In recent years, there have been increasing numbers of infectious disease outbreaks that spread rapidly to population centers resulting from global travel, population vulnerabilities, environmental factors, and ecological disasters such as floods and earthquakes. Some examples of the recent outbreaks are the Ebola epidemic in West Africa, Middle East respiratory syndrome coronavirus (MERS-Co) in the Middle East, and the Zika outbreak through the Americas. We have created a generic protocol for detection of pathogen RNA and/or DNA using loop-mediated isothermal amplification (LAMP) and reverse dot-blot for detection (RDB) and processed automatically in a microfluidic device. In particular, we describe how a microfluidic assay to detect HIV viral RNA was converted to detect Zika virus (ZIKV) RNA. We first optimized the RT-LAMP assay to detect ZIKV RNA using a benchtop isothermal amplification device. Then we implemented the assay in a microfluidic device that will allow analyzing 24 samples simultaneously and automatically from sample introduction to detection by RDB technique. Preliminary data using saliva samples spiked with ZIKV showed that our diagnostic system detects ZIKV RNA in saliva. These results will be validated in further experiments with well-characterized ZIKV human specimens of saliva. The described strategy and methodology to convert the HIV diagnostic assay and platform to a ZIKV RNA detection assay provides a model that can be readily utilized for detection of the next emerging or re-emerging infectious disease. Flavivirus and family of Flaviviridae, related to yellow fever, dengue, West Nile, Japanese encephalitis, and tick-borne encephalitis viruses. Zika virus is transmitted among humans primarily by mosquito vectors  [Multiple outbreaks of infectious diseases, including yellow fever, severe acute respiratory syndrome (SARS), Ebola, dengue, chikungunya, are occurring with increasing frequency. At least three major factors have influenced the increasing incidence of infectious disease epidemics: population concentration at the epicenter of an infection, the underlying characteristics and vulnerabilities of the population at the epicenter  and exteopictus) –8 and huopictus) . ZIKV waopictus) –12. In aopictus) –8, 13.Early pathogen identification is necessary to combat the spread of these newly emerging and re-emerging epidemics , 15. DiaIn this study we have adapted a portable microfluidic device that we developed for HIV RNA detection to detect ZIKV RNA in saliva samples. We previously reported the ability to utilize a single sample of blood and/or saliva to simultaneously detect both HIV antibodies and HIV RNA using a microfluidic device for Point-of-Care (POC) testing . We optiLAMP was first reported by Notomi et al. 2000   technique and processed by the Encompass Optimum workstation [In this study we first optimized the RT-LAMP assay to detect ZIKV RNA using a benchtop isothermal amplification device and then adapted the assay to the microfluidic device, Rheonix CARDkstation  can deteWhole Mouth Stimulated Saliva (WMSS) was selected for use in this study because it is less viscous than other oral fluids. WMSS samples were collected as previously described by Chen et al. 2016 [ZIKV strain PRVABC59, NR-50240 was obtained through Biodefense and Emerging Infections Research Resources Repository (BEI Resources), NIAID, NIH and propagated in Vero cells. ZIKV stock was obtained from infected cells supernatant.® CRL-1586TM) were maintained in tissue culture flasks at 37°C in an atmosphere containing 5% CO2 with Eagle’s minimum essential medium containing Earle’s balanced salt solution, non-essential amino acids, 2 mM L-glutamine and 1 mM sodium pyruvate, 100 units Penicillin, 0.1 mg/ml Streptomycin, 1.25% Amphotericin, supplemented with 2% fetal bovine serum .Vero E6 cells  overlay. After 5 days of incubation at 37°C in an atmosphere containing 5% CO2, cells were fixed with 10% formaldehyde, washed, and stained with 0.025% (wt/vol) crystal violet in 2% (vol/vol) ethanol for visualization of plaques (2.9 x 106 plaque-forming units/ml). Genomic purified ZIKV RNA  obtained from BEI Resources, NIAID, NIH  with a known viral RNA copy number was serially diluted ten-fold and used as a standard to generate a standard concentration curve graphing the RNA concentration (RNA copies/ml) vs amplification time in the RT-LAMP assay on the Genie III device. This allowed estimating the copy number of the ZIKV stock (2.2 x 107 RNA copies /ml).Vero cells were infected when at 70% to 80% confluence at a multiplicity of infection (MOI) of 0.01. After 1 h virus adsorption at 37°C in an atmosphere containing 5% CO13 RNA copies/ml), was donated by Dr. James Whitney, Center for Virology and Vaccine Research, Beth Israel Deaconess Medical Center, Harvard Medical School. Additional purified genomic RNA from four different ZIKV strains (Asian lineage) were obtained through BEI Resources, : PRVABC59, Puerto Rican strain, NR-50244 (2.0 x 107 RNA copies/ml); PLCal_ZV, Thailand strain, NR-50242 (5.1 x 107 RNA copies/ml); FLR, Florida strain, NR-5024 (4.4 x 107 RNA copies/ml) and R103451, Honduras strain, NR-50358 (5.4 x 107 RNA copies/ml). The BEI Resources  supplied the copy numbers of viral RNA per ml (RNA copies/ml), determined using Droplet Digital RT-PCR.Purified genomic RNA from ZIKV, strain PLCal_ZV (Thailand)  serotype 1  and serotype 2  were also obtained through BEI Resources .Primers targeting a unique conserved sequence in the ZIKV capsid were designed using the Primer Explorer V4 software  with conserved regions of the capsid gene of the PLCal_ZV, Thailand strain (GeneBank accession no. KF993678.1). A set of 3 primer pairs , includiRT-LAMP reaction was carried out in a final volume of 25 μl including the OptiGene Master mix ISO-004 with 7.5 units of WarmStart reverse transcriptase  and 3 pairs of primers targeting a unique conserved sequence in the ZIKV capsid . 3–7 μl Experiments using the Genie III  portable isothermal amplification device were performed to optimize the RT-LAMP assay. SYBR Green I interchelating dye is included in the OptiGene Master mix (OptiGene ISO-004) which allows following real-time fluorescence in a Genie III.® cartridge developed by Rheonix Inc.  is an integrated cassette that can provide both an immunological and nucleic acid (RT-LAMP) result from a single sample. It incorporates all the pumps, valves, micro channels, reactions, and reagent reservoirs on a disposable cartridge described in Chen et al. 2016 [® cartridge is controlled by the Encompass Optimum workstation developed to provide automated control of all sample preparation functions as described in Spizz et al. 2012 [Optimum workstation does not possess the ability to monitor fluorescence, we employed a novel reverse dot-blot (RDB) hybridization technique to monitor the specific ZIKV amplicons produced in the RT-LAMP assay using hybridization to nucleic acid probes immobilized on integrated DNA arrays [® [® cartridge [® cartridge.The microfluidic Rheonix CARDal. 2012 . Since tA arrays . Coupledartridge , 30 at t® cartridge.In subsequent experiments, Probe 4 was immobilized on an activated membrane as a DNA array , 30 at dDepending on the device used for the RT-LAMP assay, different detection techniques were applied .® cartridge the uninfected human saliva was spiked as above but with pre-heated inactivated ZIKV. This safety measure was required because the facilities at Rheonix Inc.  laboratory are not at a BSL-2 level.Uninfected human saliva samples were spiked with ZIKV to test the feasibility of using a RT-LAMP assay with saliva samples. Ten-fold serial dilutions of the virus in 1XPBS were prepared. Saliva was first diluted 1:5 and 10 μl of each ten-fold serial dilution of the virus was spiked into saliva and then heated at 90°C for 5 min to inactivate and lyse the virus. Inactivation of ZIKV under these conditions was confirmed with plaque assays using Vero cells as described above . For expUsing the Genie III device we first demonstrated that a RT-LAMP assay could be used for the detection of ZIKV RNA by testing serial ten-fold dilutions in 1XPBS of purified ZIKV genomic RNA  donated by Dr. Whitney) . Results6 RNA copies/ml. We determined the limit of detection of the RT-LAMP assay using ten-fold serial dilutions of purified genomic RNA from 4 different ZIKV strains of the Asian-lineage . The results indicate that the RT-LAMP assay using primers that target the capsid consistently detected down to 2 x 103 (6 RNA copies/reaction)  .3 RNA copies/ml (6.6 RNA copies/reaction) in 100% of the samples and in 75% of the samples 2.2 x 102 RNA copies/ml (0.66 RNA copies//reaction)  . Ten rep8 and 2.52 x 108 RNA copies/ml) was used to optimize the experiments on the Rheonix CARD® cartridge. The RT-LAMP assay with biotinylated labeled primers was automatically carried out in the amplification tube in the Rheonix CARD® cartridge. Representative results show  as a function of increasing concentration of the biotinylated Probe 4  and an e® cartridge, we performed an assay using pre-heated lysed ZIKV spiked in saliva samples as a template. The saliva samples containing pre-heat lysed ZIKV were dispensed into the sample reservoir of the Rheonix CARD® cartridge and then pumped into each amplification tube along with LAMP master mix for RT-LAMP reaction followed by RDB processing for visualization of the reaction product amplicons. The results indicate detection in saliva samples down to 6 ZIKV RNA copies  for results. Isothermal amplification methods have increasingly attracted attention due to their simplicity and sensitivity including nucleic acid sequence-based amplification (NASBA), loop-mediated isothermal amplification (LAMP), helicase-dependent amplification (HDA), rolling circle amplification (RCA), multiple displacement amplification (MDA) and recombinase polymerase amplification (RPA) –40 AmongMP assay .Salivary testing for diagnosis has advantages over other bodily fluids , 42 but 3 RNA copies/ml (6 RNA copies/reaction) were consistently detected in the four strains. The limit of detection of the RT-LAMP assay using spiked saliva samples was also found to be ~2 x 103 RNA copies/ml (6.6 RNA copies/reaction).We first optimized the RT-LAMP assay using ZIKV purified genomic RNA as a template on the Genie III device. We focused on the Asian-lineage ZIKV strains because they were responsible for the recent epidemics in Latin America. There is a significant variation in sequence between the African and Asian-lineages , 49. TheThere have been reports showing that ZIKV can be detected by RT-LAMP assay without the need for RNA purification , 51. Our® cartridge and processed it by the Encompass Optimum workstation. This approach successfully detects ZIKV RNA using the RDB technology on the Rheonix CARD® cartridge and coupling this with the workstation’s image analysis and software capability provides a semi-quantitative titer of viral load based on the image density of the hybridized spot. Estimating the viral load can be useful to investigate correlations of viral load with disease severity and immune responses. This would be of particular importance for monitoring fetal infections in pregnant women [® cartridge using ZIKV spiked in saliva down to 8.57 x 102 RNA copies/ml (6 RNA copies/reaction). The same limit of detection was obtained for the RT-LAMP assay on the Genie III with saliva samples spiked with ZIKV. Ongoing experiments are focused on optimizing the conditions for amplification of ZIKV RNA directly by heat-lysing the virus in the Rheonix CARD® cartridge and without purification of ZIKV RNA. Eventually the entire assay will be validated on Rheonix CARD® cartridges using well-characterized ZIKV human specimens as well as samples from people infected with other flaviviruses to confirm the reactivity and specificity of the assay.After showing that the RT-LAMP assay on the Genie III could detect purified genomic RNA as well as ZIKV spiked in saliva samples, we adapted it to the microfluidic Rheonix CARDnt women , 53. We ® cartridges is similar to other results reported using RT-LAMP and other isothermal assays [® cartridge. However these results need to be validated using ZIKV samples from well-characterized human specimens.The limit of detection of our RT-LAMP assay on the Genie III and Rheonix CARDl assays . These a® cartridge was processed in the prototype Encompass MDx SOLO workstation providing viral RNA isolation and detection via RT-LAMP as well as antibody detection [® cartridge, allowing a total of 4 samples to be simultaneously processed with real-time fluorescence monitoring. We moved to the larger Encompass Optimum Workstation because it allows 1 to 6 Rheonix CARD® cartridges (24 samples) to be processed simultaneously. Since the Encompass Optimum workstation does not employ fluorescence excitation and detection system, the RDB technique was employed to interpret the molecular results by use of hybridization to nucleic acid probes immobilized on integrated DNA arrays. A major advantage of the Encompass Optimum workstation over the Encompass MDx SOLO workstation is that the Optimum workstation automatically introduces all samples and reagents to the Rheonix CARD® cartridge, which reduces the complexity of the final assay for the end user. In addition user friendly and intuitive software is included in the workstation guiding the user. A touchscreen initiates the totally automated process and finally interprets and reports the assay results.In the HIV dual diagnostic assay that we previously developed, the Rheonix CARDetection . The comIn ZIKV infections, the virus persists in plasma or serum for only a relatively short period of time while ZIKV can be detected for extended periods in other bodily fluids such as saliva, urine or semen , 56. Thi® cartridge using a modification of the approach we used for antibody detection against HIV [® cartridges.The ZIKV serological assay is challenging because ZIKV antigens cross-react with other flavivirus amino acid sequences . We are inst HIV . Then we® cartridges. In addition, once diagnostic peptides are validated and incorporated into the Rheonix CARD® cartridges, we will be able to simultaneously detect ZIKV antibodies and ZIKV RNA. There is currently no combined antibody/nucleic acid test available commercially for ZIKV detection [There are only a few studies that have reported ZIKV detection in saliva using RT- LAMP , 59, 60.etection , 62.Adaption of this protocol for the next emerging infection requires two steps: (1) identification of the nucleic acid sequence of the infectious agent, which allows synthesis of LAMP primers and associated RDB probes and (2) discovery of diagnostic peptides that detect host immune response(s) for a protein microarray. Our results provide the proof-of-principle of a protocol for adapting an existing LAMP assay on a microfluidic device to detect HIV RNA, to an assay to detect ZIKV RNA. Further experiments with clinical samples and diagnostic peptides will validate the assay.S1 FigGraph of the amplification time (seconds) as a function of the fluorescence signal monitored with the Genie III device. No amplification detected in RT-LAMP assay using ZIKV capsid primers and DENGV serotype 1 (green) and 2 (purple) genomic purified RNA as templates. ZIKV genomic purified RNA was used as a template for positive control. Distilled water instead of RNA template was used as a negative control in the RT-LAMP assay.(TIF)Click here for additional data file.S2 FigGraph of the amplification time (seconds) as a function of the fluorescence signal monitored with the Genie III device. No amplification detected in RT-LAMP assay using DENGV serotype 1 (blue) and 2 (red) genomic purified RNA as templates. DENGV serotype 1 (green) and 2 (purple) genomic purified RNA was used as a template for positive control. Distilled water instead of RNA was used as a negative control in the RT-LAMP assay.(TIF)Click here for additional data file.S3 FigInfections at high and low MOIs with heat inactivated ZIKV at 90°C for 5 min did not show any detectable plaques. No heat inactivated ZIKV (no treatment) was used as a control for infected Vero cells. 2 replicates were used for high MOI infections and 3 for low MOI infection. The bottom well in the MOI 7 columns corresponds to Vero cells without virus used as an uninfected control.(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.


The flow of genetic information within biological systems, as described by the central dogma of molecular biology Crick, , lies atTranscriptional infidelity, i.e., the inaccurate conversion of DNA to RNA, constitutes one class of information transmission errors. By expressing an error-prone version of RNA polymerase II (RNAPII), increased rates of transcriptional promiscuity have been demonstrated to induce proteotoxicity and reduce cellular longevity in yeast  assay” and provide the first comprehensive analysis of transcription errors in eukaryotic cells , induces toxicity through interfering with protein quality control systems and mitochondrial function  and to examine the effects of aging, disease, DNA damage, and specific genes, RNAs and proteins on transcriptional fidelity.All authors listed have made a substantial, direct, and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


In most eukaryotic organisms, small noncoding RNAs called microRNAs (miRNAs) post-transcriptionally silence mRNAs to fine-tune protein expression. This mechanism, having likely evolved from a duplication of a small-RNA–mediated antiviral defense system, now orchestrates various developmental processes and is essential for maintaining cellular homeostasis . In contGiven the potential influence miRNAs can exert on their host, it is not surprising that many DNA viruses have been found to encode their own variants . CuriousViruses are obligate intracellular parasites that require the resources of the cell to amplify and spread. In many cases, the process of virus replication is optimized through the manipulation of the cell to ensure their defenses are minimized and the resources required for viral replication are maximized. These cellular alterations are most commonly orchestrated by virus-encoded proteins but can also be mediated through miRNAs. This latter strategy is best exemplified by the Herpesviridae family, which has been found to encode anywhere from one to fourteen different miRNAs . EffortsIn contrast, acute cytoplasmic viruses, regardless of genome composition or size, do not appear to usurp this aspect of host cell biology . The lacRNA-virus replication predominantly occurs in the cytoplasm. As such, generation of a virus-derived miRNA would demand production of a Dicer-compatible hairpin. While this substrate could be generated in a noncanonical manner, it is usually the product of Drosha cleavage in the nucleus. For this reason, the first efforts to address why RNA viruses do not use miRNAs focused on the engineering of influenza A virus, which replicates in the presence of Drosha. Surprisingly, this pri-miRNA–containing recombinant virus permitted generation of a functional miRNA without any evidence of attenuation , 8. SubsDespite translocation of the necessary miRNA processing machinery, it would stand to reason that the excision of a hairpin embedded in a positive-stranded RNA virus would be deleterious. Indeed, hairpin-mediated loss of viral titers has been observed with recombinant lentiviruses and has been found to demand the use of DNA-dependent RNA polymerase III in other retroviruses , 15, 16.The last theoretical constraint that might explain the lack of miRNA production from cytoplasmic RNA viruses derives from the concept of self-targeting. Because RNA virus-produced miRNAs would be perfectly complementary to the antigenome, one would imagine that this could cause dramatic attenuation. However, while small-RNA access could be hindered in both negative-stranded RNA viruses, through the formation of ribonucleoprotein complexes, or dsRNA viruses, which are already perfectly paired RNA duplexes, positive-stranded viruses should, in theory, be accessible for base-pairing with the miRNA and thus susceptible to self-targeting. However, here again, when one designs a positive-stranded RNA virus to encode a miRNA and then monitors antigenome production directly, there is no evidence of self-cleavage, likely owing to virus compartmentalization during replication. Alternatively, targeting of the antigenome could be prevented by engagement of the RNA-dependent RNA polymerase (RdRp) and parallel production of new genomes. In either instance, alphaviruses, rhabdoviruses, orthomyxoviruses, and carmoviruses have all been shown to successfully generate miRNAs without any obvious loss to viral fitness –11, 13.Given the observed experimental results, it remains a wonder as to why RNA viruses do not co-opt cellular miRNA machinery as a means of enhancing their replication. One possible explanation for the lack of miRNA utilization among acute RNA viruses could be due to kinetics. As fine-tuners of protein levels, the impact of miRNA-mediated post-transcriptional silencing occurs over the course of days, not hours . TherefoLimited host targets aside, the most compelling explanation for the absence of RNA virus-encoded miRNAs is that this strategy is simply not as advantageous as a protein with regards to cellular manipulation. By nature, the miRNA would need to be perfectly complementary in order for it to be rapidly beneficial, and this would likely render this strategy target-specific. In contrast, a viral protein can be multifunctional and engage many factors at the protein level. While it could be argued that the virus might circumvent this shortcoming by producing multiple miRNAs, a tandem array of RNA hairpins has been found to come at a fitness cost .Given the observations and current understanding of miRNA biology, it would seem safe to conclude that the selective utilization of virus-encoded miRNAs by persistent viruses is due to the limited advantages mRNA targeting can instill during an acute infection. While it remains possible that an acute RNA virus could benefit from the modulation of a single host mRNA, this is likely not observed because such an activity can be achieved more broadly and efficiently through use of a protein-based antagonist. Moreover, although initial efforts have failed to identify any obvious fitness cost from the inclusion of a single miRNA, more in-depth studies under greater selective pressures may reveal that the inclusion of a hairpin does consistently impact virus replication in a negative way. While future work may still one day identify an acute RNA virus that generates a bona fide miRNA, based on our current knowledge, it would appear that such a strategy confers a gain that does not impart any evolutionary benefit.


Igh and Igl/Igk coding sequences (CDSs) but it has been difficult to process a number of hybridomas both with accuracy and rapidness. Here, we propose a new strategy for antibody sequence determination by mRNA-seq of hybridomas. We demonstrated that hybridomas highly expressed the Igh and Igl/Igk genes and that de novo transcriptome assembly using mRNA-seq data enabled identification of the CDS of both Igh and Igl/Igk accurately. Furthermore, we estimated that only 30,000 sequenced reads are required to identify immunoglobulin sequences from four different hybridoma clones. Thus, our approach would facilitate determining variable CDSs drastically.Identification of immunoglobulin genes in hybridomas is essential for producing antibodies for research and clinical applications. A couple of methods such as RACE and degenerative PCR have been developed for determination of the  Hybridomas have been widely accepted as a method for producing large amounts of monoclonal antibodies for research and clinical application . IdentifIgh and Igl/Igk genes. Both subunits have a constant region and a variable region (V region). The constant region is conserved and codes a crystallizable region (Fc region). The V region contains the V, (D) and J segments, and codes the antigen-binding region, also known as Fab region, while Igh has only D segments. These sequences are somatically recombined in pre-B cells, and this recombination plays a key role in antigen specificity and makes it difficult to identify the genomic sequences of immunoglobulin.Antibodies are composed of two subunits; immunoglobulin heavy chain and light chain are each coded by the Igh and Igl/Igk genes. The 5´RACE method has been widely used to clone the Igh and Igl/Igk sequences from hybridomas , 8H3 [Hybridoma clone2 (HD2)], 5A10 [Hybridoma clone3 (HD3)] and 5F11 [Hybridoma clone4 (HD4)], were generated in our laboratory –11. 4E5 Total RNA was extracted from each of the six hybridoma clones  using the AllPrep DNA/RNA Mini Kit (QIAGEN). Library preparation was performed using 1 μg  or 3μg (α-Histone H3 lysine 9 acetylatioin (H3K9ac).v2, 8A2 and α-H3K9ac.v3, 13C7) of total RNA and NEBNext Ultra Directional RNA Library Prep Kit (New England Biolabs). mRNA-seq was done with an Illumina HiSeq 1500 for 50 bp  or 100bp (8A2 and 13C7) paired-end. More than 40M reads were obtained in each sample . We mainly used the mRNA-seq data of HD1 through HD4 and additionally analyzed 8A2 and 13C7 for the comparative study with Sanger sequencing.Igh and Igl/Igk constant region sequences . BWA-MEM was performed to map sequence reads with the parameter: -t 8 -P -L 10000 , rat transcripts (generated from the NCBI/Rnor5.0 refSeq GTF file in igenome reference set), and rat y TIGAR2 ). TIGAR2http://homes.cs.washington.edu/~dcjones/fastq-tools) were de novo assembled using Trinity. CPU and max_memory parameters were changed according to each read number . We extracted the Igh and Igl/Igk CDSs by filtering if the contigs contained 20–30 bp of unique sequences of the Igh and Igl/Igk constant region and had proper length . We developed an automation tool to extract immunoglobulin sequences using Trinity output, which is freely available on http://tx.bioreg.kyushu-u.ac.jp/igfinder.Total reads or subsampled reads by fastq-sample ; predenature at 94°C for 2 min; 35 cycles of 94°C, 15 s, 57°C, 30 s and 68°C, 20 s; final extension at 68°C for 5 min ; predenature at 94°C for 2 min; 35 cycles of 94°C, 15 s, 58°C, 30 s and 68°C, 20 s; final extension at 68°C for 5 min (HD4-Igh).Each hybridoma RNA was obtained by phenol/chloroform extraction. Reverse transcription was performed using the PrimeScript™ II 1st strand cDNA Synthesis Kit (TaKaRa). PCR was performed using the KOD Plus enzyme (TOYOBO) and a thermal cycler. We used the following thermal protocol: predenature at 94°C for 2 min; 35 cycles of 94°C, 15 s, 55°C, 30 s and 68°C, 20 s; final extension at 68°C for 5 min HD1-Igh For: 5’-TGAAATCCTCGCAGGAAACTC-3’ (designed on 5´UTR region)HD2-Igh For: 5’-TGATTGCCACAGCCTTCAGT-3’ (designed on 5´UTR region)HD3-Igh For: 5’-CATGAAAACCAGCCTGTCCT-3’ (designed on 5´UTR region)HD4-Igh Rev: 5’-AAATAGCCCTTGACCAGGCA-3’ (designed on constant region)HD-Igk For: 5’-GAAGGTCTTTCTCAGGGCT-3’ (designed on 5´UTR region)HD1-Igk For: 5’-GCTCAGCTGTACTCATGC-3’ (designed on 5´UTR region)HD2-Igk For: 5’-GGTTGGTTGTCATCTTACTGT-3’ (designed on 5´UTR region)HD3-Igk For: 5’-CTTGTCTTGTTGGCTTGAGAT-3’ (designed on 5´UTR region)HD4-Igk Rev: 5’-TGATGTCTCTGGGATAGAAGTT-3’ (designed on constant region)HD-mRNA-seq data were submitted to the DDBJ sequence read archive [DRA004264].Igh and Igl/Igk constant region were ranked as the highest expressed transcripts (FPKM > 10000) in all four hybridoma lines  were simply assembled with Trinity which reconstructs a full-length transcriptome from RNA-seq data without a genome [Igh CDS by filtering if the contigs contained 20–30-bp unique sequences of the Igh constant region, which can uniquely determine each gene . As the full length of IgH has more than 400 amino acids (aa) [Igh CDS containing the unique 24-bp sequence of Ighg2b after filtering if the contig has more than 1200 bp. The V region and partial constant region of the obtained Igh CDS were confirmed to be identical to the sequence obtained by Sanger sequencing of reverse-transcribed PCR (RT-PCR) products which were amplified with primer sets designed based on the 5'UTR region and constant region. Alignment of the obtained IgH protein sequence and the known rat IgH constant region (AAA60738) confirmed matching of the full length of amino acid sequence of the constant region in the known IgG2b and the 133–464 amino acids identified in the Igh CDS . As the full length of IgK has more than 200 aa [Igk CDS after filtering if the contig has more than 600 bp (Igk CDS with Sanger sequencing following RT-PCR. We demonstrated matching of the amino acid sequence of the constant region in known IgK (CAA24558) and the amino acids of the identified Igk CDS (Igh and Igl/Igk CDSs of HD2 (Ighg2a /Igk), HD3 (Ighg2a /Igk) and HD4 (Ighg2a /Igk) (data not shown). Then, these identified antibody isotypes corresponded to the results of ELISA-format isotyping assay. Mouse Igh and Igk CDSs from mouse hybridoma clones  were also identified and their amino acids sequences were identical to coding sequence determined by PCR cloning shown in [Igh and Igl/Igk transcripts was extracted by unique sequences of the mouse Igh and Igl/Igk constant region . These results indicated that simple de novo assembly using hybridoma mRNA-seq data was beneficial for identifying both the Igh and Igl/Igk genes.Next, we attempted to reconstruct the a genome ) withoutids (aa) , a 1395- Igh CDS . We alson 200 aa , a 705-bn 600 bp . We also Igk CDS . We alsoIgh and Igl/Igk genes using hybridoma mRNA-seq data. First, to estimate the required number of reads for identifying an immunoglobulin gene by our method, we randomly subsampled 5k, 10k, 30k, 50k, 100k, 500k and 1M reads from the total reads in the mRNA-seq data of four different hybridomas . Then, we repeated the de novo assembly 25 times using the randomly selected reads. We defined the Igh and Igk CDSs identified by the de novo assembly using the total reads .We implemented our immunoglobulin sequence identification strategy with a python script named Igh and Igl/Igk by de novo transcriptome assembly. Our method requires limited reads of mRNA-seq, because hybridomas highly express Igh and Igl/Igk transcripts. Our approach would be beneficial for rapid and cost-effective cloning of Igh and Igl/Igk CDSs.Here, we propose a rapid and accurate method for identifying the CDSs of Igh and Igl/Igk CDSs from hybridomas; however, it is time consuming and requires a large amount of total RNA. SMARTer RACE, which is a refinement on 5'RACE, requires only a small amount of RNA; however, SMARTer RACE and 5’RACE occasionally extract pseudo-sequences caused by annealing or mis-annealing of primers to the myeloma cell-derived Igh or Igl/Igk sequences in the hybridoma [Igh and Igl/Igk sequences were selected by filtering sequences based on the CDS length. We also surmise, on account of the remarkably high expression of Ig genes, that our method can work with as low as ~0.1μg of total RNA, which is the minimal requirement for the library prep kit used in this study.Conventionally, PCR, 5'RACE and SMARTer RACE (Clontech) have been used with degenerative primers for the determination of antibody sequences . 5´RACE ybridoma . TherefoIgh and Igl/Igk transcripts in each hybridoma. Therefore, hybridomas that express antibodies with a low level of Igh and Igl/Igk may not have enough transcripts to be assembled. In this case, increasing the read number for de novo assembly could be beneficial for identifying the Igh and Igl/Igk CDSs [Our method depends on the quantity of Igk CDSs .Igh and Igl/Igk, while other methods only identify the V region of immunoglobulin genes. Therefore, our method enables identification of the antibody isotypes and subclasses . We hope to extend our method in our future work for the detection of minor variants of antibody genes caused by somatic mutations, e.g., in clinical samples of myeloma or lymphoma cells.Another advantage of our method is the ability to identify full-length Igh and Igl/Igk CDSs, even from intact B cells or plasma cells, even at the single cell level.A recent study has demonstrated that mouse plasma cells highly express immunoglobulin genes . HybridoS1 Fig(EPS)Click here for additional data file.S1 Table(PDF)Click here for additional data file.


The persistence of long-lasting changes in synaptic connectivity that underlie long-term memory require new RNA and protein synthesis. To elucidate the temporal pattern of gene expression that gives rise to long-lasting neuronal plasticity, we analyzed differentially-expressed (DE) RNAs in mouse hippocampal slices following induction of late phase long-term potentiation (L-LTP) specifically within pyramidal excitatory neurons using Translating Ribosome Affinity Purification RNA sequencing (TRAP-seq). We detected time-dependent changes in up- and down-regulated ribosome-associated mRNAs over 2 h following L-LTP induction, with minimal overlap of DE transcripts between time points. TRAP-seq revealed greater numbers of DE transcripts and magnitudes of LTP-induced changes than RNA-seq of all cell types in the hippocampus. Neuron-enriched transcripts had greater changes at the ribosome-loading level than the total RNA level, while RNA-seq identified many non-neuronal DE mRNAs. Our results highlight the importance of considering both time course and cell-type specificity in activity-dependent gene expression during memory formation. Hippocampal long-term potentiation (LTP) serves as one of the best physiological correlates of behavioral learning and memory, with the persistence of LTP requiring new gene transcription and translation. However, the cell types within which these gene expression changes occur and the temporal regulation of these genes following LTP induction remain poorly understood. In this study, we utilized Translating Ribosome Affinity Purification to identify global changes in the ribosome-associated RNA population within excitatory hippocampal neurons during a critical, early time window following LTP induction. Through analysis of gene expression at three times points after LTP induction, our study provides a comprehensive and quantitative portrait of the gene expression changes that occur following induction of LTP within a specific population of neurons.Synaptic plasticity, the experience-dependent remodeling of neuronal connectivity, provides a means of storing memories in the brain  from adult RiboTag mice  and phosphorylation levels of ERK1/2 and eIF4E  for induction of LTP, using qPCR to monitor injury-induced up-regulation of a set of positive control immediate early transcripts . We chose a cLTP induction protocol that has been shown to be transcription- and translation-dependent  in the 5′ and 3′ untranslated regions (UTRs) of mRNAs has been shown to regulate translation of transcripts , the proportion of exclusively DE transcripts at their respective time points was 48% of DE transcripts at 30 min, 73% of DE at 60 min, and 83% of DE transcripts at 120 min. To more rigorously characterize the temporal dynamics of differential expression following LTP induction, we utilized Short Term Expression Miner ; however, the overlap was modest, with a smaller fraction of TRAP-seq DE transcripts being detected by RNA-seq at all time points. Another difference we observed was that unlike TRAP-seq, a majority of DE transcripts detected by RNA-seq at earlier time points was also DE at later time points  from the same set of mini-slice homogenates used to generate RNA for TRAP-seq, allowing a direct comparison between the two techniques from a single biological sample Table . Overalls Figure . Differes Figure .When collapsed across all time points, a total of 740 transcripts were DE only by TRAP-seq, while 176 transcripts were DE only by RNA-seq and 159 were DE in both Figure . This inMap2 and Map1b, were DE by TRAP-seq but virtually unchanged by RNA-seq. This raised the possibility that transcripts could be regulated differently at ribosome-associated and total RNA steady states; specifically, changes in the ribosome-associated population would not necessarily require corresponding changes in the total RNA concentration. To address this possibility using a more unbiased approach, we used a bioinformatics approach to identify neuron-enriched transcripts by looking for transcripts whose basal expression was overrepresented by TRAP-seq as compared to RNA-seq . We confirmed the neuron-enriched expression of these transcripts using the Allen Brain Atlas (http://mouse.brain-map.org/); of the 30 DE neuron-enriched transcripts that had detectable expression by the Allen Brain Atlas, only 1 was excluded for being non neuron-enriched. We then compared differential expression of this set of transcripts in the TRAP-seq and RNA-seq data. From this, we identified 16 transcripts that were DE at comparable levels by both RNA-seq and TRAP-seq . This greater magnitude of differential expression was observed for both upregulated and downregulated transcripts.Interestingly, transcripts known to be neuron-specific, such as Given that specific transcription factors have been shown to coordinate the transcription of multiple important transcripts for LTP and memory  fraction of transcripts was DE across multiple time points Figure . This fic-Fos are DE in neurons but are also highly expressed within astrocytes  the faster kinetics of ribosome-associated regulation compared to regulation of total RNA steady states; (2) the lower basal copy number of ribosome-associated transcript compared to total RNA copies; and/or (3) distinct stimulus-induced post-transcriptional (and transcription-independent) regulation. Indeed, the fact that we detected such a large number of downregulated transcripts at all time points by TRAP-seq but not by RNA-seq Figure  is consiThe design and scope of this study was not to hand-select candidates and assay their function in LTP. Previous studies utilizing this approach have proven invaluable in implicating different molecules important for LTP, but a unified model of the molecular requirements for LTP is lacking . Both male and female 8–12 week old Ribotag × Camk2α-Cre double heterozygotes were used to prepare hippocampal mini-slices.Both RiboTag and T29/Camk2α-Cre mice were purchased from Jackson Laboratories (2/5% CO2) ACSF containing 124 mM NaCl, 4 mM KCl, 25 mM NaHCO3, 1 mM NaH2PO4, 2 mM CaCl2, 1.2 mM MgSO4, and 10 mM glucose. LTP was induced by submerging slices in 50 μM forskolin  for 5 min, followed by 30 mM KCl/10 mM Ca2+/50 μM forskolin in 0 Mg2+ ACSF for 5 min. Control solution contained 0.2% DMSO in normal ACSF for 10 min. Slices were perfused with normal ACSF after LTP induction, with time 0 starting immediately after applying the K+/Ca2+/forskolin solution. Slices were snap frozen in crushed dry ice at the specified time points, and stored at −80°C until RNA purification.400 μm thick hippocampal CA3/CA1 mini-slices were prepared from hippocampi of RT x Camk2α-cre double heterozygotes. The dentate gyrus was microdissected following slicing and mini-slices were allowed to recover for 2 h at 30°C in interface-type chambers with oxygenated , 1 mg/mL heparin, 100 μg/mL cycloheximide, cOmplete protease inhibitor tablet (Roche). Pre-conjugated HA beads  were washed twice with IP buffer before use. After homogenization, the homogenate was centrifuged twice at 10,000× g at 4°C. 1/10 of the homogenate at this step was immediately placed into Trizol (Invitrogen) for total RNA extraction. The remaining homogenate was incubated with washed HA beads overnight at 4°C. The following day the beads were washed 3× in a high salt buffer composed of 50 mM Tris, 300 mM KCl, 12 mM MgCl2, 1% NP-40, 1 mM DTT, and 100 μg/mL cycloheximide. RNA was eluted from the beads using 350 μL Buffer RLT + BME supplementation from the RNeasy Micro kit (Qiagen). Eluted IP RNA was column purified according to the kit protocol. Total RNA was purified through a combined Trizol and RNeasy kit protocol: after the spin gradient step in the Trizol purification, the aqueous layer was taken and mixed 1:1 with 100% EtOH and purified on a spin column. For each biological replicate, four mice were used, with ~20 slices per treatment condition. TRAP purification yielded ~200 ng RNA per 20 mini-slices, and total RNA from 1/10 of this homogenate yielded ~400 ng RNA. RNA quantification was done with the Qubit RNA HS assay (Thermo Scientific).Frozen slices were homogenized using a pestle in IP buffer which contained 50 mM Tris-HCl pH 7.4, 100 mM KCl, 12 mM MgClRRID:AB_291263) and rabbit anti-MAP2  overnight at 4°C, and with secondary antibodies  at 2 h at room temperature and counterstained with Hoechst .Hippocampal slices were fixed in 4% PFA for 2 h at room temperature, rinsed 2× with PBS, covered in HistoGel (Thermo Scientific), then paraffin-embedded. 4 μm thick paraffin sections were deparaffinized and underwent heat-induced antigen retrieval. Sections were permeabilized with 0.1% TX-100 at room temperature for 30 min, then blocked in 10% goat serum at room temperature for 60 min. Slices were incubated with mouse anti-HA antibody  was used to confirm a microglial DE transcript. Images were taken from control- and LTP-treated slices from two animals at the 120 min time point, with 3–4 slices per animal per condition. ROIs were drawn around staining from cell bodies and processes, and the mean pixel intensity was taken for each ROI using ImageJ software. Each data point represents one slice. Mann-Whitney nonparametric test was used to determine significance.Rabbit anti-Il1b  using SYBR Green PCR Master Mix (Applied Biosystems) were prepared, with the cDNA being evenly divided into each reaction. Percent de-enrichment was calculated by comparing relative Ct values between total and TRAP RNA samples for each set of primer. To assay LTP induction, 20 ng of total RNA and TRAP RNA for both basal and LTP conditions were reverse transcribed. Normalization of basal/LTP Ct values with HPRT1 was done before comparing fold-change of activity-dependent transcripts  through relative Ct values. To validate sequencing results, we collected a separate biological sample for both 60 and 120 min time points and performed RT-qPCR for the indicated transcripts. Some primer sequences were obtained from PrimerBank (http://pga.mgh.harvard.edu/primerbank) while others were designed to span exon-exon junctions.To assay depletion of non-specific transcripts, 50 ng of total RNA and TRAP RNA were reverse transcribed into cDNA using SuperScript III with random hexamer primers (Invitrogen). Technical triplicate reactions for each primer set . RNA libraries were made using Illumina TruSeq RNA library preparation v2 kit. rRNA was first depleted from all samples using the Ribo-Zero Gold kit. Libraries were prepared according to manufacturer's instructions, skipping the oligodT purification step. Single-end (30 min time point) and paired-end sequencing (60 and 120 min time points) were performed using the Illumina Hiseq2500 system. Both library preparation and sequencing were done at the UCLA Neuroscience Genomics Core. Read lengths were 64 bp for single-end reads and 69 bp for paired-end reads, with an average of 45 million mapped reads per sample.2 fold-change of at least ±0.4. Hypergeometric tests were used to determine significance of overlap between DE transcripts of different variables.RNA sequencing reads were mapped to the mm10 annotation of the mouse genome using STAR aligner  were excluded as to minimize visual artifacts.p-value < 0.05 after BH correction to be significant, with a minimum of 4 genes per category. Up and downregulated transcripts from all time points were included.KEGG pathway analysis of up and downregulated transcripts was done using WebGestalt with a whole genome background list. All functional category enrichment analyses considered an adjusted http://www.cs.cmu.edu/~jernst/stem/) using the fold-changes for transcripts that were significant in at least one time point. Gene ontology analysis was done using WebGestalt  on the temporal profiles derived from STEM analysis. All transcripts that had at least an average read number of 20 normalized reads by TRAP-seq across all time points was used as the background transcript list. For enrichment of TRAP-seq, RNA-seq, or both TRAP-seq and RNA-seq DE transcript function, PANTHER (http://pantherdb.org/) was also used to identify enriched categories and the genes associated with them with a background list derived from RNA-seq expression levels of 20 normalized reads on average across all samples and time points.Time series analysis was done with Short Time-series Expression Miner (STEM) , we took the average fold-changes between all time points the transcript was DE. Significance testing of the loghttp://opossum.cisreg.ca/oPOSSUM3/) and TRANSFAC on BioBase, respectively. Significant transcripts identified through total RNA sequencing at any time point were included in the analysis, with a background list of >20 average reads transcripts at 120′ for oPOSSUM and >10 average reads for TRANSFAC. Graphical representation is for oPOSSUM results only, while results from both analyses are included in Tables A list of enriched transcription factor targets and significantly enriched binding motifs was identified using oPOSSUM 3.0 (Use of mice in this study followed the recommendations of and protocol approved by the UCLA Institutional Animal Care and Use Committee.PC and KM designed experiments; TO and JA prepared hippocampal mini-slices; PC bred and characterized Ribotag-CamKIIα mice, performed cLTP stimulations, prepared samples and performed immunohistochemistry and qPCR; PC, RK, CB, and GC performed bioinformatics analysis; PC and KM wrote manuscript, with edits from TO, RK, and GC.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


RNA nanotechnology is attracting a great deal of attention recently. As the multiple roles that RNA plays in molecular biology and physiological regulation become clearer, there are many opportunities for engineering RNA-Nanoparticle Complexes (RNA-NPCs). The high “engineerability” of RNA-NPCs comes from the ability to modify the RNA and NP chemistry. For example, the NP can be derived from materials with anticancer activity and the RNA delivered by it, designed to target cell signaling pathways that contribute to the molecular basis of these diseases. Despite this rapid advancement and the availability of new quantification and characterization techniques, a key challenge is to develop a better understanding of the RNA-nanobio interface; that is, the interactions of RNA with NP (RNA-nanobio interface) and how that impacts the structure, function, delivery, and activity of the RNA. Here, we attempt to summarize the state-of-the-art in this new and exciting field, and to lay out potential directions for bioengineering research on RNA-NPCs. Nanotechnology has emerged at the forefront of science in the past decade with many applications in engineering. In parallel, with advancements in genomics and proteomics, much of the early biomolecular nanotechnology work was done on DNA or protein-based Nanoparticles (NPs). However, with the advancement of our understanding of RNA, RNA nanotechnology has recently exploded onto the scene . As shown in RNA is more than just a source code and genetic information ,2. WhileConventionally, RNA-NPs are defined as nanostructures primarily composed of RNA ,9, with RNA-NPCs have been used successfully as bio sensors ,16,17—toThe engineerability of the RNA-nanobio interface arises in large part from the ability to specify the interactions between the RNA molecule and the NP surface. This is achieved through chemical manipulations of the RNA molecule or through chemical modifications on the surface of the NP. In the case of RNA, a variety of chemistries are available which modify either the backbone or the base. Additionally, the 5′ or 3′ termini of the RNA molecules can be synthesized with various derivatives which impart desirable properties on them such as fluorescence or stability . As shown in RNA Phosphodiester backbone modifications: The phosphodiester bond between RNA is a hotspot for stability manipulations. Some of the more common of these, that are likely to impact the RNA-nanobio interface are the Phosphorothioate (PS) and boranophosphate [hosphate  derivatihosphate . Both ofRNA ribose sugar modifications: A second hotspot for modifications is on the 2′ carbon of the ribose sugar molecule. 2′-O-Methyl and 2′-F modifications are seen to substantially improve RNAse resistance. However, 2′-O-Me synthesized modified oligos are not recognized by other enzymes. 2′-F oligos, are now being extensively used due to their ability to retain native RNA configurations [urations .RNA modifications for fluorescence: RNA possesses no intrinsic fluorescence. Some RNAs such as aptamers may need to target the cell membrane, others such as antisense and siRNA act in the cytosol, whereas SSOs and other RNAs designed for transcription modulation require nuclear delivery. Here, it can be helpful to visualize cytosolic localization by modifying the RNA with fluorescent moieties. For example, carboxyfluorescein (6-FAM) and Cy5 modifications to the RNA can be used to view the RNA location in cell imaging. Additionally, conjunction of the dyes on either end of RNA has been used as a primitive cleavage marking system [g system  in the cg system . More reg system . Similarg system . Self-asIn addition to engineering the RNA, many investigators modify NP chemistry. NP  surface modifications have been used to confer beneficial properties to NPs . Below aNP modifications with polymers: One of the prevailing types of common functionalization is polyethyleneglycol (PEG) modification, generally thought to confer physical stability upon the NP and prevent aggregation [regation . Polyethregation . Chitosaregation . NP modification with amino acid: NPs can be functionalized with amino acids, such as arginine, to enhance binding of RNA. It is well known that RNA motifs can be engineered to have specificity for certain amino groups [o groups ,45,46. So groups . Amino aNP modification with cell-penetrating peptides (CPP): NP uptake into cell is crucial for cancer targeting. Cell-penetrating peptide functionalized NPs have been used with some success to enhance NP uptake. CPP modified liposomes, however, were seen to have inefficient RNA unloading post internalization; polymer NPs, however, did not have the same effect [e effect ,49. NP surfactant capping: NP growth can be controlled with capping agents such as Citrate, Cetyltrimethylammonium Bromide (CTAB), Oleic Acid (OA) etc. Capping agents enable uniform homogenous dispersion of NPs in colloidal solutions. Additionally, capping agents act as stabilizing agents by preventing NP aggregation. However, their distribution on the NP surface also inhibits catalytic active sites, preventing accurate reactions [eactions .NP modifications with chemical functional groups: Alternatively, water-soluble NPs are modified with chemical functional groups such as carboxylic acids (that prevent aggregation due to electrostatic repulsion) or thiols [r thiols . Thiols r thiols . This haMiscellaneous NP modifications and targeting: Sugar molecules often decorate the surface of cells and tissues and are conjugated to proteins to form glycoproteins. Glycosaminoglycans of various forms and sizes are present within the extracellular matrix and form the bulk of non-proteinaceous material present therein and are widely used as biomedical materials such as heparin and many others. This represents an important targeting opportunity where, for example, one study used 2-deoxy-D-glucose modified polymer NPs to target intracranial tumors which were shown to better penetrate the blood–brain barrier and accumulate in intracranial tumors [l tumors . Here, aAnother opportunity represents the techniques necessary to characterize the RNA-nanobio interface. Engineering being principally mathematically driven, it is important to be able to have quantitative parameters by which to optimize the performance of RNA-NPC. Today, a suite of technologies has become available which can shed light on the interaction of the RNA with the surface of the NP. For example, innovations in a variety of different microscopy approaches now allow us to essentially glimpse the surface. Common techniques include microscopy: Atomic Force Microscopy (AFM), Scanning Electron Microscopy (SEM), Transmission Electron Microscopy (TEM) and others. Surface Plasmon Resonance (SPR) properties of NPs , based on free electron oscillations-which are size and shape dependent , may be Zeta potential  and Dynamic Light Scattering (DLS) are two common techniques which reveal changes in the electrostatic potential at the surface when RNA binds or in the hydrodynamic diameter of the particle respectively. These techniques, while semi-quantitative, tend to be difference measurements  and may not generate information that ultimately correlates with bio-activity. 1H NMR, Raman Spectroscopy and Fourier Transform Infrared Spectroscopy (FT-IR). Based on the functional groups present, the peaks obtained from the NMR and IR can be used to detect the presence and binding of groups on the NP surface. Additionally, shifts in NMR and IR peaks can be used to detect conformational changes caused due to NP binding. Techniques such as X-ray Photon Spectroscopy (XPS) have been used with some success in determining the configuration of RNA on NP surfaces [Techniques which can discriminate the nature of the nanobio interaction include nuclear magnetic resonance (NMR), infra-red (IR), ultraviolet (UV) spectroscopy and others. Further, surface functionalization characterization can be studied by surfaces ,55. For surfaces , provideThermal characteristics of self-assembling RNA-NPs have also been used to characterize the RNA-nanobio interface. The melting point temperature of self-assembling RNA-NPs has been used to determine controlled release as well stability over temperature ranges ,9,57. FuNPs possess physiological and chemical properties that are different than their parental bulk materials. Addition of RNA and functionalization further change the NP surface properties and how they interact with the biological milieu. RNA-NPCs introduced into biological fluid have been seen to form Protein Coronas (PC)—aggregations of proteins on the NP surface . These PIntravenous injections of NP drugs necessarily requires an understanding of the interactions between RNA-NPC and blood components-PC accumulation, some of which possess RNAse activity and have been seen to hydrolyze RNA . In caseNP-mediated therapy holds the potential for rapid advancement in diagnosis in treatment; however several shortcomings need to be overcome. Firstly, RNA is highly labile and chemically unstable, rendering it extremely sensitive to RNAse degradation. This becomes of special importance for intra-venous drug applications, as the RNA is susceptible to hydrolysis. In addition, the lack of specificity renders it prone to off-target effects due to partial matching. There have been efforts to curb these with structural modifications  or usingRNA, unlike proteins, does not possess intrinsic fluorescence. In such cases, the intrinsic fluorescence properties of certain NPs  have been used to visualize RNA-NPCs. Additionally, the RNA or NPs have been functionalized with dyes for fluorescence ,57,58,59In brief, developing a better understanding of the RNA-nanobio interface is critical to engineering better RNA-NPCs. With the myriad of chemistries available both for RNA and NP, it is now crucial to identify optimal RNA-NPC combinations that will ensure enhanced stability, biocompatibility, and therapeutic effect. Advances in RNA-nanobio characterization are tied to successful RNA-NPC engineering. These opportunities and the challenge of a complex and variable biological milieu in which the RNA-NPC is expected to perform will be important to address for the long-term of RNA-NPC therapeutics.


Lagenaria siceraria plants infected with Cucumber green mottle mosaic virus (CGMMV) were comprehensively characterized and compared. Many more vsiRNAs were present in infected leaves than in fruits. vsiRNAs from both leaves and fruits were mostly 21- and 22-nt in size as previously described in other virus-infected plants. Interestingly, vsiRNAs were predominantly produced from the viral positive strand RNAs in infected leaves, whereas in infected fruits they were derived equally from the positive and negative strands. Many leaf-specific positive vsiRNAs with lengths of 21-nt (2058) or 22-nt (3996) were identified but only six (21-nt) and one (22-nt) positive vsiRNAs were found to be specific to fruits. vsiRNAs hotspots were only present in the 5′-terminal and 3′-terminal of viral positive strand in fruits, while multiple hotspots were identified in leaves. Differences in GC content and 5′-terminal nucleotide of vsiRNAs were also observed in the two organs. To our knowledge, this provides the first high-resolution comparison of vsiRNA profiles between different tissues of the same host plant.RNA silencing is an evolutionarily conserved antiviral mechanism, through which virus-derived small interfering RNAs (vsiRNAs) playing roles in host antiviral defense are produced in virus-infected plant. Deep sequencing technology has revolutionized the study on the interaction between virus and plant host through the analysis of vsiRNAs profile. However, comparison of vsiRNA profiles in different tissues from a same host plant has been rarely reported. In this study, the profiles of vsiRNAs from leaves and fruits of  In general, 21-nt vsiRNAs usually predominate in the population, there is a strong A/U bias at the first nucleotide of vsiRNAs, and vsiRNA-producing hotspots can be identified within the viral genome  is a member of the genus Lagenaria siceraria, accession “Hangzhou gourd”) were sown in soil rich in organic matters in a greenhouse with the ambient temperatures between 20 and 25°C, and watered every 3 days to maintain ample soil moisture. At the two and a half leaf stage plants were mechanically inoculated with CGMMV virions on the two expanding leaves using sap from a previously infected plant. Approximately 100 mg of tissue was homogenized in 20 volumes of inoculation buffer , while the mock plants were only inoculated with inoculation buffer.Seeds of bottle gourd  according to the manufacturer's instructions. The presence of CGMMV infection in the tissues was confirmed with a One Step RT-PCR Kit  following the product's protocol and using CGMMV specific primers . The RNA quantity and quality from each sample was evaluated by denaturing agrose gel electrophoresis and a 2100 Bioanalyzer .Approximately 5 μg of total RNA was extracted for the preparation of a small RNA library according to the protocol of TruSeq Small RNA Sample Prep Kits . Briefly, total RNA was resolved using denatured 8% polyacrylamide gel electrophoresis (PAGE) and small RNA fragments were isolated. After ligation of the 5′ and 3′ adaptors, the short RNA fragments were reverse transcribed using SuperScript II Reverse Transcriptase  and amplified by PCR. Finally, single-end sequencing (36 bp) was performed on an Illumina Hiseq2500 at LC-BIO  following the protocol of the manufacturer.http://hannonlab.cshl.edu/fastx_toolkit/). The remaining sRNA reads were collapsed to uniread sets and the reads of > 30-nt or <18-nt were discarded. Clean sRNA reads were used for further bioinformatics analysis.After parsing small RNA sequences from the 3′ adaptor sequence, low quality and junk sequences, including transfer RNAs (tRNAs), ribosomal RNA (rRNAs), small nucleolar RNAs (snoRNAs), small nuclear RNAs (snRNAs), and repetitive sequences, were removed using the FASTX-Toolkit (L. siceraria libraries were mapped to the CGMMV reference genome (NCBI Accession No: KP868654) using Bowtie software (http://bowtie-bio.sourceforge.net) with one mismatch. To facilitate comparisons across different libraries, vsiRNA read numbers were normalized to “Reads Per Million” (RPM) based on the total small RNA read numbers of the corresponding library. All of the downstream analyses were performed using custom perl scripts and linux (Cent OS 6.5) bash script. For statistical analysis of the three biological replicates, one-way ANOVA analysis using Originpro 8.5 software was performed and values of p < 0.01 were considered significant. To avoid the inaccuracy of low copy sequences, sequences with <10 raw reads in each of the three replicates were removed . Specific (Unique) vsiRNAs were extracted from the three replicates of each sample during this analysis. RNA secondary structures were predicted using RNAfold (http://rna.tbi.univie.ac.at/cgi-bin/RNAfold.cgi) with default parameters.To identify CGMMV-derived siRNAs, processed reads from each of the 12 Total RNA was isolated from plants with Trizol  according to the manufacturer's instructions. For northern blot of CGMMV RNAs, a DNA probe targeting CGMMV CP was synthesized with primers (5′-GCTTACAATCCGATCACAC-3′ and 5′-ATTATCTATCTCAGCCCTAG-3′) and labeled with DIG according to the manufacturer's protocol . For northern blot of positive-stranded CGMMV RNAs in leaves, a sequence (5′-CAACACAGGACCGTTGAGGAAAGCGTAAAAACCCGCACCTGGGAATCTAGAATTAATATCTACGACAGACGAGGGTAACGCA-3′) was synthesized and labeled as DNA probe, and its complementary sequence was used for detecting negative-stranded CGMMV RNAs. Another sequence (5′-CATAGCTCTGAGCTTTAACTACACTAAAGTCAGTTATAGATAAATACTTAAGAATGGAAAAATAGTTAGGGAGCAACTTATC-3′) was used for detecting positive-stranded CGMMV RNAs in fruits, and its complementary sequence was used for detecting negative -stranded CGMMV RNAs in fruits. Pre-hybridization, hybridization and signal detection were done according to the protocol of the DIG High Prime DNA Labeling and Detection Starter Kit II .Tissue immunoblot was carried out as described previously -infected leaves of bottle gourd showed the typical green mottle mosaic symptom 14 days after inoculation Figure , while tAfter the removal of the junk, adapter and repeat reads, total numbers of small RNAs 18–30 nt long obtained from the three virus-infected fruits were 8,558,357 , 9,240,205  and 5,654,646 . Corresponding numbers from the mock fruits were 9,442,756 , 9,432,345  and 9,268,640 . From three virus-infected leaves, totals were 10,250,660 , 7,513,459  and 12,265,181  and from three mock leaves, there were 8,766,823 , 4,064,336  and 6,644,494 . An overview of the deep sequencing results is presented in Table The size distribution of these 12 small RNA libraries was similar. Reads with 24-nt length accounted for most (60–70%) of the total sRNAs, followed by 23-nt Figure . HoweverN. benthamiana plants infected with Beet necrotic yellow vein virus (BNYVV) showed that the antiviral response was more effective in leaves than in roots; vsiRNAs accumulated more in leaves than in roots, whereas BNYVV mRNA levels were lower in leaves than in roots  of the total sRNAs of virus-infected fruits, while the corresponding figures for unique vsiRNAs were 0.95–1.15% (1.06 ± 0.10%). These values are much lower than those from infected leaves where total vsiRNAs accounted for 20.59–30.99% (27.49 ± 5.98%) of the total sRNAs and the corresponding figures for unique vsiRNAs were 2.24–3.47% (2.89 ± 0.62%)  Figures . The incThe numbers of vsiRNAs derived from positive or negative strand viral RNA were also compared. In infected leaves, many more vsiRNAs were produced from the positive strand viral RNA irrespective of vsiRNA length Figures , which iL. siceraria according to the size and density of the bands  cover the entire CGMMV genome Figure , consistPrevious studies have shown that vsiRNAs are preferentially produced from GC-rich regions and vsiRNAs tend to have a higher GC content than that of the entire viral genome , the International Science & Technology Cooperation Program of China (2015DFA30700) and Fund for Research in the Public Interest of Zhejiang Province (2015C32042).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Bunyaviridae) are enveloped negative-sense tripartite RNA viruses. The natural hosts of hantaviruses include rodents, shrews, moles, and bats. Imjin virus (MJNV) is a shrew-borne hantavirus identified from the Ussuri white-toothed shrews (Crocidura lasiura) in the Republic of Korea (ROK) and China. We have isolated MJNV and determined its prevalence and molecular diversity in Gyeonggi province, ROK. However, the distribution and phylogeography of MJNV in other regions of ROK remain unknown. A total of 96 C. lasiura were captured from Gangwon and Gyeonggi provinces, ROK, during 2011–2014. Among them, four (4.2%) shrews were positive for anti-MJNV IgG and MJNV RNA was detected from nine (9.4%), respectively. Based on the prevalence of MJNV RNA, the preponderance of infected shrews was male and adult, consistent with the gender- and weight-specific prevalence of hantaviruses in other species. We monitored the viral load of MJNV RNA in various tissues of shrews, which would reflect the dynamic infectious status and circulation of MJNV in nature. Our phylogeographic and genomic characterization of MJNV suggested natural occurrences of recombination and reassortment in the virus population. Thus, these findings provide significant insights into the epidemiology, phylogeographic diversity, and dynamic circulation and evolution of shrew-borne hantaviruses.Hantaviruses (family  Hantavirus, family Bunyaviridae) are zoonotic, negative-sense, single-stranded RNA viruses containing large (L), medium (M), and small (S) segments that encode RNA-dependent RNA polymerase (RdRp), glycoproteins (Gn and Gc), and nucleocapsid (N) protein, respectively245Suncus murinus), Imjin virus (MJNV) from the Ussuri white-toothed shrew (Crocidura lasiura), Jeju virus (JJUV) from the Asian lesser white-toothed shrew (C. shantungensis), Camp Ripley virus (RPLV) from the northern short-tailed shrew (Blarina brevicauda), Cao Bang virus (CBNV) from the Chinese mole shrew (Anourosorex squamipes), Boginia virus (BOGV) from the Eurasian water shrew (Neomys fodiens), Seewis virus (SWSV) from the Eurasian common shrew (Sorex araneus), and Artybash virus (ARTV) from the Laxmann’s shrew (S. caecutiens)7891011121314Hantaviruses In our previous study, we first discovered and isolated a shrew-borne hantavirus, MJNV, from in vitro and in nature2526Genetic exchanges among viruses give rise to genetic diversities that are the basis for molecular evolution202223C. lasiura, showing the gender and weight-specific prevalence of hantavirus infection reported in other species of shrews and rodents. Genomic sequences of MJNV were obtained from three (75.0%) of four anti-MJNV IgG seropositive shrews and six (6.5%) of 92 seronegative shrews. Additionally, the entire coding sequence of MJNV 10–8 tripartite RNA was obtained since it was identified in 2010. In an analysis of MJNV RNA in the various tissues of seropositive and seronegative shrews, threshold cycles (Ct) values demonstrated a high viral load  and divergent distribution of MJNV RNA in IFA+PCR+ (seropositive and PCR positive) shrews by reverse-transcription quantitative PCR (RT-qPCR) using MJNV M segment-specific primers. Of the IFA−PCR+ (seronegative but PCR positive) shrews, Cl 12–2 and 14–78 harboured a low amount  of MJNV RNA in different tissues, whereas MJNV RNA was observed only in the lungs of Cl 14–37, 14–42, and 14–70. Phylogenetic analyses of partial genomic sequences of MJNV tripartite RNA demonstrated the well-supported genetic diversity of MJNV in Gangwon and Gyeonggi provinces. Analyses of MJNV open reading frames (ORFs) suggested that recombination and reassortment naturally occurred in the virus population. MJNV 12–2 showed an evidence of L segment recombination, and MJNV strains from Yeoncheon were very likely M segment reassortants. In conclusion, these observations provide better understandings of the epidemiology, phylogeographic diversity, and dynamic circulation and genetic exchanges of shrew-borne hantaviruses in natural reservoirs.This study described the distribution and phylogenetic diversity of MJNV in Gangwon province, ROK. The prevalence of MJNV from 96 shrews was comparable between Gangwon and Gyeonggi provinces. There was a clear preponderance of males and adults among MJNV-infected Trapping of animals was approved by US Forces Korea (USFK) in accordance with USFK Regulation 40–1 “Prevention, Surveillance, and Treatment of Hemorrhagic Fever with Renal Syndrome”. All procedures and handling of animals were conducted under an approved protocol by the Korea University Institutional Animal Care and Use Committee .C. lasiura, 14 C. shantungensis, 921 Apodemus agrarius, 4 A. peninsulae, 10 Myodes regulus, 16 Micromys minutus, 2 Microtus fortis, 1 Tamias sibiricus, and 16 Tscherskia triton, were captured in ROK from 2011 to 2014 using Sherman traps  baited with peanut butter-covered hardtack. Field trappings were conducted for three days and two nights. For each day, a total of 100 traps were set at intervals of approximately 1 to 2 m. Trapping sites were shown in the C. lasiura via cardiac puncture, and serum was isolated by centrifugation for 5 min at 4 °C. Lungs, livers, kidneys, and spleens were collected and stored at −80 °C.Shrews and rodents, a total of 96 Sera were initially diluted 1:32 in phosphate-buffered saline (PBS) and examined for anti-MJNV IgG antibody. The diluted sera were added to wells of acetone-fixed Vero E6 cells infected with MJNV, and the slides were incubated for 30 min at 37 °C. After washing twice, fluorescein isothiocyanate-conjugated goat antibody to rat and mouse IgG antibodies  was added, and the slides were incubated at 37 °C for 30 min. After washing three times, virus-specific fluorescence was evaluated using a fluorescent microscope .Total RNA was extracted from lung tissues of seropositive and seronegative shrews using TRI Reagent Solution . Using random hexamers, cDNA was synthesized using M-MLV reverse transcriptase  or a high capacity RNA-to-cDNA kit . First and nested PCRs were performed in a 25-μl reaction mixture containing 2.5U of Ex Taq DNA polymerase , 2 μg of cDNA, 10 pM of each primer, and 200 μM dNTP . Oligonucleotide primer sequences for the nested PCR were MJN-L942F (outer): 5′-TCAGAATATAAACCTATGAC-3′, MJN-L1636R (outer): 5′-GATCAACAAACCCATATC-3′, MJN-L942F (inner) and MJN-L1612R (inner): 5′-CTTACATGAGCAGTGGCTA-3′ for the L segment; MJN-M2235F (outer): 5′-CATGGAAGAGTGCAACATGT-3′ and MJN-M2855R (outer): 5′-TATGGTCCCTAGATGTACT-3′, MJV-M2235F (inner) and MJN-M2805R (inner): 5′-TCTATAATAGGATCAGTCAT-3′ for the M segment; MJN-S350F (outer): 5′-GTTGAAGAAGGTGAYTATYTG-3′, MJN-S1102R (outer): 5′-TATRTCCTGCATTAATGCAA-3′, MJN-S350F (inner) and MJN-S1023R (inner): 5′-GGTGCATTYGCAAAAATCCA-3′ for the S segment. Initial denaturation was performed at 95 °C for 5 min, followed by 15 cycles of denaturation at 94 °C for 30 sec, annealing at 50 °C for 40 sec, elongation at 72 °C for 1 min, and then 25 cycles of denaturation at 94 °C for 40 sec, annealing at 52 °C for 40 sec, and elongation at 72 °C for 1 min . PCR products were purified by LaboPass PCR purification kit , and sequencing was performed in both directions of each PCR product using a BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems) on an automated sequencer .cytochrome b genes of shrews were amplified by PCR and phylogenetically analysed using MEGA 5.2To identify the species of shrews, mitochondrial DNA Total RNA was reverse-transcribed using a high-capacity RNA-to-cDNA Kit (Applied Biosystems), with each 10-μL reaction containing 1 μg of total RNA from lungs, livers, kidneys, and spleens. Using a SYBR Green PCR Master Mix (Applied Biosystems) on a StepOne Real-Time PCR System (Applied Biosystems), reactions were performed at a cycle of 95 °C for 10 min, followed by 45 cycles at 95 °C for 15 s, 60 °C for 1 min. Primer sequences targeting MJNV M segment were MJNV-M828F: 5′–AATTTAGGAAAAATCCACAAGGTG–3′ and MJNV-M948R: 5′–TTGAATGCTGCTAGGGTGTTT–3′.Viral genomic sequences were aligned and edited using the MUSCLE algorithm. Phylogenetic trees were generated by neighbour joining (NJ) and maximum likelihood (ML) methods (MEGA 5.2)P-value (p) was under 0.05 and the RDP recombination consensus score (RDPRCS) was over 0.6p was under 0.05 and the RDPRCS was between 0.4 and 0.6. The likelihood of recombination and reassortment events was considered insignificant when the RDPRCS was under 0.4 with p < 0.05. Subsequently, phylogenetic relationships were reconstructed for each genetic event using the ML method in MEGA 5.2.Alignments of the concatenated MJNV L, M, and S segment ORFs were analysed using RDP, GENECONV, MAXCHI, CHIMAERA, 3SEQ, BOOTSCAN, and SISCAN in the Recombination Detection Program 4 (RDP4) packageC. lasiura) were captured in Gangwon and Gyeonggi provinces from 2011 to 2014. Using sera from the shrews, serological tests showed that four (4.2%) of 96 samples were positive for anti-MJNV IgG, with three from Gangwon province and one from Gyeonggi province (C. lasiura for rodent-borne hantaviruses including HTNV and Seoul virus (SEOV). Partial MJNV L  and M  sequences were detected in nine (9.4%) out of 96 shrews. Among them, three (75.0%) of four seropositive and six (6.5%) of 92 seronegative shrews were positive for the MJNV L and/or M segments, respectively. Seven (17.1%) of 41 males and two (3.6%) of 55 females harboured MJNV RNA. The prevalence of MJNV in the shrews showed heavier animals (≥9.0 g) were infected with MJNV, but there was no positivity of MJNV infection under the animals of 9.0 g. During 2011–2014, most of C. lasiura were captured and all of MJNV-positive shrews were observed in autumn. MJNV was not detected from nine of C. lasiura collected in spring and summer. A total of 96 Ussuri white-toothed shrews  were overall low  and varied in the different tissues, demonstrating a dynamic MJNV infection in hosts. Cl 12–2 (IFA−PCR+) showed the presence of MJNV RNA in all tissues, whereas all tissues, except for the liver, from Cl 14–78 (IFA−PCR+) contained the comparable viral loads of MJNV RNA. The IFA−PCR+ shrews, Cl 14–37, 14–42 and 14–70, showed the highest MJNV RNA loads in their lungs, whereas there were very low amounts of viral RNA in their livers, kidneys, and spleens. The amount of MJNV RNA in Cl 14–71 (IFA−PCR+) was likely less than the limit of detection of the assay.To determine the amount of MJNV RNA in IFA spleens . The Ct To investigate the genetic diversity of MJNV in Gangwon and Gyeonggi provinces, phylogenetic trees were generated by NJ, ML, and Bayesian methods. The geographic locations of MJNV strains collected in this study are shown in the p-values of the analysis ranged from 2.824E-5 to 7.821E-30, and the RDPRCS of MJNV12-2 was 0.725. Using MJNV strains representing a recombinant, parents, and in- and out-groups, phylogenetic trees of the MJNV L, M, and S segments were generated. The recombined region  of the MJNV 12-2 L segment clustered with MJNV strains from Yeoncheon, whereas the rest of the MJNV 12-2 L segment  clustered with MJNV 14–21 from Inje  and youth shrews. The preferential male infection with MJNV suggests that the horizontal transmission of MJNV may occur via aggressive behaviors such as biting or fighting. The dominant prevalence of adult infection with MJNV indicates that reproductive activities may be associated with the transmission of MJNV. The breeding of C. lasiura occurs during summer and autumnRodent-borne hantaviruses, e.g. HTNV, Sin Nombre virus (SNV), El Moro Canyon virus (EMCV), Puumala virus (PUUV), Bayou virus (BAYV), have been reported for the higher prevalence of infection in the male and adult (heavier) natural hosts39404142+PCR+ and IFA−PCR+ animals. IFA+PCR+ samples showed higher viral loads of MJNV RNA in the tissues, including lungs, kidneys, livers, and spleens. For the three IFA−PCR+ samples, MJNV RNA was the highest in the lung, reflecting an early phase of infection with MJNV in the endemic areas during autumn (September and November)46To examine the viral load of MJNV in shrews, the amount of MJNV was determined from various tissues of IFAin vitro5051in vitro23265455Bunyaviridae, has shown the evidence of an M segment reassortment in nature and in vitroin vitroGenetic exchanges, e.g., recombination and reassortment, promote genetic diversities for viral evolution in natureC. lasiura collected in Gangwon and Gyeonggi provinces, ROK. The preferential infection of MJNV was described in male and adult shrews. Based on 28 partial MJNV sequences, including 24 ORF genomic sequences, the phylogenetic analyses indicated geographic diversity of MJNV in the regions. Profiling of MJNV RNA in various tissues of the shrew specimens may reflect the dynamic circulation of MJNV in natural reservoirs. Evidences for the occurrence of recombination and reassortment suggest active genetic exchanges of shrew-borne hantaviruses in nature. These findings provide broad and deep insights into the epidemiology, virus-host interaction, and molecular evolution of hantaviruses in soricid hosts.In conclusion, this study first characterized the geographic prevalence, phylogenetic diversity, and dynamic circulation and molecular evolution of MJNV from How to cite this article: Lee, S.-H. et al. Dynamic Circulation and Genetic Exchange of a Shrew-borne Hantavirus, Imjin virus, in the Republic of Korea. Sci. Rep.7, 44369; doi: 10.1038/srep44369 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


The posttranscriptional addition of nontemplated nucleotides to the 3′ ends of RNA molecules can have a significant impact on their stability and biological function. It has been recently discovered that nontemplated addition of uridine or adenosine to the 3′ ends of RNAs occurs in different organisms ranging from algae to humans, and on different kinds of RNAs, such as histone mRNAs, mRNA fragments, U6 snRNA, mature small RNAs and their precursors etc. These modifications may lead to different outcomes, such as increasing RNA decay, promoting or inhibiting RNA processing, or changing RNA activity. Growing pieces of evidence have revealed that such modifications can be RNA sequence-specific and subjected to temporal or spatial regulation in development. RNA tailing and its outcomes have been associated with human diseases such as cancer. Here, we review recent developments in RNA uridylation and adenylation and discuss the future prospects in this research area. Nontemplated addition of nucleotides to RNA 3′ ends has long been known, with the addition of CCA to the 3′ ends of tRNAs and mRNA polyadenylation being the earliest described examples ,2. RelatHistone mRNAs are the only known metazoan mRNAs that are not polyadenylated, ending instead in a conserved stem-loop sequence. The stem-loop-binding protein (SLBP) participates in nearly all aspects of histone mRNA metabolism, such as pre-mRNA processing , mRNA exIn human cells, histone mRNA degradation begins with the assembly of a complex of factors, including SLBP and Exoribonuclease 1 (Eri1), on the 3′ end of the mRNA, resulting in the addition of uridine to the 3′ end of the histone mRNA. Following the oligouridylation event, the heteroheptameric Lsm1–7 complex binds to the oligo(U) tail to trigger subsequent histone mRNA degradation via both 5′–3′ and 3′–5′ RNA decay pathways ,12. The Arabidopsis [By regulating gene expression in a sequence-specific manner, miRNAs play important roles in numerous biological processes. miRNAs repress target gene expression through translational inhibition as well as RNA degradation in both plants and animals, but the mechanisms of miRNA-triggered RNA degradation are largely different in plants and animals ,18. In abidopsis , but degbidopsis .Arabidopsis, mouse, and Epstein-Barr virus implies that uridylation has general importance [Arabidopsis, HESO1 uridylates the 5′ fragments resulting from miRNA-guided cleavage of target RNAs to trigger their degradation, and AGO1, the effector protein of miRNAs, is associated with HESO1 in vivo [The presence of an oligo(U) signature posttranscriptionally added to miRNA-directed 5′ cleavage products in species as diverse as portance . In mammportance . In addiportance . In Arab in vivo   is a member of the evolutionarily conserved snRNA class within the eukaryotic spliceosome. Mammalian U6 snRNA is heterogeneous in size due to nontemplated 3′ uridylation . A majorArabidopsis miRNAs or small interfering RNAs (siRNAs) are methylated on the 2′ OH of the 3′ terminal ribose by the methyltransferase HUA ENHANCER 1 (HEN1) [hen1 mutants, both miRNAs and siRNAs undergo 3′ truncation and 3′ uridylation, leading to their decline in abundance [hen1 mutant animals, such as Tetrahymena [C. elegans [Drosophila [hen1 mutants; it occurs in wild type cells at a lower frequency. Studies using high throughput sequencing to profile small RNAs revealed 3′ nontemplated nucleotide addition, mainly uridylation and adenylation, to mature miRNAs from viruses [Chlamydomonas reinhardtii [Drosophila [3′ uridylation of mature small RNAs was first found in plants. 1 (HEN1) ,34. In hbundance . Later, rahymena . C. eleg elegans –39, Drososophila ,41, zebrosophila  and mousosophila . Small R viruses , Chlamydnhardtii , Drosophosophila , mouse [osophila , and humosophila .C. elegans, the nucleotidyl transferase CDE-1 uridylates siRNAs bound by the argonaute protein CSR-1 to prevent their over accumulation and loading into other argonaute proteins; CDE-1 is essential for proper meiotic and mitotic chromosome segregation [Chlamydomonas reinhardtii contributes to the presence of nontemplated uridine residues at the 3′ ends of small RNAs and loss of function in MUT68 results in elevated miRNA and siRNA levels [Arabidopsis HEN1 SUPPRESSOR1 (HESO1) is a nucleotidyl transferase responsible for most of the small RNA uridylation in hen1 mutants. HESO1 prefers UTP as the substrate nucleotide, and is completely inhibited by 2′-O-methylation in the substrate RNA [HESO1 leads to increased miRNA accumulation in hen1 mutants [hen1 heso1 mutants [in vitro and act cooperatively to tail different forms of the same miRNAs in vivo [In recent years, an increasing number of terminal nucleotidyl transferases (TUTases) that uridylate small RNAs have been identified. In humans, Zcchc11 (TUT4) uridylates the cytokine-targeting miRNA miR-26b; the uridylation appears to attenuate the target repression activity of this miRNA such that Zcchc11 promotes the expression of cytokine genes  Table 1. Zcchc6 regation   polymerase responsible for the 3′ terminal monoadenylation of miR-122 and other miRNAs in mouse livers and human fibroblast cells ,78. In GBut the stabilizing effect of 3′ adenylation on miR-122 may not be extrapolated to other miRNAs. Another study examined the effects of GLD-2 knockdown in human THP-1 cells (GLD-2 was referred to as PAPD4 in this study) and showed that GLD-2 is responsible for the 3′ adenylation of many miRNAs  Table 1. HoweverA study on human miR-21, an oncogenic miRNA implicated in numerous human diseases, concluded that adenylation of miR-21 leads to its destabilization . In humaChlamydomonas, MUT68 oligoadenylates 5′ RNA fragments generated by small RNA-mediated cleavage and leads to their degradation by a 3′–5′ exonuclease, most likely the exosome [In eukaryotic cells, the cotranscriptional addition of a poly(A) tail to the 3′ ends of mRNA molecules is nearly universal; the poly(A) tail protects mRNAs from degradation and facilitates their translation from yeast to higher eukaryotes . But in  exosome .in vivo [+ pre-rRNAs [Although rRNAs are not produced by RNA polymerase II and thus are not subjected to cotranscriptional polyadenylation as do mRNAs, in yeast, a small fraction of precursor rRNAs is posttranscriptionally modified at their 3′ ends by the addition of a poly(A) tail in vivo ,86. As tin vivo . The 5′-re-rRNAs .Arabidopsis URT1 and HESO1 and Drosophila Tailor) exhibit a preference for the 3′ nucleotide in its substrate RNA [Drosophila, the levels of uridylated miRNAs are higher, but in mature tissues, the levels of adenylated miRNAs are higher [As discussed above, uridylation or adenylation of RNAs is a widespread phenomenon found for different RNA species and in many organisms, but these posttranscriptional processes exhibit specificity and undergo regulation. For example, miRNA 3′ uridylation and adenylation exhibit sequence specificity—some miRNAs are predominantly adenylated while others are predominantly uridylated ,88. Thisrate RNA ,72,73 . Monouridylation or monoadenylation may impart different outcomes on different RNAs. Key to RNA uridylation or adenylation are nucleotidyl transferases, whose preference for UTP or ATP dictates the nature of the tail to be added and whose processivity may determine whether one or a number of nucleotides are added. Plant and animal genomes encode multiple nucleotidyl transferases ,62 Figu, many ofbidopsis , among wbidopsis . Studyin


Beet black scorch virus (BBSV) and low temperature (18°C) additively enhance the systemic infection of BBSV in Nicotiana benthamiana. Northern blotting hybridization revealed a relatively reduced accumulation of BBSV-derived small interfering RNAs (siRNAs) in presence of sat-RNA as compared to that of BBSV alone. Cloning and sequencing of total small RNAs showed that more than 50% of the total small RNAs sequenced from BBSV-infected plants were BBSV-siRNAs, whereas the abundance of sat-siRNAs were higher than BBSV-siRNAs in the sat-RNA co-infected plants, indicating that the sat-RNA occupies most of the silencing components and possibly relieves the RNA silencing-mediated defense against BBSV. Interestingly, the 5′ termini of siRNAs derived from BBSV and sat-RNA were dominated by Uridines (U) and Adenines (A), respectively. Besides, the infection of BBSV alone and with sat-RNA induce down-regulation of miR168 and miR403, respectively, which leads to high accumulation of their targets, Argonaute 1 (AGO1) and AGO2. Our work reveals the profiles of siRNAs of BBSV and sat-RNA and provides an additional clue to investigate the complicated interaction between the helper virus and sat-RNA.Co-infection of none-coding satellite RNAs (sat-RNAs) usually inhibits replication and attenuates disease symptoms of helper viruses. However, we find that the sat-RNA of  Groundnut rosette virus (GRV) is largely responsible for the groundnut rosette symptom and the transmission between natural hosts of GRV  often enhances the visible symptoms of TCV in a host-dependent manner  of plant viruses rely on the helper virus for replication and encapsidation but share little or no sequence similarity with the helper virus genome  was also found to induce silencing but could be resistant to RISC-mediated cleavage  was firstly reported in northern China in the late 1980s and lately identified as a new species of genus Betanecrovirus  that caused severe symptom in N. benthamiana and obtained after a passage of propagation  of BBSV were used for inoculation by the method reported previously . The membranes were washed in 2 × SSC (0.3 M NaCl and 0.03 M sodium citrate) containing 0.2% SDS for 30 min and then twice with 1 × SSC containing 0.1% SDS for 20 min both at 50°C.Total RNA was extracted using TRIzol® Reagent . For detection of viral genomic RNA and mRNA of BBSV and its sat-RNA, 2 μg total RNA extracted from mock or virus-infected plants was used for hybridization using indicated gene-specific GSE80694.The small RNA libraries were generated following the manufacturer's protocol . Briefly, separated by electrophoresis, RNA fractions with sizes between 18–30 nt corresponding to the small RNA population were purified, and cloned using NEBNext® Multiplex Small RNA Library Prep Set for Illumina kit. The final products were quantified on the Agilent DNA 1000 chip and sequencing was performed by use of an Illumina Hiseq 2500-SE50 . The Illumina sequencing reads were first trimmed to remove the adaptor sequence to get clean reads. The trimmed sequencing reads were then blasted to the Bm (JN635330.1) and sat-RNA (NC_006460.1) and the sequences with full matched were considered as Bm or sat-RNA small RNAs. The clean reads were also blasted in miRBase for miRNAs. The data of small RNA libraries was deposited in GenBank with accession number To measure expression levels of miR168 and miR403, stem-loop quantitative RT-PCR technique was used as previously described  causes more severe symptoms than wild-type BBSV at low temperature (18°C) , which depend on BBSV for replication and movement but share no sequence homology with the helper viral genome -derived vsRNAs in N. benthamiana plants , followed by 21-nt reads (16.3–28.9%) and other length reads Figures , which sThe previous studies have reported that the selective loading of small RNAs into specific AGOs is determined by the 5′-terminal nucleotides of siRNAs  is probably mainly targeted by AGO1-associated complex, and the sat-RNA is silenced by AGO2-associated complex. These results illustrate the sat-RNA could benefit the helper virus, especially when the helper virus is confronted with strong defense of host plants.Sat-RNAs are viral parasites and depend on their helper viruses for replication, encapsidation and movement in the host plants. Sat-RNAs are usually involved in the interaction between their helper viruses and plant hosts by modulating the accumulation level of helper viruses and symptom induction. In this study, we found that the sat-RNA of BBSV facilitated the systemic infection of the helper virus in N. benthamiana because RDR6 may be inactive at the low temperature  were involved in antiviral silencing and that high temperature enhances antiviral silencing in Although the replication of defective interfering RNA (DI RNAs), sat-RNAs and viroids produces abundant siRNAs, these sub-viral RNAs are resistant to RNA silencing, because highly structured RNAs may be poor targets for RNA cleavages by RNA-induced silencing complex .The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.