Ixodes ricinus cDNA library, and studied the possible role of SUB as a chromatin remodeler through direct interaction with histones. The Y2H screening identified Importin-α as a potential SUB-interacting protein, which was confirmed in vitro in a protein pull-down assay. The sub gene expression levels in tick midgut and fat body were significantly higher in unfed than fed female ticks, however, the importin-α expression levels did not vary between unfed and fed ticks but tended to be higher in the ovary when compared to those in other organs. The effect of importin-α RNAi was characterized in I. ricinus under artificial feeding conditions. Both sub and importin-α gene knockdown was observed in all tick tissues and, while tick weight was significantly lower in sub RNAi-treated ticks than in controls, importin-α RNAi did not affect tick feeding or oviposition, suggesting that SUB is able to exert its function in the absence of Importin-α. Furthermore, SUB was shown to physically interact with histone 4, which was corroborated by protein pull-down and western blot analysis. These results confirm that by interacting with numerous tick proteins, SUB is a key cofactor of the tick interactome and regulome. Further studies are needed to elucidate the nature of the SUB-Importin-α interaction and the biological processes and functional implications that this interaction may have.Ticks and tick-borne diseases (TBDs) represent a burden for human and animal health worldwide. Currently, vaccines constitute the safest and most effective approach to control ticks and TBDs. Subolesin (SUB) has been identified as a vaccine antigen for the control of tick infestations and pathogen infection and transmission. The characterization of the molecular function of SUB and the identification of tick proteins interacting with SUB may provide the basis for the discovery of novel antigens and for the rational design of novel anti-tick vaccines. In the present study, we used the yeast two-hybrid system (Y2H) as an unbiased approach to identify tick SUB-interacting proteins in an  Ticks are obligate blood-feeding arthropod vectors of pathogenic viruses, bacteria, protozoa and helminths, and are responsible for highly prevalent tick-borne diseases (TBDs) worldwide . Ticks aIxodes scapularis  (XP_029836069.1) and there is little information related to the role of this protein in the tick interactome. However, the B7PAA1 transforming acidic coiled-coil containing protein has been implicated in mitotic spindle organization  and it is linked to different types of cancers in humans  XP_0836069.1 n humans ,31,32. Tnfection , suggestnfection ,35.importin-α gene family has undergone broad expansion during eukaryotic evolution. In humans, the genome contains several genes coding for Importin-α . I. II. scaer 2020) C. In tic4D5RQ98) C. It hassub and importin-α gene expression in vivo in I. ricinus ticks to gain information on the SUB-Importin-α interaction and its functional implications.Tick SUB can be found either in the cytoplasm or in the nucleus, in relation to its two NLS domains involved in protein transport into the nucleus . While isub and importin-α in various I. ricinus organs including midgut, fat body, ovary, salivary glands and Malpighian tubules of unfed and fed female ticks  and importin-α (41–87%) gene knockdown was observed in all tick tissues  is 100% homologous to the human ortholog  medium) and supplemented with only 5 mM of 3-amino-1,2,4-triazole . A mating strategy was used for screening an Ixodes ricinus tick cDNA library cloned into the Gal4-AD pDEST22 vector (Invitrogen) and previously established in Y187 yeast strain (Clontech Laboratories) [HIS3 reporter gene. AD-cDNAs from [His+] colonies were amplified by PCR and sequenced to identify the proteins interacting with SUB.Our yeast two-hybrid (Y2H) protocol was described in detail by Caignard et al. . Brieflyatories) . Yeast cI. ricinus ticks from a laboratory colony  were used for this experiment. For fed female organ dissection, female ticks were fed in vivo for 4 days using rabbits as host until they were partially-fed. The five principal organs  from unfed and fed female I. ricinus  were dissected and put into TRIzol Reagent (Invitrogen) for RNA extraction. RNA was extracted using Direct-Zol RNA Miniprep Plus Kit . RNAs were cleared of gDNA and retrotranscribed using iScript gDNA clear cDNA synthesis kit . cDNAs were used to characterize the expression profile of SUB and Importin-α within different organs of unfed and fed I. ricinus females by qPCR using gene-specific oligonucleotide forward (F) and reverse (R) primers , SsoAdvanced Universal SYBR Green Supermix (BioRad) and the CFX96 Touch Real-Time PCR Detection System (BioRad). A dissociation curve was run at the end of the reaction to ensure that only one amplicon was formed, and that the amplicons were denatured consistently in the same temperature range for every sample. The cDNA levels were normalized against tick elongation factor  using the genNorm Delta-Delta-Ct (ddCt) method as previously described [p < 0.05).Unfed and fed adult female escribed . NormaliI. scapularis histidine-tag recombinant SUB (Genbank accession number AY652654.1) with optimized codon usage for Escherichia coli was produced in E. coli BL21 and purified to >95% purity by Ni affinity chromatography using 1 mL HisTrap FF columns mounted on an AKTA–FPLC system  in the presence of 7 M urea lysis buffer as previously described [I. scapularis recombinant Importin-α fused to glutathione-S-transferase (GST), also produced in E. coli , were incubated in 100 mM HEPES (pH 7.5) and 10 mM imidazole (binding and wash buffer) for 1 h with shaking, at a concentration ratio of 2:1. HisLink Protein Purification Resin  was washed following the manufacturer’s recommendations and blocked with 1% BSA  for 30 min with shaking. Then, the resin was washed with binding/wash buffer and incubated with SUB-Importin-α interaction (2:1) for 1 h with shaking. For the negative control, the same resin was incubated only with Importin-α to verify that only his-tag SUB was able to link to the resin. After the 1 h incubation, the supernatant was removed, and the resin was washed twice with binding/wash buffer. SUB-Importin-α complexes were eluted from the resin using elution buffer . The eluted fractions were recovered and Laemmli sample buffer with β-mercaptoethanol was added for Western blot analysis. Recombinant Importin-α (6 µg) was used as a positive control. Samples were separated by electrophoresis in a 12% sodium dodecyl sulfate (SDS) polyacrylamide precast gel  and transferred to a nitrocellulose blotting membrane . The membrane was blocked with 3% BSA in Tris-buffered saline  for 2 h at room temperature (RT) and washed four times with TBS-0.05% Tween 20. Human KPNA6-specific primary antibodies  were used for Importin-α detection. Antibodies were diluted in TBS and incubated with the membrane overnight at 4 °C. Goat anti-rabbit IgG (whole molecule) peroxidase antibodies  diluted in TBS with 3% BSA were used as secondary antibodies and incubated with the membrane for 2 h at RT. The membrane was finally washed five times with TBS-0.05% Tween 20, and immunoreactive proteins were visualized with chemiluminescence by incubating the membrane for 1 min with Pierce ECL Western blotting substrate (Thermo Fisher Scientific).The synthetic escribed . RecombiI. ricinus nymphs (n = 3–5 nymphs) partially fed using an in vitro feeding procedure with bovine blood [Total RNA was extracted from ne blood . Whole nI. ricinus sub and importin-α  and used for sub and importin-α dsRNA synthesis from I. ricinus nymph cDNA using the Phusion™ High-Fidelity DNA Polymerase (Thermo Fisher Scientific) and the T7 RiboMAX Express RNAi System (Promega). The unrelated gene coding for green fluorescent protein (GFP) dsRNA was synthesized from the L3790 plasmid following the same protocol and used as a negative control. L3790 was a gift from Andrew Fire . The SUB dsRNA was used as a positive control as previously described [I. ricinus adult female ticks (n = 80 per group) were injected with approximately 0.5 μL of dsRNA (7 × 1011–8 × 1011 molecules/μL) in the lower right quadrant of the ventral surface of the exoskeleton [I. ricinus males  for artificial feeding. Feeding units were made and set up as previously described by Krull et al. [sub and importin-α RNAi tick groups were compared with controls by one-way ANOVA test with post-hoc Tukey HSD .Oligonucleotide forward (F) and reverse (R) primers containing T7 promoter sequences (underlined) at the 5′-end to support in vitro transcription and synthesis of dsRNA were synthesized for escribed . The dsRskeleton . The injl et al. . Bovine l et al.  and the l et al. . Data onsub and importin-α RNAi were assessed by qPCR on cDNA samples using gene-specific oligonucleotide primers whose targets differ from those of the dsRNAs , the Luna Universal qPCR Master Mix and the CFX96 Touch Real-Time PCR Detection System (BioRad). A dissociation curve was run at the end of the reaction to ensure that only one amplicon was formed, and the amplicons denatured consistently in the same temperature range for every sample. The cDNA levels were normalized against the tick elongation factor (elf) using the genNorm Delta-Delta-Ct (ddCt) method . Cycle threshold (Ct) values for normalized sub and importin-α dsRNA-treated samples were compared with those of the GFP control samples to determine levels of gene knockdown.After three days of artificial feeding, six fed females per group were selected for RNA extraction. Dissection of fed females was performed to separate the five major organs: salivary glands, fat body, Malpighian tubules, ovary and midgut . Organs were placed in 100 μL of TRIzol Reagent (Invitrogen) and total RNA was extracted and gDNA removed using the Direct-Zol RNA Miniprep Plus Kit (Zymo Research Europe GmbH) following the manufacturer’s recommendations. cDNAs of each sample were reverse-transcribed using the ProtoScript II First Strand cDNA Synthesis Kit . Gene knockdown levels after 2.6 Gene knockdown by RNAi in ticks) were used to assess the sub mRNA levels after importin-α knockdown and the importin-α mRNA levels when sub was knocked down. The Ct values were normalized against the tick elf using the genNorm Delta-Delta-Ct (ddCt) method as described above. Normalized sub and importin-α Ct values were compared to those of the GFP control samples to determine their concentration in the absence of the corresponding interacting protein.The cDNAs obtained above (see I. scapularis SUB and Anaplasma phagocytophilum heat shock protein 70 (HSP70) as a negative control. Both recombinant proteins, SUB and HSP70, were produced in E. coli as previously described [w/v), 0.1% Tween-20) at 2 μM concentration and incubated overnight at 4 °C. The next day, arrays were washed with array buffer and incubated with a protein-specific 1:500 dilution of primary rabbit polyclonal antibodies for SUB [p < 0.05) and One-Way ANOVA  tests .EpiTriton Histone Peptide Arrays were used following the manufacturer’s recommendations  with recombinant escribed . Protein for SUB  and HSP7 for SUB  for 2.5 I. scapularis SUB was produced as described above and used for interaction with the synthetic peptide of human H4 K5,8,12 but  5GGK (but) 8GLGK (but) 12GGAKRHRKVLR-Peg (Biot), butyrylated at lysines 5, 8 and 12). Dynabeads MyOne Streptavidin T1 (Invitrogen) was used following the manufacturer’s instructions. As a negative control, the same procedure was carried out but without incubation with the H4but peptide. The recombinant SUB was used as a positive control. Eluted protein-H4 complexes and positive and negative controls were separated by electrophoresis in a 12% SDS polyacrylamide precast gel (ClearPage) and transferred to a nitrocellulose blotting membrane . The membrane was blocked with 3% BSA in TBS  for 2 h at RT and washed four times with TBS-0.05% Tween 20. SUB-specific primary antibodies diluted in TBS were used for SUB detection and incubated with membrane overnight at 4 °C. Goat anti-rabbit IgG (whole molecule) peroxidase antibodies  diluted in TBS with 3% BSA were used as secondary antibodies and incubated with the membrane for 2 h at RT. The membrane was finally washed five times with TBS-0.05% Tween 20, and immunoreactive proteins were visualized with chemiluminescence by incubating the membrane for 2 min with Pierce ECL Western blotting substrate (Thermo Fisher Scientific) and with TMB Stabilized Substrate for Horseradish Peroxidase (Promega).The recombinant importin-α has been described to be regulated by miRNAs in humans [Anaplasma phagocytophilum are able to manipulate the tick miRNA profile to facilitate infection by upregulating isc-mir-79 in I. scapularis tick cells [importin-α are still unknown. In light of our results, along with the previously described functions of Importin-α in gene regulation and epigenetic modifications, further studies will be required to unravel the significance of the SUB-Importin-α interaction in different biological contexts. Moreover, the characterization of the SUB interactome and its interacting proteins such as Importin-α opens the possible identification of new targets for interventions to control tick infestation and pathogen infection that would contribute to vaccine development.The characterization of the interactome of a co-factor regulatory protein such as SUB is crucial to understand its function in the cell regulome. The physical interaction of SUB with Importin-α suggested one pathway of entrance into the nucleus for SUB, but the results also suggested that importin-α mediated entry may be not the only mechanism. Moreover, SUB has evolved to exert several functions and to interact with various other proteins in these capacities. The interaction of SUB with histone H4 also suggested new mechanisms underlying a role for this protein in chromatin remodeling and the regulation of gene expression. The expression of n humans . For exan humans . SUB/AKRn humans ,58. Pathck cells , but the


Plasmodium falciparum has not been determined. To investigate the genome-wide RNA secondary structural features at single-nucleotide resolution in P. falciparum, we applied a novel high-throughput method utilizing the chemical modification of RNA structures to characterize these structures. Structural data from parasites are in close agreement with the known 18S ribosomal RNA secondary structures of P. falciparum and can help to predict the in vivo RNA secondary structure of a total of 3,396 transcripts in the ring-stage and trophozoite-stage developmental cycles. By parallel analysis of RNA structures in vivo and in vitro during the Plasmodium parasite ring-stage and trophozoite-stage intraerythrocytic developmental cycles, we identified some key regulatory features. Recent studies have established that the RNA structure is a ubiquitous and fundamental regulator of gene expression. Our study indicate that there is a critical connection between RNA secondary structure and mRNA abundance during the complex biological programme of P. falciparum. This work presents a useful framework and important results, which may facilitate further research investigating the interactions between RNA secondary structure and the complex biological programme in P. falciparum. The RNA secondary structure characterized in this study has potential applications and important implications regarding the identification of RNA structural elements, which are important for parasite infection and elucidating host-parasite interactions and parasites in the environment.It is widely accepted that the structure of RNA plays important roles in a number of biological processes, such as polyadenylation, splicing, and catalytic functions. Dynamic changes in RNA structure are able to regulate the gene expression programme and can be used as a highly specific and subtle mechanism for governing cellular processes. However, the nature of most RNA secondary structures in  In this study, we showed that (1) RNA secondary structures in P. falciparum have common characteristics similar to those of other diverse organisms, such as UTR regions, CDS regions, noncoding RNAs, and malaria mitochondrion. (2) Structural changes are dramatically different in the ring and trophozoite stages but less pronounced in the ring stage than in the trophozoite stage. (3) Comparing the in vivo and in vitro structures indicates the important role of RNA-binding protein (RBP) in structure formation. (4) Combined with transcriptome data, RNA secondary structure changes were significantly associated with transcriptome changes. Therefore, our results make it possible to obtain a framework for understanding how malaria parasites develop through changes in RNA structure in vivo and potential use for further investigations. Our results imply that there is a critical connection between the RNA secondary structure and the complex biological programme of Plasmodium falciparum, but the mechanism governing this process needs to be further elucidated.The major strength of this study is that it explained the landscape of the RNA secondary structure associated with parasite development at single-nucleotide resolution. Our analysis profiled the structure of more than 3,396 .For eviously  before tin vitro NAI-N3 probing, we also needed to construct total RNA/ribosomal in vitro SHAPE RNA libraries. For total RNA/ribosomal in vitro SHAPE RNA libraries, no additional processing was needed. For polyA-selected in vitro SHAPE libraries, polyA selection was performed first using polyT oligomagnetic beads before NAI-N3in vitro modification was undertaken. Heat-denatured total RNA or polyA+ RNA samples were obtained from DMSO-treated pellets at 95°C for 2 min and were later transferred to ice to cool. The NAI-N3 final concentration and times for in vitro modification were 100 mM and 37°C for 10 min. The reactions were stopped by moving the samples to ice, and the samples were purified using a Zymo RNA Clean & Concentrator-5 column according to the manufacturer’s protocol.For 3 concentration can strongly affect single-hit kinetics; therefore, we focused on optimizing these two parameters, although it is possible that other factors, such as temperature and buffer conditions, could also affect these kinetics  was treated with DNase (NEB) to remove residual genomic DNA in the RNA sample. Next, the DNA-free total RNA was reverse-transcribed using SuperScript III (Invitrogen) by a colour-coded fluorescently FAM-labelled oligonucleotide in the 271-bp-293-bp region of the 18S rRNA gene. The PCR programme was as follows: samples were heated to 65°C for 5 min, maintained on ice for at least 2 min, and incubated at 50°C for 1 h followed by 95°C for 5 min, and the samples were maintained at 4°C after the reaction ended. The reverse-transcribed cDNA products were electrophoresed using an ABI 3730XL DNA Sequencer. The results were presented by GeneMarker Incubation time and NAI-Nkinetics . We tookTotal RNA was extracted from parasite lysates without any chemical reagent modification. In a 20-μL reaction, ~2 μg of total RNA was fragmented using RNA fragmentation reagents (Ambion) for the proper time at 70°C. After the end of the experiment, 10× stop solution was added to each sample, and the samples were immediately placed on ice. Next, fragmented RNA was purified with a Zymo RNA Clean & Concentrator-5 column. The purified fragmented RNA was reverse-transcribed by primer extension with gene-specific colour-coded fluorescently FAM-labelled oligonucleotides (5’-ACCCTAACATCAAAAGCTGATAGG-3’), as described above. The length of every fragment was assessed using an ABI 3730XL DNA Sequencer (Applied Biosystems). The results were shown by GeneMarker .P. falciparum 3D7 assembly release-33 downloaded from PlasmoDB . Next, read counts were collected, and gene expression levels among different samples were estimated.For RNA-seq library construction, the NEBNext Ultra Directional RNA Library Prep Kit (NEB) was used to prepare a validated strand-specific, polyA-selected RNA sequencing library according to the manufacturer’s instructions in the kit. Briefly, mRNA was purified from 3 µg of total RNA using the Poly(A) mRNA Magnetic Isolation Module. Fragmentation was carried out at 94°C for 7 min in NEBNext First Strand Synthesis Reaction Buffer. First strand cDNA was synthesized using random primers and ProtoScript II Reverse Transcriptase (with 0.1 µg/µl actinomycin D and murine RNase inhibitor). Second strand cDNA was synthesized using Second Strand Synthesis Enzyme Mix and incubated at 16°C for 1 h. Next, the double-stranded cDNA was purified using 1.8X Agencourt AMPure XP Beads. End prep of a cDNA library was performed by NEBNext End Prep Enzyme Mix at 20°C for 30 min followed by 65°C for 30 min. NEBNext adaptors were ligated to the cDNAs with the function of Blunt/TA Ligase Master Mix at 20°C for 15 min, and fragments measuring 150–200 bp were purified using AMPure XP beads. The samples were later treated with 3 μl NEBNext USER Enzyme at 37°C for 15 min before PCR amplification. Library amplification was performed using NEBNext Q5 Hot Start HiFi PCR Master Mix, NEBNext Universal PCR Primer for Illumina, and Index (X) Primer. The primer and oligonucleotide sequences used in the experiment are shown in 3 at 37°C in a thermomixer for 2 h at 1,000 r.p.m. with 4 U/µl RiboLock RNase inhibitor. Fragmentation was performed using RNA fragmentation reagent (Ambion) at 70°C for 3 min. Next, purified fragmented RNA was end-repaired by T4 polynucleotide kinase (NEB) and incubated at 37°C for 1 h. A 3’-biotin RNA linker or a 3’-ddC RNA linker were ligated to the end-repaired RNA samples by T4 RNA ligase 1. After each above step, the reaction was purified by a Zymo RNA Clean & Concentrator-5 column according to the manufacturer’s protocol.For the icSHAPE library, we used the methods developed by Ryan A Flynn et al. as described previously , with so3-modified molecules from cDNA products, the concentrated cDNA samples were purified with a Zymo DNA Clean & Concentrator-5 column. The size-selected cDNA was circularized by 5 U/µl CircLigase II (Epicentre) and incubated at 60°C for 2 h. Amplification was performed using NEBNext High-Fidelity 2X PCR Master Mix, Solexa-P5 primer, and Solexa-P3 primer. The qPCR machine was programmed as follows: 98°C for 45 s, 98°C for 15 s, 65°C for 20 s, and 72°C for 30 s. For the in vivo and in vitro NAI-N3-treated samples, 14 cycles were employed, and for DMSO-treated samples, 11 cycles were employed. Finally, the PCR-amplified library was gel size-selected using 10% TBE PAGE to obtain a size between 200 bp and 300 bp. The primer and oligonucleotide sequences used in the experiment are shown in First strand cDNA was subsequently synthesized using 1 µl of 1 µM RT primer (with a 4-nt barcode for separate samples in one sequencing lane). After reverse transcription, isolation by MyOneC1 streptavidin beads and size selection (>70 nt) of NAI-NFinal icSHAPE library material was quantified on a Bioanalyzer High Sensitivity DNA Chip 2100 (Agilent) and Qubit and was sent for deep sequencing and Illumina HiSeq analysis at Vazyme Biotech Co., Ltd.  and GENEWIZ Company .The quality of all PF data from Illumina’s HiSeq sequencer was assessed by FastQC (Babraham Bioinformatics), and the percentages of reads with Q20, Q30, and GC content were calculated. All sequenced libraries were collapsed to remove PCR duplicates, reads containing adapters, reads containing polyN, and low-quality scores.P. falciparum genome and transcriptome  using bowtie  removed by using Trimmomatic , and reag bowtie  implemened tools . All icS library . To redu library . The finhttp://rna.tbi.univie.ac.at/) with five different colours. Specifically, the coloured base pair was placed into the known regions of the 18S rRNA secondary structure, and the experimental single/double results were subsequently checked against the known ways of pairing. For the RNA secondary structure profile, all selected secondary structure models were generated using RNAstructure 6.1 12 on a Windows operating system (http://rna.urmc.rochester.edu/RNAstructure.html) with and without hard constraints due to the extracted icSHAPE score profiles of all single RNAs that were calculated  In our approach, the nucleotides that have icSHAPE scores above a specified threshold (1.5) are forced to be single-stranded. Otherwise, the positions are treated as being inaccessible to chemical modification, i.e., double-stranded or crowded by specific protein. We also used the current best parameters  for icSHAPE-directed RNA structure prediction. Alternatively, webserver tools were also used at http://rna.urmc.rochester.edu/RNAstructureWeb/. RNA structures were drawn and coloured using StructureEditor.An increasing number of online web services or software can be used for the prediction of RNA secondary structure . In our P. falciparum, NAI-N3 should be titrated to single-hit kinetics in structure probing. In our pilot experiments, we used capillary electrophoresis (CE)  , which h8S rRNAs  (Supplevia RT-PCR and sequencing. Short RNAs (measuring~100 nt) are the most common strategies that have been optimized to achieve single-hit kinetics of chemical modification, which can result in ~10% of all transcriptions being structurally informative  has been published  with five different colours according to the icSHAPE scores generated from icSHAPE-seq . The coloured base pair was placed into the known regions of the 18S rRNA secondary structure, and the experimental single/double results were subsequently checked against the known ways of pairing.To evaluate the accuracy of our NAI-Nublished ; therefoin vivo icSHAPE scores (defined as 1.5) in our data set correspond to single-stranded regions in the phylogenetic structure (41.0%), whereas 56.1%  of the bases that show low in vivo icSHAPE scores in our dataset correspond to base-paired regions in the phylogenetic structure (55.2%) and presumably are protected by either ribosomal proteins or non-canonical base-pairing tertiary RNA structures. Our data showed that there is a high correlation between icSHAPE scores and the known 18S A-type RNA secondary structure. We set the sites with icSHAPE scores above 1.5 to be localized to regions of single-stranded RNA and were consistent with traditional primer extension analysis and known RNA secondary structures. NAI-N3 modification has no specific RT stops with almost equal numbers of four bases, consistent with the known absent or minor (3 samples for As and Us) base-specificity of NAI-N3 , approximately 41.0% of bases are single-stranded, 55.2% of bases are double-stranded, and 3.8% (80 nt) are indeterminate. In the entire 18S A-type rRNA, 43.9%  of the bases that show high or minor   were obtained from in vivo and in vitro from selected regions of 110 mRNAs , and the extent of folding varies in different regions of RNAs in ring stage and trophozoite stage. In the ring stage, the 5’ UTRs exhibited noticeable but partial unfolding, with the largest variation being observed between the CDS regions and the 3’ UTRs. These three different regions of RNAs have nearly the same icSHAPE scores in vivo, but the CDS region has higher icSHAPE scores than the 5’ UTR and 3’ UTR when the RNA structure is modified in vitro. Then, we compared the average icSHAPE score collected from three different regions of RNAs in trophozoite stage, the results also exhibited higher icSHAPE scores in vitro than that in vivo. Collectively, our data suggest that RNAs are more folded or crowded by a specific protein in vivo.The average icSHAPE score of the transcripts in vivo and in vitro across the CDS regions and UTR regions to quantify the distribution of icSHAPE reactivity profiles. Due to some nucleotides that were crowded in vivo and/or protein binding, or experimental limitations, we observed that icSHAPE scores from in vivo and in vitro are different. In our research, we found that RNAs are more folded or crowded in vivo (low icSHAPE scores), consistent with previous reports  to a level that is similar to the structural propensity of the 5’- and 3’-untranslated regions (UTRs). Additionally, our data indicated that lower icSHAPE scores in the sites from nt -25 to -21 in trophozoite stage than in the other sites of 5’UTR region of the transcriptome. This low icSHAPE scores means that those regions are more structured or protein binding sites of mRNA secondary structures. These positions may provide candidate sites for the functional conformation of mRNA, but require further investigation.Metagene analysis of the transcripts, which aligned at their start and stop codons, shows that We next investigated whether a correlation is shared between intrinsic mRNA secondary structure propensity around the translation start/stop site and whether the efficiency of protein translation correlates with mRNA abundance. In 3 which exhibited high reproducibility between biological replicates.It is clear that mRNA structure is important for a variety of biological processes, including maintenance of RNA half-life and stability . We nextin vivo and/or protein binding, they were not chemically reactive, and no complete coverage was be provided in our experiment for this long transcript, the Pearson correlations in our results seem not high but with reasonable. Therefore, we hypothesized that the RNA structure could regulate the activation/repression of gene expression in P. falciparum. Combined analysis of icSHAPE with transcriptome data demonstrated that stage-specific RNA structure plays an important role in the regulation of gene expression in P. falciparum.Comparison of the average icSHAPE score over the CDS revealed a very clear correlation with the mRNA abundance with this combined approach Figure 5P. falciparum for all four bases in vivo and in vitro at two developmental stages at single-nucleotide resolution. The major strength of this scarcity of our RNA secondary structure was its potential use and important implications for understanding parasite gene expression and development. Recent studies have established RNA structure as a ubiquitous and fundamental regulator of gene expression , using a novel bifunctional chemical probe, NAI-N3, for in vivo RNA structure profiling in the genome has become a well-established tool for the analysis of RNA structure, and labelling each accessible single-stranded nucleotide provides a higher-resolution picture of the secondary structure of each transcript  even with low coverage. In vitro structural profiles from protein coding RNAs at the trophozoite stage is for further investigation.Using the new chemoaffinity structure probing methodology icSHAPE, we generated a map of the ‘RNA structurome’ in 3-independent stops occurred at known endogenously modified rRNA nucleotides; thus, we conclude that icSHAPE can successfully and accurately map chemically modified nucleotides on large RNAs on a genome-wide basis from Plasmodium parasites. Due to some nucleotides that were crowded in vivo and/or protein binding or experimental limitations, they were not chemically reactive, and no complete coverage was provided in our single experiment for this long transcript. Our partial but accurate RNA secondary structure can provide important information at some vital position of each transcript. Our icSHAPE results are strongly correlated with the results from our capillary electrophoresis-based probing method, and it can be used for accurate mapping of the RNA secondary structure. Our in vivo icSHAPE data are in excellent agreement with known RNA structures.Although there is significant global correspondence between our icSHAPE-modified RNA structures and known structures, there are still some differences between icSHAPE and known structures; one reason for the differences is the noise in our approach, and the other reason is the known inaccuracies of folding algorithms . TherefoPlasmodium and many genes expressed in other asexual developmental stages or mosquito/liver stages could not be obtained here, this study still provides important information on RNA secondary structure and the changes in structures between the two stages. Our study may have had several limitations. The depth was not enough to cover all of the whole transcripts, and larger studies with longer follow-up are needed to obtain RNA secondary structure information from the very low-level transcripts. Recently, a method named hiCLIP  can be found in the National Center for Biotechnology Information (NCBI) Sequence Read Archive under the BioProject ID PRJNA625343.YQ designed and performed the experiments, analysed data, prepared figures, writing, review, and editing original draft. YZ and GZ designed and performed the experiments, analysed data, and prepared figures. BC and MZ prepared figures and performed analysis. JL, TP, and JH review and editing. XW designed and supervised the study. All authors performed data quantification, discussed the results, and commented on the manuscript. All authors contributed to the article and approved the submitted version.This research was funded by Natural Science Foundation of China (Grant number: 81902087), Guangdong Natural Science Fund Project (Grant number: 2017A030310535), and China postdoctoral Science Foundation (Grant number: 2016M592474).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Myotonic dystrophy is the most common muscular dystrophy in adults. It consists of two forms: type 1 (DM1) and type 2 (DM2). DM1 is associated with a trinucleotide repeat expansion mutation, which is transcribed but not translated into protein. The mutant RNA remains in the nucleus, which leads to a series of downstream abnormalities. DM1 is widely considered to be an RNA-based disorder. Thus, we consider three areas of the RNA pathway that may offer targeting opportunities to disrupt the production, stability, and degradation of the mutant RNA. DMPK gene [CNBP gene [Myotonic dystrophy is the most common and complex form of adult muscular dystrophy . There aMPK gene ,3,4 wherNBP gene . In eachCDK12 and CDK13 are metazoan orthologs of the Saccharomyces cerevisiae gene Ctk1 [CDK12 and CDK13 have evolved by duplication and the proteins they encode share a 43% sequence similarity and a largely conserved kinase domain (KD) (>93%). Based on the interaction of CDK12 with overexpressed cyclin L, cyclin L was initially reported to be the regulatory subunit, and the same studies suggested a role in the regulation of alternative splicing. However, recent studies have reported that the endogenous human CDK12 does not associate with cyclin L but rather with cyclin K [During transcription, the C-terminal domain of RNA polymerase II is subjected to multiple regulatory phosphorylation events that control its transcriptional potential. Several cyclin-dependent kinases are associated with the regulation of transcription including CDK7, CDK8, CDK9, CDK12, and CDK13 . These kcyclin K . CDK12 acyclin K ,11,12. CCDK12 transcripts are expressed in all human tissues [ tissues  and CDK1 tissues . CDK12 m tissues .CDK12 was found to be located at the promoter sites of protein-coding genes. These sites usually overlap with RNA PII occupancy sites . Hence, Inhibitors of CDK12 have been developed and one such inhibitor, THZ531, when bound to CDK12 leads to the premature termination of transcript formation for genes involved in DNA repair and DNA replication , based oLR mouse model, leading to potentially beneficial effects [Medium-throughput phenotypic assays developed in our lab, based on the identification of nuclear foci in DM patient cell lines using in situ hybridization and high-content imaging, facilitated the identification of potentially useful therapeutic compounds . Other s effects . Ketley et al. showed the level of CDK12 protein was elevated by 48% in DM1 muscle biopsies compared to healthy control muscle samples. Further immunohistochemistry experiments demonstrated co-localization of CDK12 with SC35 speckles in both DM1 and non-DM1 cells, consistent with previous studies ,31. HencCDK12 is not involved at the initiation of the transcription process and its inhibition does not lead to global transcriptional arrest . It is tThe possibility of targeting transcription for therapeutic benefit in DM1 was raised by Siboni et al. , who shoLR mice prior to the progression of skeletal muscle weakness [LR mice were treated with inhibitors of GSK3β [In addition to CDK12, other kinases have been suggested as potential drug targets in DM1. One such case is glycogen synthase kinase 3β (GSK3β) . An incrweakness ,35. The of GSK3β . Clinicaof GSK3β .One of the best-studied aspects of the molecular pathway underlying myotonic dystrophy is the nuclear retention of mutant expansion transcripts, resulting in the sequestration of MBNL proteins and activation of CELF1 to produce RNA mis-splicing in several different transcripts ,38 Figu.CELF and MBNL proteins are antagonistic regulators of alternative splicing. In DM1, MBNL protein activity is repressed due to its sequestration by toxic RNAs, while CELF protein activity is upregulated by protein kinase C (PKC)-mediated CELF1 hyperphosphorylation  Figure .In addition to a role in alternative splicing regulation, it has been shown that MBNL plays a role in poly A site selection. Studies using HITS-CLIP and minigene reporter assays revealed that MBNL binding to the 3′ untranslated regions (3′UTRs) of target RNAs has a direct impact on polyadenylation site switching. MBNL-binding sites are frequently concentrated proximal poly(A) signals (pAs). Depletion of MBNL resulted in silencing of distal pAs while proximal pAs were boosted . Thus, lCELF1 and MBNL1 have similar binding sites in 3′ UTRs and tend to cluster together, where they compete to bind their co-target mRNAs and lead them to different fates. The extent of CELF1 and MBNL1 binding to 3′ UTRs correlates with the degree of mRNA suppression or stabilization, respectively. Greater MBNL binding prevents mRNAs from CELF-mediated inhibition. However, this antagonism effect by MBNL1 and CELF1 is also dependent on binding site proximity. Wang et al. (2015) showed that a distal MBNL1 binding site in 3′ UTRs was sufficient to prevent mRNA repression in the presence of a CELF1 site, but this protective response was not observed in the presence of a proximal MBNL1 site . mRNAs wIn addition, CELF and MBNL proteins antagonistically regulate alternative splicing of over 200 exons . For insIt was discovered recently that another RNA-binding protein, heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1), which is downregulated during normal postnatal development, remains elevated in DM1. Thus, it induces fetal splicing shifts of target RNAs in this disease. Reduced lifespan and muscle pathology were observed following AAV-mediated overexpression of hnRNPA1 in a mouse DM1 model .TRIM71 is a member of the Trim-NHL protein family, which modulates alternative splicing by repressing MBNL1 in embryonic stem cells. It functions via binding to hairpin structures in the 3′ UTRs of MBNL1 mRNA . Recent MBNL proteins bind to toxic RNAs containing CUG and CCUG repeats through specific recognition of the Watson–Crick face of the GC dinucleotide , bindingHowever, other regions of the proteins may also interact with RNA and play an important role in regulating gene expression and RNA splicing . RegulatStudies by Kino et al. (2015) and Lee et al. (2013) suggested cross-regulation and splicing compensation among MBNL proteins ,55. OverAlthough nuclear localization of MBNL is crucial for its alternative splicing activity, Pei-Wang et al. (2018) showed that it is the MBNL1 cytoplasmic isoform, not the nuclear isoform, that promotes neurite morphogenesis and reverses the morphological defects caused by CUG triplet repeat RNA . Expandeexp RNA. It has been shown that a dimeric 2,9-diamino-1,10-phenanthroline derivative (DDAP) designed by Li et al. (2018) [exp RNA and inhibit the formation of (CUG)exp RNA-MBNL1 complexes. Those molecules are dimers that contain two 5′CUG/3′GUC-binding modules. DDAP binding to (CUG)exp RNA is enhanced through Π–Π stacking interaction with neighboring guanines [exp RNA-MBNL1 complex by efficient interaction with the phosphodiester backbone [exp and inhibited the (CUG)exp RNA-MBNL1 interaction [Oligomeric compounds and other small molecules have been applied to specifically target the DM1 pathobiology at the DNA or RNA level via selective recognition of the repeating array of 1 × 1 nucleotide U/U internal loops formed by (CUG). (2018)  and mono. (2018)  enable iguanines . Amongstbackbone . Some ofbackbone ,60. Simieraction ,62.Chakraborty et al. (2018) identified a drug, daunorubicin hydrochloride, which was reported to interact with double-stranded RNA . Their rAside from MBNL release, regulating MBNL expression intrinsically has also be investigated to ameliorate DM1 symptoms. Overexpressing MBNL1 improves the proliferation capability of skeletal muscle satellite cells (SSCs) in DM1 by inhibiting autophagy via the mTOR pathway . TreatmeResearchers recently discovered that tumor necrosis factor-alpha (TNF-α), which is a proinflammatory cytokine and a crucial contributor to various metabolic syndromes, modulates the expression of MBNL ,69. MBNLexp RNA directly [DMPK allele or other alleles containing C(C)TG repeat expansion, which may induce loss-of-function phenotypes, such as myopathy [Previous investigations have explored the potential of targeting the C(C)UG repeat sequence directly using antisense oligonucleotides (ASOs). For example, phosphoroamidate morpholino oligomers (PMOs) comprise a short segment of CA(G)G repeats that bind to expanded C(C)UGdirectly . Howevermyopathy .An alternative approach to reducing the expression of expansion RNA involves the application of microtubule inhibitors. Kaalak Reddy et al. (2019) identified multiple microtubule inhibitors, which selectively reduced CUG expansion RNA levels and partially rescued DM1-associated missplicing . TreatmeIn addition to the small molecules mentioned above, a variety of other small molecules have been developed or are being developed for use in DM therapy. It is beyond the scope of this review to mention them all, but two reviews provide a great deal of detail about such small molecules ,80.n-containing minigene, were able to prevent the translation of three polypeptides formed from the three reading frames of the repeat [In e repeat . Subseque repeat . RAN trae repeat ,83,84. CDespite it now being well categorized that RAN translation exists and contributes to the pathology of several repeat expansion diseases, the underlying mechanism that drives this form of translation remains largely a mystery, although insights have developed via similarities and differences with the canonical translation pathway. The main stages of eukaryotic translation are as follows: eukaryotic initiator factor 2 (eIF2) forms a ternary complex with met-tRNA. This then forms the 43s preinitiation complex with the 40s ribosomal subunit and several other eIFs. In parallel, mRNA is identified by more eIF proteins by way of its highly methylated 5′ cap and the downstream region is unwound to promote translation. The entire complex then scans the mRNA 5′-3′ for the AUG start codon in order for translation to commence . 7G cap as well as ribosomal 5′-3′ scanning to find an initiation codon, and that the initiation codon used is inconsistent between reading frames [RAN translation of the repeat sequence CGG has been shown to be dependent on both the 5′ mAlthough similar in many ways, the repeat expansion involved in DM1 and DM2 presents a key difference in RAN translation. DM1, being a trinucleotide CTG expansion , will thOf the five different polypeptides shown for DM1 in Unlike DM1, both the sense and antisense RAN polypeptides have been identified in DM2. Zu et al. used immunostaining techniques to demonstrate the presence of α-LPAC (sense) and α-QAGR (antisense) polypeptides in brain tissue samples from DM2 autopsies, although with significant differences between the two proteins. Transfected cells and autopsy tissue both showed distinct localization of the proteins: α-LPAC localizing to the cytoplasmic regions and α-QAGR localizing to the nuclear regions. Both proteins formed small aggregates in their respective regions . Further−/− Mbnl2−/− mice showed significant upregulation in RAN translation [Finally, Mbnl1 has been implicated as a direct modulator of some RAN translation in DM2. Zu et al. found that Mbnl1 co-transfection with CCUG reduces the amount of α-LPAC formed, and similarly that Mbnl1nslation . These fIn this review, we considered three phases of the repeat expansion mutation pathway that provide a focus for therapeutic intervention. The three phases: transcription, transcript retention, and degradation, are all currently under investigation as part of drug development efforts. Thus far, encouraging results have been produced in cell and animal models, but their application to patients and successful clinical outcomes are eagerly awaited.


Specific RNA sequences regulate functions essential to life. The Trans-Activation Response element (TAR) is an RNA stem–bulge–loop structure involved in several steps of HIV-1 replication. In this work, we show how RNA targeting can inhibit HIV-1 nucleocapsid (NC), a highly conserved protein known to catalyze nucleic acid melting and strand transfers during reverse transcription. Our RNA targeting strategy consists of the employment of bis-3-chloropiperidines (B-CePs) to impair RNA melting through bifunctional alkylation. Specific interactions between B-CePs and TAR RNA were analytically investigated by gel electrophoresis and mass spectrometry, allowing the elucidation of B-CePs’ recognition of TAR, and highlighting an RNA-directed mechanism of protein inhibition. We propose that B-CePs can freeze TAR tridimensional conformation, impairing NC-induced dynamics and finally inhibiting its functions in vitro. The field of RNA science has exploded in the past decade and is radically changing the understanding of the role that RNA plays in fundamental biological processes. Although RNA has been considered an “undruggable” pharmaceutical target for a long time, it is now accepted as a next-generation space for drug development.A highly investigated and well characterized RNA structure is the HIV-1 trans-activation response element (TAR), an interesting target for the development of antiviral drugs ,4,5,6,7.NC is a nucleic acid chaperone that, during reverse transcription, destabilizes the stable secondary structure of TAR-RNA and promotes its refolding into a more thermodynamically stable conformation to allow reverse transcription to proceed . Based o1 as the B-CePs prototype, owing to its high reactivity and low cytotoxicity [2 and 3, characterized by a longer alkylic linker connecting the reactive moieties, and with the lysine ester compounds 4 and 5 (Bis-3-chloropiperidines (B-CePs) are recently developed alkylating agents, thoroughly investigated for their mechanism of action toward DNA ,24,25,26toxicity ,25, and  4 and 5 B, all ofm) from the reference value, which was determined in the absence of compound. Sample solutions contained 1 μM of RNA substrate and 10, 50 or 100 μM of each B-CePs compound. m obtained by adding increasing concentrations of B-CePs 1–5.A first insight into the putative interaction of B-CePs with RNA was offered by the investigation of the ability of the selected compounds to stabilize the TAR hairpin structure. We employed fluorescence quenching assays (FQA) to measure the ability of each B-CeP to modify the thermal denaturation profile of the TAR construct shown in m variation is directly correlated with the RNA-binding activity of the compound, the results clearly revealed that the tested compounds are efficient stabilizers of the secondary structure of TAR RNA. m increase was particularly significant in the presence of B-CePs 1–3, whereas derivatives 4 and 5 produced a much weaker stabilization effect. Interestingly, at 50 μM, all compounds achieved a similar efficient stabilization with an averaged ΔTm of 21 ± 2 °C and did not raise when increasing the compound concentration to 100 μM, suggesting that the interaction of B-CePs with TAR reached completion in a narrow range of doses. These results show evidence of the recognition between B-CePs and a hairpin-structured RNA construct and prompted us to proceed in disclosing additional binding details.Assuming that the T1 with TAR. The RNA was folded in its hairpin secondary structure, incubated for 2 h at 37 °C with increasing concentrations of compound 1 , and the reaction products were resolved by polyacrylamide gel electrophoresis (PAGE). To further evaluate the interaction of B-CePs to TAR RNA, Electrophoretic Mobility Shift Assay (EMSA) was used as a simple tool to visualize the putative interaction of compound The electrophoretic mobility of the RNA substrate was clearly altered by the presence of the B-CeP. As shown in 5− in 1 with TAR at 1 μM. After 2 h of incubation, at 37 °C, the reaction mixture was submitted to ethanol precipitation to achieve quenching and desalting, which enabled a direct infusion ESI-MS analysis. The representative ESI-MS spectrum of the 1:1 compound/substrate molar ratio provided in 1 with TAR, which could be due to alkylation at specific sites. This hypothesis is based on analogies to the mechanism of bis-3-chloropiperidines reaction with DNA, which have been demonstrated to proceed through a reactive bicyclic aziridinium intermediate formed upon intramolecular nucleophilic displacement of chloride by nitrogen. In the presence of nucleic acids, aziridinium preferentially attacks N7 of guanines on the substrate, forming different types of adduct . A preliminary determination was accomplished on the individual TAR construct, and a representative ESI-MS spectrum is provided in f adduct .22 Analy1, with 1 μM TAR solution incubated with 10 and 50 μM of B-CeP 1, respectively. Direct ESI-MS analysis of the reaction mixtures allowed for the identification of multiple additional products: different combinations of mono- and bi-functional covalent adducts were readily recognized based on their mass shift from the unmodified substrate. Incremental binding stoichiometries up to five equivalents of compound 1 were detected as compound concentration increased, consistently with the trend observed in the gel system in 1 toward DNA, which produced extensive backbone cleavage [The results shown in cleavage ,25 in th2–5 to react with the TAR construct , which places G12 in close spatial proximity with G20. Considering that Gs are the most susceptible nucleophilic center in nucleic acids (1), we assigned G12 and G20 as the putative nucleotides engaged in the bifunctional product connecting the two strands (2 (G1:C3 + B1 + U26:C28), the presence of the two guanine residues, G2 and G27, opportunely placed to be bridged by compound 1, allowed us to assume the putative location of the bifunctional adduct in the species 2, as shown in It should be noted that the short base-paired region in  strands B. Once a1 unambiguously revealed the presence of alkylation products able to covalently link opposing strands within TAR RNA hairpin structure. These adducts, formed upon reaction with B-CePs, are likely those mainly responsible for the stabilization of the TAR secondary structure, preventing its melting, as observed in the experiments shown in Hence, the accurate MS-analysis of the species obtained after nuclease footprinting of TAR RNA construct reacted with B-CeP 50) on TAR. The results are reported in Having demonstrated that B-CePs bind and stabilize the RNA substrate of NC, we performed a high-throughput screening (HTS) to analyze the in vitro ability of the tested B-CePs to inhibit NC-mediated melting of the TAR RNA hairpin construct ,17,18,1950 values < 5 μM. In good agreement with the thermal melting results reported above, compound 2 is also slightly more potent in NC inhibition. Interestingly, compound 2 was also the most reactive towards the TAR construct, as seen by the comparison of the ESI-MS spectra obtained at the same B-CeP/TAR molar ratio , thus indicating that their ability to interfere with NC was subordinate to their remarkable TAR-binding properties.Finally, we tested the possibility that B-CePs may be capable of binding directly to the NC protein. Experiments were carried out by mixing NC with B-CePs TAR is the 29-mer RNA sequence 5′ GGCAGAUCUGAGCCUGGGAGCUCU CUGCC 3′ and was synthesized by Metabion International AG . Stock solutions were stored at −20 °C in 10 mM Tris-HCl, pH 7.5. Dilutions were made in DEPC-treated water (Ambion). When specified, TAR was labeled at 5′ and 3′ ends, respectively, by the fluorophore 5-carboxyfluorescein (FAM) and the dark quencher 4-(4′-dimethylaminophenylazo)benzoic acid (Dabcyl). The typical folding procedure consisted of snap-cooling: TAR RNA diluted in the proper aqueous buffer was heated to 95 °C for 5 min and then ice-cooled in order to assume the proper hairpin structure.−1 cm−1.The full-length recombinant NC protein was obtained as reported . The pro1–5 (Bis-3-chloropiperidines 1–5 B were sym) is the temperature at which 50% molecules of oligoribonucleotide are denatured. TAR RNA hairpin was formed by snap cooling in 1× BPE buffer  as described above. Prior to analysis, TAR RNA was incubated with different concentrations  of each compound at 37 °C for 2 h. Nucleic acid solutions without compound were used to measure the reference Tm value. The melting protocol consisted of a melting phase, in which the temperature increased from 25 to 99 °C in 1 h (0.02 °C/s). Fluorescence emission of FAM was read by using a Light Cycler 480 II  with emission at λ = 510 nm and correlated to the melting temperature of the oligoribonucleotide. The Tm value was mathematically derived from the thermal denaturing profile by using LC480 software. ΔTm was calculated by using the following equation: ΔTm = Tm2 − Tm1, where Tm2 and Tm1 are the Tm values measured by testing the RNA structure in the presence or absence of compound, respectively.The ability of the tested compounds to stabilize the TAR RNA structure was measured by the increase in the melting temperature of the oligoribonucleotide in the presence of different concentrations of each compound. Melting temperature  was then incubated with increasing concentrations (from 0 to 50 μM) of B-CeP 1, at 37 °C for 2 h in 1× BPE buffer (pH 7.4). After incubation, products were resolved by 12% non-denaturing polyacrylamide (PAA) gels containing 1× TBE (Tris Borate EDTA) buffer at room temperature. Unreacted and reacted RNA on the gel were stained with SybrGreen II® . Fluorescence in gel system was detected on a Geliance 600 Imaging System .Electrophoretic Mobility Shift Assay (EMSA) was used to assess the ability of compound Samples containing a final 1 μM concentration of TAR RNA in 1× BPE buffer were added with compound 1 at final concentrations of 1, 10, 50 μM, depending on the pur- pose of the experiment. Reaction mixtures were incubated at 37 °C for 2 h. Samples prepared in BPE were buffer-exchanged by performing ethanol precipitation in the presence of 1 M of ammonium acetate. In case of reaction mixtures, the treatment also served to achieve reaction quenching. Samples were re-dissolved and diluted in 150 mM ammonium acetate (pH adjusted to 7.0) to achieve a final 1 μM concentration of total RNA. All samples were analyzed by direct infusion electrospray ionization (ESI) on the Thermo Fisher Scientific  LTQ-Orbitrap Velos mass spectrometer. The analyses were performed in nanoflow mode by using quartz emitters produced in-house by using a Sutter Instruments Co.  P2000 laser pipette puller. Up to 5 μL samples were typically loaded onto each emitter by using a gel-loader pipette tip. A stainless steel wire was inserted in the back-end of the emitter to supply an ionizing voltage that ranged between 0.8 and 1.2 kV. The source temperature and desolvation conditions were adjusted by closely monitoring the incidence of ammonium adducts and water clusters. Control determinations were completed to verify the experimental masses of the TAR RNA substrate. Data were processed by using Xcalibur 2.1 software .1 at final concentrations of 10 μM, i.e., a 10:1 compound/substrate ratio. Reaction mixtures were incubated at 37 °C for 2 h and quenched by ethanol precipitation. Aliquots of unreacted and reacted TAR were submitted to digestion with ribonuclease A (RNAse A) in 150 mM ammonium acetate for 1 h at 37 °C. Samples were stored at −20 °C until immediate analysis by ESI-MS, as reported in the paragraph above.The TAR RNA was heated to 95 °C for 5 min and then ice-cooled in order to assume the proper hairpin structure. Typical reactions consist of a final 1 μM solution of TAR RNA in 1× BPE buffer with compound 50 value.16 Each experiment was performed in triplicate to calculate an average and standard deviation.High-throughput screening (HTS) was performed to identify the inhibitors of NC chaperone activity on TAR. We used a VictorIII (PerkinElmer) microplate reader with 485 and 535 nm as the excitation and emission wavelengths. The TAR RNA hairpin was formed by snap cooling in the 1× BPE buffer, as described above. A 1 μM aliquot of TAR, which bore 5′-FAM and 3′-DAB modifications, was folded in 1× BPE buffer. The samples were then diluted to 0.1 μM in 1× BPE buffer. Increasing concentrations of compound  were added to each sample and incubated at 37 °C for 2 h before introducing NC to a final concentration of 0.8 μM (for a 1:8 oligo to NC molar ratio). The plate was immediately read at room temperature three times with 1 min intervals, unless differently specified. The experimental data were fitted as reported earlier to enable calculation of the respective ICBis-3-chloropiperidines represent a recently developed class of nucleic acid reactive agents. Beyond their expected ability to induce DNA cleavage, we reveal in this work B-CePs reactivity with a hairpin structured RNA, such as the HIV-1 TAR domain, which is a substrate of several enzymatic activities relevant for HIV-1 replication. Considering the different conformations of double-stranded regions of RNA and DNA, we anticipate different effects ascribed to the tertiary structure of the helix. To shed more light on this important structural issue, a thorough analysis of B-CePs reactivity toward models of single- and double-stranded RNA substrates is underway.B-CePs covalently react with the TAR hairpin, producing peculiar bifunctional adducts bridging the stem portion of TAR construct and leading to its strong stabilization. Analogously to the mechanisms of NC inhibition previously observed for 2,6-dipeptidyl anthraquinones acting as threading intercalators, stabilization of TAR by B-CePs leads to the impairment of NC destabilizing activity in vitro. However, differently from the anthraquinone derivatives, which were also able to directly interact with NC protein, the mechanism of NC inhibition by B-CePs totally relies on their TAR targeting, thus confirming an extraordinary tropism for nucleic acids.Hence, the employment of agents able to react with the RNA partners of NC blocking structure remodeling represents an original strategy that draws the spotlight on the RNA-mediated processes. The proposed strategy may represent a springboard for the development of a new class of RNA-targeting therapeutics to fight old and new disease associated with RNA viruses.


The various effects of native protein folding on the stability and folding rate of intrinsically disordered proteins (IDPs) in crowded intracellular environments are important in biomedicine. Although most studies on protein folding have been conducted in vitro, providing valuable insights, studies on protein folding in crowded intracellular environments are scarce. This study aimed to explore the effects of intracellular molecular crowding on the folding of mutant transactivator HIV-1 Tat based on intracellular interactions, including TAR RNA, as proof of the previously reported chaperna-RNA concept. Considering that the Tat–TAR RNA motif binds RNA, we assessed the po tential function of TAR RNA as a chaperna for the refolding of R52Tat, a mutant in which the argi nine (R) residues at R52 have been replaced with alanine (A) by site-directed mutagenesis. We mon itored Tat-EGFP and Tat folding in HeLa cells via time-lapse fluorescence microscopy and biolayer interferometry using EGFP fusion as an indicator for folding status. These results show that the refolding of R52A Tat was stimulated well at a 0.3 μM TAR RNA concentration; wild-type Tat refolding was essentially abolished because of a reduction in the affinity for TAR RNA at that con centration. The folding and refolding of R52Tat were mainly promoted upon stimulation with TAR RNA. Our findings provide novel insights into the therapeutic potential of chaperna-mediated fold ing through the examination of as-yet-unexplored RNA-mediated protein folding as well as viral genetic variants that modulate viral evolutionary linkages for viral diseases inside a crowded intra cellular environment. In the intracellular environment, the structures and functions of various proteins exhibit cooperative interactions under crowded conditions, with particular folding energies and kinetics ,2,3. TheTo date, most protein-folding studies have focused on in vitro analyses and have not considered the sensitive intracellular molecular interactions induced by intracellular crowding ,9,10,11.We have demonstrated that TAR RNA allows chaperone function in a single arginine mutant, R52Tat, under conditions of HeLa cell crowding. The HIV-1 transactivator protein Tat is essential for the high-level expression of the HIV TAR element and requires transcription by RNA-acting regulatory elements ,21,22. WThe intracellular environment shows the structures and functions of various proteins under crowding effects on the energy and kinetic influence of protein folding. The crowding is an extremely complicated intracellular environment with a confined amount of free water and an almost complete lack of unoccupied space ,8,13,31.Several studies have shown that macromolecular aggregation can affect protein structure, folding, shape, conformational stability, small molecule binding, enzymatic activity, protein–protein interactions, protein–nucleic acid interactions, and pathological aggregation.Previous studies have suggested that binding to TAR RNA requires R52Tat to be soluble . SolubleRNA molecules play a central role and have functions in virtually all cellular processes. There are various ways in which RNA can have chaperoning effects on bound proteins. RNA-facilitated protein folding is closely associated with problems in protein folding and with both protein structure and the mechanisms by which proteins achieve their active conformations . The proThe presence of hydrophobic residues, such as alanine, on the interior of a mutant Tat with reduced refolding ability did not lead to a further reduction in this ability, indicating that a single arginine residue is associated with R52Tat-binding TAR RNA. A single arginine residue is associated with mutant and was constructed under the same concentration of TAR RNA that assists in mutant Tat refolding, and it was compared with wild-type Tat. These results show that the arginine residue in the conserved basic region of R52Tat is crucial for binding TAR RNA and that the affinity of this RNA may also influence the refolding and folding of its interacting partner, R52Tat.To identify the amino-acid associations that are important for TAR RNA recognition within the RNA sequence, the affinity and specificity of TAR for residues 47–58 of the arginine-rich region of mutant Tat were plotted . The HIVWe examined whether TAR RNAs assisted in folding when bound to the arginine-rich domain of the Tat protein in the cytoplasm. Through ionic interactions between the arginines at positions 52 and 53, the Tat amino groups are arranged in a particular tertiary form; Tat and TAR RNA are RNA-phosphate-binding regions where Tat transactivation occurs. These arginine residues at positions 52 and 53 in Tat are in a specific RNA-binding region where Tat transactivation occurs through the ionic interaction between the amino group of arginine and the RNA phosphate, which are arranged in a specific tertiary form as a ‘specific affinity’ contact between Tat and TAR RNA. In this study, we introduced a mutation into Tat at the binding site within the TAR RNA sequence to demonstrate that chaperna–TAR RNA binding is important for protein refolding inside the cell. The mutation of some protein residues influences the conformation and alters the protein structure. These results imply that the single arginine residue within Tat is associated with protein folding because hydrophobic residues, such as alanine, tend to be located on the internal surfaces of proteins. Additionally, the diminished refolding of the mutant Tat did not lead to a decreased ability to bind TAR RNA. Thus, a TAR RNA binding mutant protein was constructed using site-directed arginine mutagenesis and compared with wild-type Tat.E. coli (non-crowded solution) and those conducted using crowded HeLa conditions was the influence of vari ous intracellular factors . We measured the enhancement of Tat-EGFP refolding by monitoring the increase in intrinsic EGFP fluorescence under denaturing Tat-EGFP and folding conditions . As showThe influence of TAR RNA on the folding of wtTat-EGFP in vitro was further investigated by employing TAR RNA at various concentrations . In the presence of TAR RNA, the refolding enhancement factors for EGFP  were sigE. coli cells in an uncrowded environment on (purified) EGFP during a refolding period of 20 min and compared the results with the effect of a crowded environment in HeLa lysate during a refolding period of 1 h to normalize the measurement of wild-type Tat-EGFP to the EGFP response  were significantly higher than those for wild-type Tat-EGFP in HeLa lysate . These dThe refolding enhancement factor for mutant R52ATat-EGFP was 6% in 0.3 µM TAR RNA, whereas that for wild-type Tat was 0% . The yieTo investigate the chaperone function of TAR RNA for the arginine-mutant Tat-EGFP protein, the folding of mutant Tat-EGFP in live HeLa in the presence or absence of TAR RNA was tested . Previous studies have shown that fluorescence signals from the expression of EGFP alone were not affected by RNA co-expression; hence, a control was used ,51. HeLaGiven the use of live cell time-dependent fluorescence monitoring, cytoplasmic EGFP fluorescence was used to indicate the appropriate folding of the R52  and R53TThese reports are consistent with the behavior of the transdominant Tat mutant, which is confined to the nucleus but not to the nucleolus. In addition, TAR RNA like protrusions are highly structured in nucleosomes, suggesting that they are present in synthesized ribosomal RNA. Interestingly, some ribosomal proteins, such as Tat, contain arginine-rich regions, which have been proposed to mediate specific RNA binding. A vector expressing only the EGFP tag was used as a control; proteins were uniformly distributed throughout the cell and did not colocalize with speckles in HeLa cells, whereas speckles were identified in Tat-EGFP.Nevertheless, the average EGFP fluorescence intensity, considering the statistical significance of signals from single EGFP cells, indicated the notable stimulation of mutant R52Tat-EGFP signals in the presence of TAR RNA C. The enE. coli cells with that of a crowded environment on EGFP in the absence of TAR RNA by BLI at 1.3 nm during a refolding period of 20 min  in f 20 min  56]. We. WeE. cof 20 min  3,57]. ,57. E. cf 20 min . These ry-axis). By contrast, TAR RNA binding with refolding increased the levels of immobilized His-tagged TatR52A and TatR53A proteins. The treatment of the denatured mutant Tat-TAR RNA led to an initial increase in the BLI response at 1.6 nm during the 2 h refolding period. These results closely resemble those obtained using TAR RNA-mediated TatR52A-EGFP and TatR53A-EGFP refolding. This similarity was also noted in TatR52A and TatR53A refolding without EGFP. Immobilized His-tagged TatR52 and TatR53 in refolding buffer can identify superior refolding conditions by rapidly detecting aggregate formation and affecting the function of chaperna in HeLa lysate. As shown in We examined the chaperna-RNA that functions as a chaperone for Tat proteins in the folding of the single-arginine mutants TatR52 and R53. The RNA chaperone was distinguished from proteins that aid in the folding of interacting RNA; here, we propose the use of an RNA-based chaperone that aids in the folding of interacting proteins. In this study, TAR RNA, as a chaperna, showed strong evidence of a folding kinetics model being applicable. This demonstrated its contribution under crowded macromolecular conditions in living HeLa cells. Thus, TAR RNA has chaperone activity and dictates the folding of the mutant R52Tat. In addition, TAR RNA affected the kinetics of Tat folding in crowded intracellular conditions. The present results are consistent with the prediction that TAR RNA interacts with arginine residues in the basic conserved region of Tat under the conditions of intracellular crowding to increase R52Tat refolding. Despite Tat’s inherently unfolded nature, little is known about how the incorrect folding of the Tat protein can be prevented inside cells. The Tat protein has the potential to interact with several host proteins and fold into a stable form in a transactivator complex . HoweverTo confirm RNA-mediated integrative folding, we rapidly monitored the proper folding of Tat protein in the presence of TAR RNA for enhanced green fluorescent protein (EGFP) fusion to the C-terminus of Tat with EGFP. The refolding of R52Tat was promoted upon stimulation with TAR RNA under crowded conditions. Live cell imaging also showed the extent to which the folding rate of mutant R52 and R53Tat-EGFP was stimulated by TAR RNA. Considering that the main function of molecular chaperones undergoing intermolecular electrostatic repulsion by cell densification is to prevent cellular protein folding and the aggregation of unfolded proteins, the assay was related to chaperone activity.The core principle described here can be applied to various cellular-swelling- or shrinkage-activated responses that involve enzymes and RNA-mediated protein-folding substrates and can stimulate protein folding via intermolecular repulsion. Thus, protein conformational changes, conformational entropy, and the unstable intermolecular repulsion forces of cellular macromolecules that are distinct (but fully compatible) from attractive interactions need to be considered in crowded cellular environments . In particular, mutations in charged residues that reduce intermolecular electrostatic repulsion are implicated in aggregation-associated diseases. The general concept of molecular chaperones introduces auxiliary protein folding instead of spontaneous protein folding. It has been suggested that cellular macromolecules, including chaperones, exhibit an intrinsic chaperone activity that prevents the aggregation of physically linked polypeptides regardless of the type of linkage between them, usually due to their large exclusion volumes and intermolecular repulsion due to surface charges. However, genetic and biochemical analyses have shown that only a limited number of proteins are folded with the help of molecular chaperones ,67,68,69Thus, in various cell-dense environments, proteins can be greatly affected by the intermolecular repulsive forces of bulky cellular macromolecules that physically interact with the proteins. A more compact state may be thermodynamically favored, such as the folded and aggregated structures of dense cellular macromolecules. To date, proteins in cell-dense environments have largely been discussed as experiencing volume effects excluded from indirect stabilization by macromolecular clustering that do not involve physical linkages between proteins and cellular macromolecules. Evidence that macromolecular aggregation is a real intracellular phenomenon comes from the differences in protein-folding-rate measurements obtained in purified in vitro conditions in terms of the cell biochemical ratios and molecular equilibrium conditions in living cells .Along with the protein-folding rate and thermodynamic stability, protein conformation is an important determinant of protein aggregation. Anfinsen’s thermodynamic hypothesis relates to the concept of protein folding, which is the intrinsic structure of a protein that is the most stable under physiological conditions and, thus, occurs spontaneously . HoweverTat has been shown to exhibit RNA chaperone activity, which aids in the folding and structural rearrangement of RNA molecules and is possibly mediated by interactions with essentially disordered regions . The delRNA contains negative charges that cause electrostatic repulsion and block intermolecular interactions that lead to aggregation. These properties can stabilize intermediate folding and interfere with protein aggregation. This is particularly interesting considering previous reports that the single arginine region of R52Tat itself has been shown to aid in the folding and conformational rearrangement of RNA molecules . In thisIn conclusion, our study demonstrated that the net charge of the single-arginine mutant TatR52 in a cell-dense environment is associated with aggregation, as well as that the intermolecular electrostatic repulsion between charged residues in proteins affects protein refolding. These properties of RNA can stabilize intermediate folding and interfere with protein aggregation. This suggests that, although our understanding of the roles of various molecular chaperones is still limited, as the important role of virally encoded RNA in establishing proteome linkages is considered, there may be a variety of viral-encoding chaperones that have not yet been characterized ,78.These aspects could be extended to the supramolecular aggregates of genetically mutable viral diseases that have caused pandemics such as COVID-19, indicating that they should be considered for enhancing immunogenicity ,80. TherThe HIV Tat plasmid contained a 213-bp region. For PCR amplification, the primers used were Bam HI Tat forward 5′-GGATCC-ATGGAGCCAGTAGATCCTAGACTAGAG-3′ and EcoRI Tat reverse 5′-GAATTC-TTATTCGTGCCATTCGATTTTCTGAGCCTCGAAGATGTCGTTCAGACCTTCCTTCGGGCCTGTCGG-3′. BamH1 and EcoRI restriction sites were generated by inserting them into pcDNA3 using standard restriction cloning methods. The oligonucleotide forward primer was 5′-GGCACAAGCTGGAGTACAAC-3 and the reverse primer was 5′-ATGCCGTTCTTCTGCTTGTC-3′ for EGFP. A vector modified from pcDNA3  was used for the construction of the expression vectors for the human wild-type Tat protein using standard restriction cloning methods. The Tat mutants used for replacing the single arginine (R) residues R52 and R53 with alanine (A) were produced by site-directed mutagenesis. The mutants and reporter-fused forms included Tat (R52A), Tat (R53A), Tat (R52A)-EGFP, and Tat (R53A)-EGFP. Tat (R52A) and Tat (R53A) were constructed by the PCR overlapping mutagenesis of the Tat gene. A hexa-histidine tag was added to the C-terminus of HIV Tat.We used the following TAR RNA sequence for in vitro refolding: 5’- GGUCUC UCU GGU UAG ACC AGA UCU GAG CCU GGG AGC UCU CUG GCU AAC UAG GGA ACC -3′. Next, we performed RNase-free HPLC purification. TAR RNA was synthesized at a concentration of 100 nmol and with an RNA oligo length of 57 bp . 2 atmosphere at 37 °C. Cells were transfected when at 60–90% confluence using 2 µL of Lipofectamine 2000  and 500 ng of plasmid per 2 × 106 cells. The cells were washed once with growth medium, twice with 10× PBS at pH 7.4, and then once more with PBS alone. Transfections with the HIV Tat plasmid and the HIV Tat–GFP fusion plasmid were performed using Lipofectamine 2000 at 37 °C for 24 h. For the HIV Tat-GFP fusion plasmid and His-tagged HIV Tat plasmid protein expression, HeLa cells were lysed with FT Lysis Buffer containing 600 mM KCl, 20 mM Tris-Cl (pH 7.8), and 20% glycerol, as previously described [HeLa cells were grown in MEM  supplemented with 10% FBS  and 1% penicillin/streptomycin in a 5% COescribed . The lysHeLa cells were lysed with FT Lysis Buffer containing 600 mM KCl, 20 mM Tris-Cl (pH 7.8), and 20% glycerol, as previously described . The lys2.We expressed Tat fused with EGFP in mammalian cells. Tat-EGFP expression in cells was confirmed by laser-induced fluorescence using an inverted fluorescence microscope . A 488 nm laser line was used for green fluorescence. The laser was focused into the channel using a 20× objective. The fluorescence signal was collected by the same lens and optically filtered. We detected the excitation/emission wavelengths of the green filter set U-MWIBA3 EGFP shifts . For the refolding EGFP reporter assay, the EGFP fluorescence emission spectra of the HIV Tat protein fused with EGFP in lysates from HeLa cells were measured in PBS buffer using the Chromo4™ System . The EGFP reporter refolding assay measurements were performed at 30 °C. EGFP’s fluorescence kinetics were analyzed using the Opticon Monitor 3 (Bio-Rad). The multi-mode microplate reader FlexStation 3 system  was also used for the detection of the EGFP fluorescence kinetics. The HIV Tat-EGFP fusion proteins harvested or isolated from HeLa cell lysates were denatured in 6 M guanidine-HCl and 1 mM DTT for 20 min at 40 °C. Next, the denatured proteins were diluted 25-fold in refolding buffer containing 50 mM Tris-HCl (pH 7.5), 50 mM NaCl, and 5 mM MgClBLI was performed using an Octet Red instrument . The His Abs were immobilized on penta-His biosensors with varying concentrations of monomeric His-tagged Tat in HeLa lysates as the analyte in solution. The biosensors were hydrated for 10 min in PBS (pH 7.4), which was also used as a buffer in all the steps of the assay. Subsequently, the biosensors were first immersed in buffer for 120 s to establish a baseline and then in a solution containing 5 μg/mL of His Ab for 180 s to capture the Ab. After this, immersion in buffer was performed for a 60 s wash.D; on-rate, kon; off-rate, koff) were determined using the Octet Analysis version 7 software . The fold changes for each mutant compared with the WT values were calculated. The BLI experiments were performed using the general BLI analysis protocol, as described previously [Next, the His Abs biosensors were immersed in a solution containing various concentrations of His-tagged Tat for 120 s to allow analyte/ligand association, followed by immersion in refolding buffer every 30 s for 8000 s. The analyte was serially diluted 2-fold from 500 to 50 nM, with each step being performed with 1000 rpm shaking in the Octet Red instrument . The binding affinity constants  of IDPs enables the detailed characterization of their structural rearrangement of the virally encoded TAR RNA, which keeps the mutant Tat protein ‘folding competent’, enabling its interaction with multiple cellular factors for transactivation. Our findings provide novel insight into the therapeutic potential of viral mutant gene encodings that modulate viral evolution-based chaperna linkages for RNA virus diseases.


They are installed at the 5′ position of RNA via template-dependent incorporation of nucleotide analogues as an initiation substrate by RNA polymerases. However, the discovery of NAD-capped processed RNAs in human cells suggests the existence of alternative post-transcriptional NC capping pathways. In this review, we compiled growing evidence for a number of these other mechanisms which produce various non-canonically capped RNAs and a growing repertoire of capping small molecules. Enzymes shown to be involved are ADP-ribose polymerases, glycohydrolases and tRNA synthetases, and may potentially include RNA 3′-phosphate cyclases, tRNA guanylyl transferases, RNA ligases and ribozymes. An emerging rich variety of capping molecules and enzymes suggests an unrecognized level of complexity of RNA metabolism.Recent years witnessed the discovery of ubiquitous and diverse 5′-end RNA cap-like modifications in prokaryotes as well as in eukaryotes. These non-canonical caps include metabolic cofactors, such as NAD Gene expression is mainly regulated by adjusting RNA levels by production or degradation, but also by the differential utilization of RNA as defined by its sequence, structure or epitranscriptomic modifications. RNA capping is an important mechanism, affecting the overall fate of the respective RNA. Canonical eukaryotic 5′ RNA caps are attached to the nascent RNA early during its synthesis by RNA polymerase (RNAP) and can subsequently affect almost all its cellular roles. They enable initiation of protein synthesis, serve as an identifier for recruiting protein factors for pre-mRNA splicing, polyadenylation and nuclear export, and affect stability and susceptibility of RNA to nucleases .7-methyl guanosine (m7G)1 added co-transcriptionally to the 5′ ends of transcripts producing m7G-RNA , i, i+ (a) + (a) . Post-trt events b: (i) co+ biosynthesis pathway. NMNATs catalyse the transfer of the AMP moiety of ATP to nicotinamide mononucleotide (NMN) to form NAD+ (c), and they also catalyse the reverse reaction ) and potentially also for decapping of NADylated RNA.A suitable post-transcriptional NAD-capping candidate may be recruited from nicotinamide mononucleotide adenylyltransferases (NMNATs) or other enzymes of the NADorm NAD+ c, and thhosphate ). We speIt was previously shown that the enzymatic substrate-binding pocket of some NMNATs is not strictly specific to ATP, but it can also adopt phosphorylated proteins  or diffe+ in cells resides in the mitochondria . I. I+-depeADPR-RNA c 62,63],63+-depein vivo, the conditions used during in vitro experiments strongly suggest that the same reactions may occur in cells ..in vitroin vivo, ADP-ribose would be a new NC RNA cap, possibly present in all domains of life. It could be part of anti-viral immunity as ADP-ribosylation-dependent response of a host, which could be counteracted by virus-induced decapping of ADP-ribosylated RNAs . R. REscherates (b) . RtcA mib,d,e). RtcB and RtcA are structurally unrelated proteins with no similarities in their active sites [The main difference among the conventional DNA/RNA ligases, RtcA and RtcB is the amino acid used to form the reaction intermediate  producing adenylated tRNAHis (A-pp-tRNAHis) intermediate (His (ppp-G-tRNAHis) and AMP , is present in all three domains of life  in archaea and bacteria) ,80. tRNAHis [The adenylylated intermediate product of the pathway (A-pp-tRNA) can be classified as NC-capped RNA but while the process of adenylylation and subsequent guanylyl transfer is specifically bound to recognition of anticodon of tRNAHis , its wid. 9in vitro [Flavin adenine dinucleotide is one of the cellular metabolites reported as covalently bound to the RNA in prokaryotes, eukaryotes and viruses . It is rin vitro .+ capping.Whether there are other enzymes producing FAD-RNA remains an unanswered question. We speculate that enzymes from the flavin mononucleotide adenylyl transferase (FMNAT) family could be a possible candidate, analogically to NMNATs as candidates for NAD. 10+-, FAD- and CoA-capped RNA by attaching small-molecule precursors to a 5′-terminal ATP were evolved in vitro [in vivo synthesis of capped RNA [Intriguingly, not only proteins can cap RNA with metabolite cofactors. Ribozymes able to make NADin vitro . Ribozymin vitro . Hypothepped RNA .. 11Some positive sense single-stranded RNA viruses have viral protein genome-linked (VPgs) covalently attached to the 5′ end of their genome. In caliciviruses, VPgs were confirmed to interact with the cap-binding protein (eIF4E) and to be essential for translation . These v. 12Staphylococcus aureus repressed production of toxins, thus decreasing cytotoxicity of the bacterium [A number of potential physiological roles of NC capping are suggested in two recent reviews, including RNA folding and stability, cellular localization, etc. ,87. Yet acterium . Therefoacterium ,24,26.E. coli, B. subtilis and Staphylococcus aureus), the abundant NAD-capped species are 5'-terminal fragments of certain mRNAs [B. subtilis dormant spores [B. subtilis and S. aureus [+-capped RNAs are produced, whether the presence of NAD+ at the 5′-end affects either transcription initiation, termination or degradation of full-length RNAs to make species of shorter yet defined length, stable enough to be detected, or whether the short length is just an artefact of NAD-RNA identification process.It is unclear if a majority of the capped transcript are full-sized or truncated species. In bacteria  for NAD-RNA and for m7G-capped RNAs of the same gene, but the biological function of alternative TSSs remains unsolved. It was shown for E. coli transcription that 5′-NAD can stabilize short transcript and prevent their release improving chances for RNAP to escape into a productive elongation [The impact of capping on transcription and post-transcriptional processing of RNA is known only for a few examples. In ongation . Mammaliongation ,32, suggS. aureus 5′-NAD cap on RNAIII might impair translation of a small open reading frame inside this regulatory RNA in vivo, but the impact of additional factors cannot be ruled out. Both NAD+-RNAIII and ppp-RNAIII supported the formation of stable translational initiation complexes in vitro [+ capping seems to be higher than in other cell compartments or prokaryotic cells . It is . 13in vitro or in vivo addition of NC caps to RNA. All the reviewed enzymes with proven capping activity decorate RNA by 5′-5′ addition of nucleoside connected by a variable number of phosphates or phosphate-ribose-phosphate bridges. Interestingly, most of these enzymes share the principal mechanism of cap addition with canonical 5′ RNA capping: they use amino acid—nucleotide covalent linkage as an intermediate for nucleotidyl transfer to RNA/nucleotide  but did not observe any capping or decapping activity in tested conditions.In this review, we compiled information about a number of enzymes beyond RNAPs capable of cleotide . This coInitially, NC capping was perceived as RNAP-dependent stoichiometric event affected by the ratio of concentrations of NTPs to other NTP-analogues. This review focuses on the facts which challenge this perception, showing that cells are probably packed with enzymes capping and decapping RNA with NC caps. Whether all NC capping events are stochastic side reactions of moonlighting enzymes with established primary function, or there exist dedicated machinery for NC capping, remains to be shown. Expanding repertoire of caps and capping enzymes suggests if not a physiological function then at least a major physiological impact of these caps.Click here for additional data file.


They overcome the shortcomings of aggregation-induced emission (AIE)-type compounds that do not emit light in dilute solutions and aggregation-caused quenching (ACQ)-type compounds that do not emit light in a concentrated or aggregated state. This work reports a novel ionic DSE material based on rigid rod-shaped organic conjugated structure using 4,4′-bis(2-sulfonatostyryl) biphenyl disodium salt (BSBDS); the ion repulsion effect can reduce the strong π–π interaction in aggregation and achieve high-efficiency luminescence in solution and solid states. In addition to excellent DSE characteristics, BSBDS also exhibits a mechanochromic nature and sensitive detection performance for aluminum ion (Al Organic luminescent materials have been widely used in various areas, such as organic light-emitting diodes (OLEDs) and chemical sensors . Traditivia theoretical calculation and practical verification by the Gong group  materials that can effectively emit light in both the solution and solid states have been reported sporadically and applied in OLEDs, cell imaging, and other fields. For instance, Tang et al. reported conjugation-induced rigidity and through-space conjugation in twisting molecules for designing novel DSE materials . Dong etng group . Howeverng group . There s3+) in solution states.Herein, an ionic organic conjugated compound with a rigid rod shape was proposed to construct DSE luminogens. First, the rod-shaped structure can effectively inhibit the strong π–π interaction leading to the quenching of luminescence. Second, the ionic compound can avoid the formation of excimer and complex in the solution due to its own electrostatic repulsion. Combining the rod-shaped structure and ionic characteristics, novel DSE luminogens can be designed effectively to meet various application requirements. In our work, a typical fluorescent whitening agent named disodium 4,4′-bis (2-sulfonatostyryl) biphenyl (BSBDS) is used to verify our design strategy . Surpris28H20Na2O6S2) was purchased from Tokyo Chemical Industry Co., Ltd. Methanol, anhydrous ethanol, and metallic salts were purchased from Xilong Scientific Co., Ltd. Tetrahydrofuran was obtained from Guangdong Guanghua Technology. Standard stock solutions of metal ions  were prepared with ultrapure water from the respective metallic salts 2 3H2O, FeCl2 4H2O, FeCl3 6H2O, Zn(NO3)2 6H2O, CuCl, concentrated sulfuric acid (H2SO4), CdCl2 5/2H2O, NiCl2 6H2O, CoCl2 6H2O, and K2CO3). All the reagents were of the analytical reagent grade and used as received. All the aqueous solutions were prepared with distilled water supplied from a Milli-Q purication water system. Tetrahydrofuran (THF) was distilled from sodium/benzophenone under nitrogen before use.4,4-bis(2-sulfonatostyryl) biphenyl disodium salt . The size distribution of the suspensions for BSBDS in 90/10 THF–water was determined on a Malvern Zetasizer nano ZS90 instrument at 25°C. The toxicity of cells was confirmed with a Tecan Infinite F50 Microplate Reader. The fluorescence images of cells were gained on an Olympus FV 3000 laser scanning confocal microscope (LSCM).Ultraviolet–visible (UV-Vis) spectra were recorded on a UV3600 spectrophotometer. Fluorescence emission spectra and the quantum efficiency analysis were performed on a Horiba FluorMax-4 spectrofluorometer. X-ray diffraction (XRD) was measured on a X'Pert Pro X-ray diffractometer. The fluorescence lifetime was obtained using a Horiba QM-8000 fluorescence spectrophotometer. The luminescence quantum efficiency in solutions was measured using quinine sulfate as the standard solution , and the time-dependent density functional theory (TD-DFT) was applied to study the low-lying excited states with the B3LYP functional and 6-31+G(d) basis set. All above calculations were carried out with the Gaussian 16 B.01 package . Gaussia4 cells per well in DMEM. After 24 h of growth, the medium in the cells was replaced with a fresh solution (100 µL) containing BSBDS with different concentrations ranging from 12 (control) to 96 µM. Following the incubation of cells with the BSBDS for 24 h, the medium was replaced with a fresh medium containing MTT (0.5 mg/ml) and incubated for another 4 h. The medium and MTT solution were replaced with 150 µL DMSO in each well. The plate was shaken well and the absorbance at 492 nm was recorded using a microplate reader . The cell viability was calculated as the ratio of average absorbance intensity of samples to that of the control.The toxicity of BSBDS was evaluated using conventional MTT assay. HeLa cells were seeded in a 96-well plate at a density of 1 × 102 and humidity at 37°C. At 80% confluence, these cells were trypsinized, and about 2 × 105 cells were added to each well of a 6-well culture plate. After 24 h of growth, the cells were incubated with a commercial dye (Nuclear Red™ LCS1) at 37°C for 20–30 min; then, Al3+ and BSBDS were added to the culture medium for incubating (washing off the excess agent and washing cells with PBS three times before adding different reagents). Finally, the cells were fixed with 4% paraformaldehyde. HeLa cell imaging was carried out by using an LSCM .HeLa cells were seeded and grown in DMEM supplemented by 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (PS) under ambient conditions of 5% CO−4 M, the emission peak of BSBDS was at 430 nm. When the concentration reaches 2 × 10−2 M, the emission wavelength significantly red-shifted to 470 nm, which can be attributed to the formation of local excimers in BSBDS at high concentrations. The fluorescence lifetime/quantum efficiency of BSBDS at the concentration of 2 × 10−2 and 2 × 10−5 M are 5.55 ns/62.3% and 1.45 ns/74.3%, respectively, which indicates that luminescence at low concentration (≤2 × 10−4 M) originates from the intrinsic monomer emission of BSBDS, and luminescence at high concentration (2 × 10−2 M) is derived from excimer emission of BSBDS. It is worth noting that the fluorescence intensity of solution at the concentration of 2 × 10−5 M is greater than those of at 2 × 10−2 ∼ 2 × 10−4 M  as shown in −2 M, the solution with the THF content of 90% should have no excimer formed. The slight blue-shift of the spectrum with the increasing THF content is difficult to explain because there is no specific molecular packing model in the nanoparticles. In addition, the increase of luminescent intensity with the increasing THF content can be attributed to RIM in nanoparticles, which is similar to the AIE mechanism  in the solution had no effect on the luminescence of the BSBDS, so the optimized structure did not contain the content of Na+. The optimized anion structure of BSBDS is shown in f = 0.9057). f has a certain relationship with the luminous efficiency  method was used to optimize the molecular structure of BSBDS. It is worth noting that the sodium ions  and intensity. However, when Al3+ ions were added to the BSBDS solution, the emission peak red-shifted from 430 to 475 nm, which was consistent with the emission behavior at the concentration of 2 × 10−2 in the solution and crystalline powder. The results indicated that BSBDS complexed with Al3+ formed an excimer  were added sequentially to the BSBDS aqueous solution to estimate the detection limit of Al3+. Upon the addition of Al3+, the fluorescence intensity of BSBDS at around 430 nm gradually declined due to electron or energy transfer  regulations (3+ in drinking water. The dynamic quenching constant (Ksv) is calculated via the Stern–Volmer formula  for 30 min, and then treated with 20 μM BSBDS for another 1 h. The confocal images of HeLa cells showed a certain amount of a fluorescent signal in pseudo colors with blue and green from BSBDS, which mainly appeared in the cytoplasm of HeLa as displayed in 3+; it emitted intense blue fluorescence signal (3+ (3+ BSBDS emits strong light in both the cytoplasm and nucleus when excited by a 405-nm laser; Al3+ can also help BSBDS to enter into the nucleus. The results indicated that BSBDS is cell-permeable and could be used for dynamic imaging and Al3+ tracking in cells.In order to explore the biological feasibility of BSBDS, cytotoxicity of BSBDS was evaluated by MTT assay. As shown in e signal  in compae signal  comparedgnal (3+ . The flu3+ in the solution state, obvious mechanochromism properties in the solid state, and good biocompatibility, which can be used for cell imaging. The unique chemical structure characteristics and excellent optical properties of this ionic rod-shaped organic compound make it exhibit broad applications in many fields, such as organic optoelectronics, chemical sensing, ion detection, and biological imaging. This work will provide new ideas for the development of novel DSE materials.In summary, we propose an ionic rod-shaped organic compound for the construction of high-efficiency DSE luminescent materials. Due to the large rod-shaped conjugation structure, BSDBS can emit light efficiently in the state of dilute solution. At the same time, the repulsive effect of ions at high concentrations can lead the rod-shaped molecules only form excimers locally, avoiding the aggregation quenching effect and achieving highly efficient luminescence in concentration and aggregation states. BSBDS was selected as a model compound to validate the molecular design strategy, and the results showed that the compound possessed high-fluorescence quantum yield in both solution and solid states. In addition, BSDBS has recognition ability for Al