VPgmut). PVAVPgmut RNA replicated in infiltrated leaves, but RNA accumulation remained low. Systemic infection occurred only if a reversion to wild type PVA occurred. VPg was able to stabilize PVA RNA and enhance the expression of Renilla luciferase (3’RLUC) from the 3’ end of the PVA genome. VPgmut could not support either PVA RNA stabilization or enhanced 3’RLUC expression. The RNA silencing suppressor helper-component proteinase (HCPro) is responsible for the formation of PVA-induced RNA granules (PGs) during infection. While VPgmut increased the number of PG-like foci, the percentage of PVA RNA co-localization with PGs was reduced from 86% to 20%. A testable hypothesis for future studies based on these results is that the binding of eIF(iso)4E to PVA VPg via the YXXXLΦ motif is required for PVA RNA stabilization, as well as the transfer to the RNA silencing suppression pathway and, further, to polysomes for viral protein synthesis.The interaction between the viral protein genome-linked (VPg) and eukaryotic initiation factor 4E (eIF4E) or eIF(iso)4E of the host plays a crucial role in potyvirus infection. The VPg of potato virus A (PVA) contains the Tyr-X-X-X-X-Leu-phi (YXXXLΦ) binding motif for eIF(iso)4E. In order to investigate its role in PVA infection, we substituted the conserved tyrosine and leucine residues of the motif with alanine residues in the infectious cDNA of PVA (PVA Potyviridae includes the genus Potyvirus and nine other genera. Due to their economic and scientific importance, two potyviruses, namely potato virus Y (PVY) and plum pox virus (PPV), have been designated as being among the top ten most important plant viruses ) and incubated in the wells for 3 h at 37 °C. Plates were washed 3 to 4 times with a wash buffer  to remove the excess unbound antibody, and then 100 µL of leaf samples were added in each well of the coated plate before being incubated overnight at 4 °C. A non-infected plant sample extract was used as a negative control for the experiment. Plates were gently washed with thewash buffer and were then used for cDNA preparation. qRT-PCR was then performed in 96-well plates by using a CFX96 Touch real-time PCR system . Each reaction mixture of 10 μL in volume contained 5 μL of Maxima SYBR green qPCR master mix (Thermo Scientific), 0.5 μM of each forward and reverse primers, 1 μL of cDNA, and 3 μL of nuclease-free water. Three technical replicates were performed from each cDNA sample. The following primers were used: CPqPCRF′ (5′-CATGCCCAGGTATGGTCTTC-3′) and CPqPCRR′ (5′-ATCGGAGTGGTTGCAGTGAT-3′). The housekeeping gene protein phosphatase 2A (PP2A) was used as a reference gene. PP2A was amplified with the primers, as described previously [2O) as a non-template control and RT reaction mixtures that lacked the reverse transcriptase as non-RT controls. A serial dilution of PVA icDNA was used, and the quantification cycle (Cq) values were plotted against the values for the input cDNA to construct the standard curve.Infected leaf discs were collected at different time point, as mentioned in the results. Each sample set represented a pooled sample from four infection foci. The leaf samples were immediately frozen and grounded in liquid nitrogen. RNA isolation was carried out with an RNeasy plant minikit  according to the manufacturer’s protocol. One microgram of total RNA from each sample was treated with RNase-free DNase (Thermo Scientific). First-strand cDNA synthesis was performed by using a RevertAid H Minus first-strand cDNA synthesis kit (Thermo Scientific) according to the user protocol. For the quantitation of the total RNA amount, the random hexamers provided with the kit were used to prime the cDNA synthesis. For the negative-strand RNA quantification, a minus strand-specific forward primer (CPqPCRF’) was used for the cDNA synthesis. For IC-qRT-PCR, no RNA isolation was performed before cDNA synthesis; instead, CP assemblies and/or intact PVA particles were captured from the infected leaf sample. The grounded samples for this experiment were mixed with 200 µL of a sample extraction buffer . The samples were allowed to settle on ice for 30–45 min. Ninety-six-well plates that were coated with anti PVA CP antibodies were used to capture the CP assemblies from the infected leaf samples. For coating, the PVA antibody  was diluted to 1:1000 in the coating buffer (15 mM Naeviously . The ampRFP or eIF(iso)4ERFP with P0YFP, the living tissues of the infected N. benthamiana leaves were examined at 3 days post infiltration (dpi). Four leaf discs surrounding the infiltration point were cut with a cork borer and mounted in a slide with a few drops of water. Leaf discs were placed under cover glass, and the abaxial side of the leaf discs was scrutinized under confocal laser scanning microscopy, Leica True Confocal Scanning-Spectral Photometric 5 . For visualizing YFP, excitation was performed with an argon laser at 488 nm, and emission was recorded at 525–555 nm. RFP was excited with Diode Pumped Solid State (DPSS) 561 nm laser and emission recorded at 570–620 nm (DD 488/561 beam splitter). The sequential scanning mode was applied for the co-imaging of fluorescent proteins. Single cells were magnified under a 63× water immersion objective to acquire images. All images presented in the figures are acquired at the same magnification. The co-localization analysis of P0YFP with λN22RFP or eIF(iso)4ERFP were performed with the Fiji  image analysis software package by using the co-localization threshold-function. P0YFP-containing granules were selected as regions of interest for quantitation of co-localization, and the results are given as the % intensity of P0YFP co-localizing with λN22RFP or eIF(iso)4ERFP. A bar diagram was made to indicate the percentages of co-localization and a Student’s t-test was employed to calculate the significance of the differences between the experimental and control samples.In the co-localization study of λN22N. benthamiana leaves by using epifluorescence microscopy . Four leaf discs, surrounding the infiltration point were cut out from each plant. Leaf discs were mounted in a glass slide with a few drops of water and placed under a cover glass. An appropriate filter for YFP was used to visualize PGs. PGs were counted in the epidermal tissue under 20× magnification. The average number of PGs found in 1 mm2 view area from each set is presented in a bar diagram. For each condition, three different plants were used, and from each plant, PGs were counted from four separate leaf areas (n = 12). Standard deviation was calculated. PGs were quantified at 3 dpi from infected To observe the PVA particles, infected leaf samples were collected at 7 dpi from local leaves. Four leaf discs of approximately 100 mg of leaf tissue was ground in liquid nitrogen and mixed with a 0.06 M phosphate buffer. Leaf samples were incubated in ice for about 1 h to let the cell debris settle. Carbon-coated electron microscopy (EM)-grids were incubated with the anti-CP antibodies  that were diluted to 1:100 for 1 h at room temperature. The excess antibody was washed with phosphate buffer. Antibody-coated grids were incubated at 4 °C overnight with supernatant that was collected from the leaf extracts. Grids were further washed with 20 drops of a phosphate buffer and immediately stained with 2% uranyl acetate for 15 seconds. Excess stain was drenched from the grids by using filter paper, and dried grids were used for the visualization of the particles with a Jeol JEM-1400 transmission electron microscope . WT) that expresses 3’RLUC as part of the viral polyprotein from a cistron between nuclear inclusion protein b  and CP cistrons in PVA RNA [VPgmut , is not conserved throughout potyviruses. VPgs of only seven potyviruses carry this motif A. In thi PVA RNA . To enab PVA RNA . This co [VPgmut B. In ordVPgmut RNA with the aid of 3’RLUC activity. Our question here was whether PVAVPgmut gene expression resembled that of the replicating or non-replicating PVA RNA. The experiment was initiated by infiltrating Agrobacterium carrying PVAVPgmut construct (OD600 0.05) into Nicotiana benthamiana leaves. For comparison, we infiltrated similarly Agrobacterium carrying PVAWT, PVAΔGDD and PVACPmut constructs. 3’RLUC activity coming from the non-replicating PVAΔGDD RNA was translated from capped viral transcripts that were derived from nucleus, as illustrated in WT, PVACPmut and PVAVPgmut RNAs emerging from the nucleus prior to the first round of replication. We assumed that the replicated PVACPmut and PVAWT RNAs carried wild type VPg at their 5’ends. This assumption was based on studies that have shown that PVA VPg gets uridylylated by the catalytic activity of NIb and may therefore serve as an uridylylated protein primer for replication [VPgmut replicates, according to the same logic, PVAVPgmut RNA ought to carry VPgmut protein at its 5’end, as schematically drawn in VPgmut, and the other PVA RNAs were determined at 3 dpi RNA. PVAWT and PVAΔGDD were used as a positive and a negative control, respectively. We collected the infected leaf samples at 4 dpi and quantitated PVA (−)RNA by qRT-PCR. In three parallel experiments, PVAVPgmut samples contained an approximately fivefold higher amount of (−)RNA than PVAΔGDD, though this was still 1000 fold less than PVAWT -strand RNA in ΔGDD came from PVAVPgmut RNA or from its reversion to PVAWT. As the 3’RLUC expression level did not provide an ultimate proof for PVAan PVAWT C. These Nicotiana benthamiana leaves showed an accumulation of VPgs from PVAWT and PVAΔGDD, as well as VPgmut from PVAVPgmut  expressed virus-derived 3’RLUC in the systemic leaves. The 3’RLUC activities that were determined in systemic leaves at 10 dpi in a representative experiment are given in WT RNA. Thus, the data indicated that systemic infection and particle formation in PVAVPgmut-infiltrated plants can only occur after reversion to PVAWT. We assumed that reversion occurred only in few cells within the leaves infiltrated with PVAVPgmut, and, therefore, particle formation remained hard to detect from the local leaves. We reasoned that if the PVAlication . Howeverat 7 dpi A. This wat 7 dpi B. Number amounts B. When tlant see B. We susWT and PVAΔGDD RNA accumulation and gene expression [RLUC gene is located between NIb and CP encoding cistrons in PVA RNA and not if it resides in front of the HCPro encoding cistron [mut behaves in respect to the enhancement of viral RNA accumulation and 3’RLUC expression. We co-infiltrated PVAWT, PVAΔGDD and PVAVPgmut and either VPg, VPgmut or GUS with Agrobacterium. GUS expression with PVA RNAs served here as the baseline. PVA RNA accumulation and 3’RLUC activity were both quantitated from the samples that were collected from the infiltrated local leaves at 3 dpi. In line with our previous results [WT RNA accumulation by approximately 10 fold  remained two magnitudes of order lower than the PVAWT RNA copy number (>109). The accumulation of VPg and VPgmut from the corresponding expression vectors was verified by Western blotting 4E interaction is required together with HCPro to assist the replicated VPg-linked PVA RNA to silencing suppression pathway and translation. With this hypothesis in mind, we wanted to analyze how PGs behaved in the presence of PVAVPgmut and whether free VPgmut had any effect in that. Silencing suppression is one of the major strategies to protect viral RNA from degradation. One feasible possibility as to why PVAAgrobacteriumcarrying PVAWT, PVAΔGDD, and PVAVPgmut constructs along with the expression constructs for GUS, VPg, and VPgmut in different combinations (YFP), which co-localizes together with HCPro in PGs [Agrobacterium containing P0YFP expression construct was infiltrated together with each combination of the other constructs. We calculated the number of foci that contained P0YFP in a defined area of an infiltrated leaf under epifluorescence microscopy at 3 dpi  and an RFP gene to a λN22 encoding sequence in the λN22RFP construct were described in [Agrobacterium-infiltrated Nicotiana benthamiana plants with a PVAΔGDD B-box construct along with the λN22RFP and P0YFP constructs and with either a GUS or VPgmut construct. The co-localization of the λN22RFP and P0YFP signals in the PVAΔGDD B-box-infiltrated and GUS-expressing samples verified the existence of viral RNA in PGs 4E tagged with RFP was expressed together with P0YFP and either PVAWT or PVAVPgmut in N. benthamiana leaves. The eIF(iso)4ERFP-signal co-localized with P0YFP in granules within PVAWT-infected cells (n = 47). In the presence of PVAVPgmut, the percentage of the co-localization of P0 and eIF(iso)4E was reduced to 70% 4E, which is a component of PGs , is presled RNAs . No PG-lribed in , as thesVPgmut that carried similar mutations in the conserved eIF4E binding site YXXXXLΦ in PVA VPg as in [The interaction between VPg and eIF4E/(iso)4E is among the most important factors supporting potyvirus infection . HoweverPg as in ,45. In [Agrobacterium infiltration, T-DNAs that carried PVA cDNAs were transferred to the host cell nucleus, where the capped PVA transcripts entered cytoplasm and the first round of viral translation. The capped PVAVPgmut transcripts produced VPgmut before entering the replication complex. Wild type VPg can be available in PVAVPgmut-expressing cells only if a reversion to PVAWT RNA occurs. The accumulation of the (−)-strand PVAVPgmut RNA suggests that the capped transcript could serve as a template for replication. In accordance, it has been proven that in vitro transcribed capped full-length PVA RNAs replicate and consequently initiate infection, e.g., [mut can become uridylylated similarly to the wild type potyviral VPgs [VPgmut RNA carries VPgmut at its 5’end 4E interaction may have played a role here. In this assay, PVAVPgmut RNA behaved like the non-replicating capped PVAΔGDD RNA. In fact, a substantial amount of the transcripts in these cells were capped ones that were exported from the nucleus, as the viral RNA amount in PVAVPgmut-expressing cells remained close to that of PVAΔGDD RNA 4E-binding site do not affect VPg’s capacity to inhibit autophagy. We previously reported that PVA infection induces the formation of granules, PGs, which contain viral RNA and several host and viral RNA-binding proteins . We demorans see . In TuMVmut may not have been similar to those formed during wild type infection. When the localization of PVA RNA in PGs was tested, a reduction was observed in the presence of VPgmut compared to that of the wild type VPg. In the test, we used PVAΔGDD RNA that was tagged with several copies of the B-box sequence, which binds the λ22 peptide [WT may compromise infection. We demonstrated the presence of PVAΔGDD RNA in PGs with this methodology in [ΔGDD RNA and P0 in PGs of the cells that express VPgmut indicated that VPgmut negatively affected the transport of PVAΔGDD RNA in to the P0-containing PGs. The question of whether replicating PVA RNA would behave similarly to the capped PVAΔGDD RNA in this respect remains. Protection from RNA silencing is especially important for PVAWT RNA, as the replicating viruses are strong inducers of RNA silencing in planta [WT infection is the suppression of RNA silencing [VPgmut plants in spite of the capped PVAVPgmut RNA being replicated; 2) there was a significant reduction of PVAΔGDD RNA in PGs in the presence of VPgmut; and 3) VPgmut was not able to increase PVAWT RNA accumulation similarly to the wild type VPg. In accordance with this, the interactions between VPg and HCPro with eIF4E/(iso)4E were both suggested in [The PGs observed in the presence of VPg peptide . The nonology in . The sign planta . Accordiilencing . On thesested in  to be ofmut-expressing cells. An interesting network of binding sites was demonstrated in [mut may arise from the interaction of eIF(iso)4E with HCPro [WT infection, enhanced channeling is seen as the dispersal of PGs [mut to disperse PGs may be the reason for the accumulation of the PG-like aggregates seen in Though PVA RNA is associated with PGs, it is not needed for their formation . HCPro irated in , in whicth HCPro ,45. VPg th HCPro , a functth HCPro . In PVAWl of PGs . The faiIn , we deteAs a testable future model, we propose that an essential function of eIF(iso)4E-VPg binding is to protect PVA RNA from being sent to the RNA silencing pathway. We suggest that the VPg-eIF(iso)4E interaction is required for the successful assembly of a protective protein complex around PVA RNA, thus enabling its transfer to the RNA silencing suppression pathway, which involves PGs as one component, and further to polysomes for viral protein synthesis.


Beet necrotic yellow vein virus (BNYVV) is the cause of rhizomania, an important disease of sugar beet around the world. The multipartite genome of the BNYVV contains four or five single-stranded RNA that has been used to characterize the virus. Understanding genome composition of the virus not only determines the degree of pathogenicity but also is required to development of resistant varieties of sugar beet. Resistance to rhizomania has been conferred to sugar beet varieties by conventional breeding methods or modern genome engineering tools. However, over time, viruses undergo genetic alterations and develop new variants to break crop resistance. Here, we report the occurrence of genetic reassortment and emergence of new variants of BNYVV among the isolates of Thrace and Asia Minor (modern-day Turkey). Our findings indicate that the isolates harbor European A-type RNA-2 and RNA-3, nevertheless, RNA-5 is closely related to East Asian J-type. Furthermore, RNA-1 and RNA-4 are either derived from A, B, and P-types or a mixture of them. The RNA-5 factor which enhance the pathogenicity, is rarely found in the isolates studied (20%). The creation of new variants of the virus emphasizes the necessity to develop new generation of resistant crops. We anticipate that these findings will be useful for future genetic characterization and evolutionary studies of BNYVV, as well as for developing sustainable strategies for the control of this destructive disease. Beta vulgaris L.) worldwide. Since the first report of rhizomania1 numerous studies have reported the worldwide infection of sugar beet fields with this disease. Tamada and Baba2 first identified Beet necrotic yellow vein virus (BNYVV) as the cause of rhizomania when they isolated the virus from infected plants of sugar beet fields in Japan. This disease reduces sugar content by 8%, root yield up to 90%, and sugar yield up to 80%4. The BNYVV genome is multipartite and composed of four single-stranded RNA species designated as RNA-1, RNA-2, RNA-3, and RNA-4, coating with a 21-kDa protein5. In addition, a fifth RNA species (RNA-5) has been identified in some of the European and Asian BNYVV isolates12. RNA-1 and RNA-2, which contain 6746 and 4612 nt-long RNA species, respectively, encode viral “housekeeping” genes involved in virus replication, assembly, cell-to-cell movement and suppression of post transcriptional gene silencing14. Therefore, when the virus vector Polymyxa betae Keskin15 is not present, RNA-1 and RNA-2 are required for the maintenance of BNYVV in the environment16. RNA-3 consisting of a 1775 nt-long RNA species, is involved in viral pathogenicity18. RNA-4 (1431 nt) plays a key role in transmission of the virus by P. betae19. RNA-5 (1342–1347 nt in length) is associated with rhizomania severity, but is not required for virus survival21. Comparative studies revealed that the RNA-1, RNA-4, and RNA-5 contribute to the development of different rhizomania symptoms7. Moreover, the interaction between RNA-3, RNA-4 and RNA-5 increases rhizomania symptoms20. BNYVV is mainly divided into three pathogenic types designated as A, B and P types17. A and B are the most widespread types such that A-type is more prevalent than B-type virus and the P is a rare type throughout the world3. Type A is found in European countries as well as in the USA, China, and Japan, whereas, Type B occurs mainly in countries such as France, Germany, Sweden, Poland, China and Iran23. Type P is associated with RNA-5 and was first isolated from the city of Pithiviers, France24. However, RNA-5 species was previously described in East Asian isolates20. Studies also revealed that the J-type East Asian BNYVV isolates was different from French isolates for the length and the sequence of RNA-525. With more than 340 thousand hectares of sugar beet harvesting area, Turkey is the world’s fifth largest sugar beet producing country  and rhizomania is known to cause serious economic losses in sugar beet production26.Rhizomania is one of the most destructive soil-borne diseases of sugar beet .The double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) results confirmed BNYVV infection of only 38 bait plants grown in collected soil samples . The highest OD value was recorded 2.160 for samples of Kütahya; however, the other values of positive isolates ranged between 0.068–1.609 (Table Reverse transcription-polymerase chain reaction (RT-PCR) assay was successfully optimized to amplify the desired fragments using positive control isolates Fig. . Based oIsolates representing different regions  were selected to study the nucleotide sequences of RNA-1 Table . SimilarIsolates of TR-S49, TR-S58, TR-S61, TR-S91, TR-S105, TR-S5, TR-S24, TR-S2, TR-S19, TR-S86, TR-S125, TR-S67 were studied for P13, P14 and P15 open reading frames (ORF) corresponding to transport and suppressor associated proteins in the positions of between 3700–4140 nt. Although no novel nucleotide variation was identified in P13, ORF sequences of P14 and P15 contained several nucleotide variations that were not previously reported Fig. . RNA-2 sStudying the isolates of TR-S5, TR-S19, TR-S2, TR-S24, TR-S125, TR-S61, TR-S86, TR-S49, TR-S79, TR-S91, TR-S58 for RNA-3 (P25) revealed the occurrence of unique nucleotide polymorphisms between the nucleotide positions of 420–1000 Fig. . Based oRNA-4 (P31) analyses of the selected isolates  unveiled the incidence of unique polymorphism in the nucleotide position of 381–1280 Fig. . AccordiSequence analysis of RNA-5 P26) in the coding region 420–1160 nt) showed that TR-S49 and TR-S79 isolates were notably different from previous ones, unlike TR-S5. Despite the presence of polymorphism in TR-S5, the nucleotide sequence is the closest (99.58%) to the Chinese isolate CH2 (AB018614.1). Nevertheless, the genetic distance between TR-S49 and TR-S79 isolates and previously reported sequences was higher (97.11%). The results showed that the studied isolates harbored P26 similar to East Asian J-type  analyses have been widely used in detection and studies of BNYVV structure. ELISA is a routine serological assay used in diagnosis of BNYVV in plants, although concentration of the virus, soil temperature and the type of the sampling tissue affect the efficiency of this method28. Therefore, the ELISA test is less efficient particularly when virus concentration is low in infected plants29.To control losses incurred by rhizomania, cultivation of resistant sugar beet cultivars developed through conventional breeding methods and transgenic techniques is considered an effective approach. Emergence and evolution of novel variants of BNYVV threaten sustainable crop production and yield in resistant crops, however, these variants are not widespread yet34 hence, it is increasingly used in the detection of BNYVV37. The use of nucleic acid-based methods such as RT-PCR and qRT-PCR has increased the sensitivity of the virus detection by up to 100–10,000 times27.The accuracy of the ELISA is less than molecular-based detection methods, however, being fast, cheap and the presence of commercial kits make the ELISA a preferred assay for the routine detection of BNYVV. RT-PCR is a more accurate tool compared to ELISA and is considered a more reliable method in virus detection studies38. In this study, the ELISA values recorded for negative controls were below 0.02 (0.012–0.019) therefore values over 0.06 were accepted as positive samples  revealed a unique polymorphism which could be the probable causes of the failure of RFLP analysis in the previous study42. Our results are in accordance with previous studies that assigned RNA-2–3 as A-type and RNA-5 as East Asian J-type. Furthermore, this study found that RNA-1 was derived either only from A type BNYVV or from mixture of P and A-types as some of the isolates were harboring RNA-1 highly resembling both European and Japanese P and A-types . Moreover, Iğdır province in Eastern Anatolia had the highest RNA-5 occurrence (57%) among the studied regions. Analysis of RNA-5 in the coding sequences indicated that although all the TR-S5, TR-S49 and TR-S79 isolates were closely related to East Asian J-type, there was a notable nucleotide and amino acid difference between them . The DAS-ELISA assay was carried out according to the manufacturer’s recommendations. Bio-Rad Novapath Microplate Reader was used to measure absorbance values of the samples at 405 nm wavelength. The data were analyzed following the MeunierTotal RNA was isolated from root tissues using the TRI Reagent™ kit using manufacturer’s recommendations . RNA concentrations were analyzed by a NanoDrop-1000 spectrophotometer  and the quality was checked by 1% agarose gel electrophoresis and visualizing under UV (GeneGenius Gel Imaging system).BNYVV nucleotide sequences were retrieved from the GeneBank and highly conserved regions were used to design PCR primers Fig. . The pri2. PCR was initiated with a denaturing step of 3 min at 94 °C followed by 35 cycles including 94 °C for 1 min (denaturation), 60 °C for 2 min  and 72 °C for 2 min (extension), and the final extension period of 10 min at 72 °C. PCR products were electrophoresed on 1.5% agarose gel and the GeneGenius Gel Imaging system was used to visualize the amplicons.The NG dART RT kit  was used to perform the first strand cDNA synthesis and RT-PCR assay. Prior to screening, positive controls were utilized to optimize the amplification of the RNA species. cDNA concentrations and quality were assessed by a NanoDrop-1000 Spectrophotometer. PCR amplifications were performed in a 25 µL reaction mixture containing 200 ng of cDNA, 10 pmol of each primer, 2.5 mM dNTPs, 0.5 units GoTaq DNA polymerase  and 1.5 mM MgCl48. Trees were created by the Neighbor Joining method using the Jukes-Cantor distance algorithm and bootstrap support of 100. Pairwise identity and similarity of nucleotide sequences were calculated through matrix analyses performed by a SIAS online tool .Some of the isolates were selected based on ELISA and RT-PCR results to undergo further sequence and phylogenetic analyses. The resulting PCR products of RNA-1–5 species were purified from agarose gels after electrophoresis and double sequenced by the Sanger dideoxy method . Sequence analyses were performed for RNA 1–5 species in comparison with relevant sequences retrieved from the GeneBank. The sequences obtained were initially subjected to the NCBI  Blast analysis. Sequence comparisons were conducted using the CLC Sequence Viewer 7.6.1 workbench . Phylogenetic trees were constructed to study the evolutionary relationships. CLC Sequence Viewer 7.6.1 program was used to perform the phylogenetic analysisSupplementary information.


The polypyrimidine tract binding protein (PTB) is a multi-domain protein involved in alternative splicing, mRNA localization, stabilization, polyadenylation and translation initiation from internal ribosome entry sites (IRES). In this latter process, PTB promotes viral translation by interacting extensively with complex structured regions in the 5′-untranslated regions of viral RNAs at pyrimidine-rich targets located in single strand and hairpin regions. To better understand how PTB recognizes structured elements in RNA targets, we solved the solution structure of the N-terminal RNA recognition motif (RRM) in complex with an RNA hairpin embedding the loop sequence UCUUU, which is frequently found in IRESs of the picornovirus family. Surprisingly, a new three-turn α3 helix C-terminal to the RRM, folds upon binding the RNA hairpin. Although α3 does not mediate any contacts to the RNA, it acts as a sensor of RNA secondary structure, suggesting a role for RRM1 in detecting pyrimidine tracts in the context of structured RNA. Moreover, the degree of helix formation depends on the RNA loop sequence. Finally, we show that the α3 helix region, which is highly conserved in vertebrates, is crucial for PTB function in enhancing Encephalomyocarditis virus IRES activity. RNA binding proteins (RBPs) are essential in the regulation of diverse processes in RNA biology, such as mRNA splicing, RNA transport, storage, degradation, post-transcriptional modification and translation. Critical in all of these functions, is the ability of RBPs to recognize binding sites on the RNA in the proper structural context, i.e. RNA secondary structure and spacing between binding sites. It is important to understand how this contextual information is used by RBPs to determine the recognition of the binding site and to modulate RBP functions. Whereas many RBPs have been identified, the structural features of the RNA that determine where they bind are only beginning to be understood, although this is essential for elucidating their function .trans-acting factor of several cellular and viral internal ribosome entry sites (IRESs) located in the 5′ untranslated region of the mRNA , human rhinovirus (HRV), hepatitis A virus (HAV), foot and mouth disease virus (FMDV), Theiler's murine encephalomyelitis virus (TMEV) and encephalomyocarditis virus (EMCV). These IRES RNAs adopt highly complex structures, which contain short and long pyrimidine stretches identified as PTB binding sites. It has been proposed that PTB plays the role of an RNA chaperone and that it may stabilize or rearrange IRES RNA structure in order to enable, with the help of eukaryotic initiation factors, the recruitment of the ribosome (Polypyrimidine tract binding protein (PTB or PTBP or PTBP1) also called heterogeneous nuclear ribonucleoprotein I (hnRNP I) is a nucleocytoplasmic protein, which regulates diverse processes in mRNA metabolism . In altethe mRNA . PTB intribosome . It has ribosome ,12.PTB, which is 531 amino acid long, is a monomer in solution and adopts a linear arrangement . It consBecause of its RNA binding properties and domain topology, PTB can accomplish its functions by competing directly with other factors at specific RNA binding sites and/or by inducing structural rearrangements of the targeted RNA. The solution structure of each RRM in complex with a short CU repeat RNA revealed that each RRM bound canonically to the single-stranded RNA on their β-sheet surface ,17,25.in vitro  was cloned into the pTYB11 vector between the NdeI and Xho1 sites. Mutants were generated by site-directed mutagenesis using specific primers and verified by Sanger sequencing. The protein was purified as previously described . All RNAescribed . In addi riboses . To redu2PO4 adjusted to pH 6.5 with NaOH solution with protein and RNA concentrations of 1 mM) at a temperature of 40°C except for experiments used to detect NOEs involving imino proton resonances of the SL UCUUU RNA which were obtained at 5°C in order to reduce their exchange with water. Chemical shifts were referenced using internal DSS. Data were acquired on Bruker AVIII-500 MHz, 600 MHz, 700 MHz, 750 MHz and AVANCE 900 MHz spectrometers all equipped with cryo-probes with the exception of the 750 MHz spectrometer. Data were processed using Topspin 2.1 (Bruker) and analyzed with Sparky (http://www.cgl.ucsf.edu/home/sparky/)  and CARA (www.nmr.ch)  , 3D NOESY 1H–13C HSQC (mixing time of 200 ms) and 3D HCP NMR experiments with samples in NMR buffer dissolved in 99.97% D2O solution. The use of chemically synthesized RNAs, with specifically 13C-labeled sugars at different nucleotide positions in the UCUUU loop region, facilitated the resonance assignment especially in the protein-bound form. Imino resonances were assigned by sequential walk of imino–imino NOEs in the stem of the RNA, supplemented by the through-bond 2D H(NC4C5)H5 experiments in the loop  using IPAP-TROSY experiments implemented in an interleaved manner. Residual dipolar couplings were calculated from the difference between the one bond scalar couplings (1JCH) obtained in the absence and in the presence of alignment medium (13 mg/ml Pf1 phage from ASLA biotech). Chemical shift mapping of PTB RRM1 upon RNA binding was calculated according to the formula Δδ = ((ΔδN/6.51)2 + ΔδH2))1/2 where ΔδN and ΔδH are the nitrogen and proton chemical shift differences respectively, between the free and RNA bound states. Secondary 13C shifts were calculated by subtracting sequence-corrected random coil chemical shifts at the appropriate temperature and pH  . The bacng time) . The bace and pH ,42. Amid15N-edited HSQC obtained in 90% H2O/10% D2O, 3D NOESY 13C-edited aliphatic HSQC spectra acquired in both 90% H2O/10% D2O and in 99.97% D2O, and a 3D NOESY 13C-aromatic edited HSQC in 99.97% D2O. ATNOSCANDID peak lists were then used to derive distance constraints with CYANA. In addition, hydrogen bond restraints were defined between carbonyls and amides, which had slow hydrogen–deuterium exchange (visible 1 h after dissolution of a lyophilized sample in D2O). Dihedral angle constraints for the protein were derived using TALOS . In addition, Watson–Crick hydrogen bond restraints and loose dihedral angle restraints were defined for phosphodiester linkages of the RNA stem region  experiments were performed at 30°C on a VP-ITC instrument . Both protein and RNA samples were dialyzed together in the same NMR buffer. The concentration of the protein in the cell was 10 μM and the RNA concentrations in the syringe varied from 70 to 120 μM (280 μl). The titrations were performed with 30 or 50 injections. Data were analyzed with Origin 7.0 software.Dicistronic pRF (Renilla luciferase and Firefly luciferase) and pRemcvF plasmids were a generous gift from Prof. Dr A. Willis  . Mammaliin vivo translation assays were performed using human HEK293T cells. Cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated fetal calf serum (GibcoBRL) and antibiotics. Cells were plated on six-well plates the day before transfection and were transfected in duplicate with calcium phosphate precipitation using 250 ng of dicistronic emcv wild type or loop mutant reporter plasmids according to the original manufacturer protocol. All EMCV-IRES activity tests were performed in biological duplicates. For RNA interference experiments, cells were plated on 24-well plates the day before transfection. On Day 1, cells were transfected with 30 nM siRNA targeting PTB1 (P1), nPTB (N1) and control siRNA (C2)  according to the manufacturer's instructions. Light emission was measured with a Berthold MicrolumatPlus luminometer.To test EMCV IRES activity, RNA (C2)  using LiForty μg of cell extracts, prepared either in 1× Passive Lysis Buffer (Promega) or in Lysis buffer  NP-40, 20% (v/v) glycerol, 0.5 mM dithiothreitol (DTT), 1 mM phenylmethylsulfonylfluoride and protease inhibitor (Roche)), were separated with 10% SDS-polyacrylamide gel electrophoresis. PTB1 and nPTB knockdown and ectopic PTB1 expression was analyzed by western blot with anti PTB-NT (1:3000), anti nPTB-1S2 (1:500) and anti GAPDH (1:5000) antibodies (Sigma) using the Immun-Star WesternC kit from Bio-Rad. Chemiluminescence was detected on a Typhoon Trio (Amersham Biosciences).Escherichia coli and purified on a Nickel affinity column. Purified proteins were dialyzed against a buffer containing 20 mM NaH2PO4 (pH 7.2), 100 mM NaCl, 5 mM MgCl2, 10% (v/v) glycerol, 1 mM DTT. Concentrations of proteins were determined using optical density absorbance at 280 nm. Both wild type and loop mutants of EMCV 5′ RNA, were transcribed in vitro from linearized pUC-emcv-5′ vector and purified using the Total RNA isolation kit from Macherey-Nagel. RNAs were subsequently dephosphorylated using Calf Intestine Phosphatase (Fermentas) and 5′ labeled using γ-32P-ATP. Unincorporated nucleotides were removed using a IllustraMicrospin G-25 column . RNAs were incubated with a range of protein concentrations in a buffer containing 20 mM NaH2PO4 (pH 7.2), 100 mM NaCl, 5 mM MgCl2, 5% (v/v) glycerol, 1 mM DTT, 50 μg/ml yeast tRNA , 40 μg/ml BSA (New England Biolabs) and SUPERase·In RNase Inhibitor  for 20 min at room temperature. After addition of loading buffer, samples were loaded on a 4% acrylamide/bisacrylamide (29:1) gel in 0.5% TBE and electrophoresed for 1.5 h at 200 V at room temperature. Gels were dried and exposed on a PhosphoScreen .PTB1 mutants were overexpressed in BL21 DE3 Sequence alignments of PTB, nPTB and ROD1 homologues, and of PTB in many vertebrate species show residue conservation, which extends both N-terminally and C-terminally well beyond the canonical RRM, suggesting a functional role for these flanking regions . We therWe investigated by NMR titration the binding of PTB RRM1 to a 23 nucleotide RNA Figure  that embKd1 = 0.10 ± 0.02 μM and Kd2 = 3.0 ± 0.9 μM; To determine PTB RRM1 affinity for SL UCUUU, we measured the dissociation constant of the complex by ITC. Fitting of the experimental data with a single binding site model resulted in a stoichiometry close to 0.5 indicating that one RNA molecule binds two protein molecules. We therefore fit the data with a model for two independent binding sites. This gave better fits to the experimental data and we could extract two dissociation constants  induces a hinge directing α3 to run anti-parallel to the β2-strand.We determined the solution structure of PTB RRM1 in complex with SL UCUUU using a combination of 2318 distance constraints derived from NOEs and 71 orientational constraints derived from residual dipolar couplings (RDCs). The protein/RNA interface was defined by 98 intermolecular NOEs . Samplesstraints . The U12straints . The strstraints . In the straints . The UU straints . Particustraints . More sustraints  and B. Astraints . ImmediaAt the binding interface, the nucleotides C11–U12–U13 lie on the β-sheet surface and the flanking uracils U10 and U14 form a mismatched UU base pair Figure . The sugKd2 corresponding to the weaker binding affinity has a magnitude similar to the wild type (average of 4.1 ± 0.9 μM) (Kd1) of the different RNA mutants for WT protein showed only small increases compared to the SL UCUUU  . The firTΔS versus ΔH shows an excellent linear correlation  which are sensitive to the backbone conformation. Positive values of Δ13Cα−Δ13Cβ indicate helical character, negative values extended β-strand conformation and values near zero are indicative of random coil  show a similar pattern  is proportional to the entropy change upon complex formation.A comparison of the  Figures . In cont Figures . These d13C secondary shifts of the α3-region . Th. Th13C sthe stem  and embethe stem  indicatethe stem . Again, in vivo translation assays using a dicistronic luciferase reporter containing an EMCV IRES as previously described .,61.in viescribed ,32,35. Iescribed  and C. STo evaluate the effect of the apical loop mutations on PTB binding to EMCV IRES RNA, we performed electromobility shift assays (EMSAs) Figure . We usedet al.  we have shown that this α3 helix is transiently present in the free state of the protein with moderate helical character induced on binding CUCUCU single-stranded RNA  included the same C-terminal segment possibly due to the lower induced helical propensity ,15. Basended RNA  whereas nded RNA . Togethended RNA  15,62)..13C seconded RNA .Previous structural studies reported the presence of an additional helix C- or N-terminal to an RRM in the free state of RRM-containing proteins , as wellThe many contacts from PTB RRM1 to the phosphate backbone help to recognize the shape of the RNA and partially account for the increased affinity for stem–loops Figure . These cin vivo data demonstrated that the helix α3 is essential for PTB in promoting EMCV IRES activity, and that the E, F and H apical loops, which are PTB binding sites, are crucial cis-acting elements for EMCV IRES (Figure Although RRM1 shows only modest differences in affinity for different pyrimidine-rich loop sequences when binding a structured RNA, these sequence differences do impact α3 helix formation. This may be related to the degree to which the apical loop bases can form interactions, which help to stabilize the position of the RRM1 β4–α3 loop on the β-sheet and organize the binding pocket for U13 just N-terminal to α3. In this context the high stability of the UUCG loop may prevent conformational adaption of the RRM1 which help to stabilize α3. Our S Figure . It has S Figure ,70. It wS Figure . We can S Figure . PTB RRMS Figure , either S Figure .Chemical shifts and atomic coordinates of the PTB RRM1/SL UCUUU complex have been deposited under accession codes BMRB 25652 and PDB 2N3O respectively.gkaa155_Supplemental_FileClick here for additional data file.


This is the response article to correspondence article received for our published article in BMC surgery titled “A prospective single-center protocol for using near-infrared fluorescence imaging with indocyanine green during staging laparoscopy to detect small metastasis from pancreatic cancer”. Peter L. Labib, MBChB pointed out the necessity to administer indocyanine green intravenously in separate timing for detection of metastasis in liver and peritoneum. Preoperative injection is suitable to detect hepatic metastasis and intraoperative injection is reported to be well suited to detect peritoneal metastasis. However, we could not find the usefulness of intraoperative injection of indocyanine green for detecting peritoneal metastasis in cases with staging laparoscopy prior to this study. We employed this study protocol with only preoperative injection of indocyanine green to simplify the procedure with consideration of probably more frequent cases of hepatic metastasis that is difficult to detect with white-light imaging than those of peritoneal metastasis. Dear Editor,We would like to thank Peter L. Labib, MBChB, for addressing my recent article about near-infrared (NIR) fluorescence imaging with indocyanine green (ICG) during staging laparoscopy for pancreatic ductal adenocarcinoma . We alsoI agree with the author about the timing of ICG injection for detecting peritoneal metastases. We performed staging laparoscopy in three cases with peritoneal metastases using two ICG injection steps prior to this prospective study. We found apparent peritoneal metastases in these cases with white-light imaging during the staging laparoscopy, and there was no fluorescent peritoneal lesion through NIR imaging with preoperative ICG injection on the previous day. Then, we added intraoperative ICG injections of 0.25 mg/kg of patient weight after thorough exploration for hepatic metastasis with white-light and NIR imaging to check the fluorescence of peritoneal metastasis. In these cases, we could find no lesion positive for ICG fluorescence on the peritoneum. As the author described, Liberale et al. found 21 of 24 malignant solid peritoneal nodules to be positive for fluorescence with intraoperative ICG injection in colorectal cancer, and the surgery was modified based on NIR imaging in 4 of 14 patients . Based oWe will consider the author’s proposal to add intraoperative ICG injection to our protocol, depending on the frequency of occult peritoneal metastasis. A total of 20 patients were enrolled in our present study until now and no patient developed new peritoneal metastasis within at least three months after the staging laparoscopy. We understand that visually occult peritoneal metastasis during the staging laparoscopy was unusual in our cohort. Hence, we did not change our protocol up to this time. We thank the authors for their interest and productive comments.


IKZF1plus profile of gene deletions and one case of masked hypodiploidy. Genetic markers BCR-ABL1 (n = 1), ETV6-RUNX1 (n = 25), and rearrangements involving KMT2A (n = 3) or TCF3 (n = 3) were assessed by established conventional techniques such as karyotyping, FISH, and RT-PCR. Comparison of these results with RNA sequencing analysis revealed overall consistency in n=115/117 cases, albeit with one undetected AFF1-KMT2A fusion in RNA sequencing and one undetected ETV6-RUNX1 fusion in conventional analyses. The combined application of RNA sequencing, FISH, and CGH+SNP array reliably detected all genetic markers necessary for risk stratification and will be used as the diagnostic standard workflow for BCP-ALL patients enrolled in the AIEOP-BFM ALL 2017 study. Prospectively, consistent collection of genome-wide CGH+SNP array as well as RNA sequencing data will be a valuable source to elucidate new prognostic lesions beyond established markers of pediatric ALL. In this respect, RNA sequencing identified various gene fusions in up to half of the IKZF1plus (n = 6/12) and B-other (n = 19/36) cases but not in cases with hyperdiploid karyotypes (n = 35). Among these fusions, this study reports several previously undescribed in frame PAX5 fusions, including PAX5-MYO1G and PAX5-NCOA6.Risk-adapted therapy has significantly contributed to improved survival rates in pediatric acute lymphoblastic leukemia (ALL) and reliable detection of chromosomal aberrations is mandatory for risk group stratification. This study evaluated the applicability of panel-based RNA sequencing and array CGH within the diagnostic workflow of the German study group of the international AIEOP-BFM ALL 2017 trial. In a consecutive cohort of 117 children with B cell precursor (BCP) ALL, array analysis identified twelve cases with an The online version of this article (10.1007/s00277-020-03953-3) contains supplementary material, which is available to authorized users. Acute lymphoblastic leukemia (ALL) is the most frequent childhood malignancy with an assumed worldwide annual incidence of more than 50,000 cases , 2. FortAFF1-KMT2A gene fusion and a hypodiploid karyotype. The fusion BCR-ABL1 represented an exclusion criterion and those cases were treated within the EsPhALL study [ETV6-RUNX1, AFF1-KMT2A, and TCF3-HLF) as well as the IKZF1plus subtype [IKZF1plus is not defined by a single genetic aberration, but by deletions in six different genetic loci  [IKZF1plus profile of gene deletions points at the necessity that future routine diagnostic workflows, which are currently often limited to specific genes of interest using fluorescence in situ hybridization (FISH) and polymerase chain reaction (PCR) analyses, will have to assess a wider range of targets in order to detect novel and yet undefined profiles relevant to risk stratification.Risk stratification within the recently completed AIEOP-BFM ALL 2009 trial, an international collaborative treatment protocol for children and adolescents with acute lymphoblastic leukemia, was based on immunophenotype, specific chromosomal aberrations, and response to treatment assessed by minimal residual disease (MRD). The two chief chromosomal aberrations relevant to risk stratification in AIEOP-BFM ALL 2009 were the LL study . Cases sLL study . HoweverLL study . Therefo subtype , 9. It iIKZF1plus in array CGH, the major aim of this study was to compare the RNA-seq-based detection of above mentioned gene fusions with well-established techniques such as karyotyping, FISH, and PCR analyses. We also discuss expanded use of RNA-seq data beyond stratification relevant gene fusions, which in some cases identified potentially targetable lesions. Prospectively, the consistent collection of genome-wide CGH+SNP array as well as RNA-seq data within the AIEOP-BFM ALL 2017 trial is a valuable resource for elucidation of new prognostic markers of pediatric ALL.We here report on our results from implementing analysis of the leukemia transcriptome using panel-based RNA sequencing and genome-wide copy-number analysis using array CGH into the diagnostic workflow of the German study group of the international AIEOP-BFM ALL 2017 trial. In addition to assessing detection of n = 117 patients  with a median age of 4.8 years. Of these patients, 6 (5.1%) were classified with an immunophenotype of pro-B-ALL (B-I), 72 (61.5%) with a common-ALL (B-II), and 39 (33.3%) with a pre-B-ALL (B-III).This study was performed on a consecutive cohort of patients with pediatric ALL sent to our genetic reference laboratory for initial cytogenetic and molecular genetic diagnostics. All patients were treated in German centers and were enrolled in the AIEOP-BFM ALL 2009 trial (EudraCT no. 2007-004270-43). Written informed consent was obtained from the legal representatives . The study cohort consisted of Bone marrow (BM) aspirate was obtained at the time of initial diagnosis. BM from heparinized tubes was used for cytogenetic analyses (karyotyping and FISH). RNA was preferably isolated from EDTA BM, or if necessary from heparin BM, on a Chemagic 360 instrument as per the manufacturer’s instructions . RNA integrity  was measured by Bioanalyzer 2100 . Isolation of DNA from EDTA BM was performed using the QIAamp DNA Blood Midi Kit .ETV6-RUNX1, AFF1-KMT2A, and TCF3-HLF), hypodiploidy (< 45 chr.), and IKZF1plus. BCR-ABL1 is an exclusion criterion for the AIEOP-BFM ALL trial, as these cases are treated within the EsPhALL study instead.This study assisted to optimize the diagnostic standard workflow of the German study group of the AIEOP-BFM ALL 2017 trial. Using the methods listed below, the following genetic markers relevant for risk stratification were evaluated: gene fusions /RUNX1(AML1) ES Dual Color, Abbott; Vysis LSI MLL(KMT2A) Dual Color, Abbott; SureFISH 19p13.3 TCF3 DF, Agilent) [For all patients, short-term cultures (24–48 h) were set up from BM aspirates. Chromosome preparation, fluorescence R-banding, and FISH were performed as previously described . KaryotyAgilent) .BCR-ABL1, ETV6-RUNX1, and AFF1-KMT2A as published elsewhere [ABL1 gene.Reverse transcriptase PCR (RT-PCR) was performed in parallel to cytogenetic analyses whenever EDTA-material was available 74/117 cases) for the qualitative detection of fusion transcripts of 7 cases flsewhere . In caseRNA-seq was performed using the TruSight RNA Pan-Cancer Panel  according to the manufacturer’s instruction. The panel targets 1385 cancer genes (see online Tab.S1) and detects fusions between genes targeted by the panel but also fusions of targeted genes with non-target genes. Prior to this study, the RNA-seq method was successfully validated on commercially available reference material , external reference samples with defined fusions provided by the group of Giovanni Cazzaniga , and genetically characterized cell lines and samples from our laboratory (see Tab.S2 for tested fusions). Gene-pseudogene “fusions” and recurrent technical artifacts were defined during this validation process and excluded from later data analysis. Each RNA-seq run contained a pooled cDNA library of eight patient samples and was executed on an Illumina MiSeq machine using MiSeq® Reagent Kit v3 (150 cycles) with a PE MiSeq® Flow Cell. The anticipated minimum number of reads per sample was 3 million.https://basespace.illumina.com/apps). Fusion transcripts with a low number of split-reads (< 10) were excluded as likely false-positives, unless the fusion was verified with a second method. The turn-around time from RNA isolation to detection of fusion transcripts using this workflow is 4 days.Data analysis was performed using the Illumina BaseSpace apps TopHat Alignment .Array-based comparative genomic hybridization (array CGH) was performed using a 400 k SurePrint G3 Custom CGH Human Genome Microarray  according to the manufacturer’s protocol by hybridizing 500 ng of patient DNA isolated from EDTA material against sex-matched human reference DNA . Regions of genomic copy number change were analyzed and interpreted with Agilent Genomic Workbench 7.0 software under the following analysis setting: aberration detection algorithm ADM-2, threshold 6.0, aberration filter of at least four aberrant probes (resulting in an average genome-wide resolution of ~ 30 kb), and average log2 ratios of + 0.3 for gains and − 0.3 for losses.IKZF1plus gene deletion profile by Stanulla et al. has been based on MLPA analyses targeting IKZF1, CDKN2A, CDKN2B, PAX5, and CRLF2-P2RY8, as well as multiplex PCR for the detection of ERG deletions. Prior to this study, reproducibility of IKZF1plus profiles in our high-resolution array CGH was verified for selected cases (data not shown).The original description of the Cases indicative of masked hypodiploidy  were analyzed on CGH+SNP array  using sex-matched reference DNA .BCR-ABL1 fusion , ETV6-RUNX1 fusion (n = 26), AFF1-KMT2A fusion (n = 2), hypodiploidy (n = 1), and IKZF1plus (n = 12). Other observed genetic markers not relevant for risk stratification within the AIEOP-BFM ALL 2017 trial were as follows: TCF3 rearrangement , KMT2A rearrangement , and hyperdiploid karyotype with more than 50 chromosomes (n = 35). The remaining n = 36 cases were classified as B-other due to absence of the above-mentioned genetic markers ). However, this cryptic AFF1-KMT2A fusion was not reported by either of the two Illumina BaseSpace apps, despite its existence in the raw RNA-seq reads. The dependency on accurate fusion calls by alignment algorithms was also indicated by other cases in this study. Case 15 tested positive for ETV6-RUNX1 fusion in FISH and RT-PCR analyses, whereas only the RNA-seq Alignment app and not the TopHat Alignment app detected this fusion. In contrast, all nine cases with P2RY8-CRLF2 fusions were detected by the TopHat Alignment app only (cases 55–63).Fusion detection by RNA-seq was highly consistent with results obtained from cytogenetic and RT-PCR analyses for 115 out of 117 analyzed cases. The two discordant cases were case 29, which was misclassified by RNA-seq, and case 26, which was misclassified in conventional analyses . In detail, case 29 presented in FISH analysis with a split of the ETV6-RUNX1 fusion for case 26, whereas RNA-seq gave a positive result. Subsequently, the fusion of ETV6-RUNX1 could be confirmed using the HemaVision-28N multiplex RT-PCR Kit. In detail, analysis of the RNA sequence revealed a fusion of ETV6 exon 5 to RUNX1 exon 3, which should have been detectable by routine RT-PCR primers and FISH analyses. Furthermore, case 16 showed an ETV6-RUNX1 fusion in RNA-Seq and FISH resulting from a variant breakpoint in ETV6 exon 4, which could not be detected via routine RT-PCR due to a loss of the primer binding site.On the other side, conventional analyses also generated one false negative result in this study. FISH and regular RT-PCR indicated no BCR-ABL1, PBX1-TCF3) or specified in more detail by RNA-seq, as was the case for the identification of MLLT1 as the fusion partner of KMT2A .All other fusions addressed by karyogram, FISH, or RT-PCR were either confirmed . The IKZF1plus deletion profile was observed in twelve cases. With a median age of 8.7 compared with 4.8 years, the IKZF1plus cases were older than the overall cohort  and B-other , PAX5-NCOA6 (9p13.2-20q11.22), TERF2-JAK2 (16q22.1-9p24.1), HOOK3-FGFR1 (8p11.21-8p11.23), MEF2D-BCL9 (1q22-1q21.2), PAX5-DACH1 (9p13.2-13q21.33), PAX5-ETV6 (9p13.2-12p13.2), PAX5-LINC01251 (9p13.2-9p13.3), ZNF618-NUTM1 (9q32-15q14), and P2RY8-CRLF2 . Notably, all six cases with a structurally aberrant chromosome 9p showed a gene fusion involving the genes JAK2 (n = 1) or PAX5 (n = 5). Fusion transcripts found in patients with a completely normal karyotype and no other markers were EBF1-PDGFRB , PAX5-MYO1G , and P2RY8-CRLF2 (cases 59 and 62). Fusions of ETV6-IKZF1 (12p13.2-7p12.2) and P2RY8-CRLF2 were found in two cases with normal karyotypes at a reduced level of validity . No metaphases were available for case 47 (fusions ETV6-IKZF1 and IKZF1-DLG2 (7p12.2-11q14.1)), case 48 (EP300-ZNF384 (22q13.2-12p13.31) fusion), and cases 56 and 60 (both P2RY8-CRLF2 fusion). Presence of all fusions, except for TERF2-JAK2, IKZF1-DLG2, and HOOK3-FGFR1, could be indirectly confirmed by detection of an array CGH aberration in at least one of the two fusion genes , and in most cases in banding analysis .Cases with an aberrant karyotype displayed the following fusions: This study aimed to evaluate the applicability of panel-based RNA-seq and array CGH for detecting relevant gene fusions as well as chromosomal deletions for risk stratification of pediatric BCP-ALL patients. This knowledge is intended to support optimization of the diagnostic workflow of the German study group of the international AIEOP-BFM ALL 2017 trial by reducing the number of necessary diagnostic tests, while assuring robustness of aberration detection. As discussed later, the here presented results conclusively show that panel-based RNA-seq and CGH+SNP array are powerful tools for the detection of fusion genes and genome-wide copy number aberrations. However, these techniques still need to be supported by FISH analysis in order to guarantee reliable detection of relevant fusions for all cases. The second and more broadly defined aim of this study was to examine the detection of genetic lesions beyond established markers, as recent publications have demonstrated that patients of the B-other subtype frequently harbor fusion transcripts of possible therapeutic relevance –21. ProsIKZF1plus subtype itself is a good example for a new risk marker, which has been identified due to simultaneous consideration of multiple genetic loci. Notably, without analysis for IKZF1plus, ten cases of this study would have been assigned as B-other and two cases as hyperdiploid ALL, with no further genetic risk stratification in both groups. One needs to be aware that occasional IKZF1plus profiles might be present in cases not tested by array CGH in our workflow . Projected onto the whole cohort of 117 BCP-ALLs, we therefore observed IKZF1plus in at least 10% of the cases, which is higher than the originally published share of 6% by Stanulla et al. [IKZF1plus (Tab.S4) would be detectable by MLPA analyses as described by Stanulla et al. Furthermore, the ERG locus is covered by 37 array CGH probes, of which seven probes are located in the region assessed by ERG deletion PCR in Stanulla et al. Despite the satisfactory probe coverage of ERG in array CGH, the higher sensitivity of PCR for deletion detection needs to be acknowledged, which might influence IKZF1plus detection. However, the high number of IKZF1plus cases in our study is more likely to be explained by the small cohort size in our analysis as our extended array CGH data set on more than 600 cases comprises a lower number of IKZF1plus within the B-other and hyperdiploid cases (data not shown). In line with Stanulla et al., the age of onset of IKZF1plus cases was considerably higher than observed in the complete cohort  . FurtherBCR-ABL1 and ETV6-RUNX1 as well as rearrangements involving either KMT2A or TCF3, which are relevant for stratification if resulting in an AFF1-KMT2A or TCF3-HLF fusion, only two discrepant results were observed (cases 26 and 29). For case 26, RNA-seq outperformed the routine FISH and RT-PCR methods by identifying an ETV6-RUNX1 fusion, which could later be confirmed with a second RT-PCR kit. The reason for the failure of the routine methods to detect this fusion is still unclear. Case 29, with a small insertion of KMT2A into the AFF1 gene, was the only example of a clear failure in fusion detection by RNA-seq. Even though present as multiple, identical reads at the RNA-seq raw data level, both applied alignment algorithms did not report the AFF1-KMT2A fusion, as they require the presence of at least three non-identical split reads for fusion calling. In our study, these two discrepant cases are the only two examples for false negative results. However, further influences exist, which can cause relevant fusions to remain undetected. These include a low percentage of leukemic blasts, loss of blasts during cell culture, cryptic or variant translocations, poor RNA quality, and notably the sensitivity of bioinformatics algorithms. Importantly, not all methods  are equally affected by these influences and a combination of methods would be able to rectify their respective weaknesses in most cases. Therefore, the diagnostic workflow of the German study group of AIEOP-BFM ALL 2017 trial will use FISH and panel-based RNA-seq as standard techniques for the detection of fusion genes. Karyotyping and RT-PCR will still be available but restricted to inconclusive cases only.Detection of fusion genes using RNA-seq is a comparably new approach and not yet established in routine diagnostics for most malignancies. Our data demonstrate consistent results between RNA-seq and our routine diagnostic methods . Considering the stratification relevant aberrations P2RY8-CRLF2, were detected by one of the algorithms only. Furthermore, the minimum number of sequencing reads per sample will be set to 3 million reads, even though our current data indicate that some fusion transcripts, like ETV6-RUNX1, are detectable at coverages below 2 million reads. However, a higher number of reads per sample will also facilitate detection of lowly expressed fusion genes as well as detection of fusions in samples with low blast counts. We detected no correlation between RNA-seq coverage and RIN value or ABL1 copy numbers in RT-PCR analysis (data not shown). Thus, variance in RNA-seq coverage in our study might rather be a result of differences in library preparation and quantification, which can possibly be addressed by standardization and automatization of RNA-seq protocols. Another important technical aspect of our study is that RNA-seq was conducted on RNA isolated from BM in EDTA tubes sent to our laboratory within 24 h. Our results indicate that use of RNA stabilizing solutions, at least for the fusion genes of interest, seems expendable.As a technical prerequisite, both alignment tools tested in this study will be applied in future RNA-seq analyses, as some fusions, like IGH locus as well as translocations resulting in promoter or enhancer rearrangements  cannot be detected and these aberrations are being followed up on by FISH analysis on a research basis for selected cases. On the same note and in line with previous reports, our RNA-seq analysis detected additional fusion transcripts only in the B-other subtype, including cases 32–37, which all would have been deemed B-other until recently if not stratified as IKZF1plus in our study [EBF1-PDGFRB, ETV6-NTRK3, and TERF2-JAK2 were all observed within the IKZF1plus subtype [P2RY8-CRLF2, MEF2D-BCL9) [HOOK3-FGFR1, PAX5-DACH1, EP300-ZNF384, PAX5-ETV6, or ZNF618-NUTM1), even though their potential therapeutic or prognostic impact remains to be addressed in more detail [PAX5-MYO1G, PAX5-NCOA6, and possibly PAX5-LINC01251 seem to be promising new fusions, whose influence on the course of the disease warrants further consideration [PAX5 fusions has been highlighted, as well as the potentially initiating, subtype-defining character of PAX5 alterations in B-ALL [MYO1G and NCOA6, first identified in this study, appear to leave the paired box DNA-binding domain of PAX5 operational.It needs to be acknowledged that this workflow, despite panel-based RNA-seq and genome-wide array CGH, will not detect all aberrations present in the here defined B-other group. For example, fusions affecting the ur study , 21. Int subtype , 23, 24.2D-BCL9) –27. Manye detail –33. To tderation , 35. JusBCR-ABL1, ETV6-RUNX1, hypodiploidy, IKZF1plus as well as rearrangements of KMT2A and TCF3 necessary for risk stratification in pediatric BCP-ALL. Foremost, acquisition of genome-wide copy number and panel-wide fusion data within the standard diagnostic workflow is expected to support identification of new genetic markers relevant for treatment in the future.In conclusion, this study demonstrated that panel-based RNA-seq, supplemented by FISH analysis, and CGH+SNP arrays are a reliable combination of methods to detect the genetic markers ESM 1(XLSX 64 kb)


Despite a non-specific nature of self-rated health (SRH), it seems to be a strong predictor of mortality. The aim of this study is to assess the association of SRH and objective health status (OH) with all-cause mortality in 70-year-old community-dwelling older people in Finland.n = 1008) was assessed with a single question and OH (n = 962) by the Rockwood’s Frailty Index (FI). To assess the association of SRH and OH with mortality, Cox regression model was used.A prospective study with 5-, 10- and 27-year follow-ups. SRH , 319 (31.6%), and 932 deceased (86.3%) during the 5-, 10- and 27-year follow-ups, respectively. In unadjusted models, subjects with poor SRH had almost eightfold risk for mortality compared to those with good SRH during the 5-year follow-up; among those with poor OH, the risk was fourfold compared to those with good OH. In the 10-year-follow up, both poor SRH and poor OH predicted about fourfold risk for mortality compared to those with good health. During the 27-year follow-up, OH was a stronger predictor of mortality than SRH. Poor SRH, compared to good SRH, showed 95% sensitivity and 34% specificity for 5-year mortality; corresponding figures for OH were 54 and 80%, respectively.Single-item SRH seems to be able to capture almost the same as OH in predicting a short-term (less than 10 years) mortality risk among older adults in clinical settings. The use of SHR may also enhance the focus on patient-centered care. Self-rated health (SRH) is a subjective indicator of health status that integrates biological, mental, social and functional aspects of a person, including individual and cultural beliefs and health behaviors . DespiteIn the earlier meta-analysis of 22 studies, the duration of follow-ups ranged from 15 months to 25.1 year. The association between general SRH and mortality was similar for studies with greater than and for those with less than 5 years maximum follow-up. In 10 studies with follow-ups from 15 months to 12 years, participants were 65 years or older. However, no subgroups analyses by age were conducted –11.In this study, our main interest was to clarify whether a single-item SRH is valuable tool for risk assessment of older adults in busy clinical settings. Therefore, we analysed the association of SRH and objective health status (OH) with all-cause mortality in 70-year-old community-dwelling older people in Finland during the 5-, 10- and 27-year follow-up.n = 24) were excluded, leaving 1008 participants for this study. They were followed up for 27 years for all-cause mortality.The study population of this prospective follow-up study consisted of all 70-year-old community-dwelling older adults of the city of Turku (The Turku Elderly Study) in Southwestern Finland, who were born in 1920. Data were collected in 1990–1991 by using postal questionnaires, interviews and clinical examinations. The protocol of data collection and flow chart are described in detail previously . One thon = 31), creating a three-category SRH variable: 1. good, 2. rather good, and 3. poor.SRH was measured by using a single question “in general, how would you rate your health” with reply alternatives: 1. good, 2. rather good, 3. poor, and 4. very poor. Responses “poor” and “very poor” were combined because of low number of responses in category “very poor” , and education . Need for help was assessed with three variables (yes/no): need for daily help, having help from a relative at least once a week, and having someone who helps when needed.Cognitive function was measured by Mini-Mental State Examination . CognitiPsychosocial status was measured with questions concerning feelings of depression , loneliness , and life satisfaction .Weight and height were measured, and body mass index (BMI), measured as kilograms per square meter, was calculated. Weight change during the past 3 years was assessed as follows: 1. increased, 2. no change, and 3. decreased. Health behavior was measured with questions concerning smoking  and having daily outdoor activities . Physical functioning was assessed with difficulties in walking 500 m  and number of falls during the previous 12 months .The study Data from all participants who died before January 2017 were obtained from the official Finnish Cause of Death Register using unique personal identification numbers.Baseline differences by SRH were analyzed using the Chi-squared test and Fisher’s exact test. Hazard ratios (HRs) and their 95% confidence intervals (CI) for all-cause mortality were calculated using Cox proportional hazard models. Proportional hazards assumption was tested by using Martingale residuals. The follow-up periods were calculated from baseline measurements to the end of the follow-ups of 5-, 10- and 27-year or to the death of the person.Firstly, unadjusted Cox regression analyses were conducted for SRH and OH. Secondly, Cox regression analyses for OH was adjusted for gender and SRH for those possible confounding factors which were significantly associated with higher mortality risk at the follow-ups of 5-, 10- and 27- year and which did not have high between-correlation. The interaction between genders was included in Cox regression models. Thirdly, interaction between OH and SRH was analyzed, genders together and separately, in order to examine whether SRH gives any extra value in evaluation of mortality risk by OH.P-values less than 0.05 were considered statistically significant. All statistical analyses were performed by using SAS System for Windows, version 9.4 .p < 0.001) (Table p < 0.001).Overall, 10.2, 63.9, 25.9% of the participants reported having good, rather good, or poor SRH, respectively. Table Of the total of 1008 participants, 138 (13.7%), 319 (31.6%), and 932 (86.3%) deceased during the 5-, 10- and 27-year follow-ups, respectively. According to both unadjusted and adjusted Cox proportional hazard models, poor SRH and both moderate and poor OH were associated with significantly higher mortality risk during the 5-, 10- and 27-year follow-ups Tables . Accordip = 0.047) and poor OH (p = 0.015) predicted significantly higher mortality risk among men  than women  during the 27-year follow-up.The association of SRH and mortality did not significantly differ between men and women at any given follow-up; both moderate  unadjusted models, poor SRH predicted mortality stronger than having a moderate or poor OH in 5- and 10-year follow-ups. After adjustments of SRH with multiple variables, which were significantly associated with higher mortality risk at given follow-ups, the association of poor SRH with mortality was still almost as strong as the association of poor OH with mortality in 10-year follow-up. In 27-year follow-up, although the predictive value of OH was higher than that of SRH poor self-rated health still predicted higher mortality risk. In consistence with our results, several earlier studies have shown that poor SRH is associated with higher mortality risk , 24–26 bSingle-item self-reported scales, such as SRH, are often overlooked as they are perceived as less reliable and more sensitive to contextual effects –7. HowevThe findings of previous studies have been contradictory in relation to gender differences in the association of SRH and mortality . In our The strengths of this study were a large sample size with a high response rate and a long follow-up period. SRH was assessed by a global SHR question, which has found to be more accurate for prediction of mortality in old age than for example, comparative question . In our Findings of our study provide additional evidence supporting the value of incorporating a single-item measure of SRH into risk assessment of older adults in busy clinical settings. Routinely collecting SRH may also enhance the focus on patient-centered care.Additional file 1. Variables used in the assessment of objective health status


It is known that some mutant peptides, such as those resulting from missense mutations and frameshift insertions, can bind to the major histocompatibility complex and be presented to antitumor T cells on the surface of a tumor cell. These peptides are termed neoantigen, and it is important to understand this process for cancer immunotherapy. Here, we introduce an R package termed Neoantimon that can predict a list of potential neoantigens from a variety of mutations, which include not only somatic point mutations but insertions, deletions and structural variants. Beyond the existing applications, Neoantimon is capable of attaching and reflecting several additional information, e.g. wild-type binding capability, allele specific RNA expression levels, single nucleotide polymorphism information and combinations of mutations to filter out infeasible peptides as neoantigen.http://github/hase62/Neoantimon.The R package is available at  Recent technological advances in massively parallel sequencing have enabled the identification of genetic variants, e.g. single nucleotide variants (SNVs) and insertions or deletions (indels), in individual cancer patients. Furthermore, substantial evidence indicates that tumor-specific peptides that result from such variations can bind to a major histocompatibility complex (MHC) molecule and be presented to antitumor T cells on the surface of a tumor cell. Identification of such tumor-specific peptides, termed neoantigens, has been receiving increasing attention because of its numerous potential applications in cancer immunotherapy . Several computational methods such as netMHCpan  a text file storing gene sequence variations and (ii) a list of HLA types identified by, e.g. ALPHLARD  a/N, f(c)), where c corresponds to a uniform prior. They are attached to the output, as illustrated in Users can optionally provide (a) RNA expression data with and without (b) the corresponding Binary Alignment Map (BAM) file to attach total and allele-specific RNA expression levels and (c) copy number variation data to calculate the posterior probability distribution over cancer-cell fraction (CCFP) to evaluate tumor subclonality  SNPs data, and multiple SNVs on the same mutant peptides and among the frameshift region caused by indels. These cases are explained in The comparison of the functions among pVACseq , (v) chromosome number, (vi) NM_ID, (vii) amino acid changes (AAchages), (viii) reference and alternative alleles, (xi) exon start and end positions, (x) mutation position, (xi) total read depth and VAF, (xii) corresponding total and allele specific RNA expression , (xiii) tumor subclonality represented by CCFP and the priority score defined in the next subsection. From the output file, users can extract a plausible set of peptides for neoantigen by setting any threshold for binding capability, RNA expressions and CCFP . A simple overview of the package is illustrated in This package generates FASTA files consisting of mutant and corresponding wild-type (for SNVs) peptides according to the RefSeq trasncript sequences, and an integrated output file including peptide–MHC binding capability estimated by NetMHCpan4.0  Grant Number 18H03328 from Japan Society for the Promotion of Science (https://www.jsps.go.jp/ english/).Financial Support: none declared.Conflict of Interest: none declared.


In this optimized protocol, we describe a simple method for obtaining homogenous samples followed by chemically tagging the 3′ termini of natively-purified structured ncRNA domains that are longer than 200 nts. This protocol replaces traditional hazardous radioactive labeling with fluorescence tagging, and eliminates laborious and time consuming RNA purification and concentration steps and replaces them with straightforward recovery of RNA through centrifugal filtration, preserving the homogeneity and mono-dispersion of the preparations. The protocol provides:•An integrative, simple and straightforward approach for synthesis, purification and labeling of structured ncRNAs whilst maintaining their secondary structure intact.•Replacing hazardous, laborious and time-consuming radioactive labeling of RNA with much simpler fluorescence tagging, thereby facilitating potential downstream applications such as electrophoretic mobility shift assay (EMSA).•A versatile protocol that could be applicable to a wide-range of chemical tags and in principle could be used to label DNA or RNA.Noncoding RNAs (ncRNAs) comprise a class of versatile transcripts that are highly involved in the regulation of a wide range of biological processes. Functional long ncRNAs (> 200 nts in length) often adopt secondary structures that arise co-transcriptionally. To maintain the secondary structure elements as well as preparation homogeneity of such transcripts, native-like conditions should be maintained throughout the   Specifications Tablein vitro. To overcome this, a number of protocols had been developed for native purification of in vitro synthesized RNA, where disruptive or denaturing steps are entirely avoided Non-coding RNAs (ncRNAs) have been established as important regulators in a multitude of biological processes. Functional ncRNAs, including long noncoding RNAs  usually adopt elaborate secondary structures that often arise co-transcriptionally. Such structures enable their functions through interactions with other macromolecules Labeling of RNAs with either chemical or radioactive tags is an integral part of a number of lab procedures. While radioactive tags offer the advantage of sensitivity, they are hazardous, require specialized equipment as well as specific safety precautions, and offer limited lifespan. Chemical tags, including fluorophores, provide an alternative to radioactive isotopes that is safer and easier to handle while being sufficiently sensitive for an array of downstream applications, in addition to expanding the range of these applications. For example, fluorescence resonance energy transfer (FRET) and fluorescence polarization (FP) assays rely on chemical properties of fluorescence dyes and cannot be performed with radio-labels. End-labeling of RNAs is usually preferred to in-body labeling, as it is cheaper, simpler and more versatile Here, we describe an optimized straightforward protocol for 3′ end-labeling of purified structured ncRNAs while maintaining the homogeneity of the preparations and, therefore, their potential co-transcriptional folds. The native-like conditions are maintained throughout the protocol by replacing all harsh denaturing steps of RNA recovery, such as electro-elution and salt-ethanol precipitation, with centrifugal filtration throughout the purification and labeling stages. Following this protocol, we managed to synthesize and purify ncRNA fragments with lengths ranging between ~ 250 – 700 nts. These fragments were then successfully chemically-tagged through periodate oxidation while preserving the homogeneity and native-like state of our preparations.As a general rule, working with RNA requires maintaining RNase-free conditions at all times. Therefore, all solutions should be prepared with Diethyl Pyrocarbonate (DEPC) - treated water, and all consumables and glassware should be sterilized with DEPC-water and autoclaved. The most widely-accepted recipe for preparing DEPC- treated water includes addition of 0.1% DEPC, incubation for 1 hour at 37 °C followed by autoclaving for several hours/overnight to ensure the elimination of ethanol and other by-products of DEPC treatment that might interfere with enzymatic reactions.e.g. T7 or SP6 promoter) and at the 3′ end by a unique restriction site that enables linearizing the plasmid prior to in vitro transcription Generally, templates of RNAs of interest should be inserted into a suitable vector so that they are immediately flanked at the 5′ end by a promoter sequence  and carried over as an insert in the pUC57 vector between the unique restriction sites of BamHI and Sma1 . Sequences that were produced by PCR and purified with a PCR cleanup kit (Qiagen) were used as templates. In vitro transcription was carried out with 6 µg/mL of template DNA and 10x transcription buffer  in addition to T7 polymerase, 2 mM of each of the four rNTPs (Invitrogen) and RiboLock™ RNase Inhibitor  followed by incubation for 3 hours at 37 °C. Scavenging the pyrophosphates that are generated as a byproduct of the T7 polymerization activity might increase the transcription yield. This can be achieved by adding inorganic pyrophosphatase (iPPases) to the transcription reactions Transcripts of isolated fragments were generated with +2 concentrations could lead to the formation of non-specific aggregates.We found that incubating the transcription reactions for longer than 4 hours might result in producing abortive transcripts. It is worth-noting as well that the concentration of magnesium ions in the transcription mixtures could substantially impact the T7 activity as well as the homogeneity of the RNA preparation, and therefore should be optimized for every ncRNA fragment see following sections), such as the commercially-available AmiconⓇ Ultra Centrifugal Filters (Millipore). The AmiconⓇ filters are commonly utilized for the concentration and desalting of protein samples prior to various chromatography applications, such as HPLC, and they are ideal for removal of solutes, nucleotides, primers and other low molecular weight components from reactions. This step allows for buffer exchange as well as filtering out unreacted NTPs, digested proteins and DNA fragments as well as short abortive transcripts from our RNA preparations.RNA purification and recovery was performed under native-like conditions maintaining the samples at room temperature at all time, as previously described 2 and DNase I  and incubated for 30 min at 37 °C, and subsequently with proteinase K  and incubated for 45 min at 37 °C. Immediately afterwards, reactions were diluted with a HEPES-based filtration buffer  and applied to Amicon Ultra-4 centrifugal filters . Centrifugation was done at 4000 g for 15 min at room temperature. Following filtration, Nanodrop measurements were taken to ensure sample purity and homogeneity as well as sufficient elimination of enzymes and other proteins as represented by an A260/A280 nm absorbance ratio that ranges between 2.0 and 2.5.Following transcription, the reactions were supplemented with 1.2 mM CaClIt is important to maintain native conditions throughout the whole protocol. Thus, RNA purification and recovery steps that involve salt-ethanol precipitation, freezing or extreme agitation of RNA should be avoided entirely in order to keep the secondary structure of the RNA domains intact. In addition, digestion of proteins with proteinase K followed by centrifugal filtration is often sufficient for the removal of enzymes such as T7 polymerase The integrity and homogeneity of the reactions should be consistently monitored throughout this stage through gel electrophoresis using either native polyacrylamide or native agarose gel and visualization by ethidium bromide staining. A well-defined single band for each RNA species could serve as a preliminary indicator to the homogeneity of the preparation b. AdditiNonetheless, we found that using a processive T7 combined with multiple steps of centrifugal filtration was often enough to obtain concentrated homogenous samples, eliminating the need for further purification. It is also worth noting that homogenous preparations of the whole-length DACOR1 lncRNA molecule (~1800 nts) were obtained following the same synthesis and purification protocol b.The strategy of 3′ end-labeling of RNA through periodate oxidation has long been established Nonetheless, typical protocols of 3′ end-labeling do not generally consider RNA secondary structure and might involve common handling or purification steps that are disruptive to the secondary structure elements of the RNA samples, rendering such samples potentially unsuitable for secondary structural analysis. The fluorescence end-labeling protocol described in this section maintains the RNA secondary structure intact. This optimized protocol eliminates disruptive steps  and instead replaces them with direct filtration using centrifugal filters. Fluorescence dyes that could be used in this method are generally cyanine hydrazides, namely cyanine 3 (Cy3) and cyanine 5 (Cy5). For our RNA domains, we utilized Cy5 throughout this protocol.Unlike other labeling methods, such as radioactive tags, periodate oxidation is specific for RNA because it requires vicinal hydroxyl groups. Additionally, a wide range of aldehyde-reactive fluorophores could be used. Immediately after RNA recovery, oxidation reactions were assembled as described in 4 of the reaction. We found that the Amicon filtration was sufficient to stop the oxidation reaction, rendering the traditional step of precipitating the IO4− ions out of the solution by adding another monovalent salt unnecessary. For optimal concentration of our RNA preparations, we used the Amicon Ultra-0.5 centrifugal filters  and centrifuged for 30 minutes at 14,000 g.After incubation, we proceeded immediately to Amicon filtration to eliminate the NaIOAn additional advantage for using gravity-based or centrifugation- based filtration methods is the elimination of further RNA precipitation steps that might involve salt-ethanol or prolonged incubation on ice, both of which were found to disrupt the secondary structure of RNA and therefore should be avoided entirely when preparing RNA samples for secondary structural studies. It is worth-mentioning that lyophilization of RNA samples should also be avoided, as it probably leads to the formation of aggregates that might interfere with further methods To prepare Cy5 labeling solution from the Cy5 hydrazide salt stock, a vast excess of the dye salt is dissolved in neat DMSO to prepare a solution of 5 mM final concentration. The dye solution should then be divided into aliquots of 10 µl each and dried by centrifugation under vacuum (Speed-Vac). Aliquots are stored at −20 °C in the dark and, before assembling a conjugation reaction, an aliquot should be re-suspended in 10 µl of neat DMSO.Immediately after recovering the oxidized RNA, assemble the conjugation reactions as described in Similar to the step of recovering the oxidized RNA, purifying and concentrating the labeled RNA by means of Amicon centrifugal filters here replaces the traditional precipitation of RNA using salt-ethanol precipitation/ freezing methods. The integrity and purity of labeled RNA is examined by resolving the samples on either native polyacrylamide or native agarose gel, and visualized using an appropriate imaging system/ machine. We examined our labeled RNA samples  by resolving on 1% native agarose gel for ~ 1 hour. The wet gels were then visualized using a Typhoon FLA-9500 machine equipped with a 635 nm excitation laser source giving rise to an emission peak of the Cy-5 at wavelength of around 670 nm (LPR filter). Images were analyzed by the ImageQuant and Fiji Imagej software. For the total RNA, gels were stained with ethidium bromide and visualized using a UV-based gel documentation system (Bio-Rad). Image analysis of band intensities to estimate labeling efficiency as average ratios of  returned an estimated percentage ranging between 70 and 75% . AlthougIn this optimized protocol, we describe a simple method for chemically tagging the 3′ end of natively-purified structured ncRNA domains without compromising their secondary structures. Moreover, we eliminate laborious and time consuming RNA purification and concentration steps and replace them with straightforward recovery of RNA through Amicon centrifugal filtration. The whole protocol can be done on the bench top and does not require specialized equipment or specific safety precautions, compared to radioactive labeling. The resulted chemically-tagged ncRNAs could be used in a multitude of downstream applications; for example, to detect the binding potential to proteins using electrophoretic mobility shift assay (EMSA), and could be particularly useful in quantitative assays that depend on the properties of fluorescence dyes such as FRET and FP assays. Wet-gel scanning and detection could be performed relatively easily, using equipment that is readily available such as Typhoon and Fuji Fluorescent Image Analyzer (FLA-5000 or FLA-9000).Overall, replacing traditional hazardous radioactive labeling with fluorescence labeling facilitates the handling process of RNA and eliminates the need for specialized equipment as well as elaborate safety precautions. The activity of the recovered labeled ncRNA domains could subsequently be closely examined in their native conformation with a versatile wide-range of downstream experiments such as EMSA. This protocol is applicable to several types of chemical tags and, although we mainly use ncRNA fragments throughout the protocol, could be used for modifying both DNA and RNA.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.