The translocation of biological macromolecules between cytoplasm and nucleus is of great significance to maintain various life processes in both normal and cancer cells. Disturbance of transport function likely leads to an unbalanced state between tumor suppressors and tumor-promoting factors. In this study, based on the unbiased analysis of protein expression differences with a mass spectrometer between human breast malignant tumors and benign hyperplastic tissues, we identified that Importin-7, a nuclear transport factor, is highly expressed in breast cancer (BC) and predicts poor outcomes. Further studies showed that Importin-7 promotes cell cycle progression and proliferation. Mechanistically, through co-immunoprecipitation, immunofluorescence, and nuclear–cytoplasmic protein separation experiments, we discovered that AR and USP22 can bind to Importin-7 as cargoes to promote BC progression. In addition, this study provides a rationale for a therapeutic strategy to restream the malignant progression of AR-positive BC by inhibiting the high expression state of Importin-7. Moreover, the knockdown of Importin-7 increased the responsiveness of BC cells to the AR signaling inhibitor, enzalutamide, suggesting that targeting Importin-7 may be a potential therapeutic strategy. The latest global cancer statistics show that the incidence of breast cancer (BC) has become the most prevalent carcinoma worldwide in 2020. The mortality of BC also ranks first among female-related cancers . ClinicaTransportation of key proteins between the cytoplasm and nucleus is critical to mammalian cells to regulate various life processes –6. DysreDespite the conventional view that BC is mainly estrogen-dependent and ER-driven, the existence of androgen receptor (AR) in BC is also a common phenomenon, especially in ER-negative BC –17. AlthWith the informed consent of patients, we first detected the difference in protein levels in 9 cases of benign breast hyperplasia tissue and 18 cases of malignant BC tissue by LC–MS/MS analysis in DIA mode, the volcano plot from which showed all different protein expressions Fig. . In addiNext, in order to verify the expression of Importin-7 in BC, we performed immunohistochemical detection on the collected 30 cases of benign breast hyperplasia and 52 cases of malignant BC Fig. . In addiWe next investigated the impact of Importin-7 knockdown on proliferation in three BC cell lines to further reveal the biological function of Importin-7 on growth. First, we used siRNAs to knock down the expression of Importin-7 in these cell lines and then used the CCK8 assay kit to detect the trend of cell viability within five days after transfection. The results showed that, from the second day of transfection, restricting the expression of Importin-7 caused significant inhibition of viability in BC cells Fig. . The knoMoreover, we established Importin-7 stable knockdown BC cell lines by transfecting lentiviruses containing two pair Importin-7 shRNAs and further performed clone formation experiments on these BC cells. We showed that Importin-7 knockdown inhibited colony formation in these BC cell lines Fig. . The intThe malignant proliferation ability of BC is closely related to the dysfunction of cell cycle checkpoints. Thus, we first performed flow cytometry to detect cell cycle distribution in BC cells treated with Importin-7 siRNAs. Our analysis showed that the knockdown of Importin-7 mainly induced arrest in the G0/G1 phase Fig. . Next, wWe previously conducted a series of studies on the degradation mechanism of AR and proposed that promoting the protein degradation of AR by targeting USP14 induces growth arrest and apoptosis in ER-negative BC , 25. In Next, we conducted a more in-depth study of the Importin-7–AR–USP22 complex by the treatment of AR inhibitor  or AR ligands, including DHT and synthetic androgen R1881. We showed that the stimulation of AR ligands significantly up-regulated the expression level of AR but did not alter the expression of Importin-7 and USP22. Moreover, the expression levels of Importin-7, USP22, and AR were not affected by enzalutamide treatment. DHT and R1881 stimulation resulted in more AR binding by Importin-7, although this was more likely because DHT and R1881 induced an increase in AR, but it still showed that the level of AR carried by Importin-7 was unsaturated and variable. Enzalutamide treatment inhibited the interaction between Importin-7 and AR without changing the overall expression levels of these proteins. Fig. . Then, wIn order to address whether Importin-7 mediates the nuclear translocation of AR and USP22, cytoplasmic and nuclear proteins were isolated in BC cells treated with Importin-7 siRNAs or control sequences. Immunoblotting analysis was subsequently conducted to detect the expression of Importin-7, USP22, and AR in the nucleus and cytoplasm, respectively. YAP, known as an Importin-7 cargo , was useAs we have demonstrated previously, increasing the responsiveness of BC to AR signaling suppression with enzalutamide by inhibition of USP14-induced AR degradation was elaborated as a potential therapeutic strategy , 25. HerWe first confirmed that the knockdown of Importin-7 could enhance the growth inhibition of cancer cells by enzalutamide using CCK8 assay Fig.  and EdU BC, the greatest threat to women’s life and health, is complex cancer that exhibits high heterogeneity between and even within tumors , 28, 29.In order to ensure the normal progress of various life processes of cells, the subcellular spatial distribution of proteins needs to be precisely regulated, which depends on the selective and efficient transport of substrate proteins by a series of transport receptors , 9. DysrNext, we discovered that Importin-7 could physically interact with AR and regulate its nuclear translocation in BC cells. Previously, we have confirmed the importance of AR in the progression of ER-negative BC , 25. In In addition, we also observed that AR expression levels were upregulated after DHT and R1881 stimulation, and Importin-7-bound AR was also up-regulated to an almost equal extent, indicating that the level of AR carried by Importin-7 was not saturated. These results suggested that the efficiency of Importin-7 in AR delivery may be enhanced by androgen stimulation. Additionally, we also found that silencing USP22 resulted in a less pronounced downregulation of AR. Interestingly, silencing USP22 increased the level of Importin-7 binding to AR, implying a competing binding mechanism, which further supports the view that USP22 is also individually one of the cargos of Importin-7.Furthermore, we further explored and found that the knockdown of Importin-7 enhanced the sensitivity of BC to enzalutamide. Knockdown of Importin-7 synergizes with enzalutamide to induce cycle arrest and promote cancer cell growth inhibition. These results suggest that the identification and development of Importin-7 inhibitors may be promising for clinical application.Overall, through the analysis of clinical samples combined with experimental studies of cellular and molecular biology, the current study identifies a molecule key to BC progression, Importin-7. In this study, we focused on one of its mechanisms of action: forming a protein complex with USP22 and AR and promoting the nuclear transport of AR–USP22. In addition, this study also provides a rationale for a therapeutic strategy to limit the malignant progression of AR-positive BC by inhibiting the high expression state of Importin-7.Fresh tissues were kindly donated from patients with BC or benign breast hyperplasia. All samples were obtained from the discarded material utilized for routine laboratory tests at the Department of Breast Surgery, First People’s Hospital of Foshan . This study was performed with the approval of the Medical Ethics Committee of the First People’s Hospital of Foshan.The fresh benign breast hyperplasia and malignant BC tissues were stored in dry ice, and subsequent quantitative liquid chromatography–tandem mass spectrometry (LC–MS/MS) via data-independent acquisition (DIA) , 35 was BC cell lines were obtained from ATCC. Cell culture conditions are listed in Table Cell viability was detected by using a cell counting kit-8 (CCK8) assay , as we have described in the past . Cells wThe EdU staining assay  was used to detect the DNA replication rate of BC cells. The experimental operation methods and steps were carried out strictly in accordance with the kit instructions and the way we reported in the past .For the colony formation experiment, cells were reseeded in a 6-well plate for two weeks after being treated as directed. The cells were washed with PBS twice, fixed for 15 min with 4% paraformaldehyde, then stained for 5 min with crystal violet solution. Finally, from three separate studies, the number of colonies larger than 60 mm was counted.Consistent with the previous description, cell cycle distribution, and apoptosis were detected by flow cytometry , 36, 37.For apoptosis assay, cells were treated according to the indicated conditions, then collected and washed twice using 4 °C PBS. An Annexin V-FITC/PI-containing apoptosis detection kit  was used to stain the apoptotic cells according to the instruction. Flow cytometry was performed to determine the apoptotic rate from three independent experiments.The RNA interfering and plasmid transfection assays were performed in BC cells and conducted as previously reported , 36, 38.For siRNAs and plasmids transfection, the transfection mixture was prepared with siRNAs or plasmids, RPMI opti-MEM (Gibco), and TransIT-X2® Dynamic Delivery System  according to the system in the User Guide of MIR 6000. The transfection mixture was then added to plates where cells had been seeded for 24 h.For lentivirus transfection, lentivirus containing shRNAs targeting Importin-7 or control shRNAs were generated. Firstly, a complete medium containing polybrene (5 μg/ml) was used to incubate the BC cells for 15 min, and lentivirus was then added into plates for 48 h. Medium containing puromycin (2 μg/ml) was used to eliminate the unsuccessfully infected cells.An immunofluorescence experiment was carried out as previously described , 36, 38.Separation of proteins between the nucleus and cytoplasm was strictly performed in accordance with the user guide for Nuclear and Cytoplasmic Protein Extraction Kits .Protein samples were collected at low temperatures using the cell lysis buffer (Cell Lysis Buffer (#9803) , protease inhibitor mixture, phosphatase inhibitor mixture, and PMSF . The western blotting and co-immunoprecipitation (co-IP) experiments, including the preparation steps of the protein samples, were completely consistent with our previous descriptions and reports , 36, 38.t-test or one-way ANOVA is utilized. GraphPad Prism 9 and SPSS 16.0 software were utilized for statistical analysis. A p value < 0.05 was considered statistically significant. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 vs. each vehicle control.Experimental results are reported as the mean S.D. of three separate experiments. When determining statistical probability, the paired/unpaired Student’s Supplementary TablesOriginal Data File


We study the microbiome of sea water collected from two locations of the Barbadian coral reefs. The two sites differ in several environmental and ecological variables including their endogenous benthic community and their proximity to urban development and runoffs from inland watersheds. The composition of the microbial communities was estimated using whole genome DNA shotgun sequencing with adjuvant measurements of chemical and environmental qualities. Although both sites exhibit a similar degree of richness, the less urbanized site (Maycocks reef at Hangman’s Bay) has a strong concentration of phototrophs whereas the more urbanized location (Bellairs reef at Folkstone) is enriched for copiotrophs, macroalgal symbionts and marine-related disease-bearing organisms from taxa scattered across the tree of life. Our results are concordant with previous profiles of warm ocean surface waters, suggesting our approach captures the state of each coral reef site, setting the stage for longitudinal studies of marine microbiome dynamics in Barbados.The online version contains supplementary material available at 10.1007/s00338-022-02330-y. The capacity of fragile reef ecosystems to maintain key economic, social and environmental services for coastal human societies has been severely challenged by a global decline in coral reef cover of at least 50%  and the Maycocks reef at Hangman’s Bay  at 13:00–15:00 on January 30 and 31, 2018, respectively. Bellairs is a nearshore fringing reef within the no-take area of Folkstone Marine Reserve in relatively shallow waters (~ 10 m). It lies ~ 100 m from Holetown, a densely populated area with tourism and other urban infrastructure  approximately 1.5 km away from a sparsely populated coastline with little coastal development Fig.  and is t−1 lysozyme and 20 μL of 10 μg mL−1 RNAse A and left to rotate at 37 °C for 1 h. Then, after incubation, 100 μL of 10 mg mL−1 proteinase K and 100 μL of 20% SDS were added. Filters were rotated for 2 h at 55 °C and the lysate removed. Protein was then precipitated and removed with 0.583 volumes of MPC protein Precipitation Reagent (Epicentre Inc.) and centrifugation at 10,000 × g at 4 °C for 10 min. The supernatant was transferred to a clean tube. The DNA was precipitated with cold isopropanol and resuspended in low TE buffer.At time of sampling the ocean current was flowing in a northwestern direction Fig. C. Sea waFor whole genome sequencing, DNA was prepared  and sequenced on an Illumina NovaSeq PE 150 platform (2 × 150 bp reads). Kraken2 version 2.1.0  test was used to measure significance between the frequencies of child taxa  within a specific ancestral taxa from the first site versus the frequencies of these same child taxa at the second site. When the KW test was significant, Dunn’s test was used to determine which specific child taxa were significantly different. In some instances we asked if the distribution of observed genera were enriched in a specific ancestral taxa ; towards this end we used the hypergeometric test where bins correspond to the ancestral taxa and bin size was obtained from our NCBI database above. Clustering was performed by first identifying the most abundant taxa for each sample , and then transforming their abundances to ranks before use of the Kendall τ distance metric with Ward’s algorithm to construct two dimensional hierarchical clusters.p << 0.01, Pearson’s 2). Then within the bacterial superkingdom, there are more pronounced differences in the distribution across phyla , whereas Bellairs has a preference for Proteobacteria, specifically the Alpha class. We explore these differences below.We found striking and consistent differences between Bellairs and Maycocks in many dimensions. Maycocks had higher concentrations of dissolved oxygen whereas Bellairs had higher concentrations of nitrate . In total, 97% of the genera were identified at both sites through analysis of the sequencing data . However, the relative frequency of taxa is very different between the two sites. For example, Maycocks exhibits a relative enrichment of Bacteria and Chlorophyta whereas Bellairs contains more eukaryotic  and archaeal taxa , highlighting Dinoroseobacter, Tateyamaria and Jannaschia. Dinorosebacter are aerobic anoxygenic phototrophic bacteria, highly abundant in marine turf algae. Tateyamaria is a genus of coastal aerobic bacteria which can thrive under acidification conditions . This is not likely due to the depth of sequencing alone given that Maycocks in fact received 1.57 fold more reads than Bellairs and analysis in Supplemental Information 1). Several marine diatoms were identified including Licmophora, Cylindrotheca, Seminavis, and others which contribute species that grow on algae and corals or feed on diatoms in eutrophic environments and are causal in brown and red tides. Supplemental Information 3 provides a more detailed investigation of the SAR clade. Analogous to the Stramenopiles, the Foraminifera contribute a surprising number of uniquely identified genera   on the state of reef health than standard monitoring efforts , as it identifies ~ 100 distinct differentially abundant genera which may interact with different dimensions of the reef ecology. As such, the findings here may influence, for example, the design and feasibility of coral rehabilitation projects and, more generally, our capacity to identify microbiome changes consistent with known stressors and states of eutrophication. This sets the stage for systematic studies using microbiome-based markers of the rate of change to reef health along many variables . Our approach here should have relevance to other small islands in the vicinity of Barbados with similar reef systems and stressors.Supplementary file1 (DOCX 4509 KB)Below is the link to the electronic supplementary material.


Introduction: Environmental stress promotes epigenetic alterations that impact gene expression and subsequently participate in the pathological processes of the disorder. Among epigenetic regulations, ten–eleven Translocation (Tet) enzymes oxidize 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) in DNA and RNA and function as critical players in the pathogenesis of diseases. Our previous results showed that chronic stress increases the expression of cytoplasmic Tet2 in the hippocampus of mice exposed to chronic mild stress (CMS). Whether the cytoplasmic Tet2 alters RNA 5hmC modification in chronic stress-related processes remains largely unknown.Methods: To explore the role of cytoplasmic Tet2 under CMS conditions, we established CMS mice model and detected the expression of RNA 5hmC by dot blot. We verified the interaction of Tet2 and its interacting protein by co-immunoprecipitation combined with mass spectrometry and screened downstream target genes by cluster analysis of Tet2 and upstream frameshift 1 (Upf1) interacting RNA. The expression of protein was detected by Western blot and the expression of the screened target genes was detected by qRT-PCR.Results: In this study, we found that increased cytoplasmic Tet2 expression under CMS conditions leads to increase in total RNA 5hmC modification. Tet2 interacted with the key non-sense-mediated mRNA decay (NMD) factor Upf1, regulated the stability of stress-related genes such as Unc5b mRNA, and might thereby affect neurodevelopment.Discussion: In summary, this study revealed that Tet2-mediated RNA 5hmC modification is involved in stress-related mRNA stability regulation and may serve as a potential therapeutic target for chronic stress-related diseases such as depression. Epidemiological studies have revealed that chronic stress caused by environmental factors has a significant impact on diseases . AdverseChronic environmental stress induced several types of epigenetic modifications to regulate gene expression in the pathological process of depression . For insRecent studies have shown that methylation and demethylation modifications were also found in RNA cytosine, namely, RNA 5mC and RNA 5hmC . These mIn this study, we found that the expression of RNA 5hmC modification increased under CMS conditions accompanied with increased cytoplasmic Tet2. The ATP-dependent RNA helicase Upf1 was identified as a Tet2-interacting protein to modify RNA 5hmC and regulate RNA stability such as Unc5b mRNA, which is involved in axonal guidance and stress responses. Our findings, therefore, provide novel mechanistic insights into the role of epigenetic modification induced by Tet2 in chronic stress, suggesting the pathological importance of cytoplasmic Tet2 and RNA 5hmC modification in psychiatric disorders.The lists of the reagents, antibodies, cell lines, and oligos used in this study are given in All procedures and protocols in this study were approved by the Institutional Animal Care and Use Committee of Soochow University. C57BL/6 mice  were purchased from the Shanghai Research Center for Model Organisms. The mice were randomly divided into two groups: the control group and the model group and exposed to various, randomly scheduled, low-intensity social and environmental stressors two–three times a day for 5 weeks. The stressors included the following:  water deDepression in mice was assessed using the TST . First, The FST was used to assess mice for depressive behavior. The FST apparatus consisted of a transparent glass cylinder (18 cm diameter and 40 cm height) filled with water up to 15 cm at about 25°C. The cylinder was wrapped in tin foil paper to prevent interference while the mice swam. Pre-testing is required before a formal experiment. Mice were placed in a cylinder and allowed to swim and then removed from the water and returned to the cage after been dried in a heated environment for several minutes. In the formal test, the camera was placed directly above the cylinder, and the immobility time of the mice was measured during a 6-min swim after being placed in the water. At the end of each test, the warm water should be refilled so as not to affect the subsequent measurement tests. The criterion for immobility in mice is to stay afloat without struggling.In this test, mice with anhedonia were evaluated with reference to the method of 2. For transfection, HEK293T cells were transfected at 70% confluency using polyethylenimine (PEI), and N2a cells were transfected at 70% confluency using PL transfection reagent according to the manufacturer’s instructions.Neuro 2A (N2a) cells and HEK293T cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and were placed in a humidified incubator at 37°C with 5% COFor endogenous IP, three 90-mm dishes containing N2a cells at 70% confluency were lysed in 1 mL RIPA buffer  containing 100 × cocktail and 200 × DTT at 4°C under rotary agitation for 30 min. About 5% input was taken after centrifuging at 13,200 rpm for 30 min at 4°C. Total protein was divided into two groups, and each group contained not less than 2 mg. The protein supernatant was added after agarose beads were activated and was pre-cleared by rotating for 2 h at 4°C. The supernatant was transferred to another tube, and the same amount of Tet2 and IgG antibody was added into the two tubes and then rotated at 4°C overnight. The supernatant was transferred to the agarose beads and rotated at 1,000 rpm at 4°C overnight. The supernatants were removed after centrifuging at 1,000 rpm at 4°C for 3 min, 50 μL 2 × loading buffer was added to the precipitate, and the samples were boiled at 95°C for 10 min.2] containing 100 × cocktail and 200 × DTT at 4°C for 30 min; centrifuged at 4°C for 30 min at 13,200 rpm; and 60 μL of the supernatant was collected as the input sample. The remaining supernatant was put into Flag beads and rotated at 4°C overnight. The supernatant was removed by a magnetic shelf, and the beads were cleaned twice with lysis buffer. About 50 μL 1 × loading buffer was added to the precipitate, and the samples were boiled at 95°C for 10 min.For exogenous IP, HEK293T cells were transiently transfected with target plasmids using PEI. Three 90-mm dishes of HEK293T cells were used as one group. HA-Upf1 (30 μg) + Vector (30 μg) and HA-Upf1 (30 μg) + Flag-Tet2 (30 μg) plasmids were transfected for 48 h. Cell pellets were lysed using lysis buffer [150 mM NaCl, 25 mM tris-HCl (pH 7.4), 10% glycerol, 0.5% Triton X-100, 2 mM MgClFor total protein extraction, cell pellets were lysed using RIPA buffer containing 50 mM Tris (pH 8.0), 0.1% SDS, 150 mM NaCl, 1 mM EDTA (pH 8.0), 1 mM EGTA (pH 8.0), 0.5% deoxycholate, and 1% Triton X-100 on ice for 30 min and then centrifuged at 4°C at 12,000 rpm for 15 min. The supernatant was collected and boiled for 10 min at 95°C after adding 1/4 volume of 5 × loading buffer. About 30-μg protein aliquot of each sample was separated using standard SDS-PAGE and transferred onto a PVDF membrane. After transferring the protein, the PVDF membrane was blocked with 10% nonfat milk for 1 h at room temperature (RT) and incubated overnight at 4°C with target primary antibody. Immunoreactive bands were detected by ECL chemiluminescence reagent after incubating with secondary antibody for 1 h. The antibody and reagents used in this experiment are given in the The samples were fixed overnight at 4°C in 4% PFA in PBS for 30 min, treated with 0.3% Triton X-100 in PBS (PBST) for 10 min, and then blocked with 5% BSA in 0.3% PBST for 1 h at RT. Subsequently, the samples were incubated with sample-specific primary antibodies overnight at 4°C, followed by three washes with PBS, and incubated with florescent-labeled secondary antibody containing DAPI for 1 h at RT. The samples were imaged using a fluorescence microscope  after patching on the microslide.E.coli was used to transform the connection products obtained in the aforementioned experiments. After transformation, monoclonal colony was selected for plasmid amplification. The plasmids were extracted and verified by sequencing.RNA was extracted from the hippocampus of wild-type C57BL/6 mice and reverse-transcribed onto cDNA. Target fragments were amplified from this cDNA as templates. The gel extraction products of target fragments were linked with linearized pCMV-HA or pCMV-flag plasmids by homologous recombination. DH5α http://jvenn.toulouse.inra.fr/app/example.html). Metascape (http://metascape.org/gp/index.html#/main/step1) is a gene-list analysis tool for gene functional enrichment analysis . GAPDH was used as an endogenous control for real-time PCR amplification. For data analysis, fold change was calculated using the ΔΔCt method according to the threshold cycle (Ct) value obtained from RT-PCR. The sequences of the primers used in this study are given in Total RNA was extracted using RNA TRIzol and was reverse-transcribed using the Reverse Transcription Kit as per the manufacturer’s protocol. During RNA extraction, DNase I, RNase-free pipettor tips, RNase-free centrifugal tubes, and other RNase-free plastic products were used, and RNA was dissolved in RNase-free water treated by DEPC. After the extraction, the RNA purity was tested using a NanoDrop 2000 spectrophotometer. For dot blot, a 20-μL system containing 2,000 ng RNA, 10 μL 2M NaOH (denature solution), and double distilled water was prepared. Then, the mix was allowed to stand at 4°C for 20 min. We spotted 2 µL of samples onto the nitrocellulose (NC) membrane and allowed the membrane to dry at RT. RNA was fixed to the membrane by incubating at 80°C for 30 min. Then, the NC membrane was blocked with 10% nonfat milk for 1 h at RT and incubated with 5hmC antibody overnight at 4°C. The immunoreactive signal was detected by ECL chemiluminescence reagent after 2-h incubation with secondary antibody at RT. Also, the dot signal was quantified by using ImageJ software.t-test or one-way ANOVA, followed by the Tukey’s post hoc test, and were considered statistically significant at p < 0.05.All statistical analyses were performed using Prism 7.0 (GraphPad Software) and are expressed as means ± SEM. The differences with different treatments were determined by p < 0.01, p < 0.05, Our previous results showed high levels of RNA 5hmC modification in the brainstem, hippocampus, and cerebellum of mice, and RNA 5hmC modification was downregulated in the MPTP-induced mouse model , but thep < 0.01, p < 0.01, p < 0.05, p < 0.05, p < 0.01, Studies have shown that RNA 5hmC is derived from RNA 5mC and is mainly catalyzed by Tet proteins . Thus, wp < 0.0001, 0.01, p < 0.01, p < 0.0001, p < 0.05, To further validate the role of Tet2 in the oxidation of RNA 5mC to 5hmC, we examined RNA 5hmC levels in N2a cells by either Tet2 knockdown or overexpression. Tet2 knockdown markedly decreased Tet2 mRNA and protein expression in N2a cells   and Upf1p < 0.05, 0.001, p < 0.01, p < 0.05, 0.01, To investigate the mechanism by which the Tet2–Upf1 complex affects downstream target genes, we knocked down Tet2 and Upf1 in N2a cells, respectively (p < 0.05, p < 0.01, p < 0.05, p < 0.05, p < 0.05, p < 0.05, p < 0.01, Previous studies have demonstrated that RNA 5hmC modification regulates mRNA stability at the post-transcriptional level . In ordeOur previous findings showed that Tet2 protein expression was increased in the mouse hippocampus under chronic mild stress conditions and accumulated in the cytoplasm . Some stAmong the epigenetic modifications, the most widely studied are DNA methylation modification and hydroxymethylation modification . With thTo further confirm the modification of RNA 5hmC by Tet2, we performed Tet2 knockdown in N2a cells using siTet2. The results were consistent with expectations, and the expression of RNA 5hmC was reduced after knockdown of Tet2 and increased by overexpression of Tet2 CD. Taken together, we confirmed that Tet2 is the dominant protein responsible for the increased modification of RNA 5hmC under chronic stress. Studies reported that RNA-modifying enzymes and RNA-binding proteins cooperate in the regulation of transcripts . Here, wIn conclusion, we found that increased expression of Tet2 after chronic stress in the cytoplasm led to an increase in RNA 5hmC modification. We, therefore, focused on the role and mechanism of 5hmC modification of RNA under chronic stress. In addition, Tet2 and its new interacting protein Upf1 can participate in the regulation of stress-related mRNA stability in the form of a complex, and the common target genes of Tet2 and Upf1 are mainly involved in axon guidance-related genes, also indicating that neuronal development plays a critical role in chronic stress-related diseases such as depression. However, there are still some gaps in our study, such as where the increase of Tet2 and RNA 5hmC in nerve cells mainly occurs after chronic stress, and other roles of Tet2 on RNA 5hmC modification in chronic stress, which are also the focus of our future research.


The significance of RNA functions and their role in evolution and disease control have remarkably increased the research scope in the field of RNA science. Though the availability of RNA structure data in PBD has been growing tremendously, maintaining their quality and integrity has become the greater challenge. Since the data available in PDB are results of different independent research, they might contain redundancy. As a result, there remains a possibility of data bias for both protein and RNA chains. Quite a few studies have been conducted to remove the redundancy of protein structures by introducing high-quality representatives. However, the amount of research done to remove the redundancy of RNA structures is still very low. To remove RNA chain redundancy in PDB, we have introduced RNA-NRD, a non-redundant dataset of RNA chains based on sequence and 3D structural similarity. We compared RNA-NRD with the existing non-redundant RNA structure dataset RS-RNA and showed that it has better-formed clusters of redundant RNA chains with lower average RMSD and higher average PSI, thus improving the overall quality of the dataset. Ribonucleic acid (RNA) is a single-stranded macromolecule that not only carries genetic information but also performs different biological functions by forming various three-dimensional structures ,2. Despiet al. anticipated that on average a protein structure is represented more than 4 times in PDB  is an online public database for storing 3D crystal structures of biological macromolecules such as protein and nucleic acids  where they classified RNA chains available at that time into nine different classes based on their functionality  developed by Leontis et al. . Here, tet al.  shows hoKeeping all of this in mind, we developed a non-redundant RNA structural dataset based on both RNA sequence and 3D structure coordinates. We refer to this dataset as RNA-NRD. Our main goal for developing this dataset is to identify redundant RNA chains based on both sequence and 3D structure similarity and represent a group of redundant RNA chains with a single representative chain. In RNA-NRD, two RNA chains belonging to the same source organism with a sequence identity ≥ 80%, RMSD value <4 Å and structural alignment ratio ≥80% are considered to be redundant to each other and are put together in the same cluster. Finally, the chain with the best quality score is selected as the representative of the cluster. We use the 3D structural alignment tool STAR3D which globally aligns the RNA chains. This improves the overall 3D structural comparison of RNA chains. To remove chain length dependency from RNA structural comparison, we use a parameter called structural alignment ratio which is the ratio of structurally aligned nucleotides with respect to the number of nucleotides with coordinates in the shorter RNA chain. In our dataset, we consider each RNA chain as a single entity. This not only handles redundancy among PDB files, but also handles redundancy within a PDB file. As depending on applications, the definition of redundant RNA structures can vary, we have generated another variation of RNA-NRD dataset where we don’t divide the RNA chains based on source organism. We refer to this dataset as RNA-NRD-without-Organism-Division. Both RNA-NRD and RNA-NRD-without-Organism-Division are updated on a regular basis and are publicly accessible to facilitate future research endeavors.The whole process for generating the RNA-NRD dataset is illustrated step-by-step in this section. Also, the steps to generate RNA-NRD-without-Organism-Division and the pipeline for updating both the RNA-NRD and the RNA-NRD-without-Organism-Division datasets on a regular basis is discussed here in detail.The different stages for generating the RNA-NRD dataset are shown in Figure Nearly 12418 nucleic acid containing structures have been collected from PDB on 17 March 2021. From there, protein, DNA and NA-hybrid structures have been filtered out and a total of 9705 RNA chains having length ≥20 nucleotides are selected for further analysis. In PDB, each RNA chain contains a list of features. Among those, PDB ID, Experimental Method, Release Date, Resolution (Å), Sequence, Entity Polymer Type, Polymer Entity Sequence Length, Source Organism, Macromolecule Name and Chain ID, these 10 features are selected as they are more commonly used for RNA structure analysis.Rfam is a well-known database of RNA families where each family is represented by multiple sequence alignment and co-variance model . As of 1Generally, PDB provides information regarding the source of RNA chains as organism name. In the RNA-NRD dataset, RNA chains belonging to different organisms are considered to be non-redundant. Hence, similarity checks between RNA chains are only conducted if they belong to the same organism. If the RNA chains are re-engineered RNA chains, then they are put together under the organism group ‘synthetic construct’. Including this, a total of 187 source organisms have been identified containing 8967 RNA chains. However, there are 738 RNA chains with no specified source organism. These RNA chains with missing source information are put together as a single organism under the name ‘others’, making a total of 188 organisms. A similarity check is performed within the chains belonging to this ‘others’ organism to see if there is any redundancy among them.In order to figure out possible redundancy between RNA chains, we first compare the sequence of the RNA chains and generate sequence identity. The sequence of two RNA chains are compared to each other only if the length of the longer RNA chain is not greater than twice the length of the shorter RNA chain. A pairwise comparison is done between all such pairs of RNA chains belonging to the same organism. For generating sequence alignment, Needleman-Wunsch sequence alignment algorithm  is used.Comparing the structural similarity of RNA chains is of utter importance while removing redundancy. As per studies, homologous RNA molecules having similar functionality can be structurally very similar . RNA chaCricket paralysis virus is used as an example. This organism contains 9 RNA chains in total. First, these chains are divided into two Redundant Groups based on their sequence identity. RNA chains having sequence identity ≥ 80% are assigned to the same Redundant Group. Later, based on structural similarity, ‘Redundant Group 1’ is divided into four Redundant Clusters, and ‘Redundant Group 2’ forms only one Redundant Cluster. All the RNA chains belonging to the same Redundant Cluster are considered to be redundant. In total, a number of 3157 Redundant Clusters are generated in the RNA-NRD dataset.After performing pairwise structural alignment within a Redundant Group, all the RNA chains having RMSD value <4 Å and structural alignment ratio ≥80% are assigned together to a single ‘Redundant Cluster’. This is done by generating an undirected graph where the nodes represent the RNA chains and edges are drawn between two chains if their RMSD value <4 Å and structural alignment ratio ≥80%. The structural alignment ratio between two RNA chains is calculated by dividing the number of structurally aligned nucleotides with the number of nucleotides of the shorter RNA chain that have corresponding 3D coordinates in the PDB file. Here, two superimposed nucleotides are considered to be structurally aligned if their average spatial distance is <4 Å. Structural alignment ratio is used to remove the length dependency of RNA chain structural comparison. Each connected component in the undirected graph represents a Redundant Cluster. We use the concept of transitivity, meaning if two chains are redundant by definition, the other RNA chains redundant to those two chains will also be considered redundant to each other and will belong in the same Redundant Cluster. For better understanding, this whole process is demonstrated in Figure n), number of base pairs (b), structure resolution (r) and chain degree (d). For each chain, the values n and r are collected from the corresponding PDB files. The value b is generated using the tool DSSR  is assigned to resolution (r) as RNA chains with higher resolution are considered to be more accurate in general. Also, RNA chain structures containing more nucleotides (n) and base pairs (b) are more likely to be better representatives and they are both assigned a weight of 0.25 (w2 and w3). Apart from that, chain degree (d) is also considered to prioritize RNA chains that are strongly connected in a cluster as they are structurally similar to most of the RNA chains belonging to that cluster and hence are assigned a weight of 0.1 (w4). The total of all the weights assigned to these four variables is 1. The quality score calculated for each RNA chain ranges between 0 and 1. In case multiple RNA chains have the same quality score in a single Redundant Cluster, then the representative will be selected based on other criteria in the following order of priority: (i) experimental method, (ii) chain length and (iii) release date. Among different experimental methods, X-ray diffraction is considered to have higher quality  of the last 10 years shows that on average, it releases 71 new RNA-only structures per year. To incorporate the newly introduced RNA chains in the non-redundant dataset, regularly updating the RNA-NRD dataset is mandatory. Hence, a fast and optimized pipeline has been developed to update the RNA-NRD dataset every 3 months. Instead of doing a pairwise comparison within all RNA chains of a certain organism, this pipeline will only compare the newly added RNA chains of PDB to the representative RNA chains (RP) with the same organism introduced in the previous version of the RNA-NRD dataset. Thus, it will generate a new version that includes the new PDB RNA chains. Figure PDB is a highly growing database. The PDB statistics  of the last version of the RNA-NRD dataset. For a certain new RNA chain, RPs having the same source organism, sequence identity ≥80%, RMSD <4 Å and structural alignment ratio ≥80% are considered to be similar. Here, the structural alignment ratio is calculated by dividing the number of structurally aligned nucleotides by the number of residues with coordinate of the new RNA chain instead of the number of residues with coordinate of the smaller RNA chain as the main focus of this comparison is the new RNA chain. While updating the RNA-NRD-without-Organism-Division dataset, the only difference is that the new RNA chains are compared against all the RPs regardless of their source organism.For a new RNA chain, among all the similar RPs, the RP having the maximum similarity is considered to be a match. As the new RNA chain is significantly similar to this RP, so it can be considered redundant to this RP and its cluster members. Thus, the new RNA chain is assigned to the cluster of that matched RP. Later, for such modified clusters, a new representative is selected based on the quality score. The quality score is generated using Equation .If the RP of a RNA-NRD cluster does not match with any new RNA chain, then no new RNA chains will be added to that cluster and it will remain unmodified. While generating the updated version of RNA-NRD dataset, these unmodified clusters will be added just as it is to the updated version.New RNA chains, which are not similar to any of the current cluster representatives, are compared among themselves. For that, a similar pipeline is used as shown in Figure RNA-NRD is a dataset that contains the redundant RNA chains together in a cluster and presents a representative RNA chain for each cluster. The first version of the RNA-NRD dataset is shown in In order to benchmark the RNA-NRD dataset, we compared it to the most widely used RNA non-redundant structural dataset known as ‘Representative Sets of RNA 3D Structures’ (RS-RNA) . For comThe overall comparison between the RNA-NRD and the RS-RNA datasets is shown in Table To give a better idea about how the clusters in RNA-NRD have better structural similarity compared to the compatible clusters proposed in RS-RNA, we showed the comparison of the clusters for some selected well-known organisms in Table Escherichia coli, Saccharomyces cerevisiae and Thermus thermophilus contain too many RNA chains. Hence, generating their RMSD and TM-score using the structural alignment tool RNA-align , negative (‘-’), and zero (‘=’) RMSD-diff is 0, 3, 1 consecutively. Similarly, the number of clusters having positive (‘+’), negative (‘-’) and zero (‘=’) PSI-diff is 2, 1 and 0. The number of single-chain clusters is 2 as cluster 1364 and 1366 contain one chain each. So both their RMSD-diff and PSI-diff values are represented with ‘Null’. The total absolute value of positive RMSD-diff (RMSD +) is 0, negative RMSD-diff (RMSD -) is 1.08 + 1.18 + 0.17 = 2.39, positive PSI-diff (PSI +) is 0.04 + 0.01 = 0.05, and negative PSI-diff (PSI -) is 0.04 for this organism. As the total of ‘RMSD -’ and ‘PSI +’ are higher for the clusters belonging to the RNA-NRD dataset compared to their compatible clusters from the RS-RNA dataset, it can be said that RNA-NRD clusters have better structural similarity compared to the RS-RNA clusters for the organism Oceanobacillus iheyensis. As can be seen from Table Figure The RNAMotifContrast tool is used to capture the structural variations among the well-known 11 motif families  and Zenodo (https://doi.org/10.5281/zenodo.7806754). The RNA-NRD datasets and the RNA-NRD-without-Organism-Division datasets are available in the http://genome.ucf.edu/RNA-NRD).RNA-NRD source code is available on Github (lqad040_Supplemental_FilesClick here for additional data file.


Extracellular RNA (exRNA) has long been considered as cellular waste that plants can degrade and utilize to recycle nutrients. However, recent findings highlight the need to reconsider the biological significance of RNAs found outside of plant cells. A handful of studies suggest that the exRNA repertoire, which turns out to be an extremely heterogenous group of non-coding RNAs, comprises species as small as a dozen nucleotides to hundreds of nucleotides long. They are found mostly in free form or associated with RNA-binding proteins, while very few are found inside extracellular vesicles (EVs). Despite their low abundance, small RNAs associated with EVs have been a focus of exRNA research due to their putative role in mediating trans-kingdom RNAi. Therefore, non-vesicular exRNAs have remained completely under the radar until very recently. Here we summarize our current knowledge of the RNA species that constitute the extracellular RNAome and discuss mechanisms that could explain the diversity of exRNAs, focusing not only on the potential mechanisms involved in RNA secretion but also on post-release processing of exRNAs. We will also share our thoughts on the putative roles of vesicular and extravesicular exRNAs in plant–pathogen interactions, intercellular communication, and other physiological processes in plants. In this review, we discuss the biological significance of extracellular RNAs in plants and provide insights into their secretion, processing, and trafficking within the apoplast. The plant extracellular space, alternatively known as the apoplast, is a partially interconnected matrix external to the plasma membrane that includes the free space between cells (or intercellular space), xylem, cell walls, and apoplastic fluid. Many biological processes take place in this compartment, including nutrient exchange, cell wall biosynthesis, plant signaling, and defense responses (i). Due to the presence of these RNases, RNA has generally been considered to be unstable in the apoplast, unless it is protected from RNase digestion, either by encapsulation within lipid membrane-containing extracellular vesicles (EVs) or by tight association with RNA-binding proteins (RBPs) (The presence of RNA in the plant apoplast was first suggested in the 1980s and 1990s with the discovery that plant cells secrete RNases (s (RBPs) . The diss (RBPs)  ranging Bulk analyses of the composition of exRNA have generally been accomplished through gel electrophoresis and next-generation RNA sequencing . More acg (P100)], and soluble RNAs that remain in the P100 supernatant. Separation of particle-bound extravesicular RNA from intravesicular RNA is challenging , particle-bound extravesicular RNA [located outside EVs, but associated with particles that can be pelleted by centrifugation at 100 000 llenging ; howeverSmall RNA sequencing revealed that Arabidopsis EVs are enriched in tinyRNAs, a special class of very short (10–17 nt) sRNAs, while relatively few 18–34 nt sRNAs are packaged inside EVs , and it It has recently been shown that mammalian RNA-containing EVs are small (~50 nm) and relatively rare in a general EV population , Arabidopsis exRNA contains thousands of circular RNAs  by which RBPs and their associated RNAs are trafficked to and selected for loading into newly forming EVs is still largely unknown. A more recent study in mammals has uncovered a different mechanism of exRNA secretion that involves endoplasmic reticulum (ER)–plasma membrane contact sites that tether the proteins VAP-A and Ceramide Transfer protein (CERT). This study concludes that VAP-A controls intraluminal filling of MVBs with LC3 and specific miRNAs; however, the role of RBPs in this process is still unclear . TherefoRecent observations showing that extravesicular RNAs constitute the majority of exRNA suggest that plants preferentially secrete sRNAs and lncRNAs into the apoplast via mechanisms that do not involve their packaging inside EVs  relative to total cellular RNA  move via the symplastic pathway, in which plasmodesmata along with the phloem establish a cytoplasmic network that links virtually all the cells of a plant . Apart fRNA mobility through the apoplast might be facilitated by association with RBPs. In agreement with this, it has been shown that lysine-containing cell-penetrating peptides help RNAs to cross cell walls and penetrate plant cells (5C). For instance, tRNA motifs that have a high percentage of m5C (tRNAGly or tRNAMet) can trigger mRNA mobility, while mRNAs fused with a tRNA motif that has a low level of m5C (tRNAIle) are immobile  to the extracellular space, where they are processed by extracellular RNases . InteresExtracellular processing of RNA in plants is likely because plants secrete multiple RNases into the apoplast. These RNases belong to the T2 family . The memi recycling  is an essential macronutrient for plants. Since the availability of Pi is usually low in soils, recycling and remobilization of Pi within the plant are crucial mechanisms that support plant growth and development. Nucleic acids are an important source of phosphate that plants can use to recycle Pi. Many RNases, along with phosphatases and phosphodiesterases, are involved in the release of Pi from RNA to facilitate its remobilization  with EVs (P100) isolated from CYP3RNA-expressing A. thaliana plants as well as from CYP3RNA-sprayed barley plants did not have any effect on the expression of CYP51 in Fg  DCL2, 3, and 4 genes, which strongly suggests that siRNAs, rather than long hpRNAs, mediate gene silencing in this phyto-pathosystem. Collectively, these data suggest that plants secrete siRNAs into their apoplast where these RNAs can then impact the bacterial microbiome.Emerging evidence indicates that plant exRNAs may impact gene expression in bacterial pathogens through direct RNA:RNA base pairing. A preprint from 2019 reported that expression of hpRNAs in plants can silence homologous genes in the bacterial pathogen A more recent preprint provides further support for this hypothesis, reporting that Arabidopsis secretes sRNAs into the rhizosphere that were then taken up by root-associated bacteria  into Arabidopsis leaves, which led to an increase in tasi-RNAs derived from this transcript in both local and distal tissues. Mutation of AGO7, a protein necessary for tasi-RNA biogenesis from TAS3a, abolished this increase in tasi-RNAs. These observations indicate that naked exRNA can be taken up by plant cells and processed by the endogenous RNAi machinery, although we cannot rule out that cell damage caused during the infiltration process could have facilitated T-555 cellular uptake. Also in this study, it was found that injection of 6-carboxyfluorescein (FAM)-labeled tasi-RNA into a leaf resulted in movement of the labeled RNA into the vasculature and movement into distal tissues within 3 h. Remarkably, the FAM–tasiRNA could be recovered intact from distal leaves, indicating that it was protected against degradation during systemic movement. This systemic movement appeared to be mediated by phloem transport, rather than apoplastic transport, as it could be blocked by overexpression of PDLP5, which inhibits movement through plasmodesmata   . WhetherThe existence of RNAs outside plant cells has been recognized for a long time, but it was not until recently that the biological relevance and importance of this pool of RNAs began to be considered beyond the widely held assumption that they are simply cellular waste. Although much effort has been made to elucidate the packaging and release of RNAs inside EVs, recent work has shown that the majority of exRNAs, including sRNAs, are located outside EVs. How this exRNA is protected against degradation by extracellular RNases is not entirely clear but appears to require association with proteins. In addition, we speculate that extracellular RNases play an important role in shaping the exRNA pool, eliminating many of the RNAs originally released into the apoplast, with post-transcriptional modifications along with RBPs probably playing a significant role in protecting the exRNAs that remain. It should be emphasized that identification of many exRNAs is still challenging due to the technical limitations posed by conventional RNAseq protocols. Yet, the diversity of extracellular RNAs has begun to be elucidated and, to our surprise, it seems to be much more complex than anticipated. Such diversity encourages us to speculate that exRNAs contribute to multiple biological functions, such as cell to cell communication, defense against pathogens, and shaping the plant microbiome.Plant exRNA research is still in its infancy. and new and exciting knowledge will come to light as this field expands. Indeed, the repertoire of exRNAs in plant species other than Arabidopsis still needs to be characterized, which is critical for identifying exRNAs that are broadly conserved, and thus functionally important. Elucidating how exRNAs reach the apoplast, as well as assessing how they are regulated in response to various stresses will also help us understand what these RNAs are doing ‘outside’ of cells.Long non-coding RNAs (lncRNA): RNAs longer than 200 nt that are not translated into proteins. The most abundant extracellular lncRNAs described so far are intergenic RNAs (Circular RNAs (circRNA): single-stranded, covalently closed RNAs produced via back-splicing or from lariat precursors. The 3ʹ and 5ʹ ends of a circRNA covalently bond together to form a circular RNA molecule. Although the vast majority are expected to be non-coding (ncRNAs), studies in mammals demonstrate that some circRNAs can be translated into proteins  that transfer individual amino acids to ribosomes for assembly into the growing protein during translation.Small regulatory RNAs: non-coding RNAs (21–24 nt) that play a central role in RNA silencing. sRNAs can be selectively loaded into an ARGONAUTE protein (AGO) to silence target genes via nucleotide base-pairing. According to their biogenesis they can be classified into the following types.MIR genes. In miRNAs biogenesis, DICER-LIKE proteins (DCL) cut the hairpin precursors to produce mature miRNAs.miRNAs: miRNAs are originated from imperfectly paired single-stranded hairpin precursors encoded by siRNAs: in contrast to miRNAs, siRNAs originate from DCL cleavage of dsRNA molecules that were synthesized from ssRNAs by RNA-dependent RNA polymerases (RdRPs). siRNAs can be further classified into two main categories: heterochromatic siRNAs (hc-siRNAs) and secondary siRNAs, including phased siRNAs (phasiRNAs) and trans-acting siRNAs (tasiRNAs) .Other small RNAs: 18–34 nt RNAs derived from endonucleolytic processing of diverse RNA species, mainly tRNAs , rRNAs (rRFs), mRNAs, and Pol IV products (Tiny RNAs (tyRNAs): very short RNAs (10–17 nt) with unknown functions. These RNAs are likely to be degradation products derived from multiple sources, including TEs, mRNA, miRNAs, pol IV products, intergenic regions, and rRNAs (


This helps to understand transcription termination, which is one of the least understood processes in transcription. Additionally, our strategy can potentially be used to study the co-transcription behavior of general RNA, especially when continuous transcription is not desired.T7 RNA polymerase is the most widely used enzyme in RNA synthesis, and it is also used for RNA labeling in position-selective labeling of RNA (PLOR). PLOR is a liquid–solid hybrid phase method that has been developed to introduce labels to specific positions of RNA. Here, we applied PLOR as a single-round transcription method to quantify the terminated and read-through products in transcription for the first time. Various factors, including pausing strategies, Mg RNA is transcribed by RNA polymerase (RNAP) in cells, and then involves in various cellular activities, including transferring genetic information, regulating gene expression, and catalyzing reactions as enzymes and cofactors ,4,5,6,7.32P-radiolabeled NTPs are used to significantly improve the sensitivity of detecting transcription termination via polyacrylamide gel electrophoresis (PAGE) [32P radiolabels increase the risk for experimental personnel, which is not satisfactory for conventional labs. Chung et al. developed a hybridization-based method, in which double-labeled DNA is designed to hybridize the abortive and full-length transcripts [Transcription termination quantification is important for us to identify transcription termination elements or factors, understand the interaction or regulation involved in transcription and compare termination efficiency among diverse polymerases or different termination strategies. In addition, quantifying transcription termination can be used to differentiate between different species and elucidate the mechanism of transcriptional control. Quantifying transcription termination of a single-round transcription provides accuracy to evaluate the performance of a transcription system. Several methods have been developed to analyze termination in single-round transcription reactions. Heparin or rifampicin has been added to the transcriptions to prevent re-initiation and achieve single-round in vitro transcription, in which s (PAGE) ,16,17,18s (PAGE) . Additionscripts . Lim et nscripts . Passalanscripts .pbuE adenine riboswitch RNA from Bacillus subtilis, its structures and transcription-regulatory function are dependent on the presence of adenine  to quantify the transcription termination of adenine riboswitch RNA A,B. Ribo adenine A,B. Whet adenine C. We her adenine C. ThorouBacillus subtilis, and it contains an aptamer domain and an expression platform [2+ and 0.5~10X NTPs, were tested.The adenine riboswitch RNA used in this work is from platform . The secplatform ,25,26. IE = iE × eEn, where E, iE and eE represent the overall, initiation and elongation efficiency, respectively [Single-pass transcription is performed in the PLOR reactions. The enzyme used in PLOR is T7 RNAP. T7 RNAP is the most widely used enzyme in in vitro transcription and has been used to transcribe RNA that ranges from tens to thousands of nucleotides (nt) in length ,27,28,29ectively . AdditioRiboswitch can regulate gene expression by sensing its specific ligand among countless metabolites in cells ,31. As s2+ is crucial to initiate in vitro transcription and RNA folding [2+ on the termination efficiency of the adenine riboswitch. In 2+ were added to observe termination efficiency of the adenine riboswitch. To observe the transcribed RNA more sensitively, we used Cy3-labeled UTP instead of regular UTP at step four to introduce the fluorophore Cy3 to the adenine riboswitch by PLOR . The concentration of DNA templates, primers, dNTPs and Taq polymerase used in TD-PCR were 0.01 µM, 2 µM, 0.2 mM and 0.05 g/L. In our hands, TD-PCR has higher efficiency than regular PCR. TD-PCR contains the touchdown phase and the PCR phase. Twenty cycles are included in the touchdown phase to prevent nonspecific extension of primers, which begins with melting at 95 °C for 5 min, anneals at 75–45 °C for 45 s (the temperature drops 1.5 °C per cycle) and then elongates at 72 °C for 60 s. Thirty-five cycles are included in the PCR phase, which begins with melting at 95 °C for 30 s, anneals at 58 °C for 30 s and then elongates at 72 °C for 20 s. The sequences of the primers and templates used in the TD-PCR reactions are listed in DNA template with the sequence CCGCGGATGCGGAAAAAAAATCCTGATTACAAAAAATGTCATAAACAAATTTTGTAATCAGGATTTTACGGTTCCTGGTAGACACCCTCAAACCATATTATTGAGGTTATACAACTTCCCTATAGTGAGTCGTATTATGGACTAGCTGAATCAGA  was prep2SO4, and 6 mM MgSO4, pH 8.0) to generate DNA-bead templates as reported elsewhere [The biotin-DNA templates produced by PCR were then incubated with streptavidin-coated agarose beads  at 4 °C overnight in the buffer  at 37 °C for 10 min. The total reaction volume was 100 µL, and the concentration of T7 RNAP and DNA was 5 µM. At step 1 of PLOR, 400 µM ATP, 600 µM GTP and 64 µM UTP were added to the reaction and gently rotated with T7 RNAP and DNA-beads at 37 °C for 15 min, followed by filtration and rinsing the solid-phase reactants at least three times with the buffer . The residual steps were performed similarly to step 1 except different batches of NTPs were added and gently rotated with the solid-phase reactants in the buffer  at 25 °C for 10 min. Additionally, the NTPs were added as follows: 25 μM ATP, 15 μM CTP and 20 μM UTP (step 2); 5 μM ATP, 35 μM GTP and 25 μM UTP (step 3); 10 μM ATP, 15 μM CTP and 5 μM UTP (step 4); 10 μM ATP, 10 μM CTP and 15 μM GTP (step 5); 20 μM ATP, 10 μM CTP and 15 μM UTP (step 6); 10 μM ATP, 5 μM GTP and 10 μM UTP (step 7). In order to test the effect of adenine, Mg2+ and NTP on transcription termination, the experimental conditions at step 8 were different: 60 μM ATP, 45 μM CTP, 45 μM GTP and 120 μM UTP in the buffer  used for testing adenine  used for testing Mg2+  used for testing NTP concentration  for 3–5 min, then irradiated at UV 260 nm in a Tanon 2500 gel imager . The gels of Cy3-labeled RNA products were irradiated at fluorescence 550 nm without staining by the GenoSens fluorescent gel imager . The gel bands were quantified using Image J software.


Cucurbita pepo line PI 420328 with tolerance to cucurbit yellow stunting disorder virus (CYSDV) were compared with those in Gold Star, a susceptible cultivar. The lower CYSDV symptom severity in PI 420328 correlated with lower virus titers and fewer sRNAs derived from CYSDV (vsRNA) compared to Gold Star. Elevated levels of 21- and 22-nucleotide (nt) size class vsRNAs were observed in PI 420328, indicating more robust and efficient RNA silencing in PI 420328. The distribution of vsRNA hotspots along the CYSDV genome was similar in both PI 420328 and Gold Star. However, the 3’ UTRs, CPm, and p26 were targeted at a higher frequency in PI 420328. RNA silencing is a crucial mechanism of the antiviral immunity system in plants. Small RNAs guide Argonaut proteins to target viral RNA or DNA, preventing virus accumulation. Small RNA profiles in  Crinivirus in the family Closteroviridae . As with other members of the genus t manner . Followiiteflies ,21. Inoc260/280 in the range of 1.8–2.2, OD260/230 ≥ 2.0 and RIN value ≥8.0 were used to construct the cDNA library. Small RNA libraries were constructed and sequenced on the Illumina platform . A total of nine samples were prepared for sequencing: three biological replicates each of tolerant (PI 420328), susceptible (Gold Star), and mock (Gold Star) inoculated squash. Approximately 100 mg of frozen tissue was ground in liquid nitrogen, and the total RNA was extracted using Spectrum™ Plant Total RNA Kit , following manufacturer’s instructions. The integrity and purity of the total RNA was determined using a Qubit and quantified using a NanoDrop-2000 spectrophotometer . RNA samples with ODRT-qPCR assays in a quantitative system using SSOAdvanced Universal SYBR Green Supermix  were performed to quantify CYSDV. The reactions were performed in a CFX96 Touch Deep Well Real-Time PCR System . Primers and PCR profiles published earlier for CYSDV quantification were used in qRT-PCR . Each bi23)/(length of a vector in bp * 1 × 109 * 650), in which the weight of a base pair (bp) is assumed to be 650 Da [Virus titers were estimated following protocols described earlier ,19. Cycle 650 Da . Three tRemoval of low-quality sequences and adapters was performed with CLC Genomics Workbench 21 . Only reads within the size range of 20–25 nt were retained. To retrieve and count CYSDV-derived small RNAs, clean sRNA reads were aligned to the reference sequence of CYSDV , allowing zero mismatches. The number of each size class of virus-derived small RNA (vsRNA) was also determined using CLC for quantitative comparisons. The distribution of vsRNAs along the virus genome and the 5′ terminal nucleotides of the small RNAs was determined using MISIS-2 . The num®, Version 16 . Before analysis, the data for CYSDV titer were log-transformed, and the number of vsRNA in each size class was converted to a percentage. The vsRNA aligning to the UTRs and cistrons were normalized as frequency/100bp of gene/million reads. Differences in CYSDV titer, percentage of each nt size class, and frequency of vsRNA aligning to different regions between Gold Star and PI 420328 were analyzed by t-test. Treatment means were considered significant at p < 0.05. The number of CYSDV-derived reads in Gold Star, PI 420328, and mock were analyzed using one-way ANOVA. Treatment means were separated with a post hoc t-test. Data analyses were performed in JMPt-test, p= 0.03; Following whitefly-mediated transmission of CYSDV, Gold Star and PI 420328 plants were monitored for symptom expression. Interveinal chlorosis was observed as early as two weeks post-inoculation on the lower leaves of susceptible cultivar Gold Star. The symptoms progressed rapidly, with interveinal chlorosis becoming more severe and spreading onto the upper leaves. On the other hand, plants from PI 420328 remained asymptomatic until three weeks post-inoculation. Delayed symptoms of mild interveinal chlorosis were observed on the lower leaves at 21 days after inoculation (DAI). The symptoms remained mild, and only the bottom leaves displayed prominent yellowing after 30 DAI when samples were collected for sequencing. The mock-inoculated (non-viruliferous whiteflies) plants of Gold Star did not exhibit any phenotypic symptoms of interveinal chlorosis or yellowing A–C. The C. pepo, were sequenced to compare the interaction of CYSDV with small(s) RNA silencing pathways during susceptible and tolerant interactions. Read statistics from the nine small RNA libraries generated from CYSDV-infected Gold Star, PI 420328, and mock-inoculates are presented in t-test, p = 0.04) and comparable to those in mock   E. p = 0.03) and 21 nt (p = 0.04) aligning to the CYSDV genome was significantly higher in PI 420328 (The proportion of different size classes of vsRNA in Gold Star and PI 420328 were compared. The most predominant size class obtained post-infection was the 21 nt followed by the 22 nt size class in both Gold Star and PI 420328 . HoweverI 420328 . The perI 420328 . Virus-derived sRNAs originated from the entire length of RNA1 and RNA2 of the CYSDV genome and in both polarities in both PI 420328 and Gold Star . The patp = 0.02) as well as CPm (p = 0.005) and p26 (p = 0.04) located on RNA 2. In contrast, the vsRNA reads targeting RdRp and 5′ UTR of RNA 2 (p = 0.03) were significantly higher in Gold Star than in PI 420328 (p = 0.03) (The 5′ UTRs of RNA 1 and RNA 2 were the most targeted region of CYSDV genome in both PI 420328 and Gold Star. This was followed by p4.9 located on RNA2 and p25 and RdRp located on RNA1. Some regions were targeted more frequently in PI 420328 than in Gold Star. These included the 3′ UTR of RNA 1 ( = 0.03) . The predominant 5′ nucleotide was 5′ A on both 21 nt and 22 nt vsRNA from both RNAs of CYSDV in all replicates of PI 420328 . The perTolerance has been defined as a mitigation of the impact of virus infection irrespective of the pathogen load . Even thAmong the size classes important in RNAi in both Gold Star and PI 420328, 21 nt followed by 22 nt were prominent. Interestingly, however, the percentages of 21 nt and 22 nt size classes in total vsRNA were higher in PI 420328 compared to those in Gold Star for RNA2 . The eleCuscuta campestris parasitic plants [N. benthamiana [More and more studies have demonstrated that DCL2-generated 22 nt sRNAs can trigger transitive PTGS. DCL2 promotes secondary sRNA production more efficiently, thus adding to the amplification property of the RNA silencing pathway in an infected plant. This is achieved by the 3′ ends of 22 nt sRNA that protrude from the sRNA–AGO nucleoprotein and bind to the protein SGS3. This AGO–SGS3 complex stalls ribosome translocation, thus repressing viral mRNA translation. The protruding 3′ end of the 22 nt sRNA is then available as a primer for more viral dsRNA production by host RDR . The dsRc plants . There ithamiana .The vsiRNA hotspots are regions of the viral genome that are highly targeted during RNAi ,43,44,45Crinivirus, ToCV [The number of vsiRNA targeting each gene co-related with the size of the genes in both Gold Star and PI 420328 . Howeverus, ToCV .The sorting of small RNAs into Arabidopsis Argonaute complexes is directed by the 5’ terminal nucleotide . To deteTy1 gene upon infection by tomato yellow leaf curl virus [The predominance of 21 or 22 nt vsRNA during plant virus infection is well-established ,56,57. Hrl virus . Considerl virus ,38,41,59In this study, the higher abundance of 21 and 22 nt vsiRNAs in PI 420328 and a higher percentage of 21 and 22 nt svRNAs with 5′ A could indicate the role of PTGS in rendering it tolerant to infection from CYSDV. PI 420328 will be a potential source of tolerance to CYSDV in breeding programs. Future research will aim to identify the host loci associated with tolerance and introgressing these loci into summer squash.


Mycobacterium tuberculosis sncRNA with the shortened aptamer-scaffold tag. Then, we visualized it in bacteria and bacteria-infected macrophages using the new red light-emitting Mango-activated dye.Progress in RNA metabolism and function studies relies largely on molecular imaging systems, including those comprising a fluorogenic dye and an aptamer-based fluorescence-activating tag. G4 aptamers of the Mango family, typically combined with a duplex/hairpin scaffold, activate the fluorescence of a green light-emitting dye TO1-biotin and hold great promise for intracellular RNA tracking. Here, we report a new Mango-based imaging platform. Its key advantages are the tunability of spectral properties and applicability for visualization of small RNA molecules that require minimal tag size. The former advantage is due to an expanded (green-to-red-emitting) palette of TO1-inspired fluorogenic dyes, and the truncated duplex scaffold ensures the latter. To illustrate the applicability of the improved platform, we tagged   Small noncoding RNA (sncRNA) have recently emerged as essential modulators of transcription and translation. Many eukaryotic sncRNA are differentially expressed in cancer ,2, neuroDespite recent advances in encoding fluorogenic RNA or intracellular RNA labeling for fluorescent microscopy imaging ,8, availMost of the above challenges and issues have been addressed to some extent in current imaging systems based on an aptamer and a fluorogenic dye . Such syKD = 3.2 nM) to the cognate thiazole orange (TO)-derived dye TO1-biotin. The dye showed up to 1100-fold fluorescence enhancement upon binding to Mango I aptamer  and TO3-Biotin  simplification of the fluorogenic dye synthesis; (ii) enhancement of the fluorescence quantum yield in the bound state and (iii) adjustment of the emission range. The binding of dyes to two well-characterized Mango aptamers, namely Mango II and Mango IV , was stuFor a detailed description of the synthesis of fluorogenic dyes, see Supporting Information.Oligonucleotides (ONs)  were obtained from Litekh (Russia).The secondary structures of Mango-RNA were confirmed by circular dichroism (CD) spectroscopy using 5 μM solutions of free RNAs or their 1:1 complexes with the dyes in the working buffer . All samples were heated to 90°C for 5 min and snap-cooled on ice  or cooled gradually to room temperature  before all measurements. The annealing scheme had no noticeable effect on the CD spectra. The spectra were registered with a Chirascan spectrophotometer  at room temperature in quartz cuvettes of 1 cm path. In all other experiments, rapid annealing was used.4b with genetically engineered RNA constructs were characterized using 1 μM solutions in a 20 mM sodium phosphate buffer, pH 7.2, supplemented with 0.05% Tween-20 and 140 mM КСl. Absorption, fluorescence excitation, and emission spectra were registered with a Chirascan spectrophotometer equipped with the CS/SEM fluorescence accessory at room temperature. Fluorescence quantum yields ФF were calculated using equation . To register the fluorescence emission of free dyes, 50 μM dye solutions in the working buffer were analyzed, and the intensity was normalized to 5 μM concentration for comparison with the dye–RNA complexes. Complexes of Fr = 0.95 is fluorescence quantum yield of the reference dye rhodamine 6G (R6G) in ethanol, Ar and Fr are absorbance and fluorescence of R6G in ethanol, A and F are absorbance and fluorescence of the dye–RNA complex in the working buffer, and nr and n are refractive indices of ethanol and the buffer, respectively.where Ф4b-Mango binding constants, 10 nM solution of 4b in the working buffer supplemented with 0.05% Tween-20 was titrated with increasing concentrations of Mango RNA, and integral fluorescence was registered in microcapillaries using Monolith NT.115 device  equipped with a RED/GREEN detector in RED mode at 25 ºC. Normalized fluorescence was analyzed using MO. Affinity Analysis software , and KD values were obtained by fitting the experimental data to equation .4b and Mango-RNA, respectively.where [dye] and [RNA] are total concentrations of 4a–d and DMSO as negative control were prepared in cultural medium . Cell suspensions were added to the wells with compounds dilutions or DMSO control (∼2 × 104 cells per well). The final concentration series of eight dilutions started from 50 μM. The cells were incubated at 37°C in a CO2-incubator for 24 h and 7 days. After incubation, the cells were analyzed using Olympus CKX-31 microscope for cell viability. For this, cultural medium was substituted with resazurin solution (25 mg/mL) and cells were incubated at 37°C in a CO2-incubator for 4 h. Then 20 ml of 10% SDS was added to stop the reaction and fluorescence was measured with Promega GloMax-Multi Detection System at λex 525 nm and λem 580–640 nm. As additional controls, the same series of cells treated with compounds or DMSO dilutions but without resazurin solution to subtract the background fluorescence were used; and a medium with resazurin solution was used to set up a minimal value of non-reduced resazurin. All experimental procedures were performed in two replicates. Statistical analysis was performed, and fluorescence curves were plotted using MS Excel 2013. The 50% cytotoxic concentration (CC50) was calculated (compound concentration required to induce a cytopathic effect in 50% of the cells in a monolayer).Two-fold dilutions of studied compounds TO1-biotin and + ions were used. The parameters needed for interatomic energy calculation were taken from the force fields RNA.YIL (T = 300 K) and constant pressure (p = 1 atm) over 80 ns. To control the temperature, Langevin thermostat was used with a collision frequency of 1 ps−1. Energies were estimated by using the MM-GBSA approach. The polar contribution EGB was computed using the Generalized Born (GB) method and the algorithm developed by Onufriev et al. for calculating the effective Born radii . Partial charges on the dyes atoms were calculated using DFT/M06-2X /6–311+g, 6–31g*  RNA.YIL ,40 for Rrn radii . The non4b on bacteria were assessed by MSmeg cellular growth in the presence of the dye at various concentrations since the measurements of bacterial growth rate reflect the concentration of actively growing cells , and grown up at 37°C while shaken at 200 rpm. Then, the optical density of the culture (OD600) until the stationary growth phase was measured . The DNA templates for RNA synthesis were generated by PCR using the appropriate expression plasmids (see below and ) and oliMycobacterium smegmatis mc(2)155 (MSmeg) obtained from the bacterial collection of the Bach Institute of Biochemistry  was pre-cultured for 24 h at 37°C on an orbital shaker (200 rpm) in 40 ml of Luria Bertani (LB) medium supplemented with 0.05% Tween-80 (to prevent cell clumping) and used as inoculums for further experiments. MSmeg recombinant strains were grown in LB supplemented with 34 μg/ml chloramphenicol.r), between mycobacterial rrnB promoter of MSmeg and rrnB-T1 terminator  into HindIII site of pAMYC (Cmrminator . The seqEscherichia coli DH5_α grown in Luria Bertani (LB) broth and LB agar supplemented with chloramphenicol (34 μg/mL). MSmeg cells were transformed by electroporation with the constructed plasmids and empty pAMYC vector (negative control), and recombinant strains were selected on LB agar containing with 34 μg/mL chloramphenicol.Plasmids were amplified in 600 0.8). Cells were pelleted by centrifugation at 3000 × g for 15 min, washed twice with PBS and either incubated for 30 min in visualization buffer  or resuspended in RPMI-1640 medium  for infection in macrophages. M. smegmatis transformed by pAMYC without insertion was used as a negative control strain.Recombinant MSmeg strains were grown up to the logarithmic growth phase (OD4 cells/glass) were infected with ds_Mango II_MTS1338, ds_Mango II or pAMYC strains at MOI 10:1 for 1 h. After 1 h of infection, the medium was removed, and infected macrophages were washed three times with PBS and incubated for 30 minutes in the visualization buffer (see above). Then the samples were analyzed using an Eclipse TE2000 confocal microscope . ds_Mango II_MTS1338 and ds_Mango II in Mycobacteria were detected in a green channel (ex 488/em 590 nm) for TO1-biotin and in a red channel (ex 543/em 650 nm) for 4b, whereas Hoechst 33258-stained cell nuclei were visualized in a blue channel (ex 408/em 515 nm). Because the fluorescent signal from the modular RNA complexes with the dye was clearly visible after 30 min of incubation in the visualization buffer, this time period was long enough for the dye to diffuse into macrophages and phagocytized bacteria and interact with RNA aptamers. Thus, the response time of the sensor is <30 min. The fluorescent signal from the modular RNA complexes with the dye was detectable for at least 2–3 h, suggesting the RNA tag and the dye are relatively stable in the cellular medium, which makes it possible to observe the processes occurring with the tagged molecules in living cells.For the infection RAW 264.7 cells cultured in RPMI-1640 supplemented with 10% fetal calf serum (FCS) (Gibco) were seeded in the same medium on cover glasses  placed in 6-well culture plates . After 24 h, cells   -catalyzed azide–alkyne 1,3-dipolar cycloaddition (CuAAC) reaction compared to the amide bond-forming one, we analyzed crude reaction mixtures containing TO1-biotin and 4a using LC-MS (4a) gave 1.2 and 9.2% for TO1-biotin (Rf = 5.65 min) versus 22.4 and 76.5% for 4a (Rf = 5.86 min). The reaction yields were 1.8 and 3.5% for TO1-biotin and 4a, respectively, after a single HPLC purification, presumably due to the low solubility of both derivatives in the loading buffer (water with 0.1% TFA). However, after several iterations of HPLC purification, overall yield of TO1-biotin and 4a reached 6.8 and 58.1%, respectively (see Supplementary).TO1-biotin was prepared according to the literature . For the of 61%)  was condynthesis . Followiynthesis  (Scheme glycole)  were conng LC-MS . The purN,N-dimethylaminophenyl (4b), 2,3,6,7-tetrahydro-1H,5H-pyridoquinolin-9-yl (4c), or 1,2,2,4-tetramethyl-1,2-dihydroquinolin-6-yl (4d) group as an electron donor. For this, benzothiazolium salt 1 was condensed with an appropriate aromatic aldehyde via an aldol-type reaction in acetic anhydride (2b-d (Scheme 3 in the presence of copper (I) iodide, TBTA and DIPEA yielded, after preparative HPLC purification, fluorogenic dyes 4b–d as trifluoroacetate salts.Inspired by the chemical design of TO3-biotin and YO3-biotin with extended conjugated systems, which are significantly red-shifted but show drastically reduced fluorescence enhancement upon aptamer binding with respect to the parent TO1-biotin ,20, and nhydride  to affor4a–d and the control dye TO1-biotin were investigated in a free state and in complexes with aptamers Mango II and Mango IV , while equimolar mixtures with the aptamers showed up to nearly three orders of magnitude increase in fluorescence emission  compared to Mango IV (Ф = 0.32) and a slightly reduced brightness (Table 4a exhibited higher contrast (FE) than TO1-biotin, presumably due to the reducing effect of the 1,2,3-triazolyl linker on the rotational flexibility of heteroaromatic systems compared to the amide bond. Dye 4b was also shown to be a more robust probe for Mango II than Mango IV. Dye 4b had red-shifted spectra with an increased Stokes shift (Δλ = 55 nm) compared to TO1-biotin and 4a (Δλ = 35 nm). Dyes 4c and 4d were inferior to other variants and showed substantially reduced fluorescence quantum yields and brightness  compared to TO1-biotin (∼100X Mango:ODN light-up ratio). Among the new dyes, the highest selectivity was observed for 4b  . The setsequence , random-sequence ,62, the sequence , which asequence , and a Dsequence . None ofin Table . For thiy Figure .4b for MangoII/IV over randomly selected G4 RNA and its profound Stokes shift, we questioned whether it could be applied in combination with another dye/aptamer pair for multiplex imaging or FRET-based monitoring of RNA–RNA juxtaposition  moiety (TO1-biotin and 4a) or its substitute (4b and 4c) and the benzothiazolium (BT) group in the dye–Mango II complexes and thus could affect fluorescence enhancement. The biotin-containing tag can hardly make a significant contribution, so it was excluded from the analysis. The dyes were localized on the surface of the outed Mango G-quartet by docking, and the stability of the resulting complexes was tested by molecular dynamics (MD) simulations.To elucidate the molecular basis for the high light-up effects of 4b Table , we mode4b Table   moiety in 4b remained roughly planar throughout the simulation, in 4c, the 2,3,6,7-tetrahydro-1H,5H-pyridoquinolin-9-yl (THPQ) residue underwent full (180°) rotation. End-point structures of the complexes (top views of the dyes and the neighboring aptamer residues) are shown in Figure 4c, likely results from weak stacking interactions between THPQ and A17. In contrast, MQ of TO1-biotin and 4a, as well as DAP of 4b, formed stable stacking contacts with A17, as was evident from the plots of COM distances and the angles between the normal and planes of these residues  among all new dyes  in sed RMSD . The 4b–nterface , which enew dyes .4b for intracellular imaging of M. tuberculosis small RNA MTS1338, detecting this sncRNA in bacteria in vitro and tracking in infected macrophages. For this purpose, we used the modular RNA (ds_Mango II_MTS1338) consisting of MTS1338 fused with ds_Mango II (the optimized fluorescence-activating tag) that was prepared by truncating one of the stems in F30 folding scaffold , was shown . No changes in cell morphology (data not shown) or metabolic activity were observed after 24 h incubation with the dyes . We also performed cytotoxicity assays for bacterial cells. For bacteria, no differences were found in growth rate in the presence/absence of 4b in the medium  clones expressing the modular RNA, ds_Mango II, and the empty vector-transformed clone (as a negative control). The non-pathogenic MSmeg mc(2)155 was used in the study because its genome and metabolism are very similar to Mycobacteriumtuberculosis. MSmeg also has a significantly higher growth rate and much easier undergoes genetic modifications, therefore is widely used as a surrogate organism  do not always coincide with those generated by Hoechst 33258 (blue channel) due to the bacteria's movement. No bright green or red signals were detected in experiments with bacteria transformed by empty vector pAMYC and stained with TO1-biotin or 4b. Control images of bacteria with the empty pAMYC vector  are given in Visualization of the modular RNA and ds_Mango II in live bacterial cells is shown in Figure When macrophages infected with MSmeg recombinant strains expressing modular RNA or ds_Mango II were stained with TO1-biotin, the fluorescent bacteria were detected, suggesting a correct folding of the label's secondary structure .4b during 1 and 1.5 h. For this experiment we chose a field of view to visualize ∼1–2 phagocytosed bacteria  for visualization of sncRNAs within infected living cells.Fluorescence-activating RNA aptamers can selectively bind to RNA targets, followed by the light up of small molecule fluorogenic dye binders. They are the key player in RNA visualization/tracking systems and FRET constructs that allow to monitor RNA–RNA interactions and their structural rearrangements within the living cell. Among proposed aptamer–dye pairs, the Pepper-based imaging system appears to cover the entire range of visible light emission . HoweverMango-based imaging systems also look very promising for both the fluorogenic dye (TO1-biotin) and the aptamers (Mango I–IV and others) ,29,30. T4a, a TO1-biotin analog with the same thiazole orange (TO) fluorogenic moiety, was prepared in an overall yield of 25% which is ∼60 times higher than in a classical route to TO1-biotin , the use of the stable Mango II provides the opportunity for live-cell imaging, in contrast to Mango III and its variants, which have been long studied only in cuvette, whereas the applicability of Peach aptamers for intracellular imaging has not yet been evaluated.Here, an efficient scheme for the synthesis of analogs of TO1-biotin, proposed initially as a fluorogenic dye for Mango aptamers , has bee1-biotin . The usenm Table . Althoug4a for Mango II compared to genomic RNA G4 utr-z from the human KRAS 5'-UTR sequence (4b provides an increase in the selectivity to 4.9 (Table FE) upon TO1-biotin binding to these structures, low affinity (KD) determines low fluorescent efficiency (E = FE/KD) of the complexes, which is approximately two orders of magnitude lower than for TO1–biotin–Mango I complex.Another critical issue is the contrast-providing selectivity of dyes to cognate aptamers over abundant DNA and RNA components within cells, which has not been systematically studied to date. We observed the selectivity of TO1-biotin to Mango aptamers compared to model RNA corresponding to the literature data  and a gesequence  was only.9 Table . The res.9 Table . Despitein cuvette  appeared superior to other known FRET pairs, namely DFHBI-Spinach with TO3-biotin-Mango I  and DFHBI-1T-Spinach with YO3-biotin-Mango I (30% overlap) , the proposed pair is even more suitable for excitation by 488/555 nm lasers and detection using common EGFP and Texas Red filters.Recently, a FRET system was developed based on RNA aptamers Spinach and Mango I that is genetically encodable and responds to conformational changes . Mango I cuvette ,28. Here cuvette  and 4b–M) Figure . TO3–bioM. tuberculosis RNA MTS1338 may also be secreted and affects the protective cells of the immune system (4b with improved characteristics for intracellular imaging/tracking regulatory sncRNA were verified using MTS1338. We visualized the modular RNA and ds_Mango II in live bacterial cells using 4b and TO1-biotin to control the correct aptamer folding (Figure There are examples that noncoding bacterial RNAs of intracellular pathogens can be secreted into the cytoplasm of infected cells and modulate the immune response during infection ,79. Smale system . To stude system . Howevere system , resultie system . The optg Figure . The flu4b within macrophages infected with bacteria bearing modular RNA or ds_Mango II for 1 and 1.5 h (Figures Correct folding of RNA secondary structure in macrophages infected with strains expressing modular RNA or ds_Mango II was confirmed by staining with TO1-biotin . Finally Figures  and 7. IM. tuberculosis surrogate M. smegmatis. Thus, our approach can find applications in developing genetically encodable molecular tools for studying RNA–RNA interactions and conformational rearrangements, as well as sensors and advanced devices.In conclusion, a new dye with improved selectivity and red fluorescence has been developed and shown to be promising in combination with stable in living cells Mango II aptamer for RNA imaging/tracking and FRET system development. Furthermore, a shortened fluorescence-activating tag compared to one bearing a widely used F30 stabilizing scaffold for live-cell sncRNA imaging has been proposed, and its effectiveness has been tested for tracking the movement of a fluorescent signal from labeled bacterial RNA in a complex living system consisting of macrophages infected with the gkad100_Supplemental_FileClick here for additional data file.


Hematopoietic stem cells (HSCs) developing from mesoderm during embryogenesis are important for the blood circulatory system and immune system. Many factors such as genetic factors, chemical exposure, physical radiation, and viral infection, can lead to the dysfunction of HSCs. Hematological malignancies  were diagnosed in more than 1.3 million people globally in 2021, taking up 7% of total newly-diagnosed cancer patients. Although many treatments like chemotherapy, bone marrow transplantation, and stem cell transplantation have been applied in clinical therapeutics, the average 5-year survival rate for leukemia, lymphoma, and myeloma is about 65%, 72%, and 54% respectively. Small non-coding RNAs play key roles in a variety of biological processes, including cell division and proliferation, immunological response and cell death. With the development of technologies in high-throughput sequencing and bioinformatic analysis, there is emerging research about modifications on small non-coding RNAs, as well as their functions in hematopoiesis and related diseases. In this study, we summarize the updated information of small non-coding RNAs and RNA modifications in normal and malignant hematopoiesis, which sheds lights into the future application of HSCs into the treatment of blood diseases. Hematopoietic stem cells (HSCs) are one type of pluripotent stem cells that mainly exists in the bone marrow, circulating blood, and cord blood. Since their first discovery by Till and McCulloch in 1961 from the mouse spleens , researcThere are three types of cell division for HSCs: symmetric self-renewal with products of two HSCs, symmetric differentiation with products of two progenitor cells (HPCs), and asymmetric self-renewal with products of one HSC and one HPC. The HSC niche, a classical concept about the location and microenvironment of HSCs, has been verified to function in the HSC self-renewal regulation with the cooperation of various types of cells  and cytokines  . It is nRecent studies have emerged that multiple lineages have already existed in HSCs and HPCs, indicating direct fate-deciding lineage from different HSC subsets to distinct blood cell types ; Carrel. Althougvia sinusoidal CXCR2 signaling and vascular permeability  are one type of small non-coding RNA usually with 18–24 nucleotides . There avia TRIM28-dependent STAT3 activation  with a common length of 60–300 nts are mainly in the nucleolus of eukaryotic cells. Based on the structure and their location, snoRNAs can be categorized into three subtypes: H/ACA box snoRNAs, C/D box snoRNAs, and scaRNAs . Most snIn recent years, with the development of sequencing technology, more and more data show that snoRNAs are dysregulated in many diseases including hematological malignancies, solid tumors, hereditary diseases, and metabolic diseases ; Liu et; Verbee. In a stThe length of transfer RNAs (tRNAs) ranges from 76 to 90 nt, with the cloverleaf-like secondary structure and “L”-shaped tertiary structure . As an eRecent studies revealed that modifications of tRNAs that are located either on the anticodon loop or “elbow” part in the tertiary structure are vital. There is also evidence showing relationships between dysregulation of tRNA and various diseases such as cancer, neurological, and metabolic diseases . Recent It is the common feature of non-canonical sncRNAs that most of them are derived from other RNAs with longer length. Thanks to the development of novel technologies , the detectable level of modified sncRNAs experienced a dramatic increase . Recent in situ hybridization. Designing probes with higher sensitivity and specificity would greatly assist this field.Evidence that the dysregulation of tsRNA biogenesis is involved in the pathogenesis of chronic lymphocytic leukemia has been found in primary B cell samples taken from patients with the disease as compared to normal B cells obtained from healthy human donors . StudiesCircular RNAs (circRNAs) were firstly discovered under the electron microscope in 1979 . CircRNA+. Knockdown of circMYBL2 effectively inhibited the proliferation of FLT3-ITD AML cells and overcame the acquired resistance to quizartinib+. It has been demonstrated that circMYBL2 controls FLT3 translation by recruiting PTBP1 to promote the progression of FLT3-internal tandem duplication acute myeloid leukemia  domain family, IGF2BP1/2/3, and HNRNPA2B1. M6A functions by regulating translation and stability of mRNA.N6-methyladenosine (m6A) is the most common and conserved internal modification in eukaryotic RNAs. The writer complex of m6A methylation reaction, m6A methyltransferases, includes METTL3/14/16, RBM15/15B, ZC3H3, VIRMA, CBLL1, WTAP, and KIAA1429. There are two kinds of demethylases, or say erasers, fat mass and obesity-associated protein (FTO) and alpha-ketoglutarate-dependent hydroxylases homolog 5 (ALKBH5) that rely on Fein vitro  through pseudouridine synthase. The distribution of pseudouridines covers tRNA, rRNA, snRNA, snoRNA, and mRNA. There are three characteristics of ractions .Drosophila, resulting in Hoyeraal-Hreidarsson syndrome with the symptoms of immunodeficiency, growth retardation, and microcephaly  to inhibit translation in stem cells. Loss of PUS7 and mTOGs affects hematopoietic commitment and early embryogenesis in vitro. Therefore, pseudouridine on tsRNA can regulate hematopoiesis through control of specific mRNA translation in stem cells (HSPCs)    . Pseudou (HSPCs) . High cof cancer ;  is the modification of adding a methyl group (-CH3) to the 2’ hydroxyl (-OH) of the ribose moiety, which can occur either during or after the transcription. Nm has been found in many types of RNAs , and can affect RNA molecules on their structure, stability, and interactions, thus regulating a range of cellular processes, such as self vs non-self-recognition and epigenetic gene regulation .It is already known that small nucleolar RNAs (snoRNAs) can direct internal Nm modification to further control mRNA and protein expression. It is reported that two box C/D of snoRNAs and the 2’-O-methyltransferase fibrillarin act together and make Nm modification in the protein-coding region of peroxidasin (Pxdn) . The disSimilar processes also happen during the Nm modification of rRNA. Research on the zebrafish identified the importance of rRNA Nm modifications for vertebrate development. Dysfunction on any one of the three snoRNAs: SNORD26, SNORD44, or SNORD78 results in severe morphological abnormalities and embryonic death . ThroughThe breakthroughs of research on small RNA modification and related diseases cannot be separated from the development of sequencing technology. Before the introduction of high-throughput RNA sequencing, Northern Blot was used to assess the level of RNA expression . The quaSince its application 10 years ago, next-generation RNA sequencing (RNA-seq) technologies provide accurate methodologies to obtain information of the whole transcriptomes, including mRNA, small RNA (sRNA), and long non-coding RNA (lncRNA). Small RNA sequencing based on NGS technologies helps scientists to isolate and collect information about non-coding RNA molecules, which is powerful to profile and discover new types of small RNAs .However, because of the RNA modifications that interfere with adapter ligation and reverse transcription processes, there are difficulties in the detection and sequence of non-coding small RNA. As an advanced sequencing and analysis technology, PANDORA-seq has developed enzymatic treatment methods to handle certain RNA modifications. For instance, the demethylating AlkB and its mutant variants are employed to assure reverse transcription. In order to facilitate adapter ligation for RNA-seq of small and large RNAs, T4 polynucleotide kinase (T4PNK) is applied. It is also noticeable that T4PNK pretreatment of RNA should be applied according to the specific requirement (T4PNK is not recommended for research on miRNA due to the higher level of non-miRNA reads) . Based oVarious specialized techniques have been developed and refined for detecting each RNA modification type. RibOxi-seq, for instance, is effective in characterizing rRNA Nm sites with minimal input RNA material and sequencing depth while maintaining good accuracy. The production of RNA fragments with random 3′-ends, accompanied by periodate oxidation of all molecules terminating in 2′, 3′-OH groups, is crucial for this approach. As a result, only RNAs with 2′-OMe groups at their 3′ends can be sequenced in RibOxi-seq . Relevanvia RNA species linkage and a unique molecular identifier. The author used this method to research small-RNA expression in naive and primed human embryonic stem cells, as well as cancer cells , which can identify gene expression patterns in uniform cell populations . Based oer cells .Small RNAs are important components used as guide RNA (gRNA) in CRISPR/Cas9 systems. After transducing gRNA and Cas proteins into cells, the Cas9 protein binds to gRNA and attaches the target DNA sequence to perform DNA double-strand cleavage. When a break occurs in the DNA sequence, it activates the DNA repair mechanisms and results in mutations. CRISPR/Cas9-based gene screening, a large-scale unbiased genetic loss-of-function screening method designed to unbiased screen functional genes, was also applied in investigating the epigenetic regulation in hematopoiesis and leukemia . With vaIn this study, we have formulated the basic concept of HSCs and small non-coding RNA modifications. We also listed recent research results and summarized typical modifications  found on small non-coding RNAs associated with HSCs-related diseases. Many studies have indicated the important function of small non-coding RNAs that regulates gene expression to maintain homeostasis. Modifications of the small non-coding RNAs are still under research. The research area about their indirect effects on gene expression through the regulation of small non-coding RNAs remains largely unknown.Nowadays, many diseases like cancer, mitochondrial diseases, and neurological disorders have been detected to be associated with small non-coding RNA modifications. These characteristics make small non-coding RNA modifications potential diagnostic and prognostic biomarkers. For example, hypomodified tRNA was found in colorectal cancer, which induces ribosome frameshifting and further causes TYW2 epigenetic inactivation through non-sense-mediated RNA decay . Researc