Yangjing capsule (YC), a traditional Chinese compound herbal preparation, has been proven as an effective drug to improve spermatogenesis in clinical practice. However, its pharmacological mechanisms were not fully clarified. This study was designed to investigate the protective effects of YC on spermatogenesis in the mouse model of spermatogenesis dysfunction induced by cyclophosphamide (CP). The administration of YC significantly increased the epididymal index, sperm count, and sperm motility of model mice. Histopathological changes demonstrated that CP caused obvious structural damage to testis, which were reversed by the administration of YC. Results from TUNEL assay showed that treatment with YC dramatically decreased the apoptosis of spermatogenic cell induced by CP. Moreover, YC treatment could inhibit the mRNA and protein expression of Bax to Bcl-2 and also raised expression of AR at both mRNA and protein levels. These data suggest that YC might ameliorate spermatogenesis in male mice exposed to CP through inhibiting the apoptosis of spermatogenic cell and enhancing the actions of testosterone in spermatogenesis. Infertility affects 15% of couples worldwide. It is estimated that roughly half of the infertility cases are due to male factors , 3. AmonSpermatogenesis is a sophisticated multistep process involving three major phases: mitotic, meiotic, and postmeiotic phases. Other cellular events such as apoptosis of spermatogenic cell, migration, and differentiation also play vital roles in the process of spermatogenesis . The dynThe Yangjing capsule (YC), a traditional Chinese compound herbal preparation, has been used for over ten years for the treatment of male reproductive diseases, in China, including male infertility and sexual dysfunction. Clinical practice showed that YC could improve the density of sperm, its motility, and DNA integrity in infertile men , 9. ThesIn recent years, mouse models of spermatogenesis dysfunction are used widely to investigate the mechanisms of spermatogenesis. Alkylating agents such as CP are the most common agents implicated in inducing the mouse model. Furthermore, it has been demonstrated that CP can inhibit testosterone synthesis and induce apoptosis of spermatogenic cells , 13. The125I] Testosterone Radioimmunoassay Kit was supplied by Beijing North Institute of Biological Technology . The primers were synthesized by Invitrogen Life Tech . The mouse monoclonal anti-glyceraldehyde 3-phosphate dehydrogenase (anti-GAPDH) and horseradish peroxidase-conjugated secondary antibodies were purchased from Bioworld . The rabbit monoclonal anti-tubulin and rabbit polyclonal anti-Bax antibodies were procured from Abcam . The rabbit polyclonal anti-AR and rabbit polyclonal anti-B-cell lymphoma 2 (anti-Bcl-2) antibodies, goat anti-rabbit immunoglobulin G (γ-chain specific) (peroxidase conjugate), and In Situ Cell Apoptosis Detection Kit I (POD) were procured from Boster Biological Engineering Co. Ltd. .CP was purchased from Pude Medicine Co. Ltd. . Iodine  Testosterone Radioimmunoassay Kit according to the protocol described by the manufacturer. The amount of testosterone was determined from a calibration curve.Serum testosterone was examined using Iodine  of the tissue was calculated as described by Yuan et al. .−ΔΔCt method.Bax, Bcl-2, and AR mRNA expression levels in the testes were determined using real-time quantitative PCR. The total RNA of the testes samples was extracted using Trizol reagent . Sample cDNAs were used as templates for amplification to quantify the mRNA levels of target genes using SYBR Green PCR Master Mix reagent kits (TaKaRa Biotechnology). GAPDH was selected as the control. The primer sequences were as follows: GAPDH: forward, 5′-TGG CCT TCC GTG TTC CTA C-3′; reverse, 5′-GAG TTG CTG TTG AAG TCG CA-3′; AR, forward, 5′-TCC AAG ACC TAT CGA GGA GCG-3′; reverse, 5′-GTG GGC TTG AGG AGA ACC AT-3′; Bax, forward, 5′-AGA CAG GGG CCT TTT TGC TAC-3′; reverse, 5′-AAT TCG CCG GAG ACA CTC G-3′; Bcl-2, forward, 5′-GCT ACC GTC GTG ACT TCG C-3′; reverse, 5′-CCC CAC CGA ACT CAA AGA AGG-3′. Relative quantification of expression of target genes was estimated using the 2Total proteins extracted from testis were prepared following standard procedures and quantified using the bicinchoninic acid protein assay . The proteins were separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and subsequently transferred onto nitrocellulose membranes. The membranes were blocked with 5% skim milk in Tris-buffer saline containing 0.1% Tween 20 at 37°C for 1 h and then incubated at 4°C overnight with the primary antibodies of Bax (1 : 200 dilution), Bcl-2 (1 : 300 dilution), and AR (1 : 300 dilution). Tubulin or GAPDH was used as an internal control. Densitometric analysis of the scanned immunoblotting images was performed using a Quantity One image system.P < 0.05 was considered as statistical significance.All quantitative data derived from this study were analyzed using the SPSS 20.0 statistical package. The results were expressed as the mean ± standard deviation. The one-way analysis of variance was used to analyze the difference between groups, and post hoc least significant difference test was used for intergroup comparisons. P < 0.01, resp.). CP treatment also decreased the sperm count and motility by 47% and 34%, respectively, as compared to the control group (P < 0.01). After treatment with YC, the body weight and the testis index in the YC (1260 mg/kg) group significantly increased by 8.1% and 20.7% as compared to the CP group. YC (630 and 1260 mg/kg) treatments also significantly increased the epididymal index by 30.9% and 24.5% (P < 0.01) when compared with the CP-treated group. YC (630 and 1260 mg/kg) treatment also notably increased the sperm count and motility when compared with the CP group  group, in spite of thinned seminiferous epithelium, many mature spermatids appeared in the seminiferous tubules . In the YC (630 and 1260 mg/kg) groups, the values were 0.61 ± 0.26 and 0.76 ± 0.35 (mol/L), respectively. Although the serum testosterone level increased in the YC-treated groups, the difference did not reach statistical significance when compared with the CP group .TUNEL assay was performed to analyze the apoptosis of spermatogenic cell. After administration of CP, apoptotic cells were observed more frequently in the testis of the CP group mice . When trP < 0.05, resp.). Treatment with YC significantly decreased the ratio of Bax mRNA to Bcl-2 mRNA in a dose-dependent manner (P < 0.05).To further study the potential mechanism of antiapoptosis of YC, the expressions of Bax and Bcl-2, at mRNA and protein levels, in the testes of mice were examined using the quantitative real-time PCR and Western blotting. As shown in t manner , while Yt manner   as compared with the control group. YC (630 and 1260 mg/kg) treatment significantly improved the AR mRNA and protein expression levels when compared with the CP group  is used to treat infertile men with spermatogenic defects, but clinical results are limited especially for patients with idiopathic failure of spermatogenesis . TraditiCP, as an alkylating agent, is the most common agent implicated in causing dysfunction of spermatogenesis. In this study, mature male Balb/c mice were injected with CP (50 mg/kg) i.p. for 7 days to induce the dysfunction of spermatogenesis. For the next 30 days, the mice received treatment with YC. Animals were killed at 30 days after the last injection. We found that administration of CP significantly decreased the sperm count and motility as well as the relative testicular weights and body weights. The administration of CP also caused histologic lesions of the testis and epididymis. Testicular tissues from the CP-treated mice showed atrophied seminiferous tubules, thinned seminiferous epithelium, and reduced spermatogenic cells and interstitial cells. We also found that the cauda epididymis from the CP-treated mice illustrated a lower density of sperm and an increase in the number of premature sperm cells.Interestingly, we found that treatment with YC significantly restored these CP damage instances. YC (630 and 1260 mg/kg) treatment not only notably increased the sperm count and motility but also increased seminiferous epithelial layers and the number of mature spermatids and interstitial cells in testis. YC treatment also increased the density of sperm and decreased the deciduous premature sperm cells in the ductus epididymis when compared with the CP group. These results indicated that YC could ameliorate spermatogenesis in male mice exposed to CP.Mammalian spermatogenesis depends on the balance among proliferation, differentiation, and apoptosis of germ cells. Apoptosis of spermatogenic cell occurs during various stages of mammalian spermatogenesis to remove abnormal spermatogenic cells , which mTestosterone is a major regulator in spermatogenesis, which works by binding to AR. In the absence of testosterone or the dysfunctional AR, spermatogenesis is halted during meiosis so that few germ cells develop to the haploid spermatid stage and the elongated spermatid can not form –26. FurtPrevious studies have shown that administration of CP could inhibit the synthesis of testosterone , 29, 30.As we know, traditional Chinese medicine, especially those compound recipes, plays a role through multiway and multitarget with its multicomponents. YC is composed of 11 traditional Chinese drugs. Of necessity, YC contains many active ingredients. It has been demonstrated that total flavonoids of Herba Epimedii Brevicornus and polysaccharides could not only enhance testosterone actions , but alsIn conclusion, the present study suggests that treatment with YC can ameliorate the impairment of spermatogenesis induced by CP in male mice. The effects of YC on improving spermatogenesis are likely due to the inhibition of apoptosis of spermatogenic cell and enhancement in the actions of testosterone in spermatogenesis. Therefore, YC might be considered as an alternative therapeutic remedy for male infertility.


The cognitive view on deception holds that lying typically requires additional mental effort as compared to truth telling. Psychopathy, however, has been associated with swift and even compulsive lying, leading us to explore the ease and compulsive nature of lying in psychopathic offenders. We explored the costs of instructed lying versus truth telling through RTs and error rates in 52 violent male offenders, who were assessed with the Youth Psychopathic Traits Inventory (YPI). Our deception paradigm also included trials with the free choice to lie or tell the truth. By coupling monetary loss to slow and erroneous responding, we hypothesized that the frequency of lying despite likely negative consequences, would provide an index of compulsive lying. Offenders were slower and erred more often when lying than when telling the truth, and there was no robust association between psychopathy and the cognitive cost of lying. From an applied perspective, this suggests that psychopathy may not threaten the validity of computerized cognition-based lie detection. In the face of probable negative consequences, high grandiose-manipulative offenders chose to lie three times as often as low grandiose-manipulative offenders. Our new lying frequency index is a first attempt to create a much needed tool to empirically examine compulsive lying, and provides preliminary support for the compulsive nature of lying in grandiose-manipulative offenders. Alternative interpretation of the findings are discussed. Surprisingly, an early review of the literature by Hare and colleagues  found thClinical descriptions, psychopathy theory, and assessment instruments consider lying, manipulation and deceit to be at the heart of psychopathy, but empirical data do not provide unequivocal support for that notion. There are only very few studies, however, that examined the ease of lying in forensic samples. Note that the very notion of non-criminal psychopathy is debated , ), 22]), https://osf.io/w746e/The study was approved by the local ethical committee of the Psychology Department of the University of Amsterdam (2013-CP-3043). All participants provided written informed consent using a consent form approved by the local ethical committee. The script of the experimental task as well as the data are publically available on Fifty-two violent male offenders were recruited in three Dutch forensic institutions. Exclusion criteria were (1) schizophrenia, bipolar disorder or acute psychotic symptoms, (2) clinician estimated IQ < 80, (3) visual impairment, 4) use of antipsychotic medication, and 5) somatic impairments effecting muscle- or coordination functions .SD = 4%) correctly indicated whether they were about to lie or tell the truth. One participant indicated on 24% of the instructed lie and truth trials that he would behave different from what was instructed . We excluded the data of this participant, leaving n = 51. We also excluded the data of one participant because of an excessive error percentage on the instructed lie and truth trials , leaving n = 50.On instructed lie and truth trials, participants nearly always . Most patients had the Dutch nationality (90%), and were unmarried (87.5%). None had higher education at the college or university level. YPI was available for all patients and showed substantial variation across the psychopathy spectrum , and WAIS-IQ was available for 43 patients .The final sample consisted of 50 patients that were on average 42 years old  and twoYouth Psychopathic Traits Inventory , the Remorseless, Unemotionality, and Callousness scales tapping into the factor Callous-unemotional , and the Thrill-Seeking, Impulsiveness, and Irresponsibility tapping into the factor Impulsive-irresponsible traits . The YPI items are worded in a positive direction in order to eliminate the effect of social desirability. The YPI was initially developed for use in adolescents, but has no adolescent-specific items, and has been shown to have good psychometric properties in young adults  was good for YPI Grandiose-manipulative (α = .89), YPI Callous-unemotional (α = .78), YPI Impulsive-irresponsible (α = .84), and the YPI total score (α = .91).The ry  is a ses  a) aYouth s , questiThere were 40 questions: 20 questions required a YES response when telling the truth , and 20 questions required a NO response when telling the truth . Questions were tailored to assure that they required a YES or NO response. For instance, as data collection started in 2013 and ended in 2015, the question ´Is it 2014?´ was adjusted to ´Is it 2015?´ for participants tested in 2015.Participants started this task with €5, and were informed that they would lose €0.05 for behavioral errors , and €0.20 for slow responses . RTs on the cued lie and cued truth trials served as an index of the cognitive costs of lying. Reasoning that lying would be cognitively more demanding than truth telling and that fast and slow responding resulted in financial loss, lying frequency on the choice trials was taken as an index of compulsive lying (lying despite negative consequences).modified Sheffield lie test consisted of a total of 142 trials: 24 practice trials  and 120 test trials .The task was practiced in two blocks of 12 trials, using another set of very simple true  and false  questions. The first practice block had a fixed order that built up in complexity. The second practice block had a random order. After the two practice blocks, there was one block with 120 test trials: 40 instructed truth trials, 40 instructed lie trials, and 40 choice trials, presented in random order. Across individuals, but not within each individual, each question was presented about equally often with each of the 3 instruction cues. In sum, the LIE minus RTTRUTH; ErrorLIE minus ErrorTRUTH) and compulsivity of lying  on the other hand. We did not control for age or IQ in these analyses, because the psychopathy total and factor scores were unrelated to both age and IQ; all r´s <.13, all p´s > .40.Because taxometric analyses indicate that psychopathy is a dimensional construct , we useM = 32%; SD = 13%) than when telling the truth  on the instructed lie and truth trials, t(49) = 7.90, p < .001. We calculated the effect size for within-subject contrast as Cohen´s dwithin = M(ErrorLIE − ErrorTRUTH / √(SDLIE2 + SDTRUTH2 − 2*r*SDLIE*SDTRUTH), where r is the correlation between ErrorLIE and ErrorTRUTH (here: r = .42). The difference between lying error rate and truth error rate was large dwithin = 1.11 (95% CI: .74–1.47). We used (ErrorLIE minus ErrorTRUTH) as a first index of the cognitive cost of lying.Participants erred more often when lying  than when telling the truth  on the instructed lie and truth trials, t(49) = 5.52, p < .001. Controlling for the correlation between RTLIE and RTTRUTH, r = .89, the difference between RTLIE and RTTRUTH was moderate to large dwithin = 0.79 (95% CI: .45–1.38). We used (RTLIE minus RTTRUTH) as a second index of the cognitive cost of lying.Behavioral errors were excluded for RT analyses. To reduce the impact of outliers, we censored the RTs following established procedures . ). 29]). M = 22%, SD = 28%). Forty-two percent of the sample never lied on the choice trials. There was considerable variance, and Participants mostly chose to tell the truth on the choice trials, as evidenced by the low average lying frequency  and compulsive lying  on the other hand.r = -.29, p = .04 that indicated a reduce cost of lying in high callous-unemotional offenders. This association, however, was carried by two outliers with YPI-CU scores  that deviated more than 3SDs from the sample mean . After excluding the two outliers, the correlation was no longer significant, r = -.22, p = .13. We therefore think that the observed association was distorted by the two outliers, and that there is no real association between callous-unemotional traits (YPI-CU) and the cost of lying in error rates.First, there appeared an association between callous-unemotional traits (YPI-CU) and the cost of lying in error rates, r = .35, p = .01, see n = 15) versus the highest thirtile , and found that high GM offenders  chose to lie three times as often as low GM offenders . This was also the case when comparing the lowest quartile  versus the highest quartile  with a lying frequency of M = 36.34 (SD = 25.89) versus M = 12.08 (SD = 26.32).Second, the more grandiose-manipulative (YPI-GM), the more frequent the choice to lie, r = .31, p = .03. Also, the more frequent the choice to lie, the lower the cognitive cost of deception .The deception indices appeared not completely independent from each other: The higher the cognitive cost of deception in error rates, the higher the cognitive cost of deception in RTs, Laboratory evidence in undergraduates has fuelled the idea that the truth typically comes naturally, and that lying requires additional mental effort. This cognitive view on deception has gained wide attention and led to the development of cognition-based lie detection techniques . An impoWhen provided with the choice to lie versus tell the truth, we found that offenders preferred to tell the truth, and chose to lie on only 22% of the trials. An important restriction to this finding is that a penalty was coupled to slow and erroneous responding, indirectly deterring from chosing to lie. This payment system was set-up to create an index of pathological lying . Taking this important restriction in mind, the fact that offenders preferred truth telling above lying, supports the cognitive theory of lying. Specifically, it has been shown that when people are offered the option to perform one of two tasks in task switching paradigms, they typically prefer the easier task above the more difficult task . As suchactivation of the truth, the decision to lie, the construction of the lie, or acting sincere. Given the non-interactive nature of our task, the action component is not at play. The use of instructed lying versus truth telling also excludes the decision component. This implies that the activation of the truth and/or the construction of the lie were sufficient to produce a notable behavioral cost for lying. The observed cognitive cost of lying in offenders requires qualification. First, lacking a non-offender control group, we do not know whether lying is as mentally taxing for offenders as it is for non-offenders. Second, that offenders displayed more mental effort for lying than for truth telling was observed within a very specific laboratory paradigm. We chose for the Sheffield lie task because it consistently provides maximal experimental control and leads to robust lie-truth differences. A downside of the task is its highly artificial nature, that has led to concerns with regard to whether the task still measures deception . The taion  appeared smaller in offenders with high callous-unemotional traits, but that correlation was carried entirely by two outliers. After excluding those with an extreme score on the YPI-CU, the correlation was no longer significant. Clearly, those two cases are highly influential. Should the outliers be excluded from the analyses? A first possibility is that the association is real, hence that the cognitive cost of lying is only reduced in those with extremely elevated callous-unemotional traits. Such an interpretation fits with a categorical view on psychopathy . While oWhile offenders largely preferred to tell the truth on the choice trials, there was large heterogeneity: Some offenders nearly always chose to tell the truth and some lied almost all of the time. Grandiose-manipulative traits partly explains this variability. High grandiose-manipulative offenders chose to lie 3 times as often as low grandiose-manipulative offenders. Grandiose-manipulative offenders may be compulsive liars, and lie despite probably negative consequences. Grandiose-manipulative traits were, however, not related to the cognitive costs of lying. Perhaps then, this association can be explained by poor self-judgement. Whereas they do not really have superior lying skills, grandiose-manipulative offenders seem to consider themselves as swift liars, up to the point that they engage in lying when it has probable negative consequences. While we consider the higher lying frequency on the choice trials as implying compulsive lying, we are mindful of alternative explanations. For instance, random responding  may alternatively explain the higher lying frequency on the choice trials. Taking the limitations of our study into account, our findings generally support the cognitive view of lying. Offenders evidenced greater cognitive load for lying than for truth telling, and largely preferred to tell the truth in the face of negative consequences coupled to lying. Clinically, it is interesting that grandiose-manipulative offenders chose to lie in spite of probable negative consequences—pointing to compulsive lying. From an applied perspective, it is important to note that there was a substantial cognitive cost for lying, that did not vary with psychopathic traits. While this finding is restricted to an artificial laboratory deception paradigm and should be followed up with paradigms that involve social interaction, it does indicate that psychopathy may not be a major threat to such computerized cognition-based lie detection tests.S1 File(DOCX)Click here for additional data file.


Small non-coding RNAs are essential regulators of gene expression at the transcriptional and posttranscriptional levels. High-throughput sequencing has revealed thousands of predicted small RNAs; however, only a few of these have been well characterized. Northern blotting is the most convincing method for small RNA validation.Arabidopsis thaliana and Oryza sativa were investigated. The results suggest that this improved method is sensitive and efficient, with approximately 5 μg of total RNA being sufficient for detection. Furthermore, long-term storage of probes labeled in this manner is more convenient, less contaminative and degradative compared with traditional probes.In this study, we improved the Northern blot method by using biotin-labeled probes. miRNAs and siRNAs derived from both This protocol is an alternative strategy for small RNA detection and represents an efficient means of researching small RNAs.The online version of this article (doi:10.1186/s12284-014-0026-1) contains supplementary material, which is available to authorized users. In eukaryotic cells, small non-coding RNAs have been demonstrated to play fundamental roles in gene expression modification during development . Small C. elegans two decades ago, remarkable advances in the characterization of this gene family have been achieved . Recent Hannon, ; Studhol Hannon, ). To datrRNAs and tRNAs account for over 90% of the total RNA in a eukaryotic cell, whereas small non-coding RNAs account for only approximately 2%, and detection of these RNAs by some methods is difficult . BecausArabidopsis thaliana and Oryza sativa for validation, the results suggested that this method was sensitive and efficient, as it was capable of detecting as little as 1 to 5 μg of total RNA. Furthermore, storage of biotin-labeled probes is convenient, and the probes are stable; therefore, northern blots can be carried out with ease. Taken together, these data suggest that biotin-labeled probes can be widely used to investigate small RNAs and even mRNAs in the future.Biotin is another molecule that can increase the sensitivity of small RNA detection. In this study, we designed biotin-labeled oligonucleotide probes to investigate the expression of small RNAs. When we applied this improved method to In a eukaryotic cell, the abundance of rRNA and tRNA impairs the detection of small RNAs. To test the method of RNA preparation in this study, total RNA and small RNAs were extracted with two different reagents. To detect the integrity of the RNA, we loaded the RNA onto 15% PAGE gels under denaturing conditions (7 M urea) or onto 1.2% agarose gels with EtBr for staining. The results showed that the RNAs were suitable for further analysis, such as northern blotting Figure . Next, tArabidopsis. It is well known that the abundance of small non-coding RNAs is less than 2%. To address this problem, researchers usually increase the amount of input RNA to 20–60 μg depending on the expression level of the target miRNA. In this study, the input RNAs (ranging from 1 to 5 μg) were loaded onto 15% denatured PAGE gels before northern blotting to test the sensitivity of this method. We examined certain miRNAs  because they had previously been shown to be expressed in leaves  . As shon et al, ). ta-siRn et al, ). Subseqhttp://bioinformatics.cau.edu.cn/PMRD/), miR156, miR171a, miR390b, and miR168a were selected for investigation. As shown in Figure Rice is an important staple crop worldwide, and it is also an essential model crop for research. To ensure that this method can be applied to rice, we also validated established rice miRNAs and ta-siRNAs. Based on previous studies and the Plant MicroRNA Database , LED . When uArabidopsis and Oryza sativa were performed, and the results suggest that this improved method is both sensitive and efficient. Furthermore, biotin-labeled probes are more stable and safer for researchers and the environment than traditional probes. This protocol represents an important contribution to the small RNA field.To date, high-throughput sequencing has revealed thousands of predicted small RNAs; however, only a few of them have been characterized. Most researchers use isotope-labeled probes for small RNA detection and validation. Although biotin has been used in many fields, Northern blots using biotin-labeled probes have not been reported. In this study, we improved Northern blotting with the use of biotin-labeled probes. Northern blots using RNA derived from both http://bioinformatics.cau.edu.cn/PMRD/), and some have been reported in previous studies . ProbesArabidopsis and Oryza sativa. Total RNA was extracted from leaves using TRIzol reagent  following a previously described protocol . Small The 15% denatured polyacrylamide gels containing 7 M urea were 15 cm wide, 20 cm long, and 1.5 mm thick. After polymerization at room temperature for 30 minutes, the gels were pre-run with 1× TBE at 40 mA (600 V) for 30 minutes. The RNAs or oligonucleotide probes were mixed with 2× loading buffer ; the RNA samples were heated at 70°C for 5 minutes. The PAGE gels were run at 40 mA (600 V) for approximately 2 hours until the bromophenol blue reached approximately 1 cm above the bottom of the gel. The gels were then stained with 0.5 μg/ml EtBr in DEPC-treated TBE buffer for 10 minutes. The RNAs were then transferred onto Hybond-N + positively charged nylon membranes  using a Bio-Rad transblot apparatus at 200 mA (9 - 10 V) for 3 hours . Next, the membranes were further crosslinked at 1200 μjoules for 20 minutes and dried at 50°C for 30 minutes to improve sensitivity. Membranes prepared in this way can be stored at 4°C for several months.2HPO4 (pH 7.0), 5 μg/ml salmon sperm DNA (SSDNA)). A 3-hour incubation period is recommended for decreasing background noise. Next, the pre-hybridization buffer was removed, and hybridization buffer containing 50 pmol/ml labeled probes was added. The biotin-labeled probe did not require denaturation. The membranes were hybridized for 12 to 16 hours at 40°C with gentle shaking and subsequently rinsed with washing buffer  3 times for approximately 15 minutes total at room temperature.Before hybridization, the membranes were pre-hybridized for at least 30 minutes at 40°C in pre-hybridization buffer . After rinsing, the membranes were blocked again with blocking buffer for 15 minutes with gentle shaking at room temperature followed by incubation for an additional 15 minutes with hybridization buffer containing stabilized streptavidin-HRP conjugate. After washing 3 times, the membranes were equilibrated in substrate equilibration buffer for 5 minutes. Finally, the membranes were placed in a clean container, covered completely with working solution, and incubated for 5 minutes in the dark. A working solution was prepared with luminol/enhancer solution and stable peroxide solution. The membranes were placed in a cassette with X-ray films and exposed for different periods of time .QH performed the experiments and analyzed the data, ZM performed the experiments and analyzed the data, SL performed the experiments and analyzed the data, JH conceived the study, performed the experiments, analyzed the data and wrote the manuscript, YZ conceived the study. All authors read and approved the final manuscript.Additional file 1: Table S1.: Probes used in this study. (DOC 33 KB)Authors’ original file for figure 1Authors’ original file for figure 2Authors’ original file for figure 3Authors’ original file for figure 4Below are the links to the authors’ original submitted files for images.


An affinity resin-based pull-down method is convenient for the purification of biochemical materials. However, its use is difficult for the isolation of a molecular complex fully loaded with multiple components from a reaction mixture containing the starting materials and intermediate products. To overcome this problem, we have developed a new purification procedure that depends on sequential elimination of the residues. In practice, two affinity resins were used for purifying a triangular-shaped RNP (RNA-protein complex) consisting of three ribosomal proteins (L7Ae) bound to an RNA scaffold. First, a resin with immobilized L7Ae protein captured the incomplete RNP complexes and the free RNA scaffold. Next, another resin with an immobilized chemically modified RNA of a derivative of Box C/D motif, the binding partner of L7Ae, was used to capture free protein. The complete triangular RNP was successfully purified from the mixture by these two steps. Obviously, the purified triangular RNP displaying three protein-binding peptides exhibited an improved performance when compared with the unrefined product. Conceptually, this purification procedure should be applicable for the purification of a variety of complexes consisting of multiple components other than RNP. Currently, molecular complexes consisting of various, multiple biomaterials have been developed –6. TheseSophisticated artificial RNA-protein complexes with defined three-dimensional (3D) structures have been designed and constructed by combining the two substances. Previously, an RNP nano-object consisting of three L7Ae proteins bound to an RNA scaffold was constructed by employing L7Ae, a ribosomal protein bound to an RNA molecule containing a box C/D kink-turn motif . BindingThe triangular RNP can be prepared by mixing the RNA and the protein components containing L7Ae, resulting in an unrefined RNP. However, the purification of the unrefined product is cumbersome because the reaction mixture contains the fully loaded triangular RNP together with partially formed RNP complexes carrying one or two L7Ae proteins, as well as the starting materials. It is conceivable that this contamination might affect the performance of the triangular RNP adversely, so that it is difficult to determine the actual performance of the fully loaded RNP.In this study, we demonstrate a procedure for the purification of the fully loaded triangular RNP by eliminating the incomplete complexes and the starting materials by the successive use of affinity resins. Polyacrylamide gel electrophoresis based separation followed by RNP elution should be adequate for the purification , 6. Altegaattcaagg tggcAGCAAC TGGTCCCATC CGCAGTTTGA GAAGggtggc ctcgag-3’ ] was inserted into EcoRI and Xhol sites in an L7Ae expression plasmid, pET28b-His-L7Ae-cmyc-His. All synthetic DNAs were purchased from Operon Biotechnologies (Japan).L7Ae and L7Ae fusion proteins were expressed and purified as previously described , 6. For in vitro transcription , respectively, at 4°C until use.The affinity resins were prepared by the canonical amine-coupling reaction using NHS-activated Sepharose 4 Fast Flow  according to the supplier’s protocol. Typically, 750 pmol L7Ae was immobilized onto 1 μL of the resin. Chemically synthesized BCD35g 2’-fPy-RNA (fBCD35g) with a 5’-amino modification was purchased from GeneDesign (Japan), and typically, 80 pmol of the RNA was immobilized onto 1 μL of the resin. The prepared L7Ae- and fBCD35g-immobilized resins were stored in 20% ethanol and in the assay buffer -CTP (PerkinElmer). Using the labelled RNA, 30-μL of a 10-fold concentrate of triangular RNP with L7Ae or L7Ae-ST2 fusion protein was prepared. Then, the RNP samples were subjected to purification as described above. The RNA concentration of the recovered samples was measured by liquid scintillation counting , followed by dilution with the assay buffer to a final RNA concentration of 1 nM. 100-μL of the purified and diluted sample was transferred into Strep-Tactin coated microtiter plates . After 30 min incubation at room temperature, the microtiter plates were washed with 100-μL of the assay buffer twice, followed by RNP elution with 100-μL of 4.5 mM D-desthiobiotin (IBA GmbH) at room temperature for 30 min. The released RNP was recovered, and the remaining bound RNP was recovered by an additional 30-min elution under the same conditions. The first and second eluates were combined, and the total amount of RNA eluted was measured by liquid scintillation counting. EMSA was carried out as described above. For the detection of [32P]-labelled RNA, the gels were dried and autoradiographed using a phosphoimager screen. The gel images were scanned by FLA-3000 (Fujifilm).Internally labelled RNAs were prepared by i.e., Ni2+ or Co2+)-chelated resin to capture the hexa-histidine-tagged L7Ae, followed by treatment with a triplex-forming oligonucleotide-immobilized resin to capture the RNA  between BCD35 RNA and L7Ae was determined as 0.29 nM using the surface plasmon resonance (SPR) method employing a L7Ae-immobilized sensor chip and the RNA as the analyte , a very popular modification reagent for improving the stability of RNA , is appl analyte , S1 Fig. analyte , S1 Fig.D) was calculated from the ka and kd values. At least, three independent experiments were carried out, and the average values are tabulated. “n.d.” means not detectable.Kinetic parameters of the original and modified box C/D motifs. The association rate (ka) and dissociation rate (kd) of the indicated RNA for L7Ae protein were evaluated by SPR analysis with an L7Ae-immobilized sensor chip. The dissociation constant (KL1) and the C residue at the L3 position (CL3) form intramolecular hydrogen bonds, suggesting that they have a crucial role in binding  was low enough (3 nM) for the purification of the triangular RNP to be successful.For 2’-fPy-RNA, a C to A replacement (BCD35a) showed an affinity two orders of magnitude lower than that of the original BCD35 RNA. However, a C to G replacement (BCD35g) showed an affinity of one order of magnitude lower than the original , S1 Fig.L1 . AlternaPurification of the fully equipped triangular RNP by a sequential affinity elimination method was investigated by employing L7Ae- and fBCD35g-immobilized resins. 0.3 pmol of box C/D RNA was captured with 1 pmol of the immobilized L7Ae, while 1 pmol of L7Ae was captured with 1 pmol of the immobilized fBCD35g (data not shown). The resins were stable under the storage conditions for several weeks (data not shown).The triangular RNP was prepared with 100 nM of the RNA component, Tri26L/S, and 150 nM of the fluorescently labelled L7Ae. The complete triangular RNA , incomplete RNP complexes , and free materials were present in the mixture under those conditions , lane 3.Next, the recovered supernatant was applied onto the fBCD35g-immobilized resin and incubated at room temperature for 5 min. After the treatment, most of the free L7Ae was eliminated , lane 6.To confirm the enrichment of the complete triangular RNP, the purified and non-purified samples were analyzed by liquid-phase high-speed AFM . After tIn a previous study, the binding affinity of the triangular RNP with multiple antibodies bound was superior to that of the free antibody due to the avidity effect . A similWhen the triangular RNP was prepared with a lesser amount of the protein (100 nM), the resulting mixture also consisted of the fully loaded triangular RNP, incomplete RNP complexes, and the starting materials .. In thiin vitro. It might also be usable for purifying samples containing unwanted molecules from a mixture such as cell lysates after partial purification by canonical procedures.In this study, we developed a new purification procedure for a fully loaded triangular RNP with three proteins. The procedure enables selective purification of the fully loaded triangular RNP from samples containing incomplete RNP complexes that cannot be eliminated by regular affinity purification procedures as confirmed by EMSA  and AFM S1 FigIndicated concentrations of BCD35 RNA (A), BCD35a RNA (B), BCD35g RNA (C), BCD35 2’-fPy-RNA (D), BCD35a 2’-fPy-RNA (E), and BCD35g 2’-fPy-RNA (F) were injected onto the L7Ae-immobilized CM5 sensor chip  at 120 sec. The data was obtained by subtracting the signals for a flow cell without protein immobilization. The analysis was performed by BIAcore 3000 , and the kinetic parameters were calculated using BIAevaluation software .(EPS)Click here for additional data file.S2 Fig32P]-labelled RNA and 2 μM of L7Ae or L7Ae-ST2 fusion protein, and the binding assay was performed as described in (A) EMSA analysis of the RNP formation. (B) Binding profile of the ST2-displaying RNPs. Tri26-m1 and Tri26-m2 carry one and two inactively mutated box C/D motifs, respectively; that is, two and one active box C/D motifs are present. All three box C/D motifs of Tri26-m3 are mutated, and L7Ae protein cannot bind to the RNA. The RNPs were constituted with 1 nM of [(EPS)Click here for additional data file.S3 Fig32P]-labelled Tri26L/S RNA (1st and 4th lanes) and the indicated concentrations of L7Ae-ST2 fusion protein. Under the conditions, the resulting mixture consisted of the fully loaded triangular RNP, incomplete RNP complexes, and the starting materials. After the purification by sequential affinity elimination method (5th lane), the fully loaded triangular RNP was highly enriched. The [32P]-labelled RNA bands were detected by autoradiography using a phosphoimager screen.The ST2-displaying RNP complexes (2nd and 3rd lanes) were prepared with 1 nM of [(EPS)Click here for additional data file.


In vitro RNA polymerization assays and site-directed mutagenesis showed that: (1) the hPBV RdRP is fully active using both ssRNA and dsRNA templates; (2) the insertion loop likely functions as an assembly platform for the priming nucleotide to allow de novo initiation; (3) RNA transcription by the hPBV RdRP proceeds in a semi-conservative manner; and (4) the preference of virus-specific RNA during transcription is dictated by the lower melting temperature associated with the terminal sequences. Co-expression of the hPBV RdRP and the capsid protein (CP) indicated that, under the conditions used, the RdRP could not be incorporated into the recombinant capsids in the absence of the viral genome. Additionally, the hPBV RdRP exhibited higher affinity towards the conserved 5’-terminal sequence of the viral RNA, suggesting that the RdRP molecules may be encapsidated through their specific binding to the viral RNAs during assembly.During the replication cycle of double-stranded (ds) RNA viruses, the viral RNA-dependent RNA polymerase (RdRP) replicates and transcribes the viral genome from within the viral capsid. How the RdRP molecules are packaged within the virion and how they function within the confines of an intact capsid are intriguing questions with answers that most likely vary across the different dsRNA virus families. In this study, we have determined a 2.4 Å resolution structure of an RdRP from the human picobirnavirus (hPBV). In addition to the conserved polymerase fold, the hPBV RdRP possesses a highly flexible 24 amino acid loop structure located near the C-terminus of the protein that is inserted into its active site.  Viral polymerases replicate the genome of the virus, which is essential for the synthesis of the progeny. All double-stranded (dsRNA) viruses have virion-associated polymerases that catalyze RNA synthesis within an intact capsid. Picobirnavirus (PBV) is a small dsRNA virus, and it has been shown that the capsid of PBV possesses an unusual architecture suggesting an uncommon assembly pathway that it could potentially share with another group of small dsRNA viruses called partitiviruses. We have performed both structural and functional studies to look at how the PBV polymerase performs its function and how these molecules are placed within the capsid. The PBV RdRP structure was solved and revealed that the RdRP has an interesting loop structure in its interior. Additionally, activity assays showed that the RdRP possesses catalytic activity in the absence of other viral proteins. Removing the loop structure was found to change the way that the RdRP initiates RNA replication. Further experiments also showed that the RdRP did not interact with the viral capsid protein (CP) but had a strong affinity for a conserved terminal sequence of the PBV genome. This suggests that in PBV the RdRP may be encapsidated based upon both the RdRP and CP co-interacting with the viral genome. Double-stranded (ds) RNA viruses are a diverse group of viruses that vary widely in host range , genome segment number (one to twelve), and in the number of capsid layers, with many of them considered important pathogens of either agriculture or human health. A common feature of dsRNA viruses is that their capsid associated polymerase performs both of its functions, namely replicating as well as transcribing the viral genome, from within the confines of the virus capsid. This sequestration of the polymerase and the dsRNA genome prevents the activation of the host’s RNA induced antiviral response .gag-pol) fusion protein, which is then incorporated into viral particles as a minor structural component during capsid assembly ..3) in apThe heavy atom sites and experimental phases were determined by the AutoSol Wizard in the PHENIX software suite . The proin vitro transcription using the T7 RNA polymerase and complementary DNA templates amplified by polymerase chain reaction (PCR). The primers used for hPBV2-specific cDNA production were hPBV2_T7_Forward (5’ CGCGTAATACGACTCACTATAGTAAAATTTTCGAATTTTATAATAATTAAG) and hPBV2_Reverse (5’ GCAGTTGGGACTGTTAGTCCCAATG) as well as hPBV2_Forward (5’ GTAAAATTTTCGAATTTTATAATAATTAAG) and hPBV2_T7_Reverse (5’ CGCGTAATACGACTCACTATAGCAGTTGGGACTGTTAGTCCCAATG) for (+)strand and (-)strand ssRNA production, respectively. For ϕ6 specific cDNA production primers T7-1 and 3’end [The plasmids used for ssRNA and dsRNA production are presented in nd 3’end  were usend 3’end . The reand 3’end . The ϕ6 4Ac, 0.1 mM EDTA, 2 mM MgCl2, 0.1% (v/v) Triton X-100, 50 mM HEPES-KOH pH 7.5 and 0.4 U/μl RNase inhibitor RiboLock (Thermo Scientific), typically in 10 μl reaction volumes, using 55 nM concentrations of the WT or ΔLOOP polymerase and equimolar amounts of the RNA strands. The conditions were not stringent since PBV RdRP exhibited replication and transcription activity also in different divalent cation conditions (MgCl2 and MnCl2) and NTP concentrations. When back-priming and transcription time course reactions were assayed the RdRP amount was increased eight fold (without changing the molarity of the template RNA). A final concentration of 0.2 mM NTPs was used in the replication and transcription reactions. The TNTase activity was assayed in the presence of 0.03 μM UTP. For the identification of newly synthetized RNAs the reactions were supplemented with (α-33P) labeled UTP  or (γ-32P) labeled GTP . The reactions were incubated for 1 hour at 37°C and stopped with the addition of 2×U loading buffer  SDS, 6% (v/v) glycerol, 0.05% (w/v) bromophenol blue, 0.05% (w/v) xylene cyanol). For the evaluation of the back-priming activity, the reaction products were boiled for 3 minutes to denature the sample. The reaction mixtures were analyzed by gel electrophoresis in 0.8 or 1.2% (w/v) agarose. The gels were dried and the signals were collected on imaging plates (Fujifilm), which were subsequently scanned using Typhoon TRIO Imager . Quantification was done using AIDA Image Analyzer.The replication, transcription and TNTase activities of the hPBV RdRP were assayed in 6% (w/v) PEG 4000, 20 mM NHE. coli. The expression of both proteins (i.e. untagged CP and His-tagged RdRP) was then induced by the addition of IPTG. The cells were pelleted, resuspended, sonicated, and clarified in as described for the WT RdRP. After clarification, the supernatant was run through a nickel-NTA column to remove any free RdRP. The VLPs were subsequently purified using density gradient ultracentrifugation in a CsCl gradient of 1.1–1.4 g/cm3. The ultracentrifugation was performed using a SW41 Ti rotor (Beckman Coulter) at 35,000 rpm for 6 hours, after which the light-scattering VLP-zone was collected. Western blots were carried out using an anti-hPBV CP antibody obtained from Pacific Immunology  and an anti-6xHis antibody obtained from ThermoFisher . VLP formation was confirmed using TEM  as previously described [The gene encoding the CP of the hPBV Hy005102 strain  was cloned into a pET19b(+) vector without the corresponding UTRs. This construct was co-transformed along with the hPBV RdRP gene in a pET28b(+) vector into a Rosetta 2 strain of escribed .in vitro transcription as described above. RNA oligos and PBV2+ ssRNA molecules were labeled with γ-32P-ATP using T4 polynucleotide kinase (New England Biolabs) at 37°C for 30 minutes. The labeling was halted by the addition of 5 μl of 500 mM EDTA. The labeled oligonucleotides were then purified and desalted using a Sephadex G-50 Nick column (GE Life Sciences). The gel shift assays were conducted in 50 mM Tris-HCl pH 7.5, 50 mM KCl, 1 mM NaN3, 5 mM β-mercaptoethanol, and 10% (v/v) glycerol. A 1 nM concentration of 32P-labeled RNA was incubated with increasing concentrations of either the WT or the ΔLOOP hPBV RdRP for 30 min at room temperature. The samples were then loaded onto a 15% (w/v) native polyacrylamide gel and run at 50 V for 4 hours. The polyacrylamide gel setup was placed in an ice bath during the experiments to minimize heating. The radioactive RNA was visualized by phosphorimaging using the FujiFilm FLA-5000 imager and quantified using the program ImageGuage v4.0. The fraction of the RNA bound was then calculated as the amount of the bound RNA divided by the sum of the total RNA and plotted versus the corresponding concentration of the protein.The RNA oligos for the gel shift assays were commercially purchased from Sigma-Aldrich. The three RNA oligonucleotides used for these experiments were the PBV 5’-(+)UTR (5’-GUAAAAUUUUCGAAUUUUAU-3’), PBV 3’-(+)UTR (5’-GGACUAACAGUCCCAACUGC-3’), and a nonsensical CA repeat (5’-CACACACACACACACACACA-3’). The two PBV2+ ssRNA molecules, including the full-length PBV2+ and a deleted version PBV2+(Δ1–645), were made by S1 FigThe elution profiles of the WT and ΔLOOP RdRPs are displayed in red and blue, respectively. The elution positions of three reference proteins and their relative molecular weights are indicated above.(TIF)Click here for additional data file.S2 Fig(A) Alignment of ten polymerase sequences from PBV infecting various hosts. As indicated by the color bar, conserved regions are shown in pink while poorly conserved regions are shown in purple. The seven polymerase motifs are highlighted in black boxes. The six highly variable regions are shown in blue boxes. Secondary structure assignments based on the hPBV polymerase are included and the coloring schedule is the same as in (TIF)Click here for additional data file.S3 Fig33P) labeled UTP using three RNA templates: PBV2+ and PBV2- ssRNAs as well as PBV2 dsRNA. The main radiolabeled products detected were PBV2 dsRNA as shown on the left.Starting from the left are hPBV RdRP proteins with an N-terminal his-tag, a C-terminal his-tag, and without any fusion tag. The RNA synthesis activities were assayed in the presence of (α-(TIF)Click here for additional data file.S4 FigReaction products were collected from 0 minute to 240 minutes after reaction started. Templates used were the ϕ6 genomic RNA including RNA segments of three distinct sizes . Transcription products made by ϕ6 RdRP were included in the first lane for comparison. The expected positions of dsRNA  and ssRNA  products are indicated on the left of the gel.(TIF)Click here for additional data file.S5 Figin vitro produced ϕ6-specific dsRNA (lanes 2 to 5) was approximately one third of the amount of the ϕ6 genomic dsRNA (lane 1). The 3'-terminal sequences of the transcription (-)strand templates are shown at the bottom. Additional terminal sequences of ϕ6 genomic RNA can be found in The RNA templates applied are indicated at the bottom and the mobility of the RNA products on the left. The amount of the (TIF)Click here for additional data file.S6 Figrd lane). (b) RNA transcription using dsRNA templates. RdRP activity was tested using PBV2, ϕ6 S, ϕ6 L, and two mixtures of PBV2 with either ϕ6 S or ϕ6 L genomic segment. In the competition reactions (4th and 5th lanes) the amount of each RNA template was one-fourth of the equimolar amount of the RdRP. The mobilities of the expected dsRNA reaction products are indicated on the left.(a) RNA replication using ssRNA templates. RdRP activity was tested using PBV2+, ϕ6 s+, and a mixture of PBV2+ with ϕ6 s+. Equimolar amounts of each ssRNA and RdRP were applied in the competition reaction (3(TIF)Click here for additional data file.S7 FigLeft, PBV1+. Right, PBV2+. Both 5’ and 3’-ends of the RNA molecules are labeled. The predicted stem-loop structures at the 5’-end are highlighted in blue ovals.(TIF)Click here for additional data file.S1 Table(TIF)Click here for additional data file.S2 Table(TIF)Click here for additional data file.S1 References(TIF)Click here for additional data file.


Compartmentalization in a prebiotic setting is an important aspect of early cell formation and is crucial for the development of an artificial protocell system that effectively couples genotype and phenotype. Aqueous two-phase systems (ATPSs) and complex coacervates are phase separation phenomena that lead to the selective partitioning of biomolecules and have recently been proposed as membrane-free protocell models. We show in this study through fluorescence recovery after photobleaching (FRAP) microscopy that despite the ability of such systems to effectively concentrate RNA, there is a high rate of RNA exchange between phases in dextran/polyethylene glycol ATPS and ATP/poly-L-lysine coacervate droplets. In contrast to fatty acid vesicles, these systems would not allow effective segregation and consequent evolution of RNA, thus rendering these systems ineffective as model protocells.The online version of this article (doi:10.1007/s11084-014-9355-8) contains supplementary material, which is available to authorized users. The RNA world hypothesis provides a conceptual framework for the early development of life on earth in which RNA functions both as a molecule capable of propagating genetic information and as a catalyst. The capacity of RNA to transmit genetic information is exemplified by the RNA viruses, which can have genomes up to 30 kb in length consisting entirely of RNA  and charge-complex coacervates, have been proposed as model protocells Oparin , 2013. BBecause of the membrane-independent nature of ATPS and coacervate models, it is unclear whether these systems are able to compartmentalize genetic molecules such as RNA with minimal exchange between droplets. We have therefore studied the ability of ATPS and coacervate droplets to retain RNA oligonucleotides 15 and 50 nucleotides in length, and thereby gauge their effectiveness as membrane-free protocell model systems.w/v) ATPS was prepared, yielding roughly equal volumes of the dextran-rich and PEG-rich phases  system (yielding ≈ 55 % DEAE-dextran-rich phase by volume), RNA partitioned strongly into the DEAE-dextran-rich phase due to the positive charge of the DEAE-dextran and the more polar nature of that phase; the degree of partitioning was so great that the RNA concentration in the PEG-rich phase was below our detection limit (Table w/v) system (≈60 % dextran-sulfate-rich phase by volume), RNA partitioned strongly into the PEG-rich phase, presumably due to charge repulsion from the anionic dextran-sulfate. Droplets in the DEAE-dextran/PEG system coalesced more slowly than droplets in the dextran/PEG or dextran-sulfate/PEG system  and anionic dextran-sulfate Fig. . As expeit Table . Converstem Fig. , most liWe also prepared coacervates consisting of complexes of anionic ATP and cationic poly-L-lysine (pLys). Upon visual inspection, the ATP/pLys system  appeared similar to the ATPSs as two phases formed under specific concentration conditions Fig. . Followiw/v). The sizes of droplets ranged from 1 to 5 μm in diameter  microscopy to analyze the rates at which the RNA moved from the bulk phase into photo-bleached droplets. At steady state, this would be equivalent to the rate at which RNA diffused out of droplets into the bulk phase (and then into other droplets). We acquired and analyzed fluorescence recovery data for fluorescently labeled RNA in droplets from four systems Table : 16 % deter Fig. , similarter Fig. , up to 5ter Fig. , similarter Fig. .Fig. 1Ra1/2) of the fluorescently labeled RNA oligonucleotides was 8–20 s (Table In dextran-rich and DEAE-dextran-rich droplets (in their respective ATPSs) between 5 μm and 10 μm in diameter, the fluorescence recovery half-life (t s Table . In the  s Table  recovere s Table .1/2 was 5–21 s for the ATP/pLys droplets measured (3–9 μm in diameter)  Table .1/2 after photobleaching of droplets of the dextran/PEG ATPS and the ATP/pLys system containing a fluorescently labeled RNA 50-mer. For the droplets measured in both of these systems, the fluorescence recovery t1/2 was 11–76 s (4–11 μm in diameter)  Table . Comparer) Table , dropletTo compare the time scale of RNA retention between phase-separated droplet systems and fatty acid vesicles, we prepared oleic acid vesicles, similar in size to the droplets studied above, that contained the fluorescently labeled RNA 15-mer. For the vesicle experiments, a high concentration of fluorescently labeled RNA was present outside of the vesicles as well. Ten minutes after photobleaching a sample, the external solution had fully recovered in fluorescence intensity due to the diffusion of RNA from adjacent non-bleached sample regions. However, the vesicles did not regain any detectable internal fluorescence intensity Fig. . As expeWe then asked whether combining a dextran/PEG ATPS or an ATP/pLys coacervate system with current vesicle systems would allow RNA partitioning within a model protocell. Previous work has shown that it is possible to form phospholipid vesicles that contain dextran/PEG ATPSs  hydrochloride >500 kDa, poly-L-lysine (pLys) hydrobromide 1–5 kDa, poly-L-lysine hydrobromide 4–15 kDa, poly-L-lysine hydrobromide 15–30 kDa, and Sepharose 4B (45–165 μm bead diameter) beads were purchased from Sigma-Aldrich Corporation .Tris(hydroxymethyl) aminomethane (Tris), sodium chloride, magnesium chloride hexahydrate, D-(+)-glucose, 2-mercaptoethanol, adenosine 5′-triphosphate (ATP) disodium salt hydrate, adenosine 5′-diphosphate (ADP) disodium salt, adenosine 5′-monophosphate (AMP) disodium salt, guanosine 5′-triphosphate (GTP) sodium salt hydrate, guanosine 5′-diphosphate (GDP) sodium salt, guanosine 5′-monophosphate (GMP) disodium salt hydrate, uridine 5′-triphosphate (UTP) trisodium salt hydrate, 8-hydroxypyrene-1,3,6-trisulfonic acid (HPTS) trisodium salt, enzyme catalase from bovine liver, polyethylene glycol (PEG) 8 kDa, dextran 9–11 kDa from Aspergillus was purchased from Serva Electrophoresis . Glass cover slips (18 × 18 mm No. 1) were purchased from Thermo Fisher Scientific . All solutions were produced in nuclease-free water from BioExpress .RNA oligonucleotides  were purchased from Integrated DNA Technologies  and used without further purification. dNTPs and cytidine 5′-triphosphate (CTP) sodium salt were purchased from GE Healthcare . Oleic acid was purchased from Nu-Chek Prep, Inc. . rNTPs and glass microscope slides  were purchased from VWR International . Glucose oxidase from w/v dextran 9–11 kDa and 10 % w/v PEG 8 kDa solution was prepared by dissolving the solid components in a solution of 50 mM Tris-Cl pH 8 and 100 mM NaCl  was prepared by mixing respective stock solutions (200 mM ATP and 10 % or 50 % w/v pLys both in 100 mM Tris-Cl pH 8) and diluting with 100 mM Tris-Cl pH 8. All samples were prepared in 1.5 mL eppendorf tubes at room temperature. Due to the viscosity of the DEAE-dextran/PEG sample, pipet tips that were cut roughly 1 cm from the tip were used for that sample. To each sample, 5′-6-FAM-labeled RNA (5′- CCAGUCAGUCUACGC-3′ or 5′-CAUCUAGUUACCUCUAGGAUCUCAUGAUGCCUGAAGCGUAGACUGACUGG-3′) from a 100 μM stock solution in nuclease-free water was added to a final concentration of 5 μM RNA. Each solution was vortexed for 30 s. For applications that required the two phases to be separated, the sample tube was centrifuged for 15 min at 14,000 rpm. Each phase was then pipetted into separate tubes. Transmittance measurements were performed using a GE Healthcare (formerly Amersham) Ultrospec 3,100 pro UV-Visible spectrometer . RNA phase-specific partitioning measurements were performed using a Thermo Fisher Scientific  Nanodrop 2000c Spectrophotometer. For confocal microscopy, DEAE-dextran/PEG and ATP/pLys samples also contained the GODCAT system  to reduce photobleaching (Hentrich and Surrey w/v D-(+)-glucose, 0.5 mg/mL catalase, 1 mg/mL glucose oxidase, and 143 mM 2-mercaptoethanol. A 6–8 μL droplet  for each sample was applied to each glass slide (25x75mm) and a cover slip  was applied. The slide was allowed to sit at room temperature until the droplet applied was completely spread across the entire cover slip area, and then the cover slip was sealed using Valap  to avoid evaporation. Samples were covered with aluminum foil to reduce photobleaching by stray light until imaging.A 16 % ~10 mM oleic acid vesicles containing 5′-6-FAM-labeled RNA (5′-CCAGUCAGUCUACGC-3′) were prepared by mixing 1.6 μL pure oleic acid (3.17 M) with 50 μL of 10 μM RNA in 500 μL 180 mM bicine buffer adjusted to pH 8.5 with NaOH, followed by vortexing for 30 s. The sample was covered with foil and allowed to gently tumble overnight. A 3 μL droplet was applied to a glass slide as above for microscopy. The glass slide was then allowed to sit (cover slip down) at room temperature for 30 min to allow larger vesicles to rest on the surface of the cover slip.2O, 0.12 mM final concentration) and 10 μL of 100 μM 5′-Cy5-labeled RNA  were added. The solution was vigorously vortexed and visually inspected to verify that it contained only one phase. Subsequently, 3 μL of oleic acid were added to the solution and after another vigorous vortexing, the solution was tumbled over night on a rotating wheel (6 rpm) to allow vesicle formation. The next day, the vesicles were purified from unencapsulated dye and RNA using a short 1 cm Sepharose 4B gel filtration column and 1 mM oleic acid in 200 mM bicine (adjusted to pH 8.5 with NaOH) as a running buffer. 6 μL of gel-filtered vesicles were spread out (to around 1 cm2) on a 25x75 mm microscope slide and the droplet was allowed to evaporate for 6 min at room temperature. Then an 18x18mm coverslip was placed onto the droplet and the slide was sealed using Valap. Alternatively, a 3 μL droplet was placed on a slide and a coverslip was placed immediately on top of it. In this case, the coverslip was not sealed, but only fixed in the corners with Valap, and evaporation was allowed to occur through the edges over several hours. Slides were observed either with a confocal microscope (see below) or with a Nikon  TE2000 inverted fluorescence microscope with a 100× oil objective.To 840 μL of 5.95 % PEG 8 kDa, 10.7 % Dextran 10 kDa, 200 mM bicine pH 8.5 (adjusted with NaOH), 0.5 μL 200 mM HPTS . Confocal microscopy was performed using a Leica  SP5 AOBS Scanning Laser Confocal Microscope , a Carl Zeiss  LSM 510 Laser Scanning Microscope , or a Nikon  A1R Confocal Microscope . After selection of the droplet to be analyzed, a time zero image was acquired, and then a circular or square region was photobleached at high power using an Argon laser at 488 nm (or a solid state laser for the Nikon system). Each photobleaching region was chosen to be as small as possible while still containing a single, whole droplet to minimize collateral photobleaching of neighboring droplets. The fluorescence intensity  was then measured over time to track the fluorescence recovery of 5′-6-FAM-labeled RNA molecules within the droplet of interest.Curve fitting of the fluorescence recovery after photobleaching (FRAP) intensities was carried out by first obtaining intensities across all time points of a specific droplet. These intensities were normalized to the intensities of a non-bleached droplet and the background within the same frame, to correct for nonspecific photobleaching during sampling. The intensities were then normalized to the initial intensity of the droplet analyzed, to account for variable photobleaching before the recovery step across runs Movie S2(AVI 3858 kb)Movie S3(AVI 30671 kb)Movie S4(AVI 711 kb)Movie S5(AVI 1389 kb)ESM 6(PDF 3.00 mb)Below is the link to the electronic supplementary material.


O-ribose methylation. These modifications aid in the folding and stabilization of nascent rRNA molecules and their assembly into ribosomal particles. The genomes of hyperthermophilic archaea encode large numbers of C/D box sRNA genes, suggesting an increased necessity for rRNA stabilization at extreme growth temperatures.In archaea and eukaryotes, ribonucleoprotein complexes containing small C/D box s(no)RNAs use base pair complementarity to target specific sites within ribosomal RNA for 2'-O-methylation. Gene duplication, rearrangement, mutational drift and convergent evolution of sRNA genes and guide sequences were observed. In addition, several C/D box sRNAs were identified that use their two guides to target locations distant in the rRNA sequence but close in the secondary and tertiary structure. We propose that they act as RNA chaperones and facilitate complex folding events between distant sequences.We have identified the complete sets of C/D box sRNAs from seven archaea using RNA-Seq methodology. In total, 489 C/D box sRNAs were identified, each containing two guide regions. A combination of computational and manual analyses predicts 719 guide interactions with 16S and 23S rRNA molecules. This first pan-archaeal description of guide sequences identifies (i) modified rRNA nucleotides that are frequently conserved between species and (ii) regions within rRNA that are hotspots for 2'-O-methylation in archaea. The interaction between the sRNP complexes and the nascent rRNA facilitates proper folding and the methyl modifications stabilize higher order rRNA structure within the assembled ribosome.This pan-archaeal analysis of C/D box sRNA guide regions identified conserved patterns of rRNA 2'-The online version of this article (doi:10.1186/s12864-015-1839-z) contains supplementary material, which is available to authorized users. O-methylation) and the conversion of uridine to pseudouridine. In bacteria, 2'-O-methylation modifications are relatively rare and are introduced by dedicated site specific or region specific methyltransferases. In contrast, eukaryotes and archaea utilize ribonucleoprotein (RNP) complexes containing small (s)RNAs to identify targets for 2'-O-methylation. These sRNA molecules are called small nucleolar RNAs (C/D box snoRNA) in eukaryotes and the archaeal homologs are C/D box sno-like RNAs (C/D box sRNA). They are characterized by four conserved sequence elements termed box C and C' (consensus sequence: RUGAUGA) and box D and D' (consensus sequence: CUGA). These elements fold into a ubiquitous RNA structural motif, the kink-turn (k-turn) [Stable RNA molecules frequently undergo post-transcriptional modifications. The majority of these modifications involve the addition of methyl groups. The most prevalent modifications found in ribosomal RNA (rRNA) molecules are the methylation of the ribose moiety at the 2'‐hydroxyl group (2'-(k-turn) –3. The s(k-turn) . In addi(k-turn) .O-methylation to the nucleotide that is base paired with the guide five nts upstream from the start of the D or D' box sequences using S-adenosyl-methionine as methyl donor [O-methylated RNA nucleotides to support RNA folding and stabilization [In archaea, two copies of the L7Ae protein bind respectively to the C and D, and D' and C' box sequences and stabilize the k-turn structural motif. This is followed by the further addition of the proteins Nop5 and the fibrillarin methyltransferase to complete assembly of the active complex. The guide regions of the sRNA base pair with complementary sequences in the target RNA and mediate 2'-yl donor , 6–9. Thyl donor , 11. Thulization .Single-particle electron microscopy studies and the crystallization of archaeal C/D box sRNP complexes with different sRNA compositions revealed structures that either contained two or four copies of L7Ae, Nop5 and fibrillarin , 14. RecSulfolobus acidocaldarius, which led to the identification of 18 different C/D box sRNAs and their targets [S. acidocaldarius and Haloarcula marismortui [O-methylation patterns of archaeal rRNA molecules. This pan-archaeal analysis revealed methylation hotspots in functionally important rRNA regions. Furthermore, C/D box sRNAs were identified that are proposed to function as RNA chaperones assisting rRNA folding during ribosome assembly.Previous studies along with our results indicate that C/D box sRNA genes and their transcripts are highly abundant in hyperthermophilic archaea , 16. Ini targets . Physicaismortui . The advismortui , 16, 18.Nanoarchaeum equitans, a hyperthermophilic archaeon with a highly compacted genome of only 490 kb size which harbors a small set of only 26 C/D box sRNA genes under genome reduction constraints. Six of the species are thermophilic, with optimal growth temperatures of approximately 75 °C and above. We identified all abundant sRNA molecules (< 100 nt) in the RNA-Seq datasets. These sequences were manually searched for the presence of GA/AG k-turn motifs to define C and D boxes  in three organisms.The majority of hotspots (except for those in domain IV) are located in close proximity to the catalytic peptidyl transferase center (in domain V), where peptide bond formation and peptide release occurs . All sixrganisms . In our E. coli [E. coli U2552) is predicted to be modified in five of the species and the fifth nucleotide in the loop  is predicted to be modified in two species. For S. acidocaldarius, our predicted sites of modification agree with the methylation sites reported previously [Methanococcus maripaludis only a few C/D box sRNAs exist and in this species the A-loop does not appear to contain any sites of guide directed methyl modification.In general, the concentration of methylation sites near the peptidyl transferase center in archaea resembles the pattern of posttranscriptional modification of nucleotides (base methylations and pseudouridylations) in the 23S rRNA of E. coli . A MALDIE. coli . The A-lE. coli , 29. Thieviously . In the O-methylation hotspots but are not in direct proximity to the peptidyl transferase center. However, these helices are part of the interface between the large and the small ribosomal subunit [E. coli ribosome crystal structure revealed an interaction between A1912 in 23S helix 69 and C1407 and G1494 in 16S helix 44. Conserved pseudouridylation sites were shown to exist in helix 44 and the loop region of helix 69; these modifications influence RNA folding and are crucial for ribosome subunit association [O-methylation sites in helix 69 around E. coli position A1912 . In 16S helix 44 methylation predictions are relatively sparse  in two species  and at G1482 (E. coli position G1494) in the mesophilic species M. maripaludis.Helices 68, 69 and 71 in Domain IV are 2'- subunit . The E. ociation , 31. In Most of the RNA in the large subunit of the ribosome is protected and stabilized by the presence and binding of ribosomal proteins. The proteins often contain elongated termini that penetrate between RNA helices, interacting with several RNA domains to stabilize tertiary RNA structure . The excO-methylation target site predictions. The methylated nucleotides in this region likely contribute to the stabilization of the decoding center as well as the tight association of the four domains.Next, we analyzed the locations of conserved methylation sites within the 16S rRNA structure Fig. . The fouE. coli 16S rRNA) is intimately associated with the interaction of the A site tRNA anticodon with the mRNA codon; site directed mutations at this position are lethal [Helix 18  monitors the correct codon-anticodon pairing and is the core of the decoding center in the small ribosome subunit , 34. Thee lethal . Other mE. coli position G530) are predicted to be methylated, respectively, in three and four archaeal species. We predict that the high density of predicted modifications contributes to the folding, structural stabilization and translational fidelity function of this helix. It is interesting that there are no predicted modifications in this helix in either M. maripaludis (a mesophile) or in N. equitans (organism with contracted genome).In five of the seven species examined there are a total of 23 methylation predictions at 13 different positions within helix 18. Seven of the 13 sites are modified in only a single species whereas six of the sites are modified in two or more of the species. Positions A509 and A512  is predicted to be modified in all six of the thermophilic archaeal species examined in this study (See “Methylation sites in 23S rRNA” above). This means that each species contains a sRNA with a conserved guide region that exhibits complementarity to the 23S rRNA regions surrounding position U/C2880   that use D' guides to direct modification to position 16S A512 (35) Fig.  in helixThere are numerous other examples of sRNAs from the various archaeal species that share a related guide sequence  and the simultaneous interactions likely play an important role in facilitation rRNA folding events. Similarly, we identified C/D box sRNAs with targets separated by more than 100 nts that are required to be brought in close proximity during rRNA folding. These distant interactions provide further evidence for a potential RNA chaperone function of C/D box sRNAs.The pan-archaeal analysis of experimentally verified C/D box sRNA molecules was used to deduce 2'-It should also be noted that RNA-guided methylation adds to ribosome heterogeneity as the methylation of individual nucleotides is not always absolute. Elevated growth temperatures could result in altered target recognition which would increase ribosome heterogeneity and represent another level of adaption to extreme growth conditions. Future analysis of the methylation status of all individual rRNA nucleotides and the abundance of various sRNAs under different physiological conditions will be necessary to evaluate the importance of site specific methylation and the degree of rRNA heterogeneity.P. calidifontis small RNA was performed using a Roche/454 GS FLX sequencer as described [RNA extraction was performed on log-phase cells using the mirVana miRNA isolation kit (Ambion) according to the manufacturer's instructions. Illumina Hiseq 2000 RNA-Seq methodology was applied as described before , 16. RNAescribed . The samIllumina TruSeq sequencing reads with a quality score limit of 0.05 were trimmed to remove linkers and poly-A tails and mapped to the reference genomes  with CLC Genomics Workbench 5.0 . The following mapping parameters were employed . Reads below 15 nts were not considered. The program was also used to determine the coverage of individual RNA molecules. All predicted RNAs and their 5' and 3' termini were manually verified and all intergenic regions were checked for the presence of RNAs with sequence coverage of less than 1000 reads. Gene annotations (including tRNA genes) were obtained from Genbank .O-ribose methyl modification occurs in the target rRNA nucleotide that is base paired with the nucleotide in the guide regions of the C/D box sRNA that is located at the plus five position upstream from the start of either the D or D' box sequences. Methylation sites in rRNA were computationally predicted by scanning for extended complementarity between the D and D' guide regions of the sRNAs and corresponding species-specific 16S and 23S rRNA sequences. One mismatch, two GU base pairs, and no bulges were allowed in the duplex between the guide and the rRNA region containing the target nucleotide. The first nucleotide of the D or D' box was included in the guide sequence, as this position may participate in the formed duplex [Guide directed 2'-d duplex . From thFrequently C/D box sRNAs utilize both the D and D' guides to direct modification to separate targets within a region of 100 nucleotides in the rRNA sequence; these are called “double guide” sRNAs. In a small number of instances, the nine base pair complementarity criteria for target prediction was relaxed somewhat for these double guide sRNAs by allowing G:U base pairs and/or a single nucleotide mismatch base pair in the region of complementarity. The rationale for this was that simultaneous interaction of the two sRNA guides with adjacent sequences within rRNA results in thermodynamically more stable structure than a single guide-target interaction. Of the 489 sRNAs examined, approximately half (262/489) were predicted to function as double guide sRNAs. In a small number (16/262) of the double guide sRNAs, we noted that one of the two guide-target complementarities contained a mismatch base pair at the predicted site of modification. In spite of the mismatch, we believe that both guide-target interactions form in the complex but that methylation occurs only at the single position where the critical WC base pair is present , 39.From the collection of 489 sRNAs, each containing two guide regions, we identified a total of 735 guide interactions that satisfied the above significance criteria. We predict that 719 of these interactions result in methylation of the target rRNA whereas 16 of the interactions are likely non-productive because of the lack of the critical WC base pair in the guide-target interaction.M. kandleri) and GSE38821 .RNA-Seq data are available at NCBI's Gene Expression Omnibus (GEO) database as series GSE44979  [Sequencing reads of csc.edu) . C/D boxThe following additional data are available with the online version of this paper.O-methylation in 16 and 23S rRNA for the D and D′ guides of each sRNA.Additional file Additional file E. coli and the seven archaeal species listed in Table Additional file E. coli and the seven archaeal species listed in Table Additional file


Retroviridae, must selectively recognize their full-length RNA genome among abundant cellular RNAs to assemble and release particles. How viruses coordinate the intracellular trafficking of both RNA and protein components to the assembly sites of infectious particles at the cell surface remains a long-standing question. The mechanisms ensuring packaging of genomic RNA are essential for viral infectivity. Since RNA packaging impacts on several essential functions of retroviral replication such as RNA dimerization, translation and recombination events, there are many studies that require the determination of RNA packaging efficiency and/or RNA packaging ability. Studies of RNA encapsidation rely upon techniques for the identification and quantification of RNA species packaged by the virus. This review focuses on the different approaches available to monitor RNA packaging: Northern blot analysis, ribonuclease protection assay and quantitative reverse transcriptase-coupled polymerase chain reaction as well as the most recent RNA imaging and sequencing technologies. Advantages, disadvantages and limitations of these approaches will be discussed in order to help the investigator to choose the most appropriate technique. Although the review was written with the prototypic simple murine leukemia virus (MLV) and complex human immunodeficiency virus type 1 (HIV-1) in mind, the techniques were described in order to benefit to a larger community.In cells, positive strand RNA viruses, such as  Most retroviruses share the same characteristics: a spherical capsid about 110–120 nm in diameter enclosing a positive single-strand RNA genome. During infection, the genomic RNA (gRNA) is retro-transcribed into DNA, which is integrated into the host chromosomes. Transcription of this viral DNA gives rise to a primary, full-length transcript that acts as gRNA for packaging into progeny viruses. In addition, the gRNA serves as a messenger RNA (mRNA) template for the synthesis of the structural Gag and enzymatic Pol polyproteins. A common feature of most retroviruses is the selective packaging among the bulk of viral spliced and cellular RNAs of two identical copies of gRNA per viral particle. The packaging of retroviral RNA is a highly regulated process that requires structural motifs located in the 5’ untranslated region of gRNA, notably the Psi region. Psi folds into stem-loops that constitute the major packaging determinants recognized by Gag to be packaged into virions , maniew, see ,15. Furtiew, see ,17,18.In order to understand the fundamental mechanisms of retroviral replication, it is essential to accurately characterize the identity and amount of RNA packaged into virions. This includes not only the gRNA, the key molecule carrying all the viral genetic information, but also RNA from the host cell, which may contribute to the viral lifecycle . The preThe Northern blot and the ribonuclease protection assay (RPA) were among the first methods developed for measuring gene expression in cells and tissues. These techniques use complementary labeled probes for detecting RNA in a purified sample. For detecting long transcripts such as viral mRNA and gRNA, the probe is most often an internally labeled riboprobe, which has been obtained by using radioactive in vitro transcription. The signal is recorded only after several steps which obligatorily include gel electrophoresis  is then added, whose purpose is the removal of excess probe and unprotected fragments—i.e., not engaged in a duplex. Following RNase treatment, the sample is run on a denaturing polyacrylamide gel electrophoresis (PAGE) and the electrophoretic profile of the probe is recorded  Figure a. The siThe performance of RPA critically depends on the quality of probe detection with PAGE analysis. Probe length for RPA is most often comprised between 100 and 700 nt, the upper size being usually constrained by the resolution of protected fragments during PAGE analysis. The integrity of the probe being used is the first important parameter to consider. Therefore, it is often required to gel-purify riboprobes after radioactive labeling in order to get rid of extra bands caused by abortive transcription products. We will review some of the issues usually encountered with RPA, and how to address them in order to improve the sensitivity, the specificity and the scale of the assay.The RPA provides a significant improvement to sensitivity compared to the Northern blot, partly because it is much less affected by RNA degradation. Indeed, if the target sequence is cut by an endonuclease during sample preparation, the resulting fragments still have a chance to anneal and protect the probe. In addition, solution hybridization permits the testing of large amount of RNA (up to 100 µg for most protocols), whereas Northern blot is limited by the capacity of electrophoresis wells. Substantial improvements to sensitivity can be further obtained with high-specific activity probes, obtained for instance by increasing labeling efficiency. During optimization of the RPA assay, the sensitivity and the linearity of detection can be tested by annealing the probe to serial dilutions of an artificial complementary RNA. Such standards may subsequently be used to infer the absolute concentration of target RNA in the experimental sample b. They mThe RPA is a highly reproducible method, and it is much less affected by trace contaminants than polymerase chain reaction (PCR) approaches. However, the interaction of the probe with sequences that do not belong to the target RNA can create undesirable protected fragments. They can be caused for example by partially related RNA present in the sample, or by self-complementarity within the probe. Long probes have a higher risk to form stable secondary structures and repeats, which can be addressed by increasing the stringency of annealing conditions. If the RNase treatment is not efficient, extra bands, corresponding to the undigested probe, may appear and this is usually solved by testing different mixtures and concentrations of enzymes. Finally, the RNase treatment might be able to discriminate between single-nucleotide variants , which cThe concentration of target RNA can be expressed relative to another RNA whose level remains constant. This can be achieved in RPA by multiplexing the number of probes used in the assay . SeveralBy combining Northern blot and RPA, it is possible to get exhaustive information about the size, the exon arrangement and the extremities of RNA molecules. A major advantage of these techniques over others is that only partial knowledge of the target RNA sequence is required to design probes. For instance, a Northern blot using a probe corresponding only to the 5’ leader of retroviral RNA, could in theory reveal several variants corresponding to alternatively spliced RNA. In addition, RPA permits researchers to examine how the RNA sequence is arranged at a precise location, for instance splicing sites b and RNAThe Northern blot and RPA approaches are especially useful for studying RNA viruses since a single probe can simultaneously detect retroviral gRNA and its differentially spliced variants b. For incis-acting sequences during MLV or HIV-1 assembly [The RPA proved to be a useful tool for testing the packaging efficiency of gRNA mutants. It has been instrumental for deciphering the role played by assembly ,30,31. Tassembly ,32. Howeassembly , semi-quassembly . While tCt) is defined as the cycle number at which the fluorescent signal of the reaction crosses the threshold. The threshold corresponds to a fluorescence intensity significantly over the background value, which is determined for each sample during the initial cycles of PCR. Differences in threshold cycle number are used to quantify the relative amount of PCR target contained within each tube, and for each experimental sample the initial concentration of target varies inversely with the Ct value.The RT-qPCR is the method of choice to quantify RNA expressed at very low levels. It is highly specific and much more sensitive than the methods mentioned above. Its fundamental principle is based on monitoring the accumulation of the target amplicon during successive cycles of PCR. Different kinds of approaches based on fluorescence dyes permit to label the double-stranded DNA (dsDNA) being synthesized. During the exponential phase, the amount of the fluorescent signal generated is proportional to the concentration of the target RNA in the original sample. For each PCR reaction, the threshold cycle  RNA for example, β-actin mRNA was used as an endogenous control, thanks to its consistent presence inside the virus particles .Taq polymerase. Degradation of the probe releases the fluorophore and takes it away from the quencher, allowing fluorescence of the fluorophore. TaqMan performs better than SYBR Green assay in terms of sensitivity and specificity, as the fluorescence is only generated when the probe is released to the target sequence during PCR amplification. However, the approach is less economical for multiple target RNA analysis. In contrast, SYBR Green can be used with any pair of primers for any target. Nonetheless, when used during a PCR reaction, SYBR Green will bind to any dsDNA including the template, primer dimers, specific PCR products and nonspecific products. This is a major disadvantage, which can compromise the specificity of detection. There are a few ways of handling this problem, including analyzing DNA melting curves post-PCR and using approaches to reduce mis-priming.A variety of fluorescence-based strategies exists to monitor dsDNA accumulation on qPCR. The two most popular ones are the TaqMan Assay, using a fluorogenic probe specific to the target gene ; and theGood primer design is one of the most important parameters in real-time PCR for obtaining high specificity and sensitivity. Primer pairs should have compatible melting temperatures and amplicon lengths should be chosen as short as possible (less than 300 pb). The formation of primer-dimers should be avoided, as it decreases PCR efficiency and obscures analysis. Techniques such as hot start reduce mis-priming by suppressing the activity of PCR enzymes at lower temperatures, where non-specific duplexes can form.When SYBR Green is used, the specificity of the real-time PCR reaction and the absence of primer dimers can be verified using melting curve analysis. Immediately after the PCR cycles, the temperature is raised by increment until all dsDNA molecules are “melted” in single-stranded DNA (ssDNA). Individual amplicons and primer dimers have their own melting temperature. Therefore, by monitoring the change in fluorescence triggered by the release of SYBR Green upon melting, one can estimate the proportion of specific PCR products in the reaction .The RT-qPCR is a very sensitive technique capable to detect few copies of transcripts in a complex mixture. This sensitivity is critically dependent on the efficiency of the amplification and on the quality of the cDNA template. Ultrapure, intact RNA is essential for full-length, high quality cDNA synthesis.Following DNase treatment on nucleic acid extracts to remove DNA contamination, equal RNA amounts are set for reverse transcription. Two negative controls of RT-PCR reactions must be systematically performed: one to confirm the absence of DNA by running a RT reaction without enzyme, and another to evaluate background amplification level by running a real-time PCR with a sample lacking the target RNA, extracted for example from mock-infected cells. Oligo(dT) primers are a favorite choice for cDNA synthesis as they prime all RNAs having poly-A 3′ tail simultaneously. It is convenient for viral and cellular mRNAs, while reverse transcription of non-coding RNA may require specific internal or random primers ,9. SpeciStable secondary structure elements present in viral RNA may lead to incomplete cDNA generation. Conditions of RT extension, such as temperature, duration of the incubation and choice of the enzyme, can be optimized to guarantee a successful cDNA synthesis .The RT-qPCR has been useful for characterizing the content of retroviral particles and for precisely measuring RNA packaging ability. Two qPCR strategies, TaqMan ,32,35,40trans-acting factor, and to test the function of trans-acting factors participating in the assembly, such as the nucleocapsid or matrix domain of Gag protein [The RT-qPCR has been particularly useful to identify the packaging determinants in the viral RNA, such as the 5′ UTR structures including the Psi element, and the  protein ,42,43,44 protein .The RT-qPCR technique can be laborious to establish and needs numerous controls to guarantee accurate and reliable results. However, with careful optimization, it is highly sensitive and specific, allowing the detection of very small amounts of RNA, and the quantification of several different targets from a single RNA sample, and often from a unique (oligo-dT) RT reaction. The RT-qPCR allows to run high-throughput analysis, as numerous assays can be run simultaneously, and several targets can be quantified in the same assay (by using TaqMan probes for instance). Finally, the RT-qPCR has also the great advantage of being a safe technique, using neither radioactivity nor harmful chemicals products. In conclusion, it is the method of choice for quick and precise quantification of RNA packaging.Over the last few decades, fluorescence-based methods have been developed to label the RNA in cellulo with high specificity and sensitivity. While biochemical techniques based on total RNA extraction from pools of cells and viruses provide information on the average behavior of a population, fluorescence microscopy enables direct visualization of single RNA molecules within single cells and viruses. This represents a major breakthrough for the study of RNA metabolism because it accounts for cell-to-cell variation and focuses on the behavior of single RNA transcripts. Moreover, as the integrity of the sample is preserved, RNA molecules can be studied within their three-dimensional (3D) cellular environment, providing information on the spatial distribution, the surrounding molecular network and cell structure. With the live-cell fluorescence microscopy approach, a 4th dimension (time) is added and thus permits a better understanding of the dynamic nature of RNA trafficking.Different RNA labeling techniques exist depending on whether fixed or live samples are used. In this section, we describe two techniques permitting sensitive and specific detection of single RNA molecules in cells or virions: fluorescence in situ hybridization (FISH), which consists in hybridizing multiple fluorescent DNA probes to a given RNA species in a fixed biological sample ; and RNARecent advances in the field of fluorescence microscopy have provided a wide array of imaging tools for the study of single molecules at the subcellular scale with great spatial and temporal resolution . AccordiIn situ RNA labeling can be used to detect endogenous or engineered RNA in fixed cells and viruses. With this technique, specific RNA transcripts are detected by hybridizing fluorescent DNA probes that are complementary to a unique RNA sequence present in the target RNA. Multiple RNA species can be distinguished using different probe sets labeled with spectrally distinct fluorophores. The specificity and stringency of hybridization needs to be optimized by adapting the temperature of hybridization, which is directly dependent on the GC nucleotide content and length of the probe, the percentage of formamide and the concentration of monovalent cations  that are present in the hybridization mixture. Post-hybridization washes under stringent conditions are further required to remove unbound probes, reduce background and achieve specificity . FISH caSingle-molecule sensitivity can be achieved using multiply-labeled probes  or many MS2 and lacZ gene sequences  by their respective RNA-binding protein, fused to a fluorescent peptide (MS2 coat protein-GFP). The MS2 system has been extensively used due to its simplicity and sensitivity. The approach has provided important insights into many aspects of the retrovirus lifecycle including transcription dynamics, nuclear export of viral RNA, translational regulation, membrane targeting, and condensation of the gRNA during virion assembly (reviewed in ).Escherichia coli bgl operon (Bgl system) [PP7, which binds its own 23-nucleotide stem-loop RNA primer-binding site  [N system) has the advantage of a smaller tag insertion, and it consists in the 15-nucleotide stem loop of the antitermination signal (BoxB RNA) recognized by the N coat protein of bacteriophage lambda [2 [The original MS2 system consists in inserting a 19-nucleotide stem-loop structure from the MS2 genome in the RNA of interest, which binds to MS2-GFP. At least three other MS2-like systems are now available, used alone or in association with the MS2-system, for live-cell RNA imaging: (i) the BglG system consists in a 29-nucleotide stem-loop recognized by the antitermination protein in the  system) ; (ii) anled PBS) ; and , valuable information on RNA–RNA/protein interactions and their spatial orchestration with the cellular structure can be obtained. Finally, organic fluorophores are more resistant to photobleaching than fluorescent proteins , and thuBoth endogenous and engineered RNA can be followed using specific probes designed accordingly. The ability to label native RNA by FISH is a major advantage over live-cell imaging, as it does not require modification of the RNA in order to incorporate such tags. Since the RNA is unaltered by the binding of exogenous proteins, FISH should provide more reliable information about the localization and native state of RNA molecules. Indeed, binding of several bacteriophage proteins to the reporter RNA may affect intracellular movement and virion morphology. Nevertheless, FISH labeling requires fixation and permeabilization of cells, which can alter molecular conformations and disrupt the integrity of certain organelles or cellular structures . FinallyRecent breakthrough in “deep-sequencing” technologies permits exhaustive transcriptome analysis, thus facilitating the large-scale study of RNA expression and the discovery of rare transcripts. Next-generation sequencing (NGS) is based on the real-time monitoring of base extension during the DNA sequencing reaction, which is performed either on a pool of different templates  or on a single molecule (Pacbio). Depending on the platform, the data may consist either in several millions of short reads  or a relatively small number of very long reads , with outputs varying between 0.5 Gb and 100 Gb. Large numbers of reads permit to attain high coverage of the sample, which is instrumental for sequence assembly and for the discovery of rare molecules. During RNA sequencing (RNA-seq), the sample RNA is reverse transcribed and adapter sequences are included at the extremities of cDNA. These extra sequences are instrumental for the subsequent amplification and sequencing of the cDNA library. RNA-seq has been used for studying the expression of viral RNA in infected cells . NGS hasThe choice of RNA quantification method in cells and virions requires consideration of several factors: sensitivity, accuracy, relative quantification, RNA size determination and probe flexibility. Each of the techniques discussed in the present review provides specific advantages and limitations to study RNA packaging. For applications in which characterization of the packaged RNAs and their relative abundance are of chief importance, the Northern blot is the more suitable choice. Indeed, the Northern blot is the only technique that provides information about RNA size. The main advantage of the RPA over the Northern blot is its higher sensitivity (10-fold), attributed by the high specific activity of the riboprobe labeling and the hybridization of the riboprobe to the target RNA in solution without prior membrane immobilization. However, recent approaches based on RT-qPCR are most commonly used. They excel for the rapid measurement of packaging efficiency and/or ability of known RNAs for which efficient sets of primers have been developed. In contrast, RNA-seq approaches are still in their infancy for the study of viral packaging but their performance opens promising perspectives. Deep sequencing of viral RNA has to deal with the representation of target molecules in the sample, and for rare transcripts it may involve the collection of significant amounts of biological material. Among all the methods discussed, RT-qPCR offers the highest sensitivity and accuracy. In practice, qPCR can detect the cDNA derived from 10 RNA copies and therefore, requires only small amounts of biological samples. Typically, either ~10% of virions released from tissue culture or 1 µg of total cellular RNA are used for the RT reaction and only a fraction (1/20 to 1/50) of the resulting cDNA is used for qPCR. Several assays can be performed with the initial sample, thus reducing the need for collecting biological material. Similar advantages are brought by the most recent imaging approaches, which permit the detection of unique tagged-RNA molecules in single cells or virions. The methods for in vivo RNA visualization provide unprecedented access to quantity, localization and dynamics of viral RNA and will likely see their usage in constant progress for the study of packaging.