We subsequently performed short hairpin RNA and small RNA interference and reactive oxygen species (ROS) detection assays to show that SLC6A1, PCBP2, and PEX5L can improve the antioxidation capacity of renal medulla cells of camel by decreasing ROS levels. Our data suggest that camels achieve sodium homeostasis through regulating the expression of salt‐reabsorption‐related genes in the renal medulla, and this involves ceRNAs  and antioxidant genes . These data may assist in the development of treatments for diseases induced by high salt diets.Salt dietary intake is tightly coupled to human health, and excessive sodium can cause strokes and cardiovascular diseases. Research into the renal medulla of camels exhibiting high salt resistance may aid identification of the mechanisms governing resistance to high salinity. In this study, we used RNA sequencing (RNA‐seq) to show that in the renal medulla of camels under salt stress, 22 mRNAs, 2 long noncoding RNAs (lncRNAs), and 31 microRNAs (miRNAs) exhibited differential expression compared with the free salt‐intake diet group. Using fluorescence  SLC14A1 expression, which can prevent excessive sodium reabsorption via competitive binding with miRNA‐34a in the renal medulla of camel. The higher expression of SLC6A1, PEX5L, and PCBP2 decreases reactive oxygen species levels to mitigate oxidative damage, contributing to the salt resistance exhibited by camels.The present study found that the lncRNA LNC003834 contributes to the upregulation of  Sodium serves as an essential molecule in mammalian physiology for its roles as an electrolyte and osmotic solute , 2, 3. TCamelus bactrianus) developed resistance to heat, drought, and salt for long‐term exposure in the desert [Animals that live in the desert, such as the Gobi Desert, frequently confront the lack of food and tolerate high salinity water for a long period of time , 21. Duee desert . The amoe desert , 24, fore desert . As a sae desert . In the e desert , 28.SLC6A19 mRNA aimed by miR‐193b and miR‐542‐5p as well as the putative competing endogenous LNC001438, LNC003417, LNC001770, miR‐199c, and TENM1 mRNA were considered as salt‐resisting modulators [RNAs transcribed from DNAs are a class of vital molecules that have diverse biological roles. mRNAs take charge of directing protein synthesis and noncoding RNAs, such as miRNAs and lncRNAs, are involved in post‐transcriptional regulation , 31, 32.dulators .Hence, taking transcriptome sequencing as a basis, our research targeting differential gene expression in the renal medulla of camels under salt stress will help scientists comprehending how the mammals achieve sodium homeostasis through natural instinct. The result of this study may offer an alternative investigation direction for solving the health problems due to excessive salt intake.−1 and increased by 100 g every 3 days, and the daily intake of salt was 900 g at the end of the experiment, following free water and feeding. Camels ate and drank freely in control group, with daily 50 g salt consumption. Procedures involving animals, their care, and the humane kill were conducted in conformity with the Guidelines on the Humane Treatment of Laboratory Animals  and were approved by the Animal Care and Use Committee of the Inner Mongolia Agricultural University (IMAU‐IACUC‐2019‐31560313). The written informed consent of the experimental research was achieved by S. Wang .The six female camels from 7.8 ± 0.4 years old (mean ± SD age) living in the Alxa Gobi Desert of northern China were penned and randomly divided into two groups (three camels per group): The experimental group was under salt stress condition, and the control group was under free salt‐intake diet, both groups were assessed for the consecutive periods of 24 days. The mean weight of the six camels was 352.6 ± 7.1 kg Table . In the −1 xylazine  using Bowtie (parameters: ‐v 0 ‐k 1) [Bos taurus database. The prediction of target genes was performed via miRanda  and RegRNA2.0 . The lncRNA data were analyzed by quality control, reads mapping to Camelus bactrianus genome  using TopHat v2.0.9 (parameters: ‐‐library‐type fr‐firststrand) [Camelus bactrianus and Bos taurus databases, and the differential expression analysis of lncRNA and mRNA was exerted by Cuffdiff (http://cole‐trapnell‐lab.github.io/cufflinks/cuffdiff/index.html). The prediction of target gene was carried out by lncRNA gene upstream/downstream 100 kb and Pearson correlation coefficient with |Pearson correlation| > 0.95 [Total RNAs were extracted from renal medulla tissues of camels using the Qiagen RNeasy Mini Kit . Potential RNA degradation and contamination were checked on 1% agarose gels. The purity, concentration, and integrity of total RNAs were determined by the NanoPhotometer spectrophotometer , the Qubit RNA Assay Kit in Qubit 2.0 Fluorometer , and the RNA Nano 6000 Assay Kit of the Agilent Bioanalyzer 2100 system , respectively. Three micrograms of total RNA per sample were utilized for small RNA and lncRNA library construction. The sequencing libraries of small RNA and lncRNA were individually created using the NEBNext Multiplex Small RNA Library Prep Set for Illumina  and the NEBNext Ultra Directional RNA Library Prep Kit for Illumina . These steps were preceded by the removal and cleaning up of ribosomal RNA using the Epicentre Ribo‐zero rRNA Removal Kit  and ethanol precipitation. Pooled RNA‐seq was applied to the constructed libraries of small RNA and lncRNA using an Illumina HiSeq 2500 platform and 4000 platform in turn . The miR 0 ‐k 1) , and nov 0 ‐k 1)  and mird 0 ‐k 1) . The dif 0 ‐k 1) . Small Rtstrand)  and Cufftstrand) . lncRNA tstrand) , CPC 0.9tstrand) , Pfam Sctstrand)  and Phyltstrand) . Annotat| > 0.95 . Gene On| > 0.95  and KOBAin situ Hybridization Kit  through fluorescent groups (FAM and Cy3) labeled RNA probes and DAPI stain. The sequences of fluorescent group labeled RNA probes are listed in Table FISH was performed on renal medulla tissues from salt stress and control camels in accordance with the protocol of Fluorescence 2 at 37 °C. The cells migrated out around tissues were initiated into digestion via using trypsin‐EDTA solution  at a cell growth confluence of 80%. The renal medulla cells of camels those in good growth condition were selected for the subsequent experiments after passage. Besides renal medulla cells, the 293T cells were also cultured in the above‐modified DMEM/F‐12 with 5% CO2 at 37 °C.The sampled renal medullas of deceased camel were removed and cleansed by Dulbecco’s phosphate‐buffered saline  with 1% penicillin and 1% streptomycin . Following chopping, the small pieces of renal medulla tissue were seeded on the T25 culture bottle  and cultivated with Dulbecco’s modified Eagle’s medium/nutrient mixture F‐12 (DMEM/F‐12)  containing 10% fetal bovine serum , 0.1% penicillin, and 0.1% streptomycin  in a humidified chamber with 5% COThe formula of high transfection efficiency was explored in 293T cells, using NC‐FAM oligo and pcDNA3.1‐GFP plasmid . Different co‐transfected combinations, miRNA‐34a oligo (GenePharma), NC oligo (GenePharma), LNC003834 plasmid vector including wild‐type (WT) and mutant type (MUT) , SLC14A1 luciferase reporter plasmid WT and MUT  were transfected into the 293T cells by lipofectamine 2000 with a 70% transfection efficiency in line with the proportion of fluorescent cells. The cells were lysed after 48 h and the fluorescent intensity of luciferase was detected using Dual‐Luciferase Reporter Assay System . The sequences of the corresponding RNAs are listed in Table a. LNC003834S1 WT: 5’‐AGGAACATAGGGACACTGCCC‐3’,b. LNC003834S2 WT: 5’‐TTGTCATTCTTTTGCACTGCCC‐3’,c. LNC003834S1 MUT: 5’‐TCCTTGTATCCCTGTGACGGG‐3’,d. LNC003834S2 MUT: 5’‐AACAGTAAGAAAACGTGACGGG‐3’.SLC6A1, PEX5L, and PCBP2 suppression, the packaging shRNA lentivectors and small RNA oligos with attached GP‐siRNA‐Mate Plus reagent were purchased from GenePharma (China). 48 h after the transfection into camel renal medulla cells, the fluorescent intensity was detected according to the manufacturer’s protocol. The synthesized shRNA and sRNA sequences are listed in Table For shRNA‐ and sRNA‐mediated RNAs were extracted from renal medulla tissues and cells of camel using mirVana miRNA Isolation Kit . The primers were designed by Primer Express 3.0.1 Table  and qRT‐The proteins were extracted by M‐PER Mammalian Protein Extraction Reagent . SLC6A1, PCBP2, and PEX5L protein expression were severally examined using antibodies against‐SLC6A1  and PCBP2  and PEX5L . The signals were monitored via Pierce ECL Western Blotting Substrate .ROS staining was arranged after RNA interference in renal medulla cells by ROS fluorescence probe‐DHE . The red fluorescence levels were analyzed by BD FACSDiva 8.0.1.1 on BD FACS flow cytometry .Aside from RNA‐seq data statistics based on negative binomial distribution, Student’s t‐test was used to compare the mean values between groups and a P‐value of less than 0.05 was considered as statistically significant.P < 0.05) expressed in camel renal medulla by comparing high‐salt group and control group  were identified as underlying targets of downregulated miRNA‐34a, suggesting that SLC14A1 mRNA, LNC003834, and miRNA‐34a could be competing endogenous RNAs (ceRNAs). The ceRNAs function has been clarified in regulating RNA transcripts via competing for shared miRNAs at the post‐transcriptional level [SLC14A1, SLC6A1, PEX5L, and PCBP2) and the 2 noncoding genes (LNC003834 and miRNA‐34a) were screened out as candidate genes of salt resistance in camel renal medulla. When considering qRT‐PCR results, their different levels were consistent with RNA‐seq gene expression analyses , 2 lncRNAs (1 upregulated and 1 downregulated), and 31 miRNAs (18 upregulated and 13 downregulated) that were differentially  lower than the ‘SLC14A1 WT + negative control (NC) mimic’ group  higher Firefly/Renilla ratio value than the ‘SLC14A1 WT + miRNA‐34a mimic + LNC003834S1 MUT’ group  lower Firefly/Renilla ratio than the ‘SLC14A1 WT + NC mimic + LNC003834S2 WT’ group, while there was no apparent difference of Firefly/Renilla ratio between the ‘SLC14A1 WT + miRNA‐34a mimic + LNC003834S2 WT’ group and the ‘SLC14A1 WT + miRNA‐34a mimic + LNC003834S2 MUT’ group , which contain original or base‐altered for miRNA‐34a potential binding sequence  Fig. , were troup Fig. . Howeveroup Fig. . The resoup Fig. . In termoup Fig. . The resSLC6A1, PEX5L, and PCBP2, the corresponding shRNAs were designed and synthesized. In line with 70% transfection efficiency   Fig. , shRNAs ons Fig. , represeion Fig. , SLC6A1 P < 0.001) in SLC6A1, PEX5L, and PCBP2 shRNAs and sRNA‐MIX cells  when compared with the negative and blank controls  fluorescent probe in renal medulla cells. Fluorescence imaging and intensity reflected that ROS levels were significantly higher  transportation [PCBP2‐deficient mice and primary mouse embryonic fibroblasts emerge ROS production increment and cellular senescence acceleration [PEX5L is engaged in the peroxisome matrix targeting signal‐1 binding and the peroxisome targeting sequence binding in which the peroxisome dysfunction is associated with fatal oxidative damage [SLC14A1, SLC6A1, PEX5L, PCBP2, LNC003834, and miRNA‐34a to be consistent with RNA‐seq outcomes by qRT‐PCR.In the previous work of our laboratory, the camel’s complex features coupled with desert adaptations such as water and fat metabolism had been unveiled through comparative genomic analysis, as well as the unique osmoregulation, osmoprotection, and compensatory mechanisms for water reservation responding to aridity stress based on transcriptomic evidences . This stresearch , SLC14A1aporin 1 , 63. In pressure . Additioortation , 64. GABortation , 65, wheleration . PEX5L ie damage , 66, 67.SLC14A1 mRNA, LNC003834, and miRNA‐34a, several experiments were performed. The fluorescence distribution of the FISH images appeared that the functional areas of LNC003834 and miRNA‐34a were localized in the extranuclear space of the camel renal medulla. Relying on dual‐luciferase reporter assay system, we subsequently found that LNC003834 can act as a molecular sponge for miRNA‐34a to regulate SLC14A1 expression and presented that: (a) SLC14A1 expression was evidently restrained by miRNA‐34a due to the densely bound 23 seed matches of 3’ tail sequence of SLC14A1 mRNA; (b) The newly recognized 21 seed matches of LNC003834 can reverse the miRNA‐34a‐mediated SLC14A1 suppressed expression via competitively binding miRNA‐34a. These experiment results demonstrated relationships between LNC003834, miRNA‐34a, and SLC14A1 mRNA and that were identified as competing endogenous RNAs with maintaining sodium homeostasis.For ceRNAs studies which contained SLC14A1 may inhibit sodium ion reabsorption from the renal tube to the descending vasa recta through competing endogenous LNC003834, miRNA‐34a, and SLC14A1 mRNA in camel renal medulla . sRNA‐MIX indicates three classes of small RNAs  co‐interference against SLC6A1, PEX5L and PCBP2. Scale bar, 100 μm.Table S1. The details of six camels.Table S2. The sequences of fluorescent group labeled RNA probes.Table S3. The inserted SLC14A1 sequences in SLC14A1 luciferase reporter plasmids.Table S4. The inserted LNC003834 sequences in LNC003834S1 and LNC003834S2 plasmids.Table S5. The sequences of different short hairpin RNAs (shRNAs).Table S6. The sequences of different small RNAs (sRNAs).Table S7. The primer sequence of candidate genes of salt‐resistance.Table S8. The significantly differential mRNAs and lncRNAs in the renal medulla of camel under salt stress.Table S9. The significantly differential miRNAs in the renal medulla of camel under salt stress.Table S10. The sequence of novel lncRNA LNC003834 gene.Table S11. Plasma Na+ concentration.Click here for additional data file.


Together with metabolites, proteins, and lipid components, the EV cargo consists of DNA and RNA nucleotide sequence species, which are part of the intracellular communication network regulating specific cellular processes and provoking distinct target cell responses. The extracellular vesicle (EV) nucleotide sequence cargo molecules are often investigated in association with a particular pathology and may provide an insight into the physiological and pathological processes in hard-to-access organs and tissues. The diversity and biological function of EV nucleotide sequences are distinct regarding EV subgroups and differ in tissue- and cell-released EVs. EV DNA is present mainly in apoptotic bodies, while there are different species of EV RNAs in all subgroups of EVs. A limited sample volume of unique human liquid biopsy provides a small amount of EVs with limited isolated DNA and RNA, which can be a challenging factor for EV nucleotide sequence analysis, while the additional difficulty is technical variability of molecular nucleotide detection. Every EV study is challenged with its first step of the EV isolation procedure, which determines the EV’s purity, yield, and diameter range and has an impact on the EV’s downstream analysis with a significant impact on the final result. The gold standard EV isolation procedure with ultracentrifugation provides a low output and not highly pure isolated EVs, while modern techniques increase EV’s yield and purity. Different EV DNA and RNA detection techniques include the PCR procedure for nucleotide sequence replication of the molecules of interest, which can undergo a small-input EV DNA or RNA material. The nucleotide sequence detection approaches with their advantages and disadvantages should be considered to appropriately address the study problem and to extract specific EV nucleotide sequence information with the detection using qPCR or next-generation sequencing. Advanced next-generation sequencing techniques allow the detection of total EV genomic or transcriptomic data even at the single-molecule resolution and thus, offering a sensitive and accurate EV DNA or RNA biomarker detection. Additionally, with the processes where the EV genomic or transcriptomic data profiles are compared to identify characteristic EV differences in specific conditions, novel biomarkers could be discovered. Therefore, a suitable differential expression analysis is crucial to define the EV DNA or RNA differences between conditions under investigation. Further bioinformatics analysis can predict molecular cell targets and identify targeted and affected cellular pathways. The prediction target tools with functional studies are essential to help specify the role of the investigated EV-targeted nucleotide sequences in health and disease and support further development of EV-related therapeutics. This review will discuss the biological diversity of human liquid biopsy–obtained EV nucleotide sequences DNA and RNA species reported as potential biomarkers in health and disease and methodological principles of their detection, from human liquid biopsy EV isolation, EV nucleotide sequence extraction, techniques for their detection, and their cell target prediction. With the development of deoxyribonucleotides and DNA molecules, more stable genomic information was generated and gene units for independent generation biological molecules of self-sufficient units were formed  in humans and other mammals include exosomes, microvesicles, and apoptotic bodies. All classes of EVs and their heterogeneous subsets differentiate in their biogenesis, diameter, and cargo, but all have a common feature of carrying and transporting parental cell molecules to target cells. Together with metabolites, proteins, and lipid components, the EV cargo consists of DNA and RNA molecules involved in the intracellular communication network regulating specific cellular processes and inducing distinct target cell responses . Human EIn the next chapters, we will briefly sum up the characteristics of EV nucleotide sequence species with their role and basic mechanisms in human cells and organisms. In the continuation, we describe the methods and procedures of EV nucleotide sequence biomarker discovery with methodological advantages and disadvantages and describe the prediction of EV nucleotide sequence cellular functions and their potential as therapeutics.The EV cargo consists of nuclear and mitochondrial DNA, while the EV transcriptome is more diverse and includes different coding and non-coding RNA species . The EV TP53, KRAS, and PTEN genes frequently altered in cancer and metastasis  were reported. Even though the mRNAs are not highly represented in EVs due to their size, EV mRNAs were reported in several studies which suggest the intracellular horizontal cell-coding transcriptome transfer . EV lncRhttp://www.mirbase.org/; Release 22.1) (The EV research is mainly directed into EV miRNA (microRNA) due to the well-characterized miRNA regulatory function in the cellular transcription and regulation of the mRNA expression to coding proteins. However, miRNA function is even more diverse. miRNAs are 16–24 nucleotides long sncRNA, coded by the miRNA genes which are in the human genome often located in miRNA gene clusters. 2,654 miRNA human sequences and 1,917 miRNA precursors are identified so far and their sequences are listed in miRBase (se 22.1) . It is ese 22.1) . The miRse 22.1) . The RISse 22.1) . miR-127se 22.1) , while sse 22.1) . Urinaryse 22.1) , while use 22.1) . The altse 22.1) .Piwi-interacting RNA (piRNA) regulation molecules are produced by similar synthesis, and have similar silencing mRNA translation and protein expression mechanisms as miRNA. This species of 26–31 nucleotides long sncRNA is highly expressed in germline cells and embryos, where pi-RNAs are involved in silencing of transposable elements, the epigenetic regulation, while in adult humans, piRNAs are essential in the process of spermatogenesis. EV piRNAs were investigated in seminal plasma EVs, where piRNAs are associated with spermatogenic ability , while mAnother sncRNAs reported in EVs are yRNAs, which are regulators of the DNA replication  and are EV transcriptome also includes tRNAs and tRNA fragments (tRFs), reported as highly represented in T-cell–released EVs . Even thSimilar to miRNA, other sncRNAs can be loaded into EVs together with RNA binding with Ago proteins or Y-box binding protein 1 , which avia miRNA-like silencing mechanisms, with binding on the mRNA transcription regulatory sites, other noncoding RNAs, or intracellular receptor mechanisms. If the EV RNA is released in the cytosol of the targeted cell, the released RNA can activate RNA-sensing receptors. Besides cytosol RIG1 receptors  columns, where vesicles are separated on a column by the EV diameter . The SECTissue-specific EVs can be isolated based on specific surface proteins with immunoprecipitation, where antibodies attached on immunoprecipitation columns or magnetic beads specifically bind to EV membrane protein markers . Due to All the EV isolation methods, some to a larger degree than others, also isolate non-EV impurity complexes. The most common isolated EVs from blood plasma contain EV low-density lipoproteins (LDL), high-density lipoproteins (HDL), and chylomicrones whose concentration is significantly increased in postprandial blood samples . TherefoWhile there are different EV isolation procedures, there are also diverse DNA or RNA isolation techniques based on different chemistry adjusted to isolate particular molecules of interest. The EV cargo typically consists of small DNA and RNA fragments; consequently, the isolation must isolate preferentially small nucleotide sequence fragments. The amount of the isolated EV DNAs or RNAs is highly variable to the isolation procedure, and it is the limiting factor that determines the EV nucleotide sequence characterization. A reasonable conclusion is that the EV DNA or RNA isolation method must ensure minimum sample losses.In order to ensure high purity and high yield of the isolated EV RNA or DNA, it is recommended to use the standardized, commercially available reagent kits for the EV nucleotide sequence extraction. The most common RNA and DNA isolation methods include the lysis reaction with the nucleotide sequence extraction, following precipitation and purification on the silicate columns and final elution . In the The EV nucleotide sequence content is still being discussed, but studies are unified in terms of a low EV nucleotide sequence quantitative amount. It was estimated that only one per 100 EVs contains miRNA . HoweverSeveral different methods can specify EV DNA and RNA molecules. The classic approach detects EV DNA or RNA using quantitative-PCR (qPCR), where only molecules of interest are analyzed, while the technique of massively parallel sequencing allows characterization of the whole RNA or DNA EV profiles . Due to Using quantitative PCR (qPCR), also known as real-time PCR, only the selected known sequences can be detected. EV sequences of interest can be selected based on the predetermined human organ and tissue expression data or can be predicted by known regulated pathways that are involved in the physiological and pathological conditions . The conEV transcriptome can also be detected using a microarray, where RNA fragments are directly labeled by the fluorescent dye and hybridized on predesigned probes with antisense sequences attached on a solid-phase glass surface or chip-based media. After wash steps, the fluorescence signal detection with intensity on specific locations on a glass slide is collected with a dedicated reader. The relative fluorescence of analyzed samples is translated to data with software algorithms, which recognize and quantify the studied particular sequences on the array surface . With miThe complete EV transcriptome and EV DNA profiles can be assessed only by NGS . Second-To simplify the sample sequencing preparation, several library preparation kits are on the market to prepare DNA, RNA, and sncRNA libraries. As small transcriptomic RNA molecules are abundant in EVs, it is recommended that small RNA library preparation kits are used for the NGS EV transcriptome detection . The smaNGS sequencing also presents the possibility to detect epigenomic modifications, which can be tissue-specific and can be very challenging in case of EV DNA and RNA. While the DNA methylation pattern detection is supported by the commercial reagents, sample preparation requires a large amount of DNA and analysis-specialized bioinformatics pipelines . The DNAThe third NGS generation is primarily designed as long-read sequencing technology for detecting DNA or RNA with several kilobase long fragments. This technology is suitable for detecting intact transcripts and providing accurate genome assemblies with successful detection even repeat sequences throughout the genome . Two comSince the EV sample processing and NGS library preparation require a skilled wet lab practice, the NGS sequencing data analysis requires bioinformatics knowledge to extract sequence information and address the EV nucleotide sequence study problem. The first part of the bioinformatics analysis starts with raw data base-calling translation from sequencing signals to nucleotide sequences. The base-called sequencing files are stored in the compressed fastq files generated by the Illumina sequencing platform and fastq5 files in the case of Oxford Nanopore. The signal translation base-calling output are text files with sequencing run info, nucleotide sequences, and quality of reading nucleotides .The RNA and DNA sequencing data analyses require different processing pipelines in the processing bioinformatics tools. However, the basic NGS pipeline includes the same basics steps: pre-processing and input data quality control, alignment, variant calling, quality control metrics report, and comparison of the expressed RNA analysis by the studied condition . First, Like in all the steps in EV research, the NGS data analysis procedure should be reported with used bioinformatics tools and their release number. It is also required to report basic analysis parameters and raw data quality parameters, sequenced reads per sample, and raw data should be submitted to public repositories, such as the SRA server (Sequence read archive), where sequencing data are accessible publicly to other researchers. While the NGS sequencing is still costly for assessing large cohorts, it is advised to confirm the NGS findings of smaller tested groups on larger cohorts, using the qPCR method.in vitro diagnostics (in vitro diagnostic EV liquid biopsy platform was presented ExoDx™ Lung (ALK) by Exosome Diagnostics. The kit detects EML4-ALK fusion transcripts in patients with non–small cell lung cancer, while the same company has also introduced exosome-based prostate cancer test ExoDx™ CE-IVD kit, which can help the physician’s decision to defer or proceed with a biopsy  includes siRNA-targeting transthyretin, used for the treatment of polyneuropathies induced by hereditary transthyretin amyloidosis, which was first-of-its kind targeted RNA-based therapy to treat a rare disease (Several procedures allow the production of EVs with specific nucleotide sequence loading for potential therapeutic application. The nucleotide sequence–loaded EVs can be produced ectamine . The celantation . While E disease . The tec disease , malariain vivo mice animal model, enabling activation of gene transcription (While the synthetic vesicles are in use for the delivery of mRNA in vaccines, the vesicles can also be loaded with human RNA to substitute cellular-damaged mRNA sequences due to genomic mutations in coding genes. Futhermore, Lainšček et al. showed EV delivery of the CRISPR/Cas system with the transcriptional regulator in the cription . In addiFrom the first EV reports, where EVs were assumed as cell-released garbage, the EVs research revealed the complexity of EVs role in intracellular signaling and cell communication with their potential as biomarkers of physiological states and pathogenesis. In humans, most studies are focused on intracellular communication, even though EVs are also involved in the interkingdom vesicle communication between human cells and microorganisms or parasites. Gut bacterial microbiota EVs in the human organism can modulate metabolism, endocrine response, and consequently regulate the entire human organism with pro-inflammatory or anti-inflammatory response . Similarin silico differentially expressed EV-nucleotide sequences’ role in the organism, the excat biological function of these differentially expressed nucleotide sequences is often not verified with in vitro or in vivo experiments. In the future, more studies will try to characterize the EV nucleotide sequence biological effect. By collecting data from standardized compatible experimental setups, it would be possible to build deep learning algorithms and artificial intelligence in order to predict pathological differences. EVs automated processing and EV deep sequencing will make possible detection of pathological abnormalities even at early stages of the disease before the first clinical symptoms appear. With these conditions, the EVs would become reliable diagnostic markers and supportive diagnostic tools in clinical practice, driving the development of novel therapeutic approaches. The exciting field of EV nucleotide sequences research has a bright future. Advanced NGS methods with single molecule resolution will give RNA and DNA nucleotide sequences a whole new perspective and additional dimension by identification of nucleotide sequence modifications. Nowadays, the EV nucleotide sequences are commonly investigated only by their primary nucleotide sequence. While DNA nucleotide modifications can lead to gene silencing and mutation development, RNA modifications can have an essential role in cellular function regulation and immunomodulation and could be associated with wide plethora of diseases. Just as the presence of EV DNA modifications is not well understood, the same goes with the EV RNA modifications, although intracellular mechanisms of pathogen detection imply their potential function. The knowledge of the role of EV nucleotide sequence modifications will gain importance in the upcoming years and will probably reveal an additional regulatory layer of multiple biological functions and disease pathologies.More and more EV studies implement NGS in their study strategy and consequently, precise sample metadata annotation is imperative for reliable interpretation of EV-expressed nucleotide sequences. While most studies report The technology and methods in genetics are well under development, and the innovations in the methodological procedures will support low EV nucleotide sequences material input. Since the cellular nucleotide characterization techniques aim to define the expression profiles on a single cell level, similar characterization would be reasonably appreciated in EVs. Single vesicle nucleotide sequences with a simultaneous surface protein markers approach would give a whole new perspective on the EV’s complexity and diversity. Compared to present studies, single vesicle information would directly link the EV nucleotide sequences and proteomic profile to the EV-released tissues. Such an approach will allow the generation of EV multidimensional multi-omics data and facilitate a new specific biomarker discovery. Determining the roles of molecules inside vesicles and those on their surface represents another challenge for future characterization. The EV research is focused on the characterization of EV lumen nucleotide sequences, even though EVs can contain parental cell origin RNA and DNA attached on the EVs’ surface lipids or anchored by glycosylated anchors .ex vivo or even in vivo treatment for inborn genetic disorders and could substitute ex vivo cell treatment for somatic mutations with viral vectors. While the lipid nanoparticles are preferentially accumulated in the lymph nodes, spleen and lungs, the future of EV therapeutics is in specific EV target tissue delivery. This could thus be achieved by EV-specific target membrane surface proteins (Because of the regulatory role of EVs, their therapeutic possibilities are being discussed and some preclinical and clinical trials already show promising results. Autologous platelet-produced therapeutic EVs significantly improve chronic postoperative healing and regenerative medicine , while mproteins , lipids,EVs and their nucleotide sequences, with their diversity, present an exciting research field that is still practically undiscovered and with enormous innovation potential represent one of the most promising fields of biomedical research.


Nymphaea ‘Joey Tomocik’ and analyze their posttranscriptional features. With the direct evidence from Iso-seq, multiple intermediates partially or fully intron-spliced were observed, and we also found that both cis- and trans-splicing introns were spliced randomly. Moreover, by using rRNA-depleted and non-Oligo(dT)-enrichment strand-specific RNA-seq data and combining direct SNP-calling and transcript-mapping methods, we identified 98 and 865 RNA-editing sites in the plastome and mitogenome of N. ‘Joey Tomocik’, respectively. The target codon preference, the tendency of increasing protein hydrophobicity, and the bias distribution of editing sites are similar in both organelles, suggesting their common evolutionary origin and shared editing machinery. The distribution of RNA editing sites also implies that the RNA editing sites in the intron and exon regions may splice synchronously, except those exonic sites adjacent to intron which could only be edited after being intron-spliced. Our study provides solid evidence for the multiple intermediates co-existing during intron-splicing and their interplay with RNA editing in organelle genomes of a basal angiosperm.Posttranscriptional modifications, including intron splicing and RNA editing, are common processes during regulation of gene expression in plant organelle genomes. However, the intermediate products of intron-splicing, and the interplay between intron-splicing and RNA-editing were not well studied. Most organelle transcriptome analyses were based on the Illumina short reads which were unable to capture the full spectrum of transcript intermediates within an organelle. To fully investigate the intermediates during intron splicing and the underlying relationships with RNA editing, we used PacBio DNA-seq and Iso-seq, together with Illumina short reads genome and transcriptome sequencing data to assemble the chloroplast and mitochondrial genomes of  Trans-spliced group II intron arises from DNA recombination events that interrupt an initially unbroken cis-spliced group II  an_037468)  as the r_037468)  was alsoanu v2.1  to de noanu v2.1  and/or Panu v2.1 .N. mexicana (NC_024542) was used as the reference sequence. In “mito_plant” mode, four CDS sequences  from the mitogenome of Arabidopsis thaliana (NC_037304) were selected as seed sequence and the same plastome (NC_024542) was set as “Chloroplast sequence,” leaving the “Reference sequence” empty. The assembled mitochondrial scaffolds were also manually joined together guided by PacBio assembly to generate the complete mitogenome.To verify the assemblies from the PacBio long reads and obtain more accurate organellar genomes, we reassembled the organellar genomes from the Illumina NGS reads by GetOrganelle v1.7.1  and NOVON. Mexicana (NC_024542) and A. thaliana (NC_000932) as references. Mitochondrial genome was annotated by Geseq [N. colorata (NC_037468) and A. thaliana (NC_037304) as references. The missing annotations, exact gene, and intron boundaries were manually checked and edited in Geneious R9.1.8 . The assembled and annotated chloroplast and mitochondrial genomes of N. ‘Joey Tomocik’ were submitted to the GenBank . Fully annotated organelle genome diagrams were drawn by OrganellarGenomeDRAW (OGDRAW) [Chloroplast genome was annotated by PGA  using plby Geseq  using mi(OGDRAW) .Repeated sequences were analyzed by ROUSFinder2.0.py with default parameters , which u® PCR cDNA Synthesis Kit  and then amplified with optimized PCR cycles by KAPA HiFiTM PCR Kits . Equimolar fractions of cDNA products from each tissue were pooled and then eight Iso-seq libraries with insert size of 0.5~6 kb were created using the SMRTbell template prep kit 1.0. Un-ligated products were removed by exonuclease and then purified by AMPure PB beads. A single SMRT cell was used for each library and then sequenced on the PacBio Sequel system.We use single-molecule long-read isoform sequencing (Iso-seq), Illumina RNA sequencing (RNA-seq), and strand-specific RNA-seq (ssRNA-seq) to assess the transcript diversity due to posttranscriptional modifications. For Iso-seq, total RNA from five tissues  of the newly sprouted individual (less than one year) were extracted separately using RNAprep Pure Plant Plus Kit (DP411) . RNA purity, concentration, and integrity were assessed by 1% agarose gel electrophoresis , Nanodrop, and Agilent 2100 Bioanalyzer, respectively. cDNA was synthesized using the SMARTer® UltraTM RNA Library Prep Kit , and then sequenced on Illumina Hiseq X ten platform with 2 × 150 bp PE mode. Each library generated about 6 Gb data.For RNA-seq, total RNA from another six tissues  of the same individual used in Iso-seq mentioned above were also extracted using Tiangen DP411 Kit, following by the assessment procedures as mentioned above. mRNA was enriched using Oligo (dT)-attached magnetic beads. After fragmentation, mRNA was reverse-transcribed into double-strand cDNA. Six cDNA libraries (one library per tissue) with insert size of about 240 bp were constructed using NEBNextTM rRNA Removal Kit . Three strand-specific libraries using rRNA-depleted RNA were generated by NEBNext® UltraTM Directional RNA Library Prep Kit  following manufacturer’s recommendations. Each library generated about 6 Gb (150 bp PE) data after sequenced by Illumina Hiseq X ten platform.For strand-specific RNA-seq (ssRNA-seq), libraries without poly-A mRNA enrichment were additionally constructed, using RNA extracted from juvenile leaf, mature leaf and stamen as mentioned above. Ribosomal RNA was removed by Epicentre Ribo-zeroRaw reads produced by Iso-seq were processed by Iso-Seq v2 in SMRTLINK 5.0.1 . SubreadN. ‘Joey Tomocik’ organelle genomes and genes/CDSs by GMAP (version 2020-10-14) [https://github.com/Magdoll/cDNA_Cupcake/wiki/Best-practice-for-aligning-Iso-Seq-to-reference-genome:-minimap2,-deSALT,-GMAP,-STAR,-BLAT#refgmap (accessed date: 29 July 2019). Mapped reads were filtered using ‘samtools view’ with the parameter “-bhF 4”. Only Iso-seq transcripts that could be properly aligned to genes and their flanking sequences were manually selected with the help of Geneious R9.1.8 software suite .Iso-seq transcripts were mapped to the assembled 0-10-14) , ‘gmap_bN. ‘Joey Tomocik’ organellar genomes by GMAP with the same parameters mentioned above.All ssRNA-seq raw data were filtered to remove adapters and low-quality reads by using fastp  with defAll splicing boundaries of intron-containing genes were manually compared to Iso-seq/Trinity transcripts GMAP mapping results. Necessary modifications were collected to refine the organelle genome annotations.Based on the Iso-seq transcripts mapping results, those Iso-seq transcripts that mapped to more than one gene (or exon of intron-containing gene) with the same direction were kept as a potential polycistronic transcript. The adjacent and overlapped cistronic transcripts were combined into a full PTU. All PTUs identified by Iso-seq transcripts were compared to Trinity transcripts alignment. Overlapped short PTUs were combined into one PTU. The chloroplast genes clustered in the same transcription direction and occurred in a full PTU were postulated as an operon.trans-splicing introns and their protein-coding sequences (CDS) were extracted. Iso-seq transcripts were mapped to these extracted sequences by GMAP as mentioned above. Iso-seq transcripts spanned any two gene segments were rendered as direct evidence of trans-splicing, and those spanned any two exons within one gene segment were considered as cis-splicing. The identified splicing events were further confirmed by Trinity transcripts alignment. The typically bipartite structure of all identified trans-splicing introns in organelle genomes was predicted according to the conserved secondary structure of six domains in group II intron of plants [Intron types of organelle genomes were predicted by using RNAweasel . Genes cf plants .Clean ssRNA-seq reads were aligned to chloroplast and mitochondrial genome using splice-aware aligner, HISAT2 version 2.2.1  with flaTo get the editing sites in intron and intergenic regions, we call all SNPs by ‘bcftools call’ with same parameters mentioned above but adding “-v” flag. Then ‘bcftools filter’ was executed with parameters “-i ‘FORMAT/DP>=20 && FORMAT/AD>=5 && QUAL>=30’”. To get RNA editing efficiency  of all filtered sites, ‘bcftools +fill-tags’ was performed with parameters “-- -t VAF”.We also identified RNA-editing sites of organelle genomes by full-length transcripts mapping method. RNA-editing sites were recorded when more than two edited Iso-seq transcripts mapped to CDSs/tRNAs, or more than three Iso-seq transcripts mapped to introns/intergenic regions. And for Trinity transcripts, more than five edited transcripts mapped to CDSs/tRNAs or adjacent introns/intergenic regions were counted. For those sites identified by REDO but no Trinity transcripts mapped were removed, and for those sites present in filtered vcf files but not in REDO annotation were kept if they were supported by more the half of the Trinity transcripts. The editing sites identified by Trinity transcripts but not in filtered vcf were manually recovered if they were also located in protein-coding sequences.To eliminate the false positive editing sites, Illumina DNA-seq reads were mapped to chloroplast and mitochondrial genome using bwa mem  with defPREPACT3  webserveNymphaea ‘Joey Tomocik’. By accurately mapping Iso-seq reads to organelle genomes, annotations by in-silico approach were refined, and multiple partially or fully intron-spliced intermediates were observed, which implied that both cis- and trans-splicing introns were spliced randomly. We also identified the RNA-editing sites in organelle genomes of Nymphaea using strand-specific RNA-seq data by direct SNP-calling and then rechecked by transcript-mapping method. By comparing the characteristics of RNA-editing in both organelles, we inferred that a common evolutionary origin and editing machinery may share by plastid and mitochondria in Nymphaea. We further proposed that RNA-editing sites in intron and exon region may splice synchronously, except for some exonic sites which were obstructed by un-spliced intron.By utilizing genomic PacBio long reads and Illumina short reads, we performed deep analysis of plastome and mitogenome of


Huntington’s disease (HD) is caused by a CAG-repeat expansion mutation in the Huntingtin (HTT) gene. It is characterized by progressive psychiatric and neurological symptoms in combination with a progressive movement disorder. Despite the ubiquitous expression of HTT, pathological changes occur quite selectively in the central nervous system. Since the discovery of HD more than 150 years ago, a lot of research on molecular mechanisms contributing to neurotoxicity has remained the focal point. While traditionally, the protein encoded by the HTT gene remained the cynosure for researchers and was extensively reviewed elsewhere, several studies in the last few years clearly indicated the contribution of the mutant RNA transcript to cellular dysfunction as well. In this review, we outline recent studies on RNA-mediated molecular mechanisms that are linked to cellular dysfunction in HD models. These mechanisms include mis-splicing, aberrant translation, deregulation of the miRNA machinery, deregulated RNA transport and abnormal regulation of mitochondrial RNA. Furthermore, we summarize recent therapeutical approaches targeting the mutant HTT transcript. While currently available treatments are of a palliative nature only and do not halt the disease progression, recent clinical studies provide hope that these novel RNA-targeting strategies will lead to better therapeutic approaches. Short tandem repeats, such as trinucleotide-repeats, are a frequent motif in the human genome, with the trinucleotide CAG being one of the most common repeat motifs . These rUpon repeat expansion beyond a certain threshold, CAG-repeat expansion is associated with the development of neurodegenerative diseases ,4. CAG rClinical features of HD include three main groups: behavioral symptoms, cognitive difficulties and involuntary movements. The disease progresses over approximately 15–20 years and is ultimately lethal. Most patients (up to 50%) show some behavioral changes, such as depression, even before HD is diagnosed . A high In HD, the mutant CAG repeat is located within exon1 of the Huntingtin (HTT) gene on chromosome 4p16.3. The HTT gene product is a large protein of approximately 348 kDa. Upon expansion of the polyglutamine tract, the HTT protein, just like all polyglutamine proteins, tends to misfold and aggregate. HTT-polyglutamine protein aggregation in the central nervous system is a pathological hallmark of the disease and pathogenesis was consistently associated with the abnormal function of mutant HTT protein ,13,14. AIn the last couple of years, there has been emerging evidence indicating the contribution of RNA-mediated abnormal functions to pathogenesis. Several lines of evidence indicate that mutant CAG repeats can induce RNA-mediated toxicity in in vivo models expressing untranslated repeats ,28,29,30One way of explaining a toxic gain-of-function of an RNA molecule is by aberrant attachment to proteins or other RNAs, where the three-dimensional structure is the key for intermolecular binding. RNA is most often a single-stranded molecule that folds onto itself to minimize its free energy. It can form fully paired and non-canonically paired regions, such as hairpins, internal loops, bulges, multi-branch loops and pseudoknots . These sAn essential part of spatial protein translation control is the regulation of cytoplasmic mRNA localization. RNA is transcribed in the nucleus and, after maturation into mRNA, binds to specific proteins that trigger mRNA export to the cytoplasm, where the mRNA can be translated into protein. The discovery that mutant CAG repeats promote nuclear retention and formation of RNA foci brought a new vision in the field of neurological disorders. Apparently, the mutant CAG-repeat RNA gets retained inside the nucleus due to aberrant RNA–protein interactions. Such interactions may pave the way to strong sequestration of other proteins, such as muscleblind 1 (MBNL1), which is an important protein in foci formation . RNAs wiOne important phenomenon that leads to RNA toxicity upon CAG-repeat expansion and that is linked to aberrant RNA localization is RNA granulation. The maintenance of neuronal circuits and synaptic strength is largely dependent on the local translation of mRNAs . NeuronaPBs are formed via phase separation within the cytoplasm and play a role in mRNA degradation . They alSGs are dense attachments of RNAs and RNA-binding proteins that form in response to cell stress. They can be seen as a storage granule for untranslated mRNAs. The stored mRNAs can go down one of three paths to promote cell survival: further storage, re-initiation of translation or decay . To faciOne approach to identifying mechanisms of RNA-mediated toxicity of mutant CAG-repeat transcripts is to analyze the proteome that aberrantly binds to expanded CAG repeats. Schilling et al. purified proteins that are captured by HTT RNA in a repeat-length-dependent manner and identified them using mass spectrometry . Of the Aberrant splicing events that occur upon the expression of mutant CAG-repeat RNA can be generally grouped into two groups: in splicing events affecting the CAG-repeat transcript itself and in splicing events affecting other transcripts. Both groups of mis-splicing events were shown in different CAG disease models .The first group, namely, mis-splicing of the mutant CAG-repeat transcript itself, occurs in diverse HD models and disease tissues. Here, the partial splicing of mutant HTT from exon 1 to exon 2 leads to the production of a small polyadenylated transcript encoding a neurotoxic mutant HTT protein fragment, which contains the expanded polyglutamine stretch ,54. ThisCREB1 transcript is mis-spliced upon the expression of mutant HTT. Mechanistically, CREB1 mis-splicing involves recruitment of the splicing factor PRPF8 to mutant HTT RNA. Importantly, CREB1 mis-splicing is also seen in HD brains [INSR, SERCA1, CLCN1 and LDB3. Here, the overexpression of the splicing regulator MBNL1 partially reverses splicing abnormalities, suggesting that MBNL1 is one of the splice factors that are mechanistically involved here [The second group of aberrant splicing events that occur upon the expression of mutant HTT affects other transcripts; several examples of this group were found in different studies. For example, the D brains . Other eved here . In a moved here . In the ved here . Additioved here . In addived here . Interesved here .All these data indicate that the sequestration of splicing factors to mutant CAG-repeat RNA affects splicing of both the CAG-repeat RNA itself and other transcripts and, thus, one major mechanism of RNA-mediated toxicity is deregulated splicing.Upon splicing and maturation of the mRNA, protein translation can be initiated, leading to protein synthesis from the mature mRNA transcript. The translation of expanded CAG-repeat mRNA results in a neurotoxic polyglutamine protein. This event can be seen as a pathogenic function, in connection with the neurotoxicity of the mutant CAG-repeat RNA. In addition to splicing factors, translation factors also bind length-dependently to CAG-repeat mRNA such that mutant, i.e., expanded repeats, recruit significantly more protein. One example of protein complexes that aberrantly attach to the mutant CAG-repeat hairpin is a protein complex consisting of MID1 (midline 1), the 40S ribosomal S6 kinase (S6K) and the catalytic subunit of protein phosphatase 2A (PP2Ac) . PP2A isAdditionally, MID1 also influences the activity of the kinase mTOR  in the mTORC1 protein complex, which consists of the proteins mTOR, mLST8  and raptor (regulatory-associated protein of mTOR). Here, MID1 regulates the composition of the mTOR/raptor complex. MID1-deficient cells have an increased level of PP2Ac, which, in the complex with its regulatory B-alpha subunit, inhibits the mTOR/raptor binding and thus the activity of the mTOR/raptor complex. Both proteins, PP2A and mTOR, play an important role in translational regulation . Among oGenerally, the translation of mRNA is initiated at the start codon (AUG). This also applies to the open reading frame of the RNAs with expanded CAG repeats, which are translated into polyglutamine protein. Until 2011, it was assumed that, in the frame with the start codon, only the polyglutamine protein is produced from the mutant CAG repeats. However, in 2011, Zu et al. showed an AUG-independent possibility of translation since mutation of the only AUG codon in ATXN8 transcripts with CAG-repeat expansion did not prevent protein translation . The repSince both the sense and the antisense strands of the DNA can be transcribed into RNA, bidirectional GC-rich mRNAs can be formed from the same gene locus. Since no AUG start codon is required for the RAN translation, these two complementary mRNAs can theoretically be translated. Thus, two expanded repeat mRNAs (sense and antisense) can be translated in all six reading frames into six different potentially toxic proteins .Four mutant HTT-RAN translation proteins  from the sense and antisense transcripts were found in 2015 by analyzing human HD autopsy brain samples . This fiTaken together, all the above-mentioned experiments suggest that mutant CAG-repeat mRNAs can aberrantly recruit translation factors and promote their own translation, not only in the open reading frame defined by the AUG start codon but also in different reading frames starting at the repeat sequence.Besides this aberrant translation triggered by the CAG-repeat mRNA, translation in HD is also affected by two further pathogenic mechanisms that are linked to the mutant HTT protein. On one hand, mutant HTT protein interacts with ribosomal proteins and can lead to ribosomal stalling, thereby disrupting translation . On the Another group of RNA-binding proteins that get trapped by mutant CAG-repeat RNA, resulting in aberrant function, includes RNA processing enzymes that are involved in the generation of small non-coding RNAs. Interestingly, more than 90% of the human genome is transcribed but does not translate into protein . These tInterestingly, some of the enzymes involved in miRNA cleavage aberrantly bind to mutant HTT RNA at its CAG-repeat region, resulting in a DICER-dependent production of small CAG-repeated RNAs (sCAGs) with <21 nucleotides. Of note, both the CAG-repeat RNA hairpin structure and the double-stranded RNA resulting from bi-directional transcription in the sense and anti-sense directions are appropriate substrates for DICER ,96. RepoAnother layer at which the miRNA machinery can affect cellular disease-mechanisms is by changing the protein expression of RNA-binding proteins that aberrantly bind to mutant HTT RNA and exhibit abnormal function in association with the CAG-repeat RNA. For example, miRNAs that regulate the expression of the MID1 complex  could ch2+ handling contribute to HD pathogenesis [While in all the above-mentioned studies, the aberrant interaction of mutant HTT mRNA was described, abnormal RNA-function in HD also occurs at a different level, namely, in the mitochondrial RNA (mtRNA). Generally, mitochondria are important for cell viability and several lines of evidence have connected mutant HTT protein with mitochondrial dysregulation ,109. Forogenesis . Interesogenesis , indicatogenesis . Recentlogenesis . Taken t2+ imaging study in Hdh150 knock-in mice by Arnoux et al. showed neuronal hyperactivity in the visual cortex well before disease onset (VFDO), suggesting that therapeutic intervention may be required at an early stage. Small bioavailable molecules that are capable of crossing the blood–brain barrier would be ideal therapeutics to inhibit such pathogenic cellular processes, for example, by reducing aberrant recruitment of RBPs to mutant HTT RNA. One compound that was studied in the context of VFDO is dimethylbiguanide metformin, which is an FDA-approved, low-cost type II diabetes drug. Metformin suppresses mutant HTT translation via interfering with the MID1 complex. This occurs through the metformin-mediated disassembly of the MID1 complex that leads to the activation of PP2A. This, in turn, decreases protein translation and, thus, the protein level of mutant HTT both in cell cultures and in vivo. In addition, metformin treatment reverses early-onset network dysregulation in HD mice [Given the fact that in HD, at least two neurotoxic gene products  contribute to neurodegeneration, an ideal therapeutic approach would target both the mutant RNA and protein. A two-photon Ca HD mice . In 2017 HD mice . Thus, mAnother molecule that was studied in HD models is furamidine. In silico identification of CAG binding ligands led to the identification of furamidine, which binds to HTT exon1 RNA in vitro. The RNA binding of one known mutant HTT interactor, namely, MID1, was blocked by furamidine, as shown, for example, in in vitro RNA protein pulldown experiments. As a consequence of blocking MID1 binding to mutant HTT RNA, the compound also reduces HTT translation and protein levels in an HD cell-line model. However, furamidine binding is not specific for CAG-repeat RNA. In addition to CAG repeats, it also binds AU-RNAs, CUG-RNAs and the DNA minor groove . Thus, uAnother interesting compound for HD therapy is one of the flavonoids. Flavonoids are known for their health benefits and can act as antioxidants, anticancer agents, neuronal protection and neurodegenerative disease prevention. One flavonoid, namely, myricetin, was shown to interact with the CAG motif and, thus, prevent both the translation of mutant HTT protein and the sequestration of MBNL1. This happens through the interaction of myricetin with RNA via base stacking at the A–A mismatch of the hairpin structure from expanded CAG repeats. Additionally, in a 3-nitropropionic acid (3-NP)-induced HD rat model, the oral supplementation of myricetin prevented mitochondrial dysfunction and oxidative stress, along with motor deficits . FurtherIn 2019, Khan et al. designed and synthesized several pyridocoumarin derivatives to attach to CAG motifs. Two of these derivatives turned out to be promising: both molecules showed higher affinity and selectivity for expanded CAG-repeat RNA when compared to regular duplex AU-paired RNA. Interestingly, both molecules are cell-permeable and exhibit low toxicity to healthy fibroblasts. Additionally, they are also capable of reducing the level of polyglutamine aggregation in cell cultures. Further research will show whether these compounds have the same beneficial effects in vivo.Antisense oligonucleotides (AONs) are short (8–50 bases), synthetically produced nucleic acids that bind complimentarily through Watson–Crick base pairing to a specific sequence within their target RNA. Depending on their chemical design and target sequence, AONs act through different mechanisms, including mechanisms that promote degradation of the target mRNA, mechanisms that lead to translational inhibition or mechanisms affecting splicing. For example, if a DNA AON binds its target mRNA, thereby forming an RNA–DNA heteroduplex, this duplex is detected by endogenous enzymes, such as ribonuclease H (RNase H), which cleaves the RNA strand. If an AON binds to its target pre-mRNA at intron/exon junctions, it can mask splicing enhancers and repressor sequences, thereby modulating splicing events. If an AON binds to its target mRNA, it can also sterically block the 40S and 60S ribosomal subunits from attaching, thereby blocking translation (reviewed in ). AONs hAlthough a lot of research has been conducted and several attempts have been made to develop therapeutic approaches, no disease-modifying treatment options are routinely available to date, and current medical management is restricted to supportive care. This may change with the development of HTT-lowering AONs. Several reports performed in mouse models showed that AONs can be beneficial.7 AON showed beneficial effects in HD models, along with models of other CAG-repeat-expansion diseases, such as spinocerebellar ataxia type 1 (SCA1) and SCA3 [For example, Kordasiewicz et al. showed that the injection of an AON into the cerebrospinal fluid of a symptomatic HD mouse model delayed disease progression . The AONand SCA3 ,124,125.To this point, there is no therapy that can cure CAG-repeat diseases, such as HD. The currently available treatments impart only symptomatic relief but do not stop disease progression. Through physiotherapy or speech therapy, the patient learns to manage involuntary movements or speech difficulties. Neuroleptics or tetrabenazine (indirectly acting as antidopaminergic) are used as medication. These substances help to inhibit uncontrollable muscle movements . PsychotVarious therapeutic approaches for curing HD are being explored in current research projects and clinical studies. A primary goal is to develop therapies that will take effect before symptoms appear, as it is not certain that the symptom-free phenotype can be restored later in HD. During this period, the post-transcriptional level offers opportunities for promising therapeutic approaches since the targeting of the mutant transcript and, consequently, the lack of the mutant protein would deprive most of the known pathomechanisms.One option for targeting the mutant transcript involves AONs. There are two different approaches using AONs that are currently used in clinical trials for HD treatment. In the first approach, AONs targeting the HTT transcripts from both alleles  are used. An example is an AON called tominersen. In preclinical studies on model organisms, the use of tominersen led to significant changes. After 14 days of treatment of BACHD mice (which express human mutant HTT) with the AON tominersen, the HTT mRNA level was reduced by up to 80%. This reduction lasted for 16 weeks. In addition, symptomatic improvement was observed in three different HD mouse models. In this context, the motor deficits in young animals were almost completely restored. Next, this AON was tested in clinical trials. In a phase 1b/2a clinical study conducted in 2015, the tominersen doses were administered intrathecally. Notably, this Ionis-Pharmaceuticals-funded study showed that tominersen decreased levels of the mutant HTT protein in cerebrospinal fluid (CSF) in patients with early stages of HD. Thus, this attempt awakens new hope for a curative treatment. However, it is unclear whether lowering wild-type HTT in an organism has long-term consequences. The phase 3 study on tominersen , which aimed to clarify these consequences, began in January 2019. This trial was planned to end in 2022 but was terminated in March 2021 due to poor tolerance of the maximum dose . Since tOne allele-specific approach to target only the mutant allele is to design AONs directed against heterozygous SNPs on the disease allele. Many SNPs have already been found in the HTT DNA sequence. With the help of special sequencing techniques, it can be determined which allele carries which SNP. This enables the generation of allele-selective AONs that target only the mutant allele HTT. It is estimated that AONs that bind to the three most common SNPs of mutant HTT would be sufficient to treat approximately 80% of the populations of European ancestors, but the exact AON must be selected specifically for each patient. In addition, the similarity of the overall sequences  limits the selectivity of the AONs, which means that the allele specificity of the AONs may not be 100%. However, the general concept of targeting the disease allele and maintaining a functionally normal HTT is a promising approach that was tested in phase 1 and phase 2 clinical trials  until spring 2021. The therapy was generally well tolerated, with most adverse events being mild or moderate in intensity  and occurring mainly in the group receiving the highest dose. However, due to the lack of consistent and significant reductions in mutant HTT, the development of WVE-120102 was stopped. As an alternative approach, Wave life science is planning a clinical phase 1b/2a study that is supposed to start in 2021 to test WVE-003. WVE-003 is a next-generation AON that targets a specific single nucleotide polymorphism (SNP3) that is commonly found on the expanded CAG allele (approximately 40% of HD patients carry SNP3 in association with the CAG expansion mutation). This AON has different phosphate modifications in the backbone to improve its function  .Like AONs, RNAi are nucleotide-based molecules that bind to mRNAs and cause the cell to degrade the bound transcript. RNAi uses RNA-based therapeutic molecules: short interfering RNA (siRNA), short hairpin RNA (shRNA) or microRNA (miRNA). RNAi compounds are also under investigation in the area of HD treatment. It should be noted that double-stranded siRNA has limited distribution and cellular uptake in the CNS compared to single-stranded AONs. Viral vectors, such as the adeno-associated virus (AAV), offer an advantage regarding delivering the siRNA or miRNA into the brain parenchyma through stereotactic surgery . This tyTaken together, HD is an incurable autosomal-dominant inherited condition caused by a CAG-repeat-expansion mutation that encodes two neurotoxic gene products: an expanded CAG-repeat transcript and a polyglutamine protein that is cleaved into two neurotoxic fragments, namely, an aggregate-prone polyglutamine N-terminal fragment and a non-polyglutamine C-terminal fragment. Of note, both neurotoxic gene products (transcript and protein) are expressed together in disease cells; as such, they will most likely also influence each other’s production: the mutant transcript obviously encodes the mutant protein and pushes its own translation, but conversely, the protein can also influence the transcript. For example, the C-terminal cleavage fragment of mutant HTT binds and inactivates dynamin 1 at the ER, thereby causing ER dilation and toxicity . ER streRecent pre-clinical research and clinical trials show that RNA-targeting drugs have great potential with regard to HD therapy. One future goal of RNA-targeting drugs would be to overcome the administration via injection into the CNS; alternatively, a bioavailable molecule that could be used for example for oral administration. However, it may take several more years before such compounds can be used in routine therapies.Additionally, further studies are required to assess the long-term side effects and their consequences. Since HTT is an essential protein and its knockout is embryonically lethal, the long-term suppression of the HTT gene products by RNA-targeting drugs, which may be necessary over decades when patients are chronically treated, may come with unwanted side effects. Recent clinical trials targeting both normal and mutant alleles have given the first evidence that lowering HTT below a critical level may not be tolerated over a prolonged period. Thus, allele-specific targeting may be a better option. However, long-term clinical observations using such allele-specific targeting drugs are required. Similarly, although HD is a late-manifesting neurodegenerative disorder, recent studies of mutation carriers suggest that the CAG-repeat expansion within the HTT gene affects neurodevelopment. In tissues from both human fetuses and mouse embryos that carried an HTT mutation, abnormalities in the developing cortex, such as mislocalization of mutant HTT protein and other junctional complex proteins; defects in the neuroprogenitor cell polarity; and differentiation, abnormal ciliogenesis, and changes in the cell cycle are detectable. Thus, HD has a neurodevelopmental component as well and is not solely a neurodegenerative disease . TherefoFinally, several of the pathogenic mechanisms involved in HD are also detectable in other CAG-repeat-expansion disorders. These include the deregulated expression of miRNAs ,102,103,