Rapid and accurate detection of pathogens under field laboratory conditions is necessary for effective control of veterinary pathogens. Here we describe a prototype, portable, pathogen detection device developed for single tube, real-time, reverse transcription, loop-mediated isothermal amplification (RT-LAMP) using Laem-Singh virus (LSNV) as a model. LSNV is an RNA virus and a component cause of growth retardation in black tiger shrimp. We chose its RNA-dependent RNA polymerase (RdRp) gene as the target for our tests. The basis for detection was measurement of turbidity arising from formation of a white, insoluble magnesium pyrophosphate precipitate byproduct upon amplification of the RdRp target sequence from 100 ng template RNA extracted from shrimp. The measurement device consisted of a heating block to maintain constant temperature in the RT-LAMP reaction for 8 Eppindorf sample tubes, a light-emitting diode (LED) light source providing red light emission at 650 nm wavelength to pass through sample tubes, a light dependent resistance (LDR) photo-detector and a software program to report turbidity events and could potentially be marketed for under US$3000. The device was connected to a computer to display real-time results in a variety of formats. The optimized protocol for LSNV detection consisted of incubation of the sample tubes at 65°C for 1 h during which turbidity was continuously measured, and quantitative results could be obtained by reaction time measurement. The sensitivity of detection was comparable to that of conventional nested RT-PCR and there was no cross reaction with other common shrimp viruses. The device was used for quantitative measurement of relative copy numbers of LSNV RdRp in 8 shrimp tissues and they were found to be highest in the gills followed in order by the lymphoid organ and hemolymph (p≤0.05). This platform can be easily adapted for detection of other pathogens under field laboratory settings. Penaeus (Penaeus) monodon from Laem-Singh district of Chantaburi province in Thailand in 2006 Leuteoviridae that comprises mostly insect-borne plant viruses P. monodonRapid and accurate detection of pathogens under field laboratory conditions is necessary to effectively control pathogens of terrestrial and aquatic animals. Laem-Singh virus (LSNV) is a positive-sense single-stranded RNA (ssRNA) virus of shrimp that was first discovered from shotgun cloning of nucleic acids isolated from growth-retarded P.monodonEven though the detailed etiology of MSGS is still uncertain, LSNV is considered to be a component cause of the disease, and it has therefore been placed on the list of specific pathogens to be eliminated from specific pathogen free (SPF) stocks of domesticated As LAMP reactions proceed, pyrophosphate ions are released from the deoxyribonucleotide triphosphate (dNTP) reagents consumed during nucleic acid polymerization and these react with magnesium ions in the reaction mix to produce a white, insoluble magnesium pyrophosphate precipitate. This product results in progressively increasing turbidity of the reaction solution In the present study, a relatively unsophisticated portable device that combined a heating block to support RT-LAMP with a turbidimeter to continuously measure magnesium pyrophosphate precipitate was developed for LSNV detection and quantification under field laboratory conditions. It was also used to determine the number of copies of the LSNV RdRp target in various organs of infected shrimp, providing information on the severity of infection.The LAMP-turbidimeter allowed for multi-channel turbidity measurements based on spectroscopic assessment of the magnesium pyrophosphate byproduct of LAMP reactions. It consisted of a heating block unit  that maintained a constant temperature during the LAMP reaction, a light-emitting diode (LED) light source providing red light emission at 650 nm wavelength, a light dependent resistance (LDR) photo-detector and a software program to continually  report turbidity P.monodon) with a known history of LSNV infection Since the gross signs of LSNV infection are retarded growth that may also arise from many other causes, the only method for easy detection is RT-PCR. To obtain specimens for this work, broodstock from a family of black tiger shrimp (5′-TTC TCC CGA GTG GTC AGG TTTA-3′ and LSNVR1 5′-CCA GAA ACG TAT TGG CAC ACG-3′) was linearized by XhoI restriction enzyme  at 37°C for 3 h and purified by NucleoSpin Gel and PCR Clean-Up Kit  according to the manufacturer’s protocols. An XhoI enzyme-digested product was used for RNA in vitro transcription by the RiboMAX Large scale RNA production system-T7 kit  following the manufacturer’s protocol. The quantity of total RNA and in vitro RNA transcripts were analyzed by spectrophotometer and subjected to ten-fold serial dilution of 50 ng to 5 fg equivalent to 0.5×106 to 1 copies of the LSNV target per reaction vial, respectively. The RT-LAMP and nested RT-PCR reactions were carried out using 2 µl of RNA template solution.Total RNA was extracted from gills of black tiger shrimp infected with LSNV using a simple and rapid method with 2 M guanidine thiocyanate (GuSCN), and it was used directly for LAMP reactions 4 , 8 U of Bst DNA polymerase , 1x of the supplied buffer, 0.25 U of AMV reverse transcriptase  and the specified amount of RNA template. To prevent evaporation, reactions were overlaid with 15 µl of mineral oil . Temperature optimization was carried out at 60, 63 and 65°C using a portable turbidimeter RT-LAMP assays for LSNV detection were carried out according to a previously reported protocol with specific LAMP primers based on the published sequence of the LSNV RNA dependent RNA polymerase (RdRp) (GenBank accession no. DQ127905), except that unlabeled primers were used  The nested RT-PCR protocol for LSNV detection was modified from previous publications in vitro LSNV-RNA transcripts (0.5×106 to 1 copies). They were used as templates under the optimized conditions for the real-time RT-LAMP and the nested RT-PCR assays, as described above.To compare the sensitivity of the real-time RT-LAMP assay and the conventional, nested RT-PCR assay, two types of RNA template were used. One consisted of ten-fold serial dilutions of total RNA (50 ng to 0.5 fg) extracted from the gill tissues of shrimp infected with LSNV, while the other consisted of Specificity was tested using RNA templates (100 ng) extracted from shrimp infected with non-target pathogens including the RNA viruses yellow-head virus (YHV), Taura syndrome virus (TSV) and infectious myonecrosis virus (IMNV) and the DNA viruses white-spot syndrome virus (WSSV) and infectious hypodermic and haematopoietic necrosis virus (IHHNV). Controls consisted of RNA template extracted from healthy shrimp not infected with those viruses.in vitro RNA transcripts were employed. The copy numbers of the in vitro RNA transcripts were calculated based on the molecular weight and Avogado’s number and were used to construct a standard curve using the portable turbidimeter. The standard curve for the LSNV RdRp gene was generated individually for each set of samples analyzed. The reaction setup was the same as that optimized above, and the reactions were carried out in the portable turbidimeter.To quantify target LSNV RNA  by the real-time RT-LAMP assay, 10-fold serial dilutions of β-actin mRNA (GenBank accession number JN808449) was chosen as the target because of previous demonstrations that its expression did not vary greatly from tissue to tissue based on copy numbers per unit of total RNA extract β-actin mRNA was amplified by RT-PCR using F3 and B3 primers, yielding a cDNA product of 212 bp that was inserted into a plasmid using a pGEM T Easy kit  according to the manufacture’s protocol. The recombinant plasmid was subjected to 10-fold serial dilution and used to prepare a standard curve of concentration of LAMP DNA amplicon versus turbidometer reading. The same RNA extracts used for LSNV quantification were also used as 100 ng templates under the same optimal reaction conditions as the LSNV real-time RT-LAMP with the turbidimeter device to determine β-actin copy numbers. To validate the data for LSNV copy numbers, it was necessary that there be no significant difference in mean β-actin copy numbers among the tissue types examined.As a reference control for our real-time RT-LAMP assays of LSNV RdRp RNA copy numbers in 100 ng samples of shrimp RNA, shrimp A total of 10 black tiger shrimp broodstock suspected of LSNV infections were collected from SGIC and their tissues  were aseptically removed and subjected to RNA extraction for analysis by real-time RT-LAMP and by conventional nested RT-PCR assays. Raw data for copy numbers were recorded and converted to a percent relative index to account for different levels of overall infection in the 10 shrimp used for each sample. Specifically, the tissue type with the highest copy number for any shrimp specimen was set at the percent relative index 100 and all other tissues for that specimen were adjusted as a proportion of this number, i.e., Relative Index = (Specific tissue number/Highest tissue number)×100. Then mean relative index and standard deviation (SD) were calculated for each tissue in preparation for statistical analysis by ANOVA followed by all pairwise multiple comparisons using the Holm-Sidak method with Sigmastat for Windows 3.5 software. Differences with p≤0.05 were considered to be statistically significant.Testing real-time RT-LAMP by the portable turbidimeter at 60, 63 and 65°C for 60 min using 100 ng of total RNA as template revealed that RT-LAMP products could be visualized at all tested temperatures. For reaction time, the turbidity was seen first at 22 min and then at 28 and 31 min at 65, 63 and 60°C, respectively . Therefoin vitro LSNV RNA transcripts), it was able to detect templates at as little as 100 fg of total RNA  and with RNA templates from healthy shrimp gave no positive results, indicating that the real-time RT-LAMP assay was specific for LSNV. As with sensitivity, specificity of our protocol was similar to that previously reported for non-quantitative RT-LAMP for LSNV detection tT) plotted against log of the initial template quantity using 10-fold serially diluted in vitro LSNV RNA transcripts (from 106 to 10 copies per reaction tube) resulted in a line plot with a high correlation coefficient (R2 = 0.981) (6 to 10 copies) of plasmid DNA containing β-actin gene gave a straight line with a high correlation coefficient (R2 = 0.971)  . The sen= 0.981) . Similar= 0.971)  and it s= 0.971) . These s5 LSNV copies per 100 ng total RNA. Parallel tests by normal nested RT-PCR were also LSNV positive for all 80 samples while only 56 were positive by the less-sensitive, one-step RT-PCR used, which by comparison to the RT-LAMP results was only capable of detecting LSNV when virus copy numbers were relatively high .When 8 tissues each from 10 broodstock specimens were tested by the finalized real-time RT-LAMP method, it was found that all 80 tissues samples from the 10 shrimp were positive for LSNV over a range of 48 to 8.6×10β-actin was chosen as the internal control target gene. Results for individual shrimp (see example for 1 shrimp specimen in β-actin expression) among the various tissues tested. However, an ANOVA test of mean relative copy indices of β-actin per 100 ng RNA for 8 tissues in 10 shrimp specimens revealed a significant difference (p = 0.013) among tissues of the 10 shrimp specimens used. A subsequent, all pairwise multiple comparison by the Holm-Sidak method revealed that this ANOVA result arose from a significant difference only between the pair of RNA from gill tissue with RNA from hepatopancreatic tissue . When the result for hepatopancreatic tissue was left out of the ANOVA analysis, there was no significant difference in the mean copy numbers among the 7 remaining tissues by a one way ANOVA test (p = 0.180). Thus, we considered that β-actin expression was a suitable internal control and that its expression did not vary significantly among the 100 ng RNA templates obtained from the tissues tested. Thus, one way ANOVA analysis of the unadjusted mean relative copy indices for LSNV were used to compare expression of LSNV in 8 tissues from 10 shrimp.For comparison of relative LSNV copy numbers in 100 ng of total RNA extracted from various tissues of LSNV infected shrimp, β-actin in various tissues of individual shrimp, those for LSNV were quite variable  was the same as the first, with the highest expression in gill tissue, followed by the lymphoid organ and hemolymph and relatively low expression in the remaining tissues, with significant differences the same as those shown in the graph in In contrast to the relatively uniform real-time RT-LAMP curves for emolymph  while coin situ hybridization assays, where positive signals for LSNV were reported from the cytoplasm of cells in lymphoid organ (LO), gills, haemocytes, brain, eyestalks, optic lobes, ventral nerve cord, cardiac tissue and connective tissue of the hepatopanceas in situ hybridization results were reported for connective tissue of the hepatopancreas, most of which is located in its sheath, and it is probable that little or no sheath tissue was included in the hepatopancreatic tissue used to make RNA extracts in our study.Our results from real-time RT-LAMP analysis for LSNV quantity in various tissues corresponded with previously reported results from Use of our assay protocol to assess viral loads in individual tissues of shrimp infected with LSNV clearly revealed the advantage of quantitative real-time RT-LAMP over conventional non-quantitative LAMP methods. We were able to show that shrimp gill tissue had the highest LSNV loads (p≥0.05) and that they were approximately 2 times higher than those for the next most heavily infected tissue . This is the first time that quantitative tissue loads for LSNV have been reported. The result is important in terms of monitoring and detection of LSNV infections because it reveals that gills should always be used as the target tissue for preparation of RNA templates. This is fortunate, because sufficient gill tissue for analysis can usually be removed without causing undue shrimp stress or mortality. By contrast, sampling lymphoid organ tissue requires killing the shrimp, an option not possible with specimens such as broodstock. The only other suitable tissue for non-destructive sampling is hemolymph, but the level of LSNV in that tissue was approximately 3 times lower than that in gills.This is the first report of quantitative detection of LSNV using a real-time RT-LAMP assay. It proved to be superior to previously reported methods based on nucleic acid amplification techniques (NAAT) such as conventional RT-PCR Use of a relatively inexpensive, portable, integrated heating-block/turbidimeter for rapid and sensitive detection of LSNV together with indication of severity of infection by real-time RT-LAMP should allow for wider application of its molecular detection, especially in field laboratories. This is because the approximate estimated cost of the heating-block/turbidimeter at under $3000 US dollars (converted from Thai baht), which is very low when compared to that for a real-time RT-PCR thermocycler. In addition, our LAMP method employs no labeled nucleic acids or special probes that can add to reagent costs for each assay. Further, the RT-LAMP/turbidimeter platform could be adapted relatively easily for detection of other shrimp, veterinary or human pathogens by rational primer design and optimization of the appropriate, specific protocols.


One purpose of the EC funded project, SPIDIA, is to develop evidence-based quality guidelines for the pre-analytical handling of blood samples for RNA molecular testing. To this end, two pan-European External Quality Assessments (EQAs) were implemented. Here we report the results of the second SPIDIA-RNA EQA. This second study included modifications in the protocol related to the blood collection process, the shipping conditions and pre-analytical specimen handling for participants. Participating laboratories received two identical proficiency blood specimens collected in tubes with or without an RNA stabilizer. For pre-defined specimen storage times and temperatures, laboratories were asked to perform RNA extraction from whole blood according to their usual procedure and to return extracted RNA to the SPIDIA facility for further analysis. These RNA samples were evaluated for purity, yield, integrity, stability, presence of interfering substances, and gene expression levels for the validated markers of RNA stability: FOS, IL1B, IL8, GAPDH, FOSB and TNFRSF10c. Analysis of the gene expression results of FOS, IL8, FOSB, and TNFRSF10c, however, indicated that the levels of these transcripts were significantly affected by blood collection tube type and storage temperature. These results demonstrated that only blood collection tubes containing a cellular RNA stabilizer allowed reliable gene expression analysis within 48 h from blood collection for all the genes investigated. The results of these two EQAs have been proposed for use in the development of a Technical Specification by the European Committee for Standardization. Project objectives are accomplished by using evidence-based, quality assurance schemes derived from external quality assessments (EQAs) and validated technologies for the collection, transport and processing of blood samples for in vitro diagnostic testing of genomic DNA, cell-free (plasma) DNA, and intracellular RNA SPIDIA  the blood collection process, (ii) the shipping conditions and (iii) the pre-analytical specimen handling protocol for participating laboratories. Here we report the results of the second SPIDIA-RNA EQA.As we noted in our previous publication of results of the first SPIDIA EQA of intracellular RNA 0 control and proficiency specimens, the resultant specimens were identified as to donor source, and the results segregated accordingly. The participating laboratories were therefore randomized into two groups, each group receiving proficiency specimens associated with one donor. To maintain constant temperature during sample shipment, we adopted dedicated shipping containers that maintained an internal temperature of 2°C to 8°C for 48 h.Since most blood specimens are collected in EDTA tubes, blood collection for the second study was performed using bags prefilled with an EDTA solution such that the final molar concentration approximated that of EDTA tubes. This step was taken to obtain a large volume of whole blood which closely resembled in composition whole blood specimens received in clinical laboratories, i.e. EDTA whole blood. Because blood from a single donor was not of sufficient volume to provide proficiency specimens to all study participants, two blood donors were enrolled, blood from each donor was aliquoted into TThe protocol for participants for the second EQA was virtually the same as for the first EQA study. Briefly, two proficiency specimens, both either with or without an RNA stabilizing additive, were sent to participating laboratories according to whether or not they wished to receive tubes containing stabilizer. Participants were asked to extract the RNA from whole blood sample from one tube immediately after receipt by the laboratory and from the second tube 24 h later after storage at either ambient or refrigerated temperature. Storage temperature was assigned randomly. The participants were instructed to extract the RNA using their routine laboratory procedure and send the purified RNA samples back to the SPIDIA facility  for analysis.ex vivo gene expression changes in stored EDTA blood, were also included in order to improve the evaluation of highly labile RNA targets The quality and quantity of RNA in the returned samples were evaluated by means of the same methodology used in the first SPIDIA-RNA EQA. These methods included the spectrophotometric measurement of total RNA yield and purity, RIN score as measured by the Agilent Bioanalyzer in vitro diagnostics.The results of these two SPIDIA RNA EQA studies have been compiled and will be used by the European Committee for Standardization (CEN) to propose an evidence-based Technical Specification for pre-analytical handling of blood for RNA-based 2EDTA Tubes , after ethical approval of the Ärztekammer Nordrhein (German). The healthy subjects signed an informed consent. We made six specimen pools, each pool containing one tube from each of three randomly selected subjects. From each pool as well as from one of the remaining EDTA tubes from each donor, a 2.5 mL aliquot was transferred into PAXgene Blood RNA Tubes (PAXgene) , incubated for 6 h at room temperature, and then frozen at −20°C. The remaining three tubes from the individual subjects, as well as the six sample pools were incubated at room temperature for up to 3 days. After one, 2, and 3 days, a 2.5 mL aliquot of blood from each sample was transferred into PAXgene tubes to stabilize the transcript profile, incubated for 6 h at room temperature, and then frozen at −20°C. At the end of the time course, RNA from all specimens stored in PAXgene tubes was extracted according to the PAXgene Blood RNA Kit Handbook Version 2 and analyzed for individual transcript levels.Blood from 18 healthy, consented subjects was collected into five Vacutainer KReverse Transcription quantitative PCR (RT-qPCR) was performed using 3 µL of the RNA eluate. The one-step qRT-PCR reactions were performed as duplex qPCR (FOS/18SrRNA and IL1B/18SrRNA) for 40 cycles on a TaqMan 7700 cycler (ABI) using the QuantiTect Probe RT-PCR Kit  and specific primers. Relative transcript levels of FOS and IL1B gene transcripts were determined by duplex RT-qPCR, using 18S rRNA as an internal standard and ΔΔCq calculation.2(RQ) values as dependent variables where RQ = 2−ΔΔCq and −ΔΔCq =  of the four genes tested are graphically represented in section C.1  = 0 corresponding to the T0 value that is expected in the absence of up- or down-regulation.In section F  we repor0 values of FOSB and TNFRSF10c transcripts from blood collected in PAXgene tubes were close to zero even 48 h post-phlebotomy. Messenger RNA species from EDTA tubes, however, showed time- and temperature-dependent expression levels. In particular, at 24 h post-phlebotomy   and 42% (D2) laboratories were within non-critical proficiency limits  whereas 29% (D1) and 30% (D2) laboratories presented one “out of control” rating and/or one or more “missing data” responses. The remaining participating laboratories  presented two or more “out of control,” with or without “missing data” quality parameters .An overall increase in the quality of lab proficiency from the first to the second SPIDIA-RNA EQA was observed with the percentage of laboratories with “good” proficiency ratings increasing from 26% in the first EQA to 43% (by considering both donors) in the second EQA.For both FOS and IL8, a statistically significant difference was observed depending upon the blood collection tube used  for bothOne of the main purposes of the SPIDIA project was to investigate the role of pre-analytical variables in the handling of blood samples for molecular testing. In order to obtain evidence-based guidelines for the pre-analytical processing of blood samples for RNA analysis, two EQAs were planned (first and second SPIDIA-RNA). For each EQA, a survey and a proficiency testing program was implemented to evaluate current sample handling parameters and technologies for blood RNA-based analysis in laboratories in Europe. In addition to providing objective measurements for laboratory proficiency, we designed this EQA to approximate blood collection, specimen storage, and RNA extraction methods currently used by participating laboratories and compare the quality of the RNA produced by these methods.Pursuant to these goals, we collected blood into EDTA-filled blood collection bags for this second EQA instead of in the CPDA blood bags used in the first EQA Because blood from one donor was not of sufficient volume to provide specimens for all of the participating laboratories, we determined the effect of blood pooling on gene expression. The results  demonstrRelative to the first SPIDIA-RNA EQA One hundred twenty-two applications were received from 21 different European countries, 109 laboratories returned the extracted RNA to the SPIDIA facility by the established deadline. During the first SPIDIA-RNA EQA, there were 124 applications, and 93 laboratories returned RNA samples to the SPIDIA facility 2EDTA tubes (66%), whereas others (21%) used PAXgene tubes. The quality of the extracted RNA samples was evaluated for yield and purity by UV analysis. Purified RNA was most often stored at −80°C, and the predominant downstream analytical methods were PCR technologies . Other aspects of sample handling and analysis protocols were more variable and included the volume of blood used and time and temperature of specimen storage post-phlebotomy.The survey queried current laboratory policies and practices specific to specimen handling. Respondents were asked to provide information on blood collection and extraction protocols . The ana260/A280) close to 2.0 indicating high quality RNA Using the same approach adopted in the first SPIDIA EQA, we evaluated the quality of RNA returned to the SPIDIA facility by participating laboratories. An individual report for each laboratory was produced which described the proficiency of the laboratory respect to the results of the other participants. The distribution of the results, similar within each donor  and D (stored 48 h post-phlebotomy)  . In contllection , Tube C.llection . SimilarAn analysis of individual participant reports according to our proficiency classification scheme revealed that the distribution of the overall proficiency ratings was similar within the two donors with almost 40% of laboratories receiving “in control” assessment for all the considered quality parameters. This result was an overall increase in proficiency level in comparison to those obtained in the first SPIDIA-RNA EQA. This improvement was most likely due to the changes in the second EQA study design  and pre-analytical specimen handling protocol (stringent time/temperature conditions).ex vivo changes in the gene expression profile. These changes include gene induction, gene down-regulation, and RNA degradation, all of which could result in erroneous measurements of gene transcript levels In conclusion, the SPIDIA-RNA EQAs identified the most critical steps in the pre-analytical procedure concerning blood collection and processing for RNA testing. Furthermore, due to the improvements we adopted in the second EQA, we were able to make important conclusions regarding pre-analytical conditions, which affect When using blood collection tubes which do not contain any RNA profile stabilizer , it is strongly recommended to investigate whether a specific RNA species intended to be analyzed in the analytical test is stable after blood draw for the duration of the entire pre-analytical workflow.The results of these two SPIDIA RNA EQAs studies have been proposed for use in the development of a Technical Specification by the European Committee for Standardization (CEN).Figure S1Distribution of participant laboratories (n = 124) through European Countries (A), Structures (B) and the main Research area (C).(TIF)Click here for additional data file.Figure S2Schematic comparison of the general workflow of the two SPIDIA-RNA EQAs: the first SPIDIA-RNA EQA study published by Pazzagli M et al. (TIF)Click here for additional data file.Table S1Questionnaire: distribution frequencies. Usual procedures performed by participant laboratories (n = 92).(DOC)Click here for additional data file.Appendix S1Report for participant. Report, related to Donor1, produced for each participant containing the overall distribution of the analyzed RNA quality parameters and the specific evaluation of the performance for each parameter and overall evaluation.(PDF)Click here for additional data file.Protocol S1Protocol A- PAXgene Blood RNA tube. Procedures and protocol for blood storage and RNA extraction for participants receiving blood collected in PAXgene Blood RNA tubes.(PDF)Click here for additional data file.Protocol S2Protocol B- EDTA tubes (+4°C). Procedures and protocol for blood storage and RNA extraction for participants receiving blood collected in EDTA tubes, which had to store the blood at +4°C.(PDF)Click here for additional data file.Protocol S3Protocol B- EDTA tubes (RT). Procedures and protocol for blood storage and RNA extraction for participants receiving blood collected in EDTA tubes, which had to store the blood at RT.(PDF)Click here for additional data file.Protocol S4Result form – Protocol A - PAXgene Blood RNA tubes. Form to fill by experimental data performing RNA extraction from blood collected in PAXgene Blood RNA tubes.(PDF)Click here for additional data file.Protocol S5.Result form – Protocol B - EDTA. Form to fill by experimental data performing RNA extraction form blood collected in EDTA tubes(PDF)Click here for additional data file.


Normalization of human RNA-seq experiments employing chimpanzee RNA as a spike-in standard is reported. Human and chimpanzee RNAs exhibit single nucleotide variations (SNVs) in average 210-bp intervals. Spike-in chimpanzee RNA would behave the same as the human counterparts during the whole NGS procedures owing to the high sequence similarity. After discrimination of species origins of the NGS reads based on SNVs, the chimpanzee reads were used to read-by-read normalize biases and variations of human reads. By this approach, as many as 10,119 transcripts were simultaneously normalized for the entire NGS procedures leading to accurate and reproducible quantification of differential gene expression. In addition, incomparable data sets from different in-process degradations or from different library preparation methods were made well comparable by the normalization. Based on these results, we expect that the normalization approaches using near neighbor genomes as internal standards could be employed as a standard protocol, which will improve both accuracy and comparability of NGS results across different sample batches, laboratories and NGS platforms. Ultra-high-throughput sequencing of RNA (RNA-seq) employing next-generation sequencing (NGS) technology is providing unchallengeable performances in transcriptome analyses12456189111213In analytical chemistry, the isotope dilution mass spectrometry (ID-MS) is regarded as the most authentic method for quantification of organic and inorganic materials1517Analogically, we aimed to develop a conceptually solid normalization method for genomic analysis, which will not only enable correction of sequence-dependent biases and equalization of different reaction efficiencies resulting from physically separate analyses but also simultaneously offer genome-scale coverages. As an effort to establish such normalization method, we suggest a ‘use of a genome from a closely related species as a spike-in standard for NGS’ and present a proof of the concept by employing chimpanzee RNA as a spike-in standard for human transcriptome analysis. It has been reported that use of internal standards carrying a few discriminable sequence variations could effectively normalize inter-sample variations in amplicon sequencing leading to accurate and reproducible quantification of gene expressionThe first consideration in selecting a spike-in standard for the RNA-seq analysis was the frequency of SNVs. Too many SNVs in a read may result in a decreased resemblance of chemical properties and reaction efficiencies between the target and the standard RNA. On the other hand, reads lacking SNVs cannot be normalized by our SNV-based strategy. In this regard, we postulated that one variation per read would be optimal for our strategy. Assuming read lengths of 150-bp by contemporary NGS platforms, one SNV per 150-bp will be the optimal frequency. SNV frequencies were estimated to be about one in 150–200-bp in chimpanzees and gorillas while much more (one in 20–50-bp) SNVs were estimated in orangutans and macaques by alignments of several ape and human mRNA sequences. Based on this estimation, we selected chimpanzee RNA as the spike-in standard for the current human transcriptome analysis.http://birch.cau.ac.kr/pub/Informative-Position.txt).To obtain informative SNVs between humans and chimpanzees, we performed RNA-seq with pure human and chimpanzee RNAs and compared the data to discover SNVs between the two species. A total of 166,310 SNVs were identified from comparisons of 40 million common reads. Only homozygous SNVs  were chosen from the initial data because heterozygous SNVs could result in ambiguity when discriminating read sources. The homozygous SNV loci were distributed over 10,119 unique transcripts representing the frequency of 14 SNVs per transcript with an average interval of 210-bp. The selected reads representing homozygous SNVs between humans and chimpanzees were reconstructed as a reference database for discrimination of the species sources of NGS reads  and induced pluripotent stem cells (HI). First we checked whether spike-in of the chimpanzee RNA caused any interference in RNA-seq results. It is an essential requirement that a spike-in standard should not lead to any interferences in results of original samples. 7Differential gene expression estimated for transcripts will not be necessarily equivalent to differential expression estimated for a specific region of the transcript due to existence of various isoforms for a gene and potential bioinformatics-driven errors during assembly to transcript information. Therefore we investigated the accuracy of estimates for differential gene expression by the SNV normalization approach not in transcript levels but for individual SNV loci. 4,264 out of 146,611 commonly detected SNV loci exhibited more than two-fold differences between the two estimation methods. The number of discordantly estimated loci was only 60 when the 85,770 loci over the read cutoff value of 40 were analyzed. To examine estimates from which methods are correct, we selected 10 discordantly and 3 concordantly estimated loci for further validation by qPCR and quantitative amplicon sequencing. Differential gene expression estimated by unnormalized read counts, SNV-normalized reads, qPCR and amplicon sequencing are compared in 2 = 0.78) implying they could not be directly compared by standard methods  while the other was directly processed (HICL) for library preparation. Degradation of RNA by RNase A may lead to preferential enrichment of 3′ regions of transcripts during purification of mRNAs based on oligo-dT since 5′ regions would not be captured by oligo-dT if any of the downstream regions were degraded by RNase A. Then gene expression profiles estimated from the 3′-biased reads from a degraded sample would not be highly comparable with those from relatively unbiased reads from a normal sample. As expected, unnormalized reads exhibited a very low correlation between the two data sets .Successful normalization of RNA-seq procedures by the spike-in standard could also be monitored by changes in read distributions. Sequence-dependent biases in fragmentation of RNA, reverse transcription and PCR would result in a skewed distribution of reads over a transcript11SPARC transcript are distinctly higher than the other peaks  gene-by-gene normalization, ii) comprehensive normalization for the whole NGS procedures, and iii) genome-scale coverage for gene expression analysis. Consequently, accuracy and comparability were significantly improved by application of the method. The improved accuracy and comparability were not only seen in well-controlled experiments but also across different library preparation methods and different in-process degradation effects. Based on these results, we expect the SNV-based normalization methodology could be used as a standard protocol to minimize biases and variations arising from differences of samples, experimental batches, laboratories and NGS platforms.A chimpanzee lymphoblastoid cell line, EB176 (JC) was purchased from Public Health England and cultured in RPMI1540 containing 15% FBS. A human foreskin fibroblast cell line purchased from System Biosciences was cultured in DMEM containing 15% FBS. Human iPSCs were derived from a human immortalized lymphoblastoid cell line under the approval of Institutional Review Board (IRB) committee in Korea Institute of Oriental Medicine (I-1210/002/002-02). The IRB approval defined uses of previously reported experimental protocols26Normal NGS library was constructed using TruSeq RNA Sample Preparation Kit v2 (Illumina) following the manufacturer’s instruction starting from 2 μg of total RNA. NexTera library was constructed following the protocol in TotalScript RNA-Seq Kit (Epicentre) with a few modifications. A mixture of 2 μg of human and chimpanzee total RNA was reverse transcribed by combined primers of 50 pmol oligo-dT and 17 ng random hexamers using Superscript III (Life Technologies). After second strand synthesis using the second strand master mix in the TruSeq RNA Sample Preparation Kit, cDNA was subjected to the library preparation procedure using NexTera DNA Sample Preparation Kit (Illumina). For preparation of partially degraded RNA, 2 μg of human and chimpanzee total RNA mixture was treated with bead-bound RNase A. The bead-bound RNase was prepared by attaching 0.1 μg of RNase A (Sigma R4642) to carboxyl magnetic beads (Spherotech CM-50-10) using the 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (Sigma) chemistry. The RNase-attached beads were washed and resuspended in 100 μL of 0.5 X PBS buffer (pH 7.0). Five μL of the RNA-attached beads solution was used for partial degradation of the RNA in 0.2 X PBS buffer for 3 minutes at 25 °C. About 70% of the 28S rRNA band was degraded and smeared by this partial digestion condition. To prevent further degradation 10 unit of RNaseOUT (Life Technologies) was added to the RNA solution separated from the beads. Then the RNA solution was directly subjected to the mRNA isolation and library preparation procedure using TruSeq RNA Sample Preparation Kit. RNA-seq was performed on HiSeq 2500 (Illumina) platform with a paired-end read length of 150-bp.Primers for qPCR and amplicon sequencing are listed in http://www.ebi.ac.uk/~stijn/reaper/src Reads were sorted according to the sample source, each of which had different barcode sequence. Forward- and reverse-end reads were separately stored as ‘R1’ and ‘R2’ files, respectively. The barcode sequence was removed from the 5′ end of the read. The 3′ end adapters and trailing sequences were trimmed using reaper (version 14-020) available at ftp://ftp.ncbi.nlm.nih.gov/refseq/H_sapiens/mRNA_Prot) in February 11, 2014. The well-annotated RefSeq mRNAs of which accession number starts with ‘NM’ were extracted. To reduce database complexity and computational complication in this pilot analysis, only one transcript per gene, the one that appeared first in the data, was selected. The human mRNA sequences were indexed for BWA and SAMtools.The human RefSeq mRNA sequences were downloaded from the National Center for Biotechnology Information (NCBI) web site  which was available at To identify informative positions showing single nucleotide difference between human and chimpanzee mRNAs, reads from pure human fibroblasts (HF), human iPSCs (HI), and chimpanzee lymphocytes (CL) were comparatively analyzed. To increase the read depth, R1 and R2 reads for each of HF, HI, and CL reads were merged. SAMtools and BCFtools were used to call variations in HF, HI, and CL data compared to human RefSeq mRNAs. The command line was “samtools mpileup –d 1000000 –u –f FASTA_FILE BAM_FILE | bcftools view –bvcg - > BCF_FILE” where FASTA_FILE was the human RefSeq mRNA data. In order to increase the depth reported by SAMtools, the maximum depth count was increased from 8,000 to 1,000,000 (the 438th line of the ‘bam_pileup.c’). Each of the three BCF files was filtered to collect variations supported by at least 10 reads. The command line was “bcftools view BCF_FILE | vcfutils.pl varFilter –d 10 –D 1000000 > VCF_FILE”. The three VCF files were analyzed to collect informative positions that passed the following conditions: (i) read depth was at least 10 in all the three reads; (ii) each of HF, HI, and CL was homozygous ; (iii) HF and HI had the same sequence but CL had a different sequence. There were 146,611 informative positions identified from 10,119 transcripts. The informative positions were stored as a BED file.To quantify human and chimpanzee transcripts in a mixed sample, numbers of human and chimpanzee alleles at each informative position of a transcript were counted. First, read sequences at each informative position were extracted; the command line was “samtools mpileup –d 1000000 –f FASTA_FILE –l BED_FILE BAM_FILE” where BED_FILE contained coordinates of the informative positions. The output files were parsed to distinguish and count human and chimpanzee alleles at each position. Ratios of the human to chimpanzee allele counts were calculated for each informative position. The R1 and R2 reads were analyzed separately and showed almost the same results.http://genome.uscs.edu). The transcript counts were calculated using HTSeq (http://www-huber.embl.de/HTSeq/doc/overview.html). Three conventional normalization methods; upper quartile (UQ)For transcriptome analysis by conventional methods, the RNA-Seq reads were mapped to the reference human genome hg 19) using TopHat using ToAccession Codes: The sequencing data from this study have been submitted to the NCBI Sequence Read Archive (SRA) under accession no. SRP046066.How to cite this article: Yu, H. et al. Normalization of human RNA-seq experiments using chimpanzee RNA as a spike-in standard. Sci. Rep.6, 31923; doi: 10.1038/srep31923 (2016).


Tangle-predominant dementia (TPD) is characterized neuropathologically by numerous neurofibrillary tangles in the limbic areas with no or occasional senile plaques throughout the brain. TPD is an under-recognized disease, while it is a common cause of dementia in those over 80 years of age. In the present study, we describe hyperphosphorylated tau (tau) accumulation in the nucleus accumbens (Acb) in patients with TPD.We investigated immunohistochemically the brain tissues from 7 patients with TPD, 22 with Alzheimer disease (AD) and 11 non-demented aged subjects. In the Acb of all 7 TPD patients, a considerable number of tau positive neurons were found together with many neuropil threads. The tau deposits in the Acb were labeled with all the anti-tau antibodies used in the present study. They included conformational change-specific, phosphorylation-specific and phosphorylation-independent antibodies. The Acb consists of the predominant medium-sized neurons with a small number of large neurons. Both the cell types were affected by tau pathology in TPD. Tau accumulation in the majority of such neurons appeared to be pretangle-like, diffuse deposits with only occasional paired helical filament formation. Tau positive neurons were also found in the Acb in some AD and non-demented aged subjects but much fewer in the majority of cases. The immunoblot analyses of fresh frozen samples of the Acb and parahippocampal cortex from 3 TPD and 3 AD patients revealed that the insoluble tau in the Acb was a mixture of the 3- and 4-repeat isoforms.To our knowledge, this is the first report on the occurrence of tau accumulation in the Acb in TPD. The Acb receives direct and massive projections from the hippocampal CA1 and subiculum where neurofibrillary tangles are known to occur more frequently in TPD than in AD. The prevalence of abnormal tau accumulation in the Acb in TPD may support the idea that abnormal tau aggregation propagates via neural circuits. In all but one TPD cases used in this study, delusion was a consistent clinical feature. Whether the Acb tau accumulation is related to the psychiatric symptoms in TPD may be an issue for further investigation. Tangle-predominant dementia (TPD), which is also referred to as neurofibrillary tangle predominant dementia, limbic neurofibrillary tangle dementia or senile dementia of the neurofibrillary tangle type, is a poorly understood and under-recognized tauopathy. TPD has been reported to comprise 0.7 to 5.8% of elderly patients with dementia -3. TPD iThe etiology of TPD is unknown. NFT in TPD consist of both 3-repeat (3R) and 4-repeat (4R) isoforms of hyperphosphorylated tau (tau), and the neuronal cell types bearing NFT in TPD are similar to those in AD. TPD seems to be a disorder that is related to AD, if it is not an atypical form of AD. TPD, as a subtype of tauopathy, is also included in the group described as neuropathologically-defined frontotemporal lobar degeneration . In factA neuropathological characteristic of TPD is the heavy accumulation of NFT in the hippocampal regions, with few or occasional NFT in neocortical areas beyond the collateral sulcus. Compared with AD patients in which a similar number of NFT occurs in the hippocampal regions, neuronal cell loss, tissue rarefaction and gliosis are less prominent in TPD, even in NFT rich areas. Changes in the neocortex are modest, with well-preserved laminar structures and unremarkable neuronal cell loss. The cortical expansion of NFT in TPD is considered to follow in principle the hierarchical pathway described in AD by Braak and Braak  but to bThe nucleus accumbens (Acb) is located in the region where the caudate head and the rostral putamen meet near the septum pellucidum Figure . The AcbIn the present study, we found the frequent and consistent tau accumulation in the Acb in TPD. Tau positive neurons were also found in the Acb in some AD and on-demented aged subjects but much fewer in the majority of such cases. We speculate that the lesions in the Acb play a role in some psychiatric symptoms such as delusion, which is often conspicuous in TPD.We used brain tissues, archived in our laboratory, from 7 patients with TPD, 22 with AD and 11 subjects without dementia or other neurological disease. The demography, Braak and Braak’s NFT stages and brain weight in each patient group are summarized in Table For routine neuropathological examinations, formalin-fixed, paraffin-embedded brain blocks were cut into 10 μm thick sections and stained with hematoxylin and eosin (HE), Klüver-Barrera, modified Gallyas-Braak and methenamine silver staining. Tissue sections of the mediobasal temporal cortex containing the hippocampus, entorhinal cortex and temporal neocortex were stained for tau and amyloid β protein (Aβ) by immunohistochemistry. Sections of the rostral striatum with the Acb and the septal nuclei were stained for tau. In TPD cases, additional tau immunohistochemistry was performed for the nucleus basalis of Meynert, amygdala and substantia nigra. The hippocampus, parahippocampal gyrus and adjacent temporal neocortex were also stained for phosphorylated α-synuclein and phosphorylated TDP-43 in TPD cases.4 for 20 min and, subsequently, with sodium borohydride for 4 min [2HPO4 and 5.2 mM KH2PO4, pH 7.2 at 4°C for 30 min, sections were briefly rinsed with distilled water and observed by fluorescence microscopy.For more detailed immunohistochemical analyses, small blocks of brain the tissues were dissected at autopsy and fixed in 4% paraformaldehyde (PFA) for 2 days. The cryocut sections of 30 μm thickness were used for the high sensitive, free-floating immunhistochemical staining . The antor 4 min . SectionFor immunoelectron microscopy, both post-embedded and pre-embedded procedures were used. For the former, the 4% PFA-fixed small tissues were embedded in LR White Resin®  without further fixation. The ultra-thin sections were stained with AT8, which was followed by incubation with anti-mouse IgG conjugated with 10 or 20 nm gold colloidal particles . For the pre-embedding procedure, the 4% PFA-fixed free-floating sections were stained with AT8 in combination with Alexa Fluor 488 FluoroNanogold anti-mouse IgG . Following examination by fluorescence microscopy to localize the positive labeling, the sections were postfixed with 2% glutaraldehyde and then treated with HQ Silver Enhancement Kit . After the treatment with 1% osmium tetroxide, which was followed by 2% uranyl acetate, the sections were embedded in epoxy resin . Ultrathin sections were cut and observed by a transmission electron microscope .For immunoblot analyses, fresh frozen samples of the Acb and the parahippocampal cortex were obtained from 3 TPD cases  and 3 AD cases. The Braak and Braak’s NFT stages of the AD cases were 4, 5 and 6, respectively. Brain tissue was homogenized in 2 vol of TS buffer , with a mixture of protease inhibitors and centrifuged at 200,000 g for 20 minutes at 4°C. The supernatant was taken as the soluble fraction and the pellet was used to further extract the sarkosyl-insoluble fraction as described previously . DephospU test was used for statistical analyses using Graph Pad Prism 4 software .For semiquantitative analyses of immunohistochemically stained tissue sections, the density of AT8 positive tau accumulation was graded to be 0 for absent, 1 for low, 2 for intermediate and 3 for high, based on microscopic observations at ×200 magnification. The Acb, septal nuclei, caudate nucleus, hippocampal CA1, entorhinal cortex and temporal neocortex were assessed in TPD, AD and non-demented aged subjects. The Mann–Whitney The demographic, pathologic, and clinical information of the TPD cases used in the present study is summarized in Tables In addition to the previously reported tau distribution, we found a considerable number of tau positive neurons in the Acb in all TPD cases used in this study Figures  and 2. SThe density of tau positive neurons and neuropil threads varied somewhat among the TPD cases. In TPD, no clear association was seen between the degree of Acb tau pathology and the Braak and Braak’s NFT stage or the presence or absence of Aβ deposits, argyrophilic grains  and vascImmunoelectron microscopy of the Acb in TPD with a tau antibody, AT8, revealed positive labeling of granular structures in the neurons Figure A. Small We then investigated the Acb in AD and non-demented, aged subjects. Tau positive neurons were found in some, but not all, AD patients and non-demented, aged subjects  as well as the AD cases with NFT stage IV (P = 0.0192) and those with NFT stage V (P = 0.0022). Two non-demented, aged subjects with tau accumulation in the Acb were both over age 90. These 2 cases, similarly to TPD, lacked tau accumulation in the caudate nucleus and showed more NFT in the subiculum than in the entorhinal cortex. The results of semiquantitative analyses confirmed our observation that tau accumulation in the Acb was a remarkable finding in TPD. We performed similar analyses for a number of brain regions. The results are summarized in Table The density of neuronal tau accumulation was graded to be 0 (absent) through 3(high) in AT8 immunostained tissue sections. Figure The results of immunoblot analyses of samples from TPD and AD patients are shown in Figure p = 0.033). Similarly, the less conspicuous but still significant difference was seen in the group of all NFT stage AD cases (p = 0.049).Finally, we examined if the degree of the Acb tau pathology was different between the subjects groups with and without the history of delusion in the clinical records : Tangle-predominant dementia; (NFT): Neurofibrillary tangles; (AD): Alzheimer’s disease; (3R): 3-repeat; (4R): 4-repeat; (Acb): Nucleus accumbens; (VTA): Ventral tegmental area; (HE): Hematoxylin and eosin; (PFA): Paraformaldehyde; (PHF): Paired helical filaments; (Aβ): Amyloid β protein; (TH): tyrosine hydroxylase.The authors declare that they have no competing interests.IK carried out the microscopic observation, immunoblot and statistical analyses. IK also drafted the initial manuscript. MHa conducted the sample preparation and immunoblot and carried them out with IK. TA participated in the design and coordination of the study. KI carried out the microscopic observation with IK. KO, KN and NA organized the brain archives including clinical information, selected appropriate cases, and performed neuropathological analyses of all cases used in this study. OK participated in the design of the study and performed statistical analyses with IK. SH conceived of the study and participated in the initial design. MHo contributed to the reagents, materials and analysis tools, and conducted free-floating immunohistochemistry. YH participated in the design of the study and helped to draft the manuscript. HA supervised the design and coordination of the study and worked up the manuscript. All authors read and approved the final manuscript.IK is a graduate student of Department of Psychiatry, Graduate school of Medicine, Yokohama City University School of Medicine. MHa is the senior director of Department of Neuropathology and Cell Biology, Tokyo Metropolitan Institute of Medical Science (TMIMS). TA, KI, NA and SH contributed to this study as visiting scientists of Dementia project, TMIMS. TA is also an associated professor of Department of Psychiatry, Graduate School of Comprehensive Human Sciences, University of Tsukuba. KO and KN are psychiatrists and neuropathologists in Tokyo Metropolitan Matsuzawa Hospital (TMMH), visiting scientists of Dementia project, TMIMS and in charge of the brain archive of TMMH/TMIMS. OK is an associate professor of Department of Psychiatry, Graduate school of Medicine, Yokohama City University School of Medicine. MHo is a chief researcher of Dementia project, TMIMS. YH is a professor of Department of Psychiatry, Graduate school of Medicine, Yokohama City University School of Medicine. HA is the senior director of Dementia project, TMIMS.The primary antibodies used in this study.Click here for fileTau accumulation in the Acb in AD and non-demented, aged subjects. Immunohistochemistry with AT8. A: absence of tau positive neurons in an AD case in Braak and Braak’s NFT stage IV. Scale bar = 100 μm in A-C. B: a diffuse cytoplasmic staining, neuropil threads and duystrophic neurite in a senile plaque in an AD case in NFT stage VI. C: a tau positive neuron and neuropil threads in a non-demented, aged subject.Click here for fileA graph of the density of neuronal tau accumulation in the Acb. The left plots: the group of Braaks’ NFT stages III and IV, which includes non-demented aged subjects, TPD cases and AD cases with Braaks’ NFT stage IV. Cases with delusion in the clinical history show higher tau score than those without delusion in the Acb. The right plots: the group of AD cases with Braaks’ NFT stages IV through VI. Again, cases with delusion show higher tau score than those without delusion.Click here for fileThe serial section immunohistochemistry for tau and tyrosine hydroxylase (TH). Forty micrometer thick, free floating sections were cut serially from two tangle predominant dementia (TPD) cases, in which the remnants of Acb blocks were available after the initial sectioning for the main body of the study. A set of every other section was stained for TH and the other set for tau with AT8. A and C: AT8 staining in a TPD case 1. B: TH staining of the section between A and C. In B, two types of areas are distinguished based on the modest difference in the density of fine, mesh-like TH staining. There is a propensity that tau pathology preferentially occurs in areas where the fine, mesh-like TH staining is relatively light . Scale bar = 2 mm in A . D: higher power photomicrographs of the boxed areas in B and C. The left half is the staining with AT8 and the right half staining for TH. Scale bar = 400 micro-m (D).Click here for file


Mn2+ decreased kon and increased koff for IRP1 binding to both FRT and ACO2 IRE-RNA, with a larger effect for FRT IRE-RNA. In order to further understand IRE-mRNA regulation in terms of kinetics and stability, eIF4F kinetics with FRT IRE-RNA were determined. kon for eIF4F binding to FRT IRE-RNA in the absence of metal ions was 5-times slower than the IRP1 binding to FRT IRE-RNA. Mn2+ increased the association rate for eIF4F binding to FRT IRE-RNA, so that at 50 µM Mn2+ eIF4F bound more than 3-times faster than IRP1. IRP1/IRE-RNA complex has a much shorter life-time than the eIF4F/IRE-RNA complex, which suggests that both rate of assembly and stability of the complexes are important, and that allows this regulatory system to respond rapidly to change in cellular iron.Metal ion binding was previously shown to destabilize IRE-RNA/IRP1 equilibria and enhanced IRE-RNA/eIF4F equilibria. In order to understand the relative importance of kinetics and stability, we now report rapid rates of protein/RNA complex assembly and dissociation for two IRE-RNAs with IRP1, and quantitatively different metal ion response kinetics that coincide with the different iron responses  Noncoding mRNA structure can make a major contribution to protein synthesis rates and gene expression in eukaryotic cells. An example is the ability of IRE-RNA to bind, competitively, two regulatory proteins: IRE-RNA binds both the generic protein synthesis enhancer, eIF4F  binding to IRE-RNA, which decreased IRP1/IRE-RNA binding and increased protein synthesis initiation factor eIF4F/IRE-RNA binding. Our working model (2+) with a 1:2 stoichiometry  IRE-RNA/IRP  of the free IRE-RNA, the crystal structure of the FRT IRE-RNA/IRP complex ,37 and fx Figure ; IRE-RNAIsolation of recombinant rabbit IRP1 from yeast used methods previously described in , except FIIRE-RNA), ferritin IRE-RNA and mitochondrial aconitase IRE-RNA were purchased from Genelink  and, after dissolving, were melted and reannealed as described in  were 490 nm and 520 nm, respectively. The temperature of the flow-cell and solution reservoir was maintained at 25°C unless otherwise stated. IRP1 binding induced an increase in FIIRE-RNA anisotropy. After rapid mixing of 0.1 μM  FIIRE-RNA with varying concentrations  of IRP1 protein, the time course of the anisotropy change was recorded by computer data acquisition. All measurements were performed in titration buffer, 40 mM HEPES/H+, pH 7.2, 1 mM MgCl2, 100 mM KCl, 5% (v/v) glycerol and 2% (v/v) 2-mercaptoethanol. In each experiment, 1000 pairs of data were recorded and sets of data from 5 to 7 shots were averaged to improve the signal-to-noise ratio. Each averaged set of stopped-flow anisotropy data was then fitted to nonlinear analytical equations using Global analysis software provided by OLIS.Stopped-flow anisotropy measurements for the binding of 2+ effects, MnCl2 was added to both RNA and protein solutions, at the same concentration, and the solutions were incubated separately for 15 min before adding to titration buffer containing the same metal ion concentration as the RNA and protein solutions. Experiments were performed as described above. Data were evaluated by fitting to the single and double exponential functions as described previously  on the binding rates of eIF4F  to FRT IRE-RNA  was carried out under the same conditions as described above.As described elsewhere ,45 RNA bFIIRE-RNA when equal volumes of the FIIRE-RNA/IRP1 complex and buffer alone or buffer containing 50 μM Mn2+ were mixed in the stopped-flow cell. The concentrations of the FRT IRE-RNA or ACO2 IRE-RNA and IRP1 protein in the solution were 0.05 μM and 1 μM, respectively, after mixing. The dissociation rates were determined for the relaxation experiment from the fits of the nonlinear analytical equations using Global analysis software provided by OLIS.To measure the dissociation rate constants of the pre-formed IRE-RNA/IRP1 complexes, RNA and protein mixtures were incubated in titration buffer for 15 min at 25°C to ensure complex equilibrium. Dissociation of pre-formed IRE-RNA/IRP1 complex was followed by measuring the decrease in anisotropy of tr is the observed anisotropy at any time, t, and rf is the final value of anisotropy, Δr is the amplitude and kobs is the observed first order rate constant. The double-exponential equation isr1 and Δr2 are the amplitudes for the first and second components of the reaction with observed rate constants kobs1 and kobs2, respectively. The residuals were measured by the differences between the calculated fit and the experimental data. The observed rate constants were further analyzed as relaxation experiments to give kon and koff from the equation kobs = kon [IRP1] + koff. A plot of kobs versus [IRP1] at varying IRP1 concentrations was used to give kon (slope) and koff (intercept). For dilution experiments of the IRE-RNA/IRP1 complex, the koff value was obtained from the previously determined Keq values and using KD = koff/kon gives kobs = koff ([IRP1]/KD +1). The life-time for the eIF4F/FRT IRE-RNA and IRP1/FRT IRE-RNA complexes were calculated using equation  is predicted to be a linear function of IRP1 concentration as shown in Equation (4),FIIRE-RNA increased with increasing IRP1 concentrations  compared to ACO2 IRE-RNA  with IRP1. These results are consistent with the FRT and ACO2 IRE-RNA binding to IRP1 experiments in the fluorescence steady-state, where binding differed by 9-fold. The Kd values calculated from the kinetic constants computed here agree well with the Kd value obtained by steady-state equilibrium method  for the binding of FRT and ACO2 IRE-RNA with IRP1 decreased 6.2- and 4.8-fold, respectively. In addition, metals ions increased the dissociation (koff) rate of IRP1 for FRT IRE-RNA 2-fold and 1.1-fold increase for ACO2 IRE-RNA  and to the dissociation rate determined from concentration-dependent reaction kinetics (koff = 6.2 ± 0.3 s−1 for FRT and 7.0 ± 0.4 s−1 for ACO2 IRE-RNA/IRP1 complex). In the presence of Mn2+, the dissociation rates for FRT (koff = 24 ± 1.5 s−1) and ACO2 (koff = 16.4 ± 1.3 s−1) IRE-RNA/IRP1 complexes increased 3-fold and 2-fold, respectively. Thus, Mn2+ can selectively increase the dissociation rates of RNA/protein complexes. Since the same protein has different dissociation rates with two IRE-RNAs  decreased 7-fold for FRT IRE-RNA and 3-fold for ACO2 IRE-RNA binding to IRP1  The ionic strength dependence of Kd values calculated from kinetic parameters (kon and koff) at 100 mM KCl was in close agreement with the values obtained by equilibrium methods . (iv) Kd values for IRP binding increased about 8-fold for FRT IRE-RNA and 4-fold for ACO2 IRE-RNA over a range of KCl concentration 100–300 mM, largely due to changes in the association rate constant  increases 3-fold and the dissociation rate (koff = 1.8 ± 0.06 s−1) decreases 2.5-fold as compared to the association and dissociation rates of eIF4F/FRT IRE-RNA at zero Mn2+ concentration  in the presence of 50 μM Mn2+ as compared to eIF4F/FRT IRE-RNA (Kd = 59 ± 2.4 nM) complex without metal ion. Kd values determined from the kinetics agree well with the Kd value determined from fluorescence steady-state measurements in the presence and absence of metal ion  relative to the lower stem in the region of the C8, U6 bulge bases. Although similar structural information on the ACO2 IRE-RNA/IRP1 complex is currently lacking, the structure of the TfRB IRE-RNA (which like ACO2 IRE-RNA lacks the U6 bulge) bound to IRP1 revealed a smaller helix distortion (∼8º)   than tha2+ differentially decrease IRE-RNA/IRP1 association rates, Mn2+ differentially increased the dissociation rates with the larger increase for FRT IRE-RNA compared to ACO2 IRE-RNA. These results suggest that the interactions with Mn2+ affect the intermediate conformational change in complex formation and reflect a direct sensing of the ion by IRE-RNA, analogous to the larger riboswitches. We reported earlier  lowered the stability of the FRT IRE-RNA/IRP1 complex; deletion of the U6 bulge greatly reduced the effect (8 bulge and the helix bend. Metal ions not only neutralize negative charges and reduce the phosphate repulsion, transition metal ions including Fe2+, the physiological signal, form specific coordination complexes with bases that change conformations in the region of both the C bulge and the hairpin loop (Tm, nuclease sensitivity and translation efficiency and affect the hairpin loop as well (6 may be a metal ion binding site.Metal ions affected both the association and the dissociation rates for IRP1 binding to FRT and ACO2 IRE-RNA. The metal ion-induced decrease in IRP1/RNA association rates was larger for the FRT IRE-RNA, about 6-fold, compared to the ACO2 IRE-RNA, where the effect was about 4.8-fold reduction Tables . Not onl earlier  that mete effect ,26–29, spin loop . Structu as well . Such ob2+ within the physiological ranges of the labile iron pool concentration (2+ concentration suggests that the reaction is largely kinetically controlled in vivo with IRP1 outcompeting eIF4F at low Fe2+ and the reverse being true at higher Fe2+ concentrations. Similarly, the longer life-time of the eIF4F/IRE-RNA complex at both high and low Mn2+ concentrations suggests that IRP1 responds more rapidly to cellular Fe2+ concentration changes while eIF4F forms a more stable platform for ribosome binding. In vivo life-times of the complexes will depend on the cellular concentrations of IRP and eIF4F and may be influenced by other cellular components.Metal ion destabilization of messenger IRE-RNA/IRP1 complexes competes with the stabilization conferred by the very large number of bonds between the protein and the RNA and the stability of the RNA fold. Selective, metal-induced destabilization of FRT and ACO2 IRE-RNA/IRP1 complexes as well as other selective, metal-RNA interactions ,26–29, entration . The metntration , and fasntration  and 4 cantration . CompariRiboswitches in eubacteria frequently involve larger RNA structures that modulate translation initiation  and use Supplementary Data are available at NAR Online.National Institutes of Health ; National Science Foundation [MCB 1157632 to D.J.G.]. Source of open access funding: National Science Foundation, National Institutes of Health.Conflict of interest statement. None declared.


StBEL5, and enhance stability and trafficking of the RNA to select organs. This protein–RNA interaction leads to enhanced tuber production.PTB proteins of potato bind to the mobile RNA,  StBEL5. The promoter of StPTB1 was active in companion cells of phloem in both stem and petioles. Expression of both types was evident in phloem cells of roots and in stolons during tuber formation. RNA accumulation of both PTB proteins was induced by short days in leaves in correlation with enhanced accumulation of StBEL5 RNA. StPTB suppression lines exhibited reduced tuber yields and decreased StBEL5 RNA accumulation, whereas StPTB overexpression lines displayed an increase in tuber production correlated with the enhanced production in stolons of steady-state levels of StBEL5 transcripts and RNA of key tuber identity genes. In StPTB overexpression lines, both the stability and long-distance transport of StBEL5 transcripts were enhanced, whereas in suppression lines stability and transport decreased. Using a transgenic approach, it is shown that the StPTB family of RNA-binding proteins regulate specific stages of development through an interaction with phloem-mobile transcripts of StBEL5.Polypyrimidine tract-binding (PTB) proteins are a family of RNA-binding proteins that function in a wide range of RNA metabolic processes by binding to motifs rich in uracils and cytosines. A PTB protein of pumpkin was identified as the core protein of an RNA–protein complex that trafficks RNA. The biological function of the PTB–RNA complex, however, has not been demonstrated. In potato, six PTB proteins have been identified, and two, designated StPTB1 and StPTB6, are similar to the phloem-mobile pumpkin type. RNA binding assays confirmed the interaction of StPTB1 and StPTB6 with discrete pyrimidine-rich sequences of the 3′-untranslated regions of the phloem-mobile mRNA,  The polypyrimidine tract-binding (PTB) family of proteins represent a multifaceted group of proteins that binds numerous mRNAs and have been implicated in a wide range of RNA metabolic processes including stability , splicinArabidopsis, two of which are involved in pollen tube development , GIBBERELLIC ACID-INSENSITIVE (GAI), a SCARECROW-LIKE protein, SHOOT MERISTEMLESS, an ETHYLENE RESPONSE FACTOR, and a MYB transcription factor. Gel mobility shift assays confirmed that specific binding of CmRBP50 to two of its target RNAs, GAI and PP16-1 of pumpkin, was facilitated by polypyrimidine tract motifs located within the transcript sequences , represented by six genes, two containing four RRMs and designated StPTB1 and StPTB6, and four three-RRM types, have been characterized. Based on amino acid sequence and function, StPTB1 and StPTB6 appear to be orthologues of AtPTB3 and CmRBP50, and serve as chaperones to full-length mRNAs that are transported through the sieve element system. RNA binding assays confirmed the interaction of StPTB1 and StPTB6 with the 3′-untranslated regions (UTRs) of the mobile RNA, StBEL5. In novel analyses using transgenic whole-plant systems, it is demonstrated that PTB proteins of potato enhance stability and regulate transport of a mobile RNA and specifically control tuber development.Through functional and genomic analyses, the PTB family of potato  was searched using the CmRBP50 and CmGAI sequence as query, respectively. From partial cDNA sequences, the rapid amplification of cDNA ends (RACE) was performed using the First Choice RLM-RACE kit (Ambion) to identify the 5′- and 3′-untranslated sequences. Full-length cDNA sequences were then obtained by PCR amplification with 5′ and 3′ gene-specific primers from RACE experiments. The amplified PCR fragments were cloned into pCRII-TOPO vector (Invitrogen) and verified by sequencing. StPTB1, StPTB6, and StPTB7 were identified in this manner. The primers used in this study (Supplementary Table S1 available at JXB online) were synthesized at the DNA Facility, Iowa State University.To obtain the full-length S. tuberosum cv. Désirée were used for most of the studies reported here. The photoperiod-responsive potato  was used for the quantitative reverse transcription–PCR (qRT–PCR) and protein analyses. RNA extraction and qRT–PCR were performed as previously described (Supplementary Table S3 at JXB online.Transgenic and wild-type plants of StPTB1:GUS (β-glucuronidase) and PromStPTB6:GUS plasmids, the constructs include DNA fragments containing 2519bp and 3593bp of sequence upstream of the translational start codon, respectively (Supplementary Table S4 at JXB online and cloned into pBI101.1-GUS (Clontech). For transformation of the 35S:StPTB–GFP (green fluorescent protein) constructs, the pBI121-GFP vector was generated from pBI121-GUS (Clontech) by replacing the GUS gene with the GFP reporter gene in BamHI/SacI sites in the pBI121-GUS vector. The 35S:StPTB1–GFP and 35S:StPTB6–GFP constructs include the coding sequences of StPTB1 and StPTB6 without the stop codon and fused in-frame to GFP. The inserts for the constructs were obtained by PCR amplification and directly cloned into the pBI121-GFP vector (Supplementary Table S4).For the Promectively . PromoteStPTB1 and StPTB6 transcript levels, both antisense and RNAi constructs were designed. The antisense construct was made from the 611bp SacI–SpeI fragment of the StPTB6 cDNA , cloned in the antisense direction into the binary vector, pCB201 (Cauliflower mosaic virus (CaMV) 35S promoter. For generating the RNAi construct (Supplementary Table S1 at JXB online), the same conserved 611bp fragment of the StPTB6 cDNA was cloned into the pENTR/D-TOPO plasmid (Invitrogen) and inserted in opposite orientations by recombination with the LR Clonase II enzyme  into the pBIN19RNAi destination vector (S. tuberosum cv. Désirée as described by StPTB1 and StPTB6 in RNA samples from leaves and stolons.To suppress –1 X-gluc (5-bromo-4-chloro-3-indolyl-β-d-GlcUA) solution and cleared using 70% ethanol. Fixation of stained materials  plasmid and transformed into Escherichia coli BL21-Codon (DE3) cells . After induction with 0.4mM isopropyl-β-d-thiogalactopyranoside (IPTG), the recombinant proteins were purified using Ni-NTA agarose  and quantified. These purified proteins were used for EMSA and for preparation of polyclonal antibody. Polyclonal antibodies against StPTB6 protein were raised at the Hybridoma Facility at Iowa State University. The purified proteins (500–800 μg) were repeatedly injected into two rabbits, and standard protocols were used for bleeding and screening to obtain reliable antibodies. The sera containing specific polyclonal antibodies against StPTB6 were used for the studies without purification. Pre-immune controls are shown in Supplementary Fig. S6 at JXB online.Supplementary Table S4 at JXB online). These probes were derived from either coding sequence or the 3′-UTR, and were selected based on the yeast three-hybrid results . RNA bait was generated using in vitro transcription with the MAXIscript® T3 kit (Ambion) and biotin-labeled UTP . Biotin-labelled RNA probes were purified using RNA Clean & Concentrator™-5 (Zymo Research) and quantified by NanoDrop 1000 (Thermo Scientific). Biotin-labelled RNAs (3fmol) were used for the binding assay. Binding reactions with labelled RNA and purified recombinant proteins (0–500nM) were incubated in 20 µl for 1.0h at room temperature in the presence of a binding buffer consisting of 40mM Tris (pH 8.0), 30mM KCl, 1mM MgCl2, 0.1% NP-40, and 1mM DTT. The binding reactions were resolved on a 6% (v/v) non-denaturing polyacrylamide gel, and then transferred onto a BrightStar®-Plus positively charged nylon membrane (Ambion). For detection of biotinylated RNA, the Chemiluminescent nucleic acid detection module kit (Thermo Scientific) and CL-XPosure™ film (Thermo Scientific) were used.For RNA-EMSAs, target sequences were amplified from potato leaf cDNA or genomic DNA using corresponding primers (S. tuberosum ssp. andigena plants grown under short-day (SD) conditions were fixed using 1.0% formaldehyde and 125mM glycine under a vacuum. The fixed samples were frozen and ground in liquid nitrogen, and total cell extracts were prepared with 0.7g of tissue sample in 2.0ml of RIP-LB buffer (® Protein A (Invitrogen). For immunoprecipitation (IP), 20 µl of Dynabeads® Protein A were coated with 2.0 µg of pre-immune serum, αStPTB6 antibody purified using the Melon Gel IgG purification kit (Thermo Scientific), or αGFP monoclonal antibody (Santa Cruz), and washed as previously described  were incubated with antibody-coated magnetic beads for 2h at 4 °C under rotation. After intensive washings (New tubers (~4.0–8.0mm in diameter) or petioles of B buffer , and cleescribed . For IP washings , to rele® Green qRT-PCR kit (Quanta Biosciences) with the Eco Real-Time PCR system (Illumina) was used. Each real-time PCR was performed in duplicate. For quantification and comparison of RNA levels between samples, the 2−ΔΔCt method was used (® RNA RIP Kit). Primer sequences used are listed in Supplementary Table S5 at JXB online.Diluted input (1.0%) of purified RNA from the IP was used for gel-based RT–PCR. For qRT–PCR, the qScript™ One-Step SYBRwas used . To accoStBEL5 and StBEL14 were amplified using NEBNext High-Fidelity 2X PCR Master Mix (NEB), and cloned into MluI and EcoRV sites of the PVX/ΔCP vector (StBEL constructs were designed to include a histidine-tag sequence for inoculum-specific detection (Supplementary Table S5 at JXB online). Transcripts for mobility assays were prepared by in vitro transcription using a T7 High Yield RNA Synthesis kit (NEB), as described in the instruction manual, and purified using LiCl precipitation. A 10 µg aliquot of in vitro transcripts was mechanically inoculated onto each leaf (2–3 leaves per plant) of wild-type S. tuberosum cv Désirée or the StPTB Désirée transgenic lines. Inoculated leaves, and non-inoculated roots and stolons were harvested at 8 d post-inoculation, and total RNA was extracted using an RNeasy Plant Mini kit (Qiagen) and followed by RNase-free DNase (Qiagen) treatment. Total RNA (150ng) was used for the gel-based RT–PCR. For qRT–PCR, the qScript One-Step SYBR Green qRT-PCR kit (Quanta Biosciences) with the Eco Real-Time PCR system (Illumina) was used. After normalization using StAct8 as an endogenous control, the relative gene quantification approach , StPTB6 (1624 nucleotides), and StPTB7 (1678 nucleotides), were identified that encode for StPTB proteins. The lengths of these three proteins are 441, 444, and 467 amino acids, respectively. StPTB1 and StPTB6 closely match the amino acid sequence of the PTB protein of pumpkin, CmRBP50 . Each of the proteins contains conserved RRMs, the conventional set of four for StPTB1 and StPTB6 but only three for StPTB7 (a three-RRM type). StPTB7 contains 11 extra residues at the N-terminus and lacks a conserved RRM4 . The conserved motifs of the RRM . One other StPTB protein has been identified from the DFCI Potato Gene Index (TC201749) and two others from the potato genome . Based on phylogenetic analysis , StPTB7 and these other three-RRM types belong to the same clade with AtPTB1 and AtPTB2 . Because of our interest in phloem-mobile mRNAs .Using RT–PCR and 5′ and 3′ RACE, three full-length RNAs, designated d AtPTB2 . PhylogeStBEL5 (T1) and StGAI . To verify protein–RNA interactions, RNA gel-shift assays with StPTB1 and StPTB6 were performed . For the T1 bait, an interaction was observed for StPTB6 with a concentration as low as 25nM . Results with both the yeast three-hybrid and the RNA gel-shift assays suggest that the interaction of StPTB6 with both T1 and the StGAI UTR fragments was stronger than the interaction with StPTB1 under the current experimental conditions. RNA IP confirms that StPTB proteins bind to StBEL5 RNA in planta . Immunoprecipitation of the new tuber extract with the StPTB6 antibody yielded a 30-fold enrichment of StBEL5 RNA relative to the control sample . These results were reproducible in both replicate tuber- and petiole-derived fractions.To test their putative function as chaperones to phloem-mobile RNAs, the ability of StPTB1 and StPTB6 to bind to RNA sequence was assessed. Preliminary analysis utilizing the yeast three-hybrid system suggested that StPTB1 and StPTB6 proteins interacted with specific 3′-UTR RNA sequence of both erformed –C. Both  as 25nM , whereas as 25nM . For the as 25nM , whereas as 25nM . No band as bait  and evenn planta , E. Crudl sample . No tranStPTB promoter activity coincides with StBEL5 expression, StPTB1 and StPTB6 activity was assayed in transgenic potato lines (cv. Désirée) by fusing ~3.0kb of upstream sequence of both types to a GUS marker. StBEL5 promoter activity was observed in leaf veins, petioles, and in phloem cells of the petiole  but not sieve elements (SEs) of petiole phloem . Discrete staining was observed for the StPTB1prom line throughout the external phloem  layers, with some staining within the internal phloem . The activity of the StPTB1prom was specific to CCs but absent from SEs in the vascular bundles of these stem sections . In contrast, StPTB6prom activity was more widespread and irregular in the stem. StPTB6prom activity was not as abundant in vascular bundles of stems as the StPTB1prom construct , but was observed in some cells of the internal phloem , in interfascicular regions, and in clusters of cells along the epidermis .Transverse sections of stained internodes from stems of StBEL5 RNA and the process of tuberization, StPTB1prom and StPTB6prom expression was investigated during tuber development. Stolons and new tubers were harvested from mature soil-grown plants and separated into four categories based on their developmental stage , whereas GUS staining of StPTB1prom stolons and tubers was essentially the same . Manual dissection of t2 tubers revealed that both the StPTB1prom and the StPTB6prom were active in vascular strands of new tubers . Quantification of the activities in these organs revealed a peak of activity for the StPTB6prom in early, induced stolons that steadily decreased as tubers formed , whereas StPTB1prom activity exhibited no such developmental regulation .Because of the functional association of al stage . GUS staStPTB6 activation during early tuberization, the StPTB6 promoter sequence was screened for tuber and sucrose inductive motifs. Three putative tuber-specific sucrose-responsive (TSSR) elements present in the promoters of class I patatin and proteinase inhibitor II genes . These are designated TSSR1, TSSR2, and TSSR3  and in the patatin promoter direct tuber-abundant expression. As an example of conservation, alignment of the putative TSSR2 motif from StPTB6 with the proximal patatin TSSR motif revealed nucleotide sequence matches in the B-box, the repression region, and the A-box of 59, 69, and 74%, respectively  conditions, there were no significant differences among the five organs tested . Under SD conditions, however, enhanced accumulation of both StPTB1 and StPTB6 was observed concomitantly with StBEL5 accumulation in leaf mesophyll and leaf veins . No SD-enhanced accumulation occurred for either PTB type in stolons or stems . Despite the observation that StPTB transcript accumulation was not induced by SDs in stems, levels of the StPTB proteins increased in both stems and stem exudate under SD conditions , suggesting some degree of photoperiod-mediated post-transcriptional regulation is in effect. StPTB protein levels were also enhanced by SDs in petioles, roots, and stolons .af veins . SD-induSupplementary Fig. S14 at JXB online). With three representative lines each, the average increase for root growth was 71% for the StPTB1-OE lines and 101% for the StPTB6-OE lines. Tuber yields from both StPTB soil-grown OE lines were more than twice that of the GFP control line, R1  lines of potato cv. Désirée were generated for both protein types, screened, and evaluated. Several high expressing lines were identified for each construct, and three to four were used in evaluating effects on RNA metabolism and phenotypes of soil-grown plants. Overall shoot fresh weight was not affected in both OE lines, but both PTB-OE types exhibited enhanced root growth .In both StPTB1- and StPTB6-OE lines, lon tips –D. LevelTB types . Based oA levels –D. Consit levels . Highligt levels , D, wherStBEL5 RNA, an RNA stability assay was performed using in vitro grown shoot tips of the overexpression lines and wild-type Désirée. RNA stability of StBEL5 was enhanced in both StPTB-OE types. Significant differences were observed for the rate of StBEL5 degradation in all four StPTB-OE lines  to both roots and stolons was enhanced relative to movement in wild-type plants  but not to sequence without these motifs  strongly suggest that this interaction is pivotal for regulating StBEL5 RNA movement. Several reports have shown that PTB proteins increase the stability of target mRNAs (StBEL5 is a very abundant phloem-mobile RNA that functions in regulating tuber formation in potato (StBEL5 in stolon tips and leaf veins (data not shown) and increased tuber production, whereas suppression lines affected the opposite trend. In petioles, the promoter of StPTB1 was active in CCs of phloem but not the SEs . Similar to this pattern of expression, RBP50 transcripts were detected only in CCs, whereas RBP50 protein was detected in both CCs and SEs . Concordant with their role in animal systems  leading to an increase in steady-state levels of its mRNA .In this system, it is proposed that SD-enhanced expression of the potato PTB proteins leads to induced transport of tabolism . This ovStBEL5, and to the activity of its transcriptional targets. The use of transgenic plants in this study illuminates the function of StPTB proteins and provides an enlightening tool for understanding how they regulate a specific phase of plant growth. The phloem-associated promoter activity of StPTB1 and StPTB6, the regulated activity during early stages of tuberization, the binding affinity of both StPTB proteins for the 3′-UTR of StBEL5, and the dramatic tuberization phenotypes of the StPTB overexpression and suppression lines unequivocally support the premise that the PTB1 and PTB6 proteins of potato function prominently in post-transcriptional events associated with signalling the onset of storage organ formation.PTB proteins have been observed to function in a diverse array of processes associated with RNA metabolism .


Recent studies showed that Rai1 and its homologs are a crucial component of the mRNA 5′-end capping quality control mechanism. They can possess RNA 5′-end pyrophosphohydrolase (PPH), decapping, and 5′-3′ exonuclease (toward 5′ monophosphate RNA) activities, which help to degrade mRNAs with incomplete 5′-end capping. A single active site in the enzyme supports these apparently distinct activities. However, each Rai1 protein studied so far has a unique set of activities, and the molecular basis for these differences are not known. Here, we have characterized the highly diverse activity profiles of Rai1 homologs from a collection of fungal organisms and identified a new activity for these enzymes, 5′-end triphosphonucleotide hydrolase (TPH) instead of PPH activity. Crystal structures of two of these enzymes bound to RNA oligonucleotides reveal differences in the RNA binding modes. Structure-based mutations of these enzymes, changing residues that contact the RNA but are poorly conserved, have substantial effects on their activity, providing a framework to begin to understand the molecular basis for the different activity profiles. Several other members of the Nudix family also possess decapping activity  from 5′ triphosphate RNA (such as the primary Pol II transcript)  . Rai1 alnscript) , in contnscript) . We subsnscript) –19.4ΦhQ (motif V), and GhR . On the other hand, the amino acid sequences outside of these motifs are generally poorly conserved among these proteins . For example, S. pombe Rai1 has 26% overall sequence identity with mouse DXO    ,17, Kluy KlDxo1)  and mous KlDxo1) , show di KlDxo1) . HoweverIn this study, we have characterized the catalytic activity of Rai1 homologs from a collection of fungal species and determined the high-resolution crystal structures for three of them, two of them in complex with RNA oligonucleotides. We have found substantial variations in the activity profiles of the Rai1 homologs, and discovered that some of them possess a new 5′ triphosphonucleotide hydrolase (TPH) activity. The binding modes of the first two nucleotides of the RNA body are similar among the Rai1 homologs, and generally involve conserved residues. On the other hand, large differences are observed in the binding modes of the rest of the RNA. Mutations of residues that contact the RNA but are poorly conserved among the Rai1 homologs had significant impacts on the activity of the enzymes, suggesting that they play important roles in determining the activity profile of each enzyme.Ashbya gossypii (Ag), Scheffersomyces stipitis (Ss), Candida albicans (Ca), Candida glabrata (Cg), Lachancea thermotolerans (Lt), S. cerevisiae (Sc), S. pombe (Sp) and Vanderwaltozyma polyspora (Vp) Rai1 were cloned into the pET28a  and pET26b  vectors (Novagen). The proteins were overexpressed in Escherichia coli. BL21 Star (DE3) cells at 16°C and purified by Ni-NTA Superflow (Qiagen) and gel filtration  chromatography. The proteins were concentrated to 25–30 mg/ml in a buffer containing 20 mM Tris (pH 7.5), 250 mM NaCl and 5% (v/v) glycerol, frozen in liquid nitrogen and stored at –80°C.Full-length 2+ complex crystals were obtained by using 1:2 protein:RNA molar ratio, and 10 mM MnCl2, in the same condition as the CaRai1 free enzyme crystals.CaRai1 crystals were obtained by the sitting-drop vapor diffusion method at 20°C with a reservoir solution containing 0.1 M Bis–Tris (pH 5.5) and 18% (w/v) PEG 3350. The CaRai1–pU5–Mn4)2SO4 and 10% (v/v) glycerol. The AgRai1–pU(S)6–Mn2+ complex was obtained by soaking the free AgRai1 crystals with 10 mM pU(S)6 RNA and 10 mM MnCl2 overnight at 20°C in the presence of 20% (w/v) PEG 3350 and 10% (v/v) ethylene glycol.AgRai1 crystals were obtained by the sitting-drop vapor diffusion method at 20°C with a reservoir solution containing 0.1 M phosphate-citrate (pH 4.2), 20% (w/v) PEG 300, 0.2 M  PEG 3350 and 200 mM NHX-ray diffraction data were collected at the National Synchrotron Light Source (NSLS) beamline X29A. The diffraction images were processed and scaled using the HKL package . The strStructure-based site-specific mutations were generated following the QuikChange Kit (Stratagene) and sequenced for verification of the correct incorporation of the target mutations.32P-labeled RNAs were generated with Vaccinia virus mRNA capping enzyme in the presence of [α-32P]GTP with or without S-adenosyl-methionine (SAM) as descripted previously .The 5′-end-cap eviously ,30. The 32P-labeled RNA were performed as previously reported , 1 mM MnCl2, 20 U RNasin RNase inhibitor (Promega), 5′-end radio-labeled RNA (∼100 counts per second) and 100 nM of the indicated histidine-tagged Rai1 proteins. Reactions were stopped with 40 μl stop buffer  and resolved by 7 M urea denaturing PAGE (5%). Dried gels were exposed to Phosphor Screen and 32P-labeled RNA detected by a Molecular Dynamics PhosphorImager (Storm860) and quantitated with Image Quant software.Exoribonuclease assays in Figures reported ,19. Brie32P-labeled RNAs and 50 nM of the indicated protein for 30 min at 37°C analogous to the same buffer conditions as the exonuclease reactions above. Decapping products were resolved on polyethyleneimine (PEI)-cellulose thin-layer chromatography (TLC) plates developed in 0.45 M (NH4)2SO4 or 1.5 M K2SO4 , C. albicans (CaRai1), C. glabrata (CgRai1), L. thermotolerans (LtRai1), S. cerevisiae (ScRai1), S. stipitis (SsRai1) and V. polyspora (VpRai1). This subset of Rai1 proteins share stronger overall sequence conservation than other Rai1 proteins . For example, ScRai1 and AgRai1 share 47% sequence identity, while AgRai1 and SsRai1 share 39% sequence identity. We also included SpRai1 in these experiments. The homologs of Dxo1, a weak sequence homolog of Rai1 found in a collection of fungal organisms, were studied earlier , and this activity was weakly stimulated by the presence of SpRat1 (lane 15). AgRai1, CgRai1 and SsRai1 also showed activity toward this substrate, with AgRai1 having the strongest activity among all the homologs. However, these enzymes released GTP (pppG) as a product rather than PPi , releasing the GpppG cap structure as a product Figure . AgRai1 7GpppG as a product   Figure  11). Sp. Sp7GpppG Figure . The othNext, we tested the 5′-3′ exoribonuclease activity of the different Rai1 proteins, using a body-labeled 5′ monophosphate RNA as the substrate Figure ,19. The Overall, our biochemical characterizations of the fungal Rai1 homologs indicate that they have diverse activity profiles Table . The res2+ and an RNA penta-nucleotide with a 5′ monophosphate (pU5) (2+ and an RNA hexa-nucleotide with a 5′ monophosphate and with the phosphodiester group between nucleotides 1 and 2 and 2 and 3 replaced with a phosphorothioate group to inhibit hydrolysis (pU(S)6) 6–Mn2+ complex was prepared by soaking AgRai1 free enzyme crystals with 10 mM pU(S)6 and 10 mM MnCl2 overnight. All the structures have good agreement with the X-ray diffraction data and the expected bond lengths, bond angles, and other geometric parameters  , and AgR(pU(S)6) . The CaRThe overall structures of CaRai1 Figure , AgRai1 2+ ternary complex, clear electron density was observed for the first four nucleotides of the RNA , III  and IV   as its only ligand from the protein or RNA and one of its coordinating water molecules is shared with the second Mn2+ ion  Figure , as obse complex . Similarys Table , suggestn Figure . It is nThe structure revealed that Glu104 (αB) is located near the expected binding site for the 5′ segment of the substrate Figure . The sid3 is stacked with that of U2, but this stacking interaction is not maintained by U4 and U5, and the RNA backbone is relatively straight in this complex  and Arg284 , while that of U5 has ionic interactions with Arg284 (motif VI) and Arg159 (αD). Arg159 is not conserved among Rai1 homologs , and the unique interaction with this residue may have helped define the bound conformation of pU5 to CaRai1. The ribose and base of U5 are placed near helix αF, and have weak interactions with a few of its side chains  in the second monomer assumes a different rotamer and is not coordinated to the second Mn2+ ion .There are two copies of the CaRai1–pU5–Mn2+ complex (2 and U3) are similar 6–Mnx Figure . The twos Figure , in conts Figure . There i1 Figure  and it i1 Figure .4, interacts with Lys254 (motif V) but is about 5 Å away from Arg275 (motif VI). The positions of its ribose and base show clear differences to those of U4 in the MmDXO complex, probably because the side chain of Trp154 (αD) in AgRai1 clashes with the position of U4 in the MmDXO complex 6 is similar to that of pU5 in the MmDXO complex but rather different from that in the CaRai1 complex Figure . The firx Figure . This rex Figure .We also observed a sulfate or phosphate ion (modeled as sulfate) in the active site region of this structure, bound through interactions with the dipole of helix αB (backbone amides of Arg106 and Gly107), the side chain guanidinium group of Arg106 (motif I), and the side chain ammonium ion of Lys198 (β9) Figure . This su5), Trp154  and Tyr159   Figure . In addi) Figure  as beingFor both CaRai1 and AgRai1, the mutants were expressed and purified following the same protocol as the wild-type enzyme, and showed the same profile on a gel filtration column (data not shown). The mutants also had essentially the same melting curves as the wild-type enzyme in thermal shift assays (data not shown). These data suggest that the mutations did not disrupt the overall structures of the proteins.2+ complex suggested that Glu104 6, we created the K110T, Y159E and K198I mutants, changing the residues to their equivalents in another Rai1 protein , and replaced Trp154 with Ala (W154A). As discussed earlier, Lys198 likely interacts with the leaving group of the substrate. The W154A and Y159E mutations greatly reduced the decapping activity, while the K110T and K198I mutations had only a small effect Figure , e. We a2 and U3), but could affect that of the rest of the RNA body. The large effect of this mutation suggests that the binding mode of the RNA body could impact the catalytic activity of Rai1, which could have wider implications given the differences in the binding modes of this region of the substrates among the Rai1 homologs  from 5′ triphosphate RNA. This 5′ triphosphonucleotide hydrolase (TPH) activity is distinct from that of the canonical 5′-3′ exonucleases, which are active toward 5′ monophosphate RNA but are inactive toward 5′ triphosphate RNA. Currently we do not understand why AgRai1, CgRai1 and SsRai1 can accommodate GTP as the leaving group, while SpRai1 uses PPi as the leaving group. Having structures of these enzymes in complex with the substrate will be important in revealing the mechanism(s) of these different activity profiles. Interestingly, our earlier studies with the H163G and H163G/D167K mutants of KlDxo1 also showed TPH activity . In addiThe atomic coordinates have been deposited at the Protein Data Bank, with accession codes 5BTB, 5BTH, 5BTO, 5BTE and 5BUD.Supplementary Data are available at NAR Online.


Bovine leukemia virus (BLV), a B-lymphotropic oncogenic retrovirus sharing common biological and structural features with the human T-cell leukemia virus I and II (HTLV-I and II), is the etiologic agent of enzootic bovine leucosis. One major feature of the BLV infection is the absence of viremia. It is widely accepted that BLV latency, due to the RNA polymerase II 5’-LTR-driven transcriptional and epigenetic repression is a viral strategy to escape from the host immune system and allow tumor development. Recently, it has been demonstrated by deep sequencing and bioinformatics analysis that the BLV genome encodes a cluster of micro-RNAs which is predicted to be transcribed by RNA polymerase III suggesting that the silencing dogma in BLV transcriptional regulation is only partially correct. Here, we demonstrated by chromatin immunoprecipitation assays that BLV miRNAs were indeed transcribed in vivo by RNA polymerase III through a type II RNAPIII promoter similar to the one directing tRNAs transcription. We also showed that an activating epigenetic environment surrounds the miRNAs cluster. Overall, our results provide new insights into a better understanding of the molecular mechanisms regulating gene expression of oncogenic retroviruses.