10832–10844. doi: 10.1093/nar/gku764The authors wish to apologize for errors in the last paragraph of the DISCUSSION section in this paper. The “RNA DQ” should read as “intramolecular RNA G-quadruplex (RQ)” at its first appearance and as “RQ” at the followings. The “DNA DQ” should all read as “DQ”.With these corrections, the correct version appears below (starting from “Alternatively…”).3 loop and bulge, the RQ might also be unstable as was the DQ. With the participation of G3 tracts from the DNA and RNA, the HQ of three G-quartets was much more stable and should be more competitive to form and to play a role in transcription termination.Alternatively, the RNA could also form an equivalent intramolecular RNA G-quadruplex (RQ) of three G-quartet layers by using the 13G-15G as the non-template DNA strand did in this work. However, the mutation in the 13G-15G in our work is expected to disrupt (M14/16) or promote (M3G) the formation of RQ, respectively, as to the DQ. But these showed little effect on the termination . This suggested that the RQ was largely irrelevant to the termination. On the other hand, because of the bulky T


Segment 8 has thermodynamically stable and evolutionarily conserved RNA structure and encodes essential viral proteins NEP and NS1. This suggests that vRNA self-folding may generate helixes and loops that are important at one or more stages of the influenza life cycle.Influenza A is a negative sense RNA virus that kills hundreds of thousands of humans each year. Base pairing in RNA is very favorable, but possibilities for RNA secondary structure of the influenza genomic RNA have not been investigated. This work presents the first experimentally-derived exploration of potential secondary structure in an influenza A naked (protein-free) genomic segment. Favorable folding regions are revealed by  RNA was purified on a 6% polyacrylamide denaturing gel and the radioactivity was measured using scintillation counter MicroBeta (PerkinElmer). Before hybridization, pure labeled RNA was folded as for chemical mapping (65°C for 5 min and slowly cooling to room temperature) in buffer A  or B  or C  or D . The RNAs in these conditions formed one band on native agarose gels, consistent with formation of one structure  of radioactively labeled RNA was used similar as described earlier , 60, 67.As for chemical mapping, vRNA8 (6 pmol) was annealed for 5 min at 65°C in folding buffer A and slowly cooled to room temperature. Then, 3 pmol of DNA oligonucleotide  (All influenza A segment 8 vRNA sequences were folded  5’ end) . These h 5’ end) .2, 50 mM HEPES, pH 7.5) at 23°C. NMIA reacted strongly and moderately, respectively, with 65 and 166 of 875 nucleotides. DMS reacted strongly and moderately with 58 and 115 nucleotides, respectively , which binds the heterotrimeric viral polymerase. This region is thought to be dynamic with at least two secondary structure [To assign probability to each base pair and unpaired nucleotide, the partition function module  in the Rtructure , 10. Thu37 of roughly -9.0 kcal/mol at 37°C  for binding to unstructured pentamer or hexamer RNAs [The chemical mapping and bioinformatics provide a robust model for the secondary structure of vRNA8. Often, antisense oligonucleotides are used to inhibit RNA function. Therefore, microarray mapping  was usedmer RNAs , 63, 64.The isoenergetic microarray contained 454 oligonucleotides complementary to potential binding sites on segment 8 vRNA . vRNA8 hSeveral approaches were used to assign unambiguous strong binding sites and the relevant data for strongly binding probes are summarized in 37 favorable enough for binding their probe if the site was completely single stranded. Moreover, DNA 9-mers targeted to sites 143 and 163 produced strong RNase H cleavages at nucleotides 143–145 and 165–166 , region 35–159 (part of domain II), and region 687–789 (part of domain IV). Base pairing of mini-vRNA8 differs from analogous fragments in vRNA8 only in the region connecting two preserved vRNA8 structural motifs and with three small hairpins—none of these differences affect the conserved local structures , but similar regions are modified in both RNAs Figs .The accessibility of mini-vRNA8 to probes in isoenergetic microarrays was compared to that of the complete vRNA8 under the same condition (37°C in buffer A) Figs . SimilarInfluenza stores its genetic information in RNA and RNA base pairs are particularly stable thermodynamically. For example, RNA and DNA nearest neighbor interactions between Watson-Crick base pairs on average stabilize a double helix by 2.1 and 1.4 kcal/mol, respectively, at 37°C. Therefore, the equilibrium constant for forming an RNA helix will be 3-fold larger per base pair than for an equivalent DNA helix. Thus RNA has a strong propensity to self-fold and form short helixes. Here, the self-folding propensities of an influenza A segment 8 vRNA are modeled on the basis of thermodynamics, NMIA and DMS chemical mapping, and sequence comparison in order to provide insight into modules that may be important at some stage of the viral life cycle.The secondary structure model of vRNA8  agrees wpanhandle structure that is essential for viral replication [The modeling successfully recapitulates the one currently known functional pairing in vRNA8—annealing of the 5’ and 3’ ends to form the lication , 10. EviIn general, the vRNA8 base pairing in 5’CAGAGGCAAUGGUCA3’) can expand and increase the stability of the hairpin in region 611–625, while leaving the rest of the model unchanged.Although the biochemical analysis focused on an H5N1 strain of influenza A, the base pairs uncovered are common throughout influenza A . The H5NRegions with low base pair probabilities may be important for function. On the basis of gel retardation experiments on protein-free RNAs, segment 8 was found to pair with segment 2, which suggests an interaction important for packaging . Region One application that benefits from determining RNA secondary structure is the design of oligonucleotide therapeutics , 72, 73.The mini-vRNA8 construct is devoid of domain III and part of domain IV , but conin vitro and explain the lack of reactivity in some regions.The single stranded regions in the vRNA8 model, especially hairpins, are generally well-supported by chemical mapping and oligonucleotide binding. In domain III, however, there are predicted single stranded regions in three hairpins and a large bulge loop (452–462) that are not reactive to chemicals. This suggests there could be alternative secondary structure for the region or possible tertiary interactions  thaThere are several possible binding sites for probes in region 409–405. From experiments with complementary DNA and RNase H , this reNMIA and DMS, respectively, interrogate ribose flexibility  and acceIn cells, influenza vRNA is thought to be largely, but not completely coated with NP protein that destabilizes base pairing , 36, 83.in vitro was determined on the basis of thermodynamics and chemical mapping coupled with sequence comparison. A shorter fragment, mini-vRNA8, was also mapped. The vRNA8 self folds into many highly probable helixes. While all the structural motifs identified may not be simultaneously present in cells, the very favorable free energies for RNA folding [The self-folding base pairing of an entire naked influenza RNA genomic segment  folding , 49 woulS1 Dataset(XLSX)Click here for additional data file.S2 Dataset(XLSX)Click here for additional data file.S1 File(Table A). Primers for polymerase chain reaction used to obtain mini-vRNA8 (Table B). Primers for reverse transcription (Table C). Heptamer probes complementary to vRNA8 (Table D). RNase H cleavage of vRNA8 in the presence of selected DNA oligonucleotides (Table E). Isoenergetic microarrays probes that bind strongly and moderately to vRNA8 and mini-vRNA8 and their thermodynamic properties (Table F). Deduced strong and medium binding sites in mini-vRNA8 for microarray probes (Table G).Primers for polymerase chain reaction using to obtain DNA template for vRNA8 and mini-vRNA8; (PDF)Click here for additional data file.S2 File. Self-folding vRNA8 predicted by RNAstructure 5.3 without any constraints .vRNA8 analysis by agarose gel electrophoresis (PDF)Click here for additional data file.


Listeria monocytogenes has been shown to be induced under infection-relevant conditions, but its physiological role and mechanism of action is not understood. In an attempt to pinpoint the exact terms of LhrC expression, cell envelope stress could be defined as a specific inducer of LhrC. In this process, the two-component system LisRK was shown to be indispensable for expression of all five copies of LhrC. lapB mRNA, encoding a cell wall associated protein that was recently identified as an important virulence factor, was disclosed to be directly bound by LhrC leading to an impediment of its translation. Although LhrC binds to Hfq, it does not require the RNA chaperone for stability or lapB mRNA interaction. The mechanism of LhrC-lapB mRNA binding was shown to involve three redundant CU-rich sites and a structural rearrangement in the sRNA. This study represents an extensive depiction of a so far uncharacterized multicopy sRNA and reveals interesting new aspects concerning its regulation, virulence association and mechanism of target binding.The multicopy sRNA LhrC of the intracellular pathogen  Throughout the last decade it has been recognized that small non-coding RNAs in bacteria are not rare and singular cases, but rather play an important and extensive regulatory role ,2. TheseListeria monocytogenes were discovered in 2006  was proven to depend on Hfq in terms of stability . A putative role of the LhrC sRNAs during listerial infections can be anticipated from later studies where they were reported to be highly expressed in blood  LisRK. Using bioinformatics tools, the lapB gene, encoding a cell-wall anchored virulence adhesin, was predicted as a target for all five LhrCs. Analyses of its regulation show that expression of lapB is targeted by the sRNAs at the post-transcriptional level. Interestingly, interaction of LhrC to lapB mRNA does not follow the predicted binding scheme involving two complementary sequences in sRNA and mRNA. Apparently, the LhrC molecule contains three redundant CU-rich motifs, residing in a stem-loop structure, a single-stranded region and the terminator structure, respectively, which are all capable of target recognition. Although LhrC was originally identified as an Hfq-binding sRNA, we find that it dispenses with Hfq for the regulatory effect on lapB.In this study we provide evidence that the LhrC sRNAs are highly induced in response to cell envelope stress and find that expression of all five L. monocytogenes serotype 1/2c strain LO28  and vigorously shaken at 37°C; when appropriate, kanamycin (50 μg/ml) was added. For northern blot experiments, the following concentrations were used to induce cell envelope stress: 4 μg/ml cefuroxime (Sigma-Aldrich), 0.03% bile salts , 4% NaCl, pH5 adjusted with HCl and 2% ethanol. In stress tolerance assays overnight cultures were diluted 1000-fold into BHI adjusted with cefuroxime (4 μg/ml), bile salts (0.07%), NaCl (8%), pH5 and ethanol (5%); growth was monitored for 24 h.The wild type strain used in this study was ain LO28 . Isogeniain LO28 . PrimersEscherichia coli TOP10 and E. coli DH5α (Invitrogen) were used and grown in Luria Broth  at 37°C. When appropriate, LB was supplemented with kanamycin (50 μg/ml), ampicillin (30 μg/ml), chloramphenicol (25 μg/ml) or erythromycin (150 μg/ml).For cloning purposes and green fluorescent protein (GFP) reporter experiments, L. monocytogenes was grown to OD600 = 0.35, cultures were split and one of them stressed. Samples were taken from stressed and control cultures after 30 min. To assimilate an induction profile of LhrC several time points of stress were sampled . In order to test RNA stability cells were treated with rifampicin (10 μg/ml) after 30 min of stress and samples collected at indicated time points. Cells were disrupted using the FastPrep instrument  and total RNA was extracted using TRI Reagent (Ambion) and purified as described previously  and incubated at 90°C for 4 min. RNA was subsequently extracted with acidic phenol (pH4.5) and chloroform using Phase Lock Gel Heavy 2.0 ml tubes (5Prime). Supernatants were precipitated with 2.5 volumes of ethanol (96%) and 1/10 volume 3M Na-acetate (pH4.5). RNA integrity was confirmed by agarose gel electrophoresis; concentration and purity determined on a NanoDrop ND-1000 (Saveen Werner). Northern blotting of 10 μg total RNA separated on an 8% denaturing polyacrylamide gel was performed as previously described  and analyzed with IQTL 8.0 quantification software .For testing the induction of LhrC in response to cell envelope stress, eviously . Total Rescribed . DNA pri600 = 0.35, split, and one stressed with 4 μg/ml cefuroxime. After 30 and 60 min samples were taken from stressed and control cultures and swiftly pelleted by centrifugation. Pellets were shock-frozen in liquid nitrogen and stored at −80°C. Total RNA was extracted according to  and labelling by polynucleotide kinase (New England Biolabs (NEB)), all in PNK buffer (NEB). Labeled transcripts were purified before gel shift employing Nucleospin miRNA kit (Macherey–Nagel) according to the manufacturer's instructions. Secondary structures of mutated LhrC4 molecules were predicted to resemble the one of wild type according to Mfold . For experiments testing mut_9 and mut_10, RNAs were mixed and incubated 5 min at 70°C, slowly cooled to 37°C and incubated for 40 min at 37°C before putting them on ice. Samples were loaded with current running and separated at 4°C in a 5% non-denaturing polyacrylamide gel.Templates for rding to  with minto Mfold . The gel . For an alkaline hydrolysis ladder 0.2 pmol labeled RNA was incubated in the presence of 10 μg yeast tRNA and alkaline hydrolysis buffer at 95°C for 5 min. The reaction was placed on ice and stopped by the addition of loading buffer type II. For RNase T1 sequencing ladders 0.1 pmol of labeled RNA was denatured  in the presence of yeast tRNA (10 μg) and structure buffer. The reaction was placed at 37°C for 1 min, provided with 0.1 U of T1 RNase and incubated for another 5 min at 37°C. Structure probing of RNA-RNA interaction generally started with denaturation of 0.1 pmol labeled RNA  which was afterwards placed on ice. In T1 RNase and lead(II)acetate experiments the labeled RNA was mixed with yeast tRNA (10 μg) and non-labeled RNA in the presence of structure buffer and kept at 37°C for 10 min. To provoke RNA cleavage the mixture was subsequently incubated with 0.1 U T1 RNase and fresh lead(II)acetate (5 mM) (Fluka), respectively. For RNase V1 or RNase A digestion, labeled denatured RNA was incubated in 10× structure buffer  before the addition of yeast tRNA, non-labeled RNA and water. After another 10 min at 37°C, 0.05 U RNAse V1  or RNase A (AM2274) was added and samples were incubated for 2 or 5 min, respectively. Details on fold excess of non-labeled RNA, amount of RNase and differing incubation times of cleaving reactions are indicated specifically. Samples were mixed with 2× loading buffer type II, denatured at 95°C for 3 min and placed on ice. A total of 2 μl of each sample was separated on an 8% polyacrylamide gel in 7 M urea at 45 W.L. monocytogenes EGD-e chromosome. The top five hits of each of the five result lists were chosen and merged into one final table comprising 13 putative targets (Supplementary Table S3).The RNApredator software  was usedlhrA core promoter, as well as upstream regions of lapB and lmo1669, were also ligated into pTCV-lac, respectively. For an in-frame translational lacZ fusion pCK-lac was used . All primers and details for transcriptional and translational fusions are listed in Supplementary Table S1. Beta-galactosidase assays were carried out as previously described  and subsequent western blot analysis were performed as described previously . Cells were lysed in HEPES buffer containing complete protease inhibitor (Roche) using a FastPrep instrument . Extracts were centrifuged  and supernatants ultracentrifuged at 100 000 × g for 1 h. Pellets comprising the membrane protein fraction were resuspended in HEPES buffer with the help of a hand homogenizer (VWR) and protein concentration determined via a BCA assay (PIERCE) according to the manufacturer's instructions. Western blot analysis was performed as described earlier  proving the specificity of the northern blot probe and a full lhrC mutant to be actually on hand.Induction of the five LhrC sRNAs (LhrC1–5) was recently reported for an blood  as well an blood . In ordes Figure . The siglhrC copies was determined using transcriptional fusions of each of the five lhrC promoters to the reporter gene lacZ in vector pTCV-lac . Previous studies have shown that L. monocytogenes defective in the TCSs LisRK or CesRK shows growth defects in the presence of several such stressors . However, in the presence of 4 μg/ml cefuroxime the ΔlhrC1–5 mutant strain exhibited a defect in growth compared to wild type  . Detailed RT-qPCR results of two biological replicates on all 13 targets are provided in Supplementary Table S4. A third biological replicate was performed on four of the targets: lapB (formerly known as lmo1666), lmo1041, lmo1993 and actA .A bioinformatics approach was pursued to search for putative direct RNA-targets of LhrC1–5 using the software RNApredator . All fivytogenes . lapB mRL. monocytogenes were prepared from wild type and ΔlhrC1–5, both from cultures stressed with cefuroxime and from unstressed samples. Cefuroxime stress was exerted on the cells for 2 h justified by the peak expression of LhrC1–5 not before 1 h of stress . Whereas LapB levels in unstressed samples were comparable between the wild type and ΔlhrC1–5, there was a clear down-regulation of the protein in the wild type strain in response to cefuroxime stress but not in the LhrC mutant ) according to the L. monocytogenes operon structure  to lacZ, yielded any activity in β-gal assays in our hands (Supplementary Table S5). Notably, lapB mRNA could be detected in RT-qPCR experiments, so the β-gal assay was assumed to be not sensitive enough to report transcription of lapB from its natural promoters at the tested conditions. To still make use of a reporter gene fusion strategy, the intergenic lmo1667-lapB sequence (100 bp) and additional 32 bp of lapB's coding region were fused downstream to a moderate promoter, and in-frame to lacZ in the translational reporter vector pCK-lac (-lapB-lacZ) in wild type and ΔlhrC1–5 cells showed increased β-gal activity in the mutant after cefuroxime stress (more than 3-fold), but not under non-stress conditions . The ΔlhrC1–4 and ΔlhrC5 cells were also transformed with pC-lapB-lacZ, and β-gal activity measured under control conditions and after cefuroxime stress . None of the mutants showed an increase in translation as observed for ΔlhrC1–5, but in both mutants β-gal activity increased compared to the wild type after cefuroxime stress, only slightly in ΔlhrC5, but clearly in ΔlhrC1–4.A reporter gene fusion strategy was employed to provide evidence for a post-transcriptional effect of LhrC1–5 on A Figure . The gen of lapB . However pCK-lac   was expressed from a constitutive promoter in pXG-10, a vector featuring GFP as reporter (lapB mRNA with the highest energy of interaction (−18.48 kJ/mol) according to RNApredator, and was therefore chosen for cloning into pNDM220. Induction of LhrC4 led to a reduction of the GFP signal on a western blot  indicating an interaction of the two RNAs in this E. coli reporter system. In order to ensure that reduction of the GFP signal indeed resulted from binding of LhrC4 to lapB mRNA, the lapB sequence was mutated to disrupt interaction with LhrC4 . The mutation reduced the negative effect LhrC4 had on GFP expression . Curiously, the interaction of the two RNAs could not be disrupted by expressing wild type lapB and correspondingly mutated LhrC4 . A similar expression level of LhrC4 and mutated LhrC4 in E. coli was assured by detecting both versions of the sRNA via northern blot analysis . A gel mobility shift experiment was consulted as another independent method to clarify the interesting discrepancy that relieving the proposed RNA-RNA interaction worked only unidirectionally. The gel mobility shift . These gel shift experiments demonstrated the ability of LhrC4 to bind Hfq, but with an increasing concentration of lapB mRNA, the presence of Hfq had no effect on sRNA-mRNA complex formation. Moreover, the translational fusion vector pC-lapB-lacZ in a Δhfq background gave rise to a β-gal activity equivalent to wild type . Finally, LhrC did not require Hfq in terms of stability in strain LO28 . Collectively, these results demonstrated that Hfq is dispensable for the regulatory action of LhrC on lapB.lapB. As loops have been extensively described as being best-suited for an initial interaction of two RNAs  disturbed sRNA-mRNA interaction even more than the sole mutations of the terminator loop (LhrC4_mut_3) and the single-stranded stretch (LhrC4_mut_2). Mutating both loop A and the single-stranded stretch (LhrC4_mut_6) resulted in a binding resembling that of the sole loop A mutant to lapB mRNA (LhrC4_mut_4). When both loops of LhrC4 were mutated in combination (LhrC4_mut_8) there was almost no detectable binding to lapB mRNA. Eventually, the concerted mutation of all three homologous sites in LhrC4 (LhrC4_mut_7) entirely abolished interaction with the target RNA. In conclusion, both loops of LhrC4 as well as the single-stranded stretch are capable of mediating binding to lapB mRNA, however, the capability of the latter seems to be much smaller compared to the loops.A closer inspection of the sequence of LhrC4 revealed similarity of the sequences of the two loops and the proposed site of interaction in the single-stranded stretch between the two stem loops Figure . The seqtwo RNAs , we tooks Figure . The seqlapB mRNA, structural probing was employed on 5′ labeled lapB mRNA as well as on 5′ labeled LhrC4. RNA cleavage of lapB mRNA was provoked by RNase T1 at single-stranded G residues or lead(II) acetate treatment at single-stranded sequences in general. The lapB mRNA sequence originally predicted to be involved in LhrC4 binding  could be confirmed by RNA probing, since it was partially protected from cleavage in the presence of LhrC4  treatment did not show a change in secondary structure in any of the two loops or in the single-stranded stretch of LhrC4, however, RNase V1 could clearly reveal loop A to change from unbound- to double-stranded state after addition of lapB mRNA, and also the stretch between the loops was subject to a change in secondary structure  before a structural rearrangement in the sRNA takes place and the final sRNA-mRNA duplex forms involving stem loop A in LhrC4 and the sequence −21 to +7 of lapB  to keep the structure of LhrC4 . However, mutation of stem A did not result in any reduction of binding efficiency of the two RNAs in vitro . This observation endorsed our theory that the stem opening and binding represents a later event in the course of sRNA-mRNA complex formation, meaning that the two RNAs are still capable of binding, but the final duplex cannot be formed. In an attempt to still mutate and compensate, lapB was mutated (lapB_mut_9) to prevent most of its interaction with LhrC4. Likewise, all three putative interaction sites in LhrC4 were substituted to match lapB_mut_9 (LhrC4_mut_9) . The interaction capability of lapB_mut_9 and LhrC4_mut_9 could not be fully restored to wild type levels, but the lapB mutation could indeed be compensated for partially by the mutations in all three LhrC4 interaction sites  and lead(II) treatment, but additionally by RNase V1 cleaving double-stranded RNA and single-stranded stacked RNA. Structural probing of unbound LhrC4 revealed that loop A might exist in the form proposed in Figure A Figure  and C: Ie Figure . The mats Figure .lapB mRNA binding all contain a UCCC motif which was reported in the literature to be a common loop motif for target recognition in sRNAs of several Gram-positive bacteria , which was mutated to keep the secondary structure of the compensatory LhrC4 mutant mentioned below. Strikingly, this minimal mutant (lapB_mut_10) lost almost all of its binding capability to LhrC4 demonstrating the importance of the implied motif for target recognition .The three sites in LhrC4 important for bacteria . We wondn Figure  and H. Ag Figure . LhrC4_mA Figure . The come Figure , since LL. monocytogenes during cell envelope stress. An involvement of sRNAs when dealing with cell envelope stress has been frequently described for Gram-negative bacteria. In fact, a disproportionately high number of trans-encoded sRNAs regulate proteins of the outer membrane (OMP) or transporters  and demonstrated lapB, which is absent from non-pathogenic Listeria species, to encode a sortase-dependent, cell wall-anchored adhesin required for entry into eukaryotic cells . Although all five LhrC molecules are quite similar, they still show slight differences in their C-rich segments and flanking regions which could supply LhrC with a lot of flexibility in terms of target recognition as shown for the Qrr sRNAs in V. harveyi /Csr (carbon storage regulator) family which have been discovered and characterized in several γ-proteobacteria . OriginaE. coli . CsrA, asyringae . The facein CsrA .S. aureus and for FsrA in B. subtilis , and subsequently this first complex is transferred into the ultimate RNA–RNA duplex in an irreversible and mostly not as rapid reaction (lapB mRNA already inhibiting translational initiation (lapB mRNA with loop A of LhrC4, as illustrated in Figure lapB molecules will take this path. In our study we were able to abolish binding of LhrC4 and lapB mRNA by mutating the three sites in LhrC4 capable of an interaction with lapB mRNA. A compensatory mutation of the lapB sequence could only partially restore interaction which we assume is caused by structural issues proven to be highly relevant for such RNA–RNA interactions (in vivo have to be performed to corroborate the suggested interactions between LhrC and lapB mRNA.The three sites containing the UCCC motif are located in both loops and in the single-stranded stretch between the two stem loops of the LhrC molecule. This appears plausible as loops and single stranded regions are known to be best suited for an initial contact of two RNA molecules . Besidesposition  and for position . In bothreaction ,79. In titiation . The irrractions ,79. FutuL. monocytogenes virulence and the sophisticated way of target binding calls for further in-depth analyses on these five homologous sRNAs.A plethora of non-coding sRNAs in scores of bacterial species have been identified throughout the last decade. With respect to pathogenic bacteria one hopes to exploit these regulatory sRNAs in anti-microbial strategies one day. However, there is still a long way to go since most of them are completely uncharacterized. This study unveiled details on the sRNAs LhrC1–5 concerning conditional and regulatory requirements for their expression, presents the mRNA of a virulence-associated adhesin as their first direct target, and shows interesting new mechanistic aspects on sRNA-mRNA interaction. The association with Supplementary Data are available at NAR Online.


The existence of mRNA in bacteria was firstsuggested in 1961 by Jacob and Monod, based on genetic studies [ The research discussed in this paper was initiated in my group at I.B. ZbarskyLaboratory at the A.N. Severtsov Institute of Animal Morphology of the SovietAcademy of Sciences. However, the bulk of the research was conducted in my labat the Institute of Molecular Biology of the Soviet Academy of Sciences, towhich I was invited by its director, V.A. Engelhardt, whose name the Institutenow bears.Director of the Institute of Molecular Biology, RAS, V.A. Engelhardt with theauthorFig. 1).Since phenol inhibits enzyme activity, we believedthat “phenolic” nuclei can be a good source of nuclear RNA. Later,it was shown that nuclear RNA can indeed be extracted from“phenolic” nuclei by this procedure if it is performed at 65°C[Fig. 1).It seemed that nuclear RNAcontained ribosomal RNA and a new type of RNA whose nucleotide composition wassimilar to that of DNA: i.e., informational RNA. The first experiments on thefractionation of nuclear RNA, conducted in 1961, confirmed this hypothesis[My main collaborator in the discovery of nuclear dRNA was V.L. Mantieva, whowent on to earn a PhD in biology. We were interested in the nature of nuclearRNA  and used at 65°C. The isopothesis.32P-labeled RNA experimentsrevealed that the nucleotide compositions of the total RNA of the isolatedfraction and that of the nascent RNA present in it were identical[Table). Thediscovered and purified DNA-like RNA was named dRNA. Three years later,in 1965, American authors described this type of RNAand called it heterogeneous nuclear RNA (hnRNA)[The best separation was achieved by thermal phenol fractionation, developed in1962, that included treatment of “phenolic” nuclei with a 0.14 MNaCl–phenol mixture at pH 6.0 and stepwise increase in temperature[dentical, 8(Tabl (hnRNA).Fig. 2).Next, we described the properties of nuclear dRNA. Its molecular mass washighly heterogeneous and reached very high values. The nascent nuclear dRNA hada significantly higher molecular mass than the total nuclear dRNA, whichimplied its cleavage in the cell nucleus (processing), 8.We also determined the size of dRNA in the cytoplasm, presumably, mature mRNA.We developed a method of partial blocking of RNA synthesis by actinomycin D,which in small doses selectively inhibits rRNA synthesis without affecting dRNAsynthesis. The molecular weight of the nascent nuclear dRNA significantlyexceeded that of cytoplasmic dRNA ,8.Finally, we conducted DNA hybridization-competition experiments with nucleardRNA and cytoplasmic mRNA. The addition of nuclear dRNA completely inhibitedmRNA hybridization with DNA, whereas an excess of mRNA only partially inhibitedhybridization of nuclear dRNA with DNA. It wascontrary to the data on the very high molecular mass of hnRNK isolated byphenol fractionation. To resolve this contradiction, we performed extraction inthe presence of a RNAase inhibitor from rat liver supernatant. This extractionproduced a completely different pattern of ultracentrifugation: a series ofpeaks ranging from a small 30S peak all the way up to material withsedimentation coefficients of 200S and above.Obviously, this pattern was much closer to the native one[After ultracentrifugation, most of the hnRNA was detected in a homogeneous 30Speak, which contained particles ca. 20 nm in diameter. The molecular weight ofRNA isolated from the 30S peak was low: ca. 1.4 g/ml, whichcorresponds to a RNA/protein ratio of about 1:4–1:5.Fig. 4).Next, we characterized the larger particles. Mild ribonuclease A treatmentquantitatively transformed them into 30S particles with 20-nm diameter, whichwere, therefore, monomers of the larger polyparticles. Electron microscopydemonstrated that 30S particles were monomers, 45S particles were dimers, 70Sparticles were pentamers, and that the 90–100S peak containedpolyparticles built up of 9 monomers. The measurement of RNA obtained fromvarious peaks demonstrated that in all cases the monomer was a RNA fragment ca.700 nucleotides in size. This is consistent with the fact that the buoyantdensity of all hnRNP peaks was identical.To better understand the structure of hnRNP particles, we elucidated thestructure of the 30S monomer. Intense ribonuclease treatment completelydestroyed the 30S particle RNA, which suggests that it is localized on theparticle’s surface. The 30S proteins were labeled with 34 g/cm3Fig. 5).In the presence of hnRNP of about 1.4 kDa in size, dimeric hnRNPs are formedduring the reconstruction [When protein particles were mixed with hnRNA and 2 M NaCl was removed bydialysis, hnRNP particles were reconstructed and were indistinguishable fromthe initial ones in a variety of tests. The initial 30S particles, proteinparticles, and reconstructed hnRNP particles look the same in electron microscopy andchromatin. However, regulation of hnRNA synthesis and mRNA export has beenactively studied at the Institute of Gene Biology of the Russian Academy ofSciences that was organized 15 years ago and for which it is the main focus ofresearch. Another important area of research at the Institute is new approachesto cancer therapy. This author is now engaged is this research. Some keystudies related to mRNA are summarized below.cis-elements in transcription regulation, have been discovered. Theirrole has been found to be highly dependent on their ability to bind tightly toeach other [Fig. 6).First of all, new properties belonging to insulators, importantch other , 19.Thich other Fig. 6).In contrast to enhancers, insulators are polar; they only interact with eachother if they have the same orientation. This determines conformation of a loopformed as a result of insulators interaction and may define which genes will be activated, 22.Super-long-distance interactions have been discovered in the genome, 24.TheFig. 8).Finally, it was discovered that insulators can interact with promoters (withlow selectivity), activating them, and with enhancers (more selectively).Therefore, an insulator located between an enhancer and a promoter may interactwith both, forming a non-productive complex. This may explain the well-knownuncoupling effect of an insulator ,262/ENY2 and SAYP, have been discovered. They play animportant role in the control of hnRNA transcription and subsequent stages ofmRNA formation and export , 28.Fig. 9).SAYP binds the protein initiating complex TFIID and chromatin remodelingcomplex SWI/SNF into a single supercomplex. Knockdown SAYP blocks therecruitment of TFIID and SWI/SNF at the promoter and represses thetranscription of many genes. It can be assumed that the fusion of the complexesallows TFIID to immediately bind to the promoter as soon as theSWI/SNF-activated movement of nucleosomes along the DNA release it from thenucleosomes Fig. 10).Finally, ENY2 is part of some insulator complexes, performing the barrierfunction of insulator [E(y)2/ENY2 was found to be multifunctional. It is part of the DUB module of theSAGA complex and participates in the activation of transcription initiation. ENY2 isnsulator .This is only part of the Institute’s work in the field of regulation ofmRNA synthesis and export.Therefore, early work on the identification of messenger RNA in eukaryotessuccessfully continues.


Transportation into the host cell nucleus is crucial for replication and transcription of influenza virus. The classical nuclear import is regulated by specific cellular factor, importin-α. Seven isoforms of importin-α have been identified in human. The preference of importin-α3 of avian influenza virus and -α7 isoform of human strains during replication in human cells was previously identified. In addition, both avian and human influenza viruses were shown to use importin-α1 isoform for their replication.p-value < 0.0001) and importin-α3  isoforms in human nasal mucosa while importin-α1 was detected as the highest expression importin-α isoform in lung tissues.The mRNA levels of importin-α1, −α3, and –α7 isoforms in human respiratory tract was determined by real-time RT-PCR. The results indicate that mRNA level of importin-α7 was significantly higher than that of importin-α1 (These results may explain the preference of importin-α7 isoforms in seasonal influenza viruses in human upper respiratory tract and may suggest a selective pressure toward importin-α7 in human respiratory tract infection of an avian virus. Influenza’s lifecycle requires nuclear import of the viral ribonucleoproteins (vRNPs) for viral replication and transcription, however, the large viral ribonucleoprotein (vRNPs) complexes exceed the size limitation of nuclear pore of the host cells , 2. TherIt has been previously shown that seasonal and avian influenza viruses require different importin-α isoforms for this process. Silencing of importin-α7 in human lung cells reduced seasonal influenza virus replication, while growth of avian strains was limited in importin-α3 silenced cells. On the other hand, growth of both avian and seasonal influenza viruses was reduced by importin-α1 silencing. Moreover, a reduction of viral load of human influenza viruses in lung was observed in importin-α7-knockout mice [The fact that mammalian influenza viruses showed the preference of importin-α7 isoform during viral replication prompted us to ask whether human upper airway expresses importin-α isoforms in resemblance to the known pattern of susceptibility of human respiratory tract tissues to human influenza viruses. It was previously shown that importin-α1 and -α7 was widely expressed in most human tissues , while iIn this study, the mRNA copy numbers of importin-α1, −α3 and –α7 isoforms in human respiratory tract were determined. The nasal mucosae and lung tissues were derived from 10 and 5 autopsy cases, respectively, who died accidentally at the age of 20–60 years old. This part of the study was approved by the Ethics Committee of Siriraj Institutional Review Board, Faculty of Medicine Siriraj Hospital, Mahidol University (Protocol number 805/2554 (EC2)). The parents, relatives or spouse of the dead person was informed by participant information sheet and signed in the consent forms for participation voluntarily.In order to define the expression level of importin-α1, −α3 and –α7 isoforms in human nasal mucosa, epithelial cells of nasal mucosae were collected by blade scraping. Then, the proportion of epithelial cells in mucosal samples was defined by immunofluorescence assay using anti-human cytokeratin antibody. Approximately 85 % of collected cells were shown to be epithelial cells by positive cytokeratin staining in all mucosal samples as indicated in Fig. p-value of <0.05 was considered statistically significant.The amounts of importin-α1, −α3 and –α7 mRNA copy number in human nasal mucosa were obtained by extrapolation of the cycle number against the standard curve. Importin-α1,-α3 and –α7 mRNA expression levels were normalized with glyceraldehyde 3-phosphate dehydrogenase (GAPDH) expression. The correlation coefficients of the standard curves were 0.992 for GAPDH, 0.9941 for importin-α1, 0.951 for importin-α3, and 0.94 for importin-α7 mRNA expression (data not shown). The RT-PCR was performed in three independent experiments with a total of 5 repeats, excepted for the detection of importin-α1 mRNA expression level in nasal mucosae because of limited availability of the sample. Importin-α1 measurement was done in duplicate of 2 nasal mucosa samples while the others 3 samples were done in 5 repeats as in other experiments. Statistical analysis was performed using unpaired t-test. A 2 copies with SD of 1.23 × 101 copies in all nasal mucosa samples.In order to ensure the RNA quality of individual samples, 1 μg of each extracted RNA were amplified with GAPDH specific primer. The mRNA expression levels of GAPDH were comparable among sample. The mean and standard deviation (SD) of copies numbers were found to be 5.35 × 107 copies/μg RNA, ten times greater than that of importin-α3 isoforms , and one hundred times greater than the expression level of importin-α1 . The expression ratio between importin-α7 and importin-α3 isoform of each samples were shown in Fig. Figure Besides human upper respiratory tract tissue, nasal mucosa, the expression level of importin-α1, −α3 and -α7 isoforms were also tested in human lower respiratory tract, lung tissues. The expression level of importin-α isoforms was determined in grinded lung tissues from 5 individuals. While the expression level of importin –α3 and –α7 isoforms were comparable to that in nasal mucosa, approximately one hundred times higher expression of importin-α1 was found in lung tissues when compared with nasal mucosa (Fig. This study provides an explanation for selective pressure driving the previously described human influenza virus preference for importin-α7, the interspecies barrier caused by importin alpha preference, and the different tissue tropism between seasonal and avian influenza viruses. Higher expression level of importin-α7 isoforms in human nasal mucosa than that of importin-α1 and -α3 isoforms with the known tissue distribution of the α2,3- and α2,6-linked sialic acid as the viral entry receptor may indicates a two-fold interspecies barrier at the cell and nuclear entry steps with similar tissue distribution.


To provide information about the effects of increased confusion or memory loss (ICML) in households in the United States, we describe primary respondents’ reports (proxy reports) about another person in their household experiencing ICML, using 2011 Behavioral Risk Factor Surveillance System (BRFSS) data. We used proxy reports on type of assistance needed, effects on functioning in daily activities, and whether confusion or memory was discussed with a health care professional, stratifying by age of the household member with ICML (18–50 y vs ≥65 y). About 3%  of primary respondents reported living with a household member with ICML; 75% of these household members needed some type of assistance, and nearly 60% had discussed ICML with a health care professional. Collecting proxy data about individuals in households may help paint a clearer picture of the characteristics of those experiencing cognitive decline and the potential needs of individuals and families. The Behavioral Risk Factor Surveillance System (BRFSS) has collected nationwide data on health risk behaviors and preventive practices associated with the leading causes of morbidity and mortality for more than 20 years . In 2011Data were included from 13 states  that had available household weights and included ICML questions on their 2011 surveys .Data were weighted using household weights based on the number of occupied households per state, derived from the American Community Survey Public Use Microdata Sample (PUMS) from the 2010 US Census . HousehoFunctional difficulties were assessed by asking the proxy whether ICML caused the household member to give up household activities or chores they used to do or interfered with their ability to work, volunteer, or engage in social activities . Additional questions asked about type of assistance most needed, if any, and if confusion or memory loss was discussed with a health care professional. Analyses were performed with SPSS version 22 (IBM Corp) to obtain weighted point estimates. Pairwise deletion was used for all analyses.There were 3,075 proxies  who reported about a household member who experienced ICML. For the 3,045 household members with ICML for whom age information was available, about 74% were aged 60 years or older . About one-quarter were aged 18 to 59 .For household members aged 18 to 59, 68.5% (n = 463) were reported to need some type of assistance in 1 of 5 areas compared with about 76.5%  of those aged 60 or older . HousehoThe BRFSS collected information from a primary respondent about another adult in a household for the first time in 2011. BRFSS data on children have been collected by randomly selecting a child in the household and obtaining information about that child from the primary respondent. This approach is a reliable way to obtain information on the health status of children who would not normally be included in BRFSS . In our Household members whose ICML is reported by proxy respondents appear to be more impaired by ICML than are respondents who report their own ICML. For example, we found that nearly three-quarters of household members aged 60 or older were reported by proxies to need some type of assistance. In contrast, a recent study using BRFSS data reported that for primary respondents aged 60 or older, only about half reported needing assistance because of ICML (Limitations to these findings should be considered. Data on ICML are self-reported and are distinct from data on the prevalence of Alzheimer’s disease and related dementias. BRFSS respondents were drawn from households with a telephone landline, thereby limiting the population to those who can afford one and to those who do not live in institutionalized settings. Additionally, data cannot be construed as a national average. Furthermore, research indicates selective bias by age in over- and underreporting disabilities for others . Collect


Gene transcription produces a wide variety of ribonucleic acid (RNA) species in eukaryotes. Individual types of RNA, such as messenger, structural and regulatory RNA, are known to play distinct roles in the cell. Recently, researchers have identified a large number of RNA-mediated toxicity pathways that play significant pathogenic roles in numerous human disorders. In this article, we describe various common RNA toxicity pathways, namely epigenetic gene silencing, nucleolar stress, nucleocytoplasmic transport, bi-directional gene transcription, repeat-associated non-ATG translation, RNA foci formation and cellular protein sequestration. We emphasize RNA toxicity mechanisms that involve nucleotide repeat expansion, such as those related to polyglutamine (polyQ) disorders and frontotemporal lobar degeneration-amyotrophic lateral sclerosis. Ribonucleic acids (RNAs) are polymeric macromolecules composed of a wide variety of nucleotide building blocks. Various classes of RNA have been reported to date, of which major examples are messenger RNAs (mRNAs), ribosomal RNAs (rRNAs), transfer RNAs, and non-coding RNAs. Each RNA class is distinct in terms of synthesis, properties and functions. It is widely acknowledged that most RNAs have cellular roles in gene regulation and protein translation. However, a few RNAs are known to have less common functions such as serving as genetic materials for RNA viruses. In cells, RNAs are transcribed by RNA polymerase according to sequences on the DNA template. A subset of RNAs is further modified through various processing steps such as cleavage, base modification and editing. Such modifications are crucial to the generation of mature and fully functional RNA molecules.In addition to the well-established roles of various classes of RNA molecules in neuronal  and huntingtin (htt) transcripts 20, have been shown to induce alternative splicing defects in mammalian cells , RNAs that carry expanded CAG repeats are enriched in the nuclear fraction  of the rRNA promoter, which results in UCE DNA hypermethylation. This leads to the down-regulation of rRNA transcription. We further showed that p53 protein becomes stabilized in cells and concentrated in the mitochondria. This causes the release of cytochrome c from the mitochondria, caspase activation and apoptosis induction  transcripts were detected in SCA8 patients  RNAs, were reported to form double-stranded hairpin structures , also known as double-stranded RNA-dependent protein kinase, regulates protein translation by mediating the protein phosphorylation of eukaryotic initiation factor 2α  has been identified as a cause of ~40% of hereditary ALS and 25% of familial frontotemporal dementia (FTD) cases. Together, these conditions are generally termed C9FTD/ALS -containing proteins composed of GA, GP, GR, PR and AP amino acid repeats (Ash et al., Although GGGGCC repeat expansion is located in non-coding exon of Drosophila models have been used to demonstrate the neurotoxicity of DPR polypeptides (Mizielinska et al., in vivo animal disease model.The neurotoxicity of DPR proteins is determined by their subcellular localization and by the sequestration of cellular proteins to DPR aggregates. Kwon et al.  demonstrC9or72 RNA expression and foci formation (Donnelly et al., C9orf72 RNA (Zamiri et al., Based on our current understanding of nucleotide repeat expansion disease pathogenesis pathways, various therapeutic approaches have been developed. Oligonucleotide-based therapeutics (Fiszer and Krzyzosiak, Although protein toxicity was considered to play a major role in the pathogenesis of repeat expansion diseases, we now know that mutant transcripts also induce cell dysfunction and death via multiple mechanisms, such as the alteration of gene expression levels and splicing patterns, the generation of small RNAs, the induction of nucleolar stress, the promotion of bi-directional transcription and repeat-associated non-ATG translation of the disease locus, the activation of apoptotic signaling, and the sequestration of cellular components to RNA foci. The above RNA-mediated mechanisms are predicted to operate in conjunction with the protein-mediated pathways to confer overall neurotoxicity. We now know that mutant RNAs confer cytotoxicity by sequestering cellular components to RNA foci (Wojciechowska and Krzyzosiak, The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.