Different RNA species, such as ribosomal RNAs (rRNAs), transfer RNAs (tRNAs), messenger RNAs (mRNAs), and noncoding RNAs (ncRNAs), are frequently posttranscriptionally modified. In particular, studies in the past decade have suggested that these modifications influence almost all aspects of RNA metabolism, including stability, splicing, localization, RNA–protein interaction, and translation1. Indeed, these RNA modifications exhibit essential functions in many biological contexts, including normal development, adult tissue homeostasis, and cancer3.Nascent transcripts are subject to extensive processing, such as 5ʹ capping, splicing, 3ʹ polyadenylation, RNA editing, and RNA modifications. The transcriptome plasticity conferred by these posttranscriptional regulations can alter gene expression, which is a major driver of proteomic diversity. Similar to epigenetic modifications on DNA and histone proteins, RNA is also subject to various chemical modifications, called epitranscriptomic modifications. Since the first chemical modification of RNA was identified approximately 60 years ago, more than 170 RNA modifications have been identified, including N4. To understand the functions of epitranscriptomic marks, information on the precise location and abundance of various chemical modifications in the transcriptome is critical. Multifaceted approaches, including thin-layer chromatography (TLC), liquid chromatography–mass spectrometry (LC-MS), and next-generation sequencing-based methods, have been developed. The aforementioned review summarizes the current knowledge on the history of the development, advantages, and limitations of RNA modification detection tools and discusses future challenges to a comprehensive understanding of the epitranscriptome.Despite early biochemical studies showing the prevalence of chemically modified RNA in eukaryotes, the functional contribution of these marks to biological processes has been ignored for a long time. The discovery of molecular machinery that writes, erases and recognizes RNA modifications has helped in the characterization of the functional roles played by these modifications in various contexts. In addition, advances in transcriptome-wide detection methods have expanded our knowledge related to the precise position and abundance of RNA modifications in the transcriptome, leading to a greater understanding of epitranscriptomic regulation. Xiaoyu Li and colleagues reviewed advanced technologies used to detect epitranscriptomic marks on RNA6A is the most frequent internal mRNA mark and has been extensively studied in diverse cellular contexts. The epigenome and transcriptome in pluripotent stem cells (PSCs) undergo drastic changes during differentiation, and these changes are essential to control gene expression during the early development phase. In addition to genetic and epigenetic regulation, epitranscriptomic regulation shapes the expression profile of critical factors in the maintenance and fate specification of PSCs. For example, m6A RNA modification is essential for the formation of heterochromatin that silences retrotransposons, and this silencing is necessary to maintain embryonic stem cell identity6. In addition, the gene expression of developmental regulators during cell differentiation is controlled by m6A modification in embryonic stem cells7. Yong Jun Kim and colleagues reviewed gene expression control mediated through m6A and other RNA modification marks, especially in human PSCs and during early embryonic development8.Among the various RNA modifications, m6A marks are deposited on other RNA species, including rRNAs. Caroline Vissers and a colleague summarized the current knowledge of m6A modification on 18 S rRNAs, which is mediated by the methyltransferase METTL59. In particular, m6A methylation of rRNAs regulates ribosomal activity by shaping the structure of the ribosomal decoding center, thus influencing protein translation efficiency. Interestingly, m6A abundance on rRNAs shows variability among cell types and species, suggesting that the m6A rRNA mark may participate in the dynamic regulation of protein translation in a context-dependent manner. In addition, mutations in the human METTL5 gene are associated with the risk for intellectual disability and developmental abnormalities, suggesting potential roles played by rRNA m6A modification in neurodevelopment.m8G. Inappropriate o8G•A base pairing impacts RNA structure and functions at the posttranscriptional level, such as increasing the number of translational errors and reducing protein synthesis. Many studies have suggested that o8G can also alter RNA–RNA interactions in a redox-dependent manner. For example, a position-specific o8G mark on microRNA (miRNA) may be an epitranscriptomic mechanism that coordinate pathophysiological redox-mediated gene expression in cardiac hypertrophy10. In this special issue, Sung Wook Chi and colleagues present an overview of the function of 8-oxoguanine in DNA and RNA, from oxidative damage to the regulatory mechanisms of redox-mediated gene expression in both an epigenomic and epitranscriptomic manner11.Reactive oxygen species affect various cellular processes by oxidizing biomolecules, including guanine bases in DNA and RNA. The resultant 8-oxoguanine in DNA (8-oxo-dG) can pair with adenine, causing a mutation (G > T) that is prevalent in the cancer genome, and this mutation is actively repaired by DNA repair pathways. In addition to damaging DNA, 8-oxo-dG is an epigenetic mark that changes gene expression by altering promoter activity and the distribution of histone modifications. In addition, guanine in RNA is frequently oxidized to produce o6A, m5C, and Ψ. Moreover, several additional questions will be an exciting avenue of research in the future: How do these epitranscriptomic modifications distinctly regulate RNA metabolism in different cell types in the complex tissue milieu? Do different types of epitranscriptomic modifications coexist and engage in interplay to regulate the metabolism of the same RNA molecule? What are the dynamic properties of the modifications that govern regulatory pathways in response to environmental changes or pathological conditions? Do species differences in epitranscriptomic regulation offer insight into human evolution? Can we leverage the modulation of the epitranscriptome as a therapeutic intervention for various human disorders? Advanced technologies that enable the precise detection and manipulation of the epitranscriptome in diverse cellular systems will comprise the foundation needed to answer these questions.Despite the extensive efforts and remarkable recent progress made in the field, we have only started to explore the vast unknown landscape of the posttranscriptional regulation of gene expression. To date, the scientific community has focused on the functional relevance of only a few types of epitranscriptomic modifications, including m


The accurate “base pairing” in RNA molecules, which leads to the prediction of RNA secondary structures, is crucial in order to explain unknown biological operations. Recently, COVID-19, a widespread disease, has caused many deaths, affecting humanity in an unprecedented way. SARS-CoV-2, a single-stranded RNA virus, has shown the significance of analyzing these molecules and their structures. This paper aims to create a pioneering framework in the direction of predicting specific RNA structures, leveraging syntactic pattern recognition. The proposed framework, Knotify+, addresses the problem of predicting H-type pseudoknots, including bulges and internal loops, by featuring the power of context-free grammar (CFG). We combine the grammar’s advantages with maximum base pairing and minimum free energy to tackle this ambiguous task in a performant way. Specifically, our proposed methodology, Knotify+, outperforms state-of-the-art frameworks with regards to its accuracy in core stems prediction. Additionally, it performs more accurately in small sequences and presents a comparable accuracy rate in larger ones, while it requires a smaller execution time compared to well-known platforms. The Knotify+ source code and implementation details are available as a public repository on GitHub. RNA and its functions play a significant role in a variety of biological operations. DNA molecules, where the genetic information is stored, are transcribed into mRNA, which carries the information into the cytoplasm, where translation takes place and leads to the production of a protein. Due to its utmost importance, this procedure is also called the “central dogma” of molecular biology . Apart fRecent RNA secondary structure prediction methods have been based mainly on a scoring function that may rely on a thermodynamic, probabilistic, or Artificial Intelligence (AI)-based algorithm. The majority of the methods have utilized or adopted a partially minimum free energy algorithm introduced by Zuker, which facilitates dynamic programming enhanced with parameters from experiments . The NusIn an RNA secondary structure, the pseudoknot’s prediction is the most demanding task in terms of prediction. Other common motifs are stems, hairpins, bulges, internal loops, and multibranch loops, which a variety of algorithms are able to predict with high accuracy. On the other hand, the prediction of a pseudoknot is quite complicated because dynamic programming and minimum free energy algorithms are not constructed in such a way as to facilitate the interconnection of a pseudoknot. Another important reason is that with the increase in the length of the RNA, these algorithms need an exponential execution time. Thus, the need to achieve an accurate prediction for pseudoknots led our research towards constructing a platform that predicts H-type pseudoknots combined with bulges and internal loops with an accuracy similar to well-known methods and, at the same time, an efficiency in terms of execution time, called Knotify+. The H-type pseudoknot  consistsBulge loops or bulges form when a helix is interrupted by unpaired nucleotides on one strand, and they are frequently observed in the secondary structures of RNA ,11, as tIn this work, we suggest a new version of Knotify , which iMost of the algorithms have encapsulated dynamic programming in their pipeline process in order to determine the most likely secondary structure of an RNA, trying to minimize the free energy ,17. OtheOther approaches such as Pfold ,29, PPfoMachine learning algorithms have also been proposed in the literature. They endeavor to unveil hidden patterns by applying supervised and unsupervised methods in training datasets. The majority of these need large datasets because they use deep learning techniques which require a significant amount of data for the training process to avoid overfitting. In , for exaNext, we provide background information about the core theoretical concepts such as RNA, pseudoknots, bulges and internal loops, and parsers. This information is necessary for the illustration of the proposed methodology in RNA is a single-stranded molecule that folds, forming a specific set of RNA base pairs, the Watson–Crick base pairs (A–U and G–C),  and, lesOne of the least frequent patterns in RNA sequences, but challenging in terms of prediction, is the pseudoknot. A pseudoknot is said to exist when two base pairs intersect. This motif was initially observed in the Turnip Yellow Mosaic virus . The simA bulge is constructed by unpaired bases  and its size may be from one to many unpaired bases. Their appearance in all types of structured functional RNAs  emphasizThe proposed framework, Knotify+, is an extension of the work presented in , includiS is the start nonterminal symbol, terminal symbols and nonterminal symbols form the sets T and NT, respectively, and the syntactic rules form the set R. The notation of the syntax rules is L is capable of producing a string of symbols In order to construct a CFG , four seDue to their high expressiveness, there is a considerable number of parsers in the literature. The most cited and widely used algorithms are the CYK  introducKnotify+, similarly to , encapsuIn this section, the methodology proposed by the Knotify+ platform is presented. Knotify+ is an extension of the Knotify platform presented in , includiConsequently, the proposed implementation receives as input a string representing an RNA sequence of nitrogenous bases and produces the base pairing of the given RNA sequence in extended dot-bracket notation. The Knotify+ source code and implementation details are available as a public repository on GitHub .Knotify+’s methodology is based on the platform proposed in . Hence, The proposed platform makes use of the CFG ribed in . Knotifyribed in  aiming tribed in  and presDuring the first task, all parse trees were constructed by the parser. By the use of these trees, all possible pseudoknots and their core stems were allocated. The second task dealt with the traversing of all these trees to locate further stems. The CFG proposed in  was dediIn maximum_bulge_size. For each pair of sets, there may be a bulge of length 0 to maximum_bulge_size at one set and 0 to maximum_bulge_size at the other set. In the case where the bulge’s length is zero on one side and greater than zero on the other side, then a bulge is located. Otherwise, if the bulge’s length is greater than zero on both sides, then an internal loop is located. The Cartesian product of those cases was executed, and multiple dot-brackets strings were produced. By applying the criteria of the minimum free energy and the greatest number of base pairs of the pseudoknot, the optimal case was selected. The result of this procedure is shown in stage 5 of In both loops of the pseudoknot, the base pairs at positions 9–18, 8–19, 4–12, and 3–14 were sequentially detected during stages 1 to 4, respectively. maximum_bulge_size.Knotify+ allows the user to choose the option of the base pairs, U–G, as an argument from the command line, as well as the value of the Knotify+ incorporated a hybrid model to choose the optimal tree among the trees that were produced from the CFG. This task facilitated the maximum base pairing and the MFE (Minimum Free Energy) principles. In the first stage, it ranked all the produced trees according to the count of base pairs around the pseudoknot, excluding the stems formed after the bulges or internal loops. The next stage consisted of the application of the MFE in the trees that were ranked at the top in the first stage, i.e., the trees with the most base pairs around the pseudoknot. After extensive experiments, we observed that including all the possible detected stems after the bulges or internal loops may lead to excluding the correct RNA sequence from the top-ranking sequences (regarding base pairs count) that were promoted to the second stage of selection, that of the MFE calculation. Consequently, the first task of the proposed tree selection, that of maximum pairing, was applying it to the RNA sequences without taking into consideration the stems detected after the bulges or internal loops.Finally, the secondary structure with the minimum free energy was selected. A module derived from HotKnots  calculatation in , we set  9.6 see .To evaluate Knotify+’s accuracy against other methodologies, a dataset  consistimaximum_bulge_size equal to 3.In measuring our framework’s performance, three methods were chosen: (a) the percentage of the pseudoknot’s core stems prediction, (b) the confusion matrix including the precision (PPV), recall, F1-score, and MCC (Matthews correlation coefficient), and (c) the execution time. Concerning the Knotify+ platform, all experiments were implemented with the parameter In k, l) was equivalent to  or . The results of predicting the pseudoknots’ core stems are also shown in Adopting the methodology of locating the pseudoknot proposed in , we permtp (true positive) expresses the count of the correctly predicted base pairs, fp —thecount of the incorrectly predicted base pairs, fn —the count of the base pairs that were not predicted, and tn (true negative)—the count of those correctly not predicted.The performance of all platforms regarding the precision, the recall, the F1-score, and the Matthews Correlation Coefficient (MCC) is presented in As shown in tp, tn, fp, and fn in detail. Knotify+ counted more tp and lower or equal fp and fn for the sequences smaller than 40 (L < 30 and 30 ≤ L < 40) compared to the evaluated methods. Its prediction capability in sequences larger than 40 (40 ≤ L < 50 and L ≥ 50) was better than Knotify’s and comparable to but still lower than Knotty’s, which increased its prediction capability when the length increased.In The third metric was the execution time, where the proposed methodology was tested against the other platforms in terms of time efficiency. The second column of The prediction of the RNA secondary structure is quite a challenging task, especially for pseudoknotted structures. In this context, we proposed an intelligent grammar-based algorithm that predicted H-type pseudoknots with bulges and internal loops. It detected the secondary structure performant, and its accuracy was comparable to well-known platforms. Especially for sequences smaller than 30 bases, it outperformed all the examined methods, showing that the enhancement of its expressiveness led to an important advancement of our previous version. The most notable finding was that the proposed methodology outperformed our previous version Knotify regarding the recall, F1-score, and MCC in all sets, showing a significant improvement for sequences larger than 40. In addition, Knotify+ continued to outperform Knotty for small sequences, while it was comparable for sequences between 30 and 50, and significantly decreased the gap with Knotty for sequences larger than 50 bases. Meanwhile, Knotify+ maintained the highest percentage of core stems prediction compared to all the examined methods and was approximately eight times faster than Knotty.


Aspergillus fumigatus. The results indicated that treatment with the drugs removed RNA viruses of diverse proportions in the families Chrysoviridae, Mitoviridae, Partitiviridae, Polymycoviridae, and an unclassified RNA virus group. Viruses belonging to Narnaviridae were hardly eliminated by these antiviral treatments when they were the sole infectious agents. We found that 2CMC showed activity against a wider range of RNA mycoviruses compared to ribavirin, 2CMA, and 7d2CMA, although 7d2CMA also efficiently removed dsRNA viruses from the families Chrysoviridae, Partitiviridae, and Polymycoviridae. These results indicated that removal of mycoviruses depends on the specific viral species and antiviral drug. This is the first report demonstrating a preferential antiviral effect against mycoviruses, which will enhance research on microbial RNA viruses and support their elimination from economically important fungi such as edible mushrooms.RNA viruses in fungi (mycoviruses) are model systems for understanding the relationships between eukaryotic microorganisms and RNA viruses. To reveal the effects of mycoviruses on host fungi, it is essential to compare the phenotypes between isogenic fungal isolates with or without RNA virus infection. Since active entry machinery for RNA mycoviruses has never been identified, introducing mycoviruses to fungi is a difficult and time-consuming process. Therefore, most studies have tried to generate virus-free isolates from infected strains by eliminating the mycovirus. However, methods of elimination have not been evaluated in a quantitative and comparative manner. In this study, we established a method to remove mycoviruses from host cells using the antiviral drugs ribavirin, 2′-C-methylcytidine (2CMC), 2′-C-methyladenosine (2CMA), and 7d2CMA, and compared the efficiency of removal in virus-infected strains of  RNA viruses have long been identified and isolated from humans and economically important and diseased organisms such as livestock and crops . HoweverA. fumigatus IFM 41362, and compared the phenotypes of these two strains to reveal the effect of AfuCV  from Tolypocladium cylindrosporum with 100% efficiency (Polymycoviridae (dsRNA) and Narnaviridae (ssRNA) using a combinational approach of protoplast regeneration and ribavirin treatment , Mitoviridae (ssRNA), and Hypoviridae , a guanine nucleoside analog, is a broad-spectrum inhibitor of RNA virus replication . The modficiency . Howeverreatment . Howevere ssRNA; . These sTotiviridae) in the protist Leishmania by 2CMA and 7d2CMA  is a member of the 2’-C-methylribonucleoside class of compounds. This class includes 2′-C-methyladenosine (2CMA) and the modified form 7-deaza-2′-C-methyladenosine (7d2CMA), which has previously been shown to inhibit RNA-dependent RNA polymerase (RdRp), the only universal gene among RNA viruses, of HCV . Kuhlmand 7d2CMA . In thatchomonas , and 2CMAspergillus fumigatus  plate media for 5 days at 37°C. Conidia were harvested with 0.05% Tween 20 and then passed through a 40-μm nylon mesh. For treatment with antiviral compounds, the conidia were dropped on the center of PDA containing antiviral compounds and incubated for 5 days at 37°C. The final concentrations of each compound were as follows: ribavirin , 0.1 mg/ml; 7-Deaza-2’-C-Methyladenosine , 0.2 mg/ml; 2’-C-Methyladenosine , 0.2 mg/ml; 2’-C-Methylcytidine , 0.2 mg/ml. DMSO was used as the solvent for these drugs, and DMSO without compounds  was used for the negative-control PDA. In these conditions, clear fungal growth inhibition was not observed with any compound. For liquid cultures, the strains were cultured in potato dextrose broth (PDB) with reciprocal shaking (120 rpm) for up to 5 days at 37°C. P. chrysogenum).Drug-treated spores were obtained from five mycelial plugs. Conidia were harvested with 0.05% Tween 20 and then passed through a 40-μm nylon mesh. The concentration of spores was adjusted to 50 spores/ml. To obtain single colonies, 500 μl of spore solution was spread on yeast extract peptone dextrose agar  media and incubated for 1 day at 37°C  were disrupted using FastPrep 24  in 2× STE  containing 0.1% (v/v) β-mercaptoethanol. Total nucleic acids were manually extracted with sodium dodecyl sulfate-phenol.For the detection of RNA viruses in fungal colonies, direct one-step RT-PCR was applied as described previously . The colvia conidia, to judge the efficacy of antiviral drugs in virus elimination. The scheme of antiviral drug treatment is shown in Fungi are known for their multicellular and elongated cell structures. These features make it difficult to quantitatively evaluate virus elimination efficiency in each cell. Here, we took advantage of the persistent-type lifecycle of mycoviruses, namely vertical transmission of viruses A. fumigatus and identified six host strains with seven different viruses , was treated with ribavirin. When 22 colonies from ribavirin-treated spores were checked, 15 colonies were determined to be virus-free  and Aspergillus lentulus IFM 62627 , Kitrinoviricota (AfuRV1 can be classified into this phylum), Lenarviricota , Negarnaviricota (AfuNSRV1 can be classified into this phylum), and Duplornaviricota . These five phyla ranks cover all non-retro RNA viruses , which harbor AfuNV2. In the case of AfuNV2 infecting IFM 63147, 2CMC successfully enhanced the removal of the virus (17/66). However, in the case of AfuNV2 infecting IFM 63431, no effect of 2CMC was observed , which harbor AfuPmV-1. The rates of 2CMA to remove AfuPmV-1 from the A. fumigatus strains IFM62632 and Af293 were different  are widespread among cultivated species for food production . All sources of funding received for this research was submitted.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Atherosclerosis (AS) is a common cardiovascular disease with a high incidence rate and mortality. Endothelial cell injury and dysfunction are early markers of AS. Oxidative low-density lipoprotein (Ox-LDL) is a key risk factor for the development of AS. Ox-LDL promotes endothelial cell apoptosis and induces inflammation and oxidative stress in endothelial cells. Small non-coding RNAs (sncRNAs) mainly include Piwi-interacting RNAs (piRNAs), small nucleolar RNAs (snoRNAs), small nuclear RNAs (snRNAs), microRNAs (miRNAs) and repeat-associated RNAs. Studies have shown that small non-coding RNAs play an increasingly important role in diseases.We used ox-LDL to treat rat endothelial cells to simulate endothelial cell injury. The expression changes of sncRNA were analyzed by small RNA high-throughput sequencing, and the expression changes of piRNA, snoRNA, snRNA, miRNA and repeat-associated RNA were verified by quantitative polymerase chain reaction (qPCR).Small RNA sequencing showed that 42 piRNAs were upregulated and 38 piRNAs were downregulated in endothelial cells treated with ox-LDL. PiRNA DQ614630 promoted the apoptosis of endothelial cells. The snoRNA analysis results showed that 80 snoRNAs were upregulated and 68 snoRNAs were downregulated in endothelial cells with ox-LDL treatment, and snoRNA ENSRNOT00000079032.1 inhibited the apoptosis of endothelial cells. For snRNA, we found that 20 snRNAs were upregulated and 26 snRNAs were downregulated in endothelial cells with ox-LDL treatment, and snRNA ENSRNOT00000081005.1 increased the apoptosis of endothelial cells. Analysis of miRNAs indicated that 106 miRNAs were upregulated and 91 miRNAs were downregulated in endothelial cells with ox-LDL treatment, and miRNA rno-novel-136-mature promoted the apoptosis of endothelial cells. The repeat RNA analysis results showed that 4 repeat RNAs were upregulated and 6 repeat RNAs were downregulated in endothelial cells treated with ox-LDL.This study first reported the expression changes of sncRNAs in endothelial cells with ox-LDL treatment, which provided new markers for the diagnosis and treatment of endothelial cell injury. Atherosclerosis (AS) is a common cardiovascular disease with a high incidence rate and mortality , 2. It iAfter LDL enters VECs, it combines with reactive oxygen species in cells to form ox-LDL . Ox-LDL Small non-coding RNAs (sncRNAs) mainly include Piwi-interacting RNAs (piRNAs), small nucleolar RNAs (snoRNAs), small nuclear RNAs (snRNAs), microRNAs (miRNAs) and repeat-associated RNAs . PiRNA iIn this study, we used small RNA sequencing and quantitative polymerase chain reaction (qPCR) to analyze the effect of ox-LDL on the expression of sncRNAs in VECs and identified sncRNAs related to ox-LDL-induced VEC dysfunction, thus providing a new therapeutic target for the diagnosis and treatment of AS.3 arterial implants with a very sharp scalpel and planted in a culture dish. The intimal surfaces of the arterial grafts were contacted with the bottom of the culture dish, and the planting density was approximately 1 piece/cm2. The culture dish was placed in the incubator for 2 h. Culture medium containing 25% fetal bovine serum was added from the top of the tissue to slightly cover the intimal surface of arterial grafts. The plate was transferred into an incubator for 24 h, and after 24 h, the culture medium was changed. On the 3rd day, the endothelial cells were fully grown, and fibroblasts were just about to grow. Then, the arterial grafts were gently removed, and the culture medium was changed every 2 days.The rats were killed after cervical dislocation, and the whole rats were soaked in 75% ethanol for disinfection. The chests were opened, and the aortas were dissected in sterile PBS. After excision of the fat and fibrous tissue of the vascular adventitia, the aortas were rinsed with PBS to remove the blood and coagulation in the vascular cavity. The aortas were then dissected into 1 mmThe vascular endothelial cells were inoculated into the culture plate, 2 ml of medium containing 100 μg/ml ox-LDL was added to the culture plate for 12 h, and the cell samples were collected after 12 h of culture.The lysed sample was placed at room temperature for 10 min to completely separate the nucleoprotein from the nucleic acid. Chloroform (0.2 ml) was added to the lysed sample, and the mixture was violently shaken for 15 s and placed at room temperature for 3 min. Then, the mixture was centrifuged at 12,000 rpm, 4°C, for 10 min. The upper aqueous phase was collected and mixed with an equal volume of isopropanol. The mixture was placed at room temperature for 20 min and then centrifuged at 12,000 rpm, 4°C, for 10 min. The supernatant was discarded, and 1 ml of 75% ethanol was added to wash the precipitate. RNA was purified after centrifugation at 12,000 rpm and 4°C for 3 min. Purified RNA was eluted in 30–50 μl of RNase-free ddH2O.A Qubit RNA detection kit was used to accurately quantify total RNA to determine the amount of total RNA added to the library construction. The 3′ end of RNA was connected to the connector with T4 RNA ligase 2. The 5′ end of RNA was connected to the connector with T4 RNA ligase 1. The connecting product was reverse-transcribed to obtain the cDNA strand of the connecting product. In this step, the small RNA library was reverse-transcribed into DNA. The reverse transcription product was amplified by PCR to obtain the final library product. The NextSeq 550 (Illumina) system was used for sequencing analysis. Raw reads were obtained after sequencing and then filtered, spliced and decontaminated to obtain clean reads, which were compared with the reference genome for subsequent information analysis. The data were removed with the original program. Clean data were obtained by removing low-quality sequences using the trimmatic program. The FastQC program was used to count the data volume of clean data. Fragments larger than 15 nucleotides were retained for subsequent analysis. The Bowtie program was used to compare clean data to the non-coding RNA database and genome. The edgeR program was used to analyze the differences in non-coding RNA expression. The databases used in this study were list as follow: PiRNABANK (piRNA), miRbase (miRNA) and Ensembl database (snoRNA and snRNA). As for repeat RNA were analyzed by RepeatMasker. The differentially expressed small RNAs are listed in –ΔΔCt method. The primers used are listed in Total RNA was extracted from vascular endothelial cells by TRIzol. The EZ-press microRNA Reverse Transcription Kit was used for miRNA and piRNA reverse transcription. PrimeScript RT Master Mix was used for snoRNA and snRNA reverse transcription. SYBR Premix Ex Taq was used to detect the expression of sncRNA in VECs. U6 was used as the internal control. The relative expression of sncRNA was calculated by the 22 incubator for 4–6 h, and then the medium was changed and cultured for 18–48 h.For piRNA and miRNA overexpression and knockdown, piRNA and miRNA mimics were used for overexpression, and piRNA and miRNA inhibitors were used for knockdown. The piRNA and miRNA mimics and inhibitors were designed and synthesized by General Biosystems (Anhui) Co., Ltd. A total of 50 μl of Opti MEM and 0.2 nmol of RNA were gently mixed to obtain RNA diluent. A total of 50 μl of Opti MEM and 2.0 μl of Lip2000 were gently mixed to obtain Lip2000 diluent, and the Lip2000 diluent was then incubated at room temperature for 5 min. RNA diluent and Lip2000 diluent were mixed and incubated for 20 min at room temperature to form the RNA-Lip2000 complex. RNA-Lip2000 complex was added to the incubated cells, and the culture plate was then gently shaken. Cells were incubated in a 37°C COFor snRNA and snoRNA overexpression, the target sequences of snRNA and snoRNA were cloned into the pLVX vector to construct lentivirus, which was used to transfect endothelial cells.Rat aortic endothelial cells were treated with ox-LDL for 12 h and then subjected to the indicated treatment. After that, cell apoptosis was measured using an Annexin V-FITC/propidium iodide kit (BD Biosciences) according to the manufacturer’s protocol. Apoptotic cells were observed using a flow cytometer. CellQuest software was used to analyze the apoptotic rate.T-tests and ANOVA were used to compare two groups by Excel 2017; p < 0.05 was considered a significant difference, and P < 0.01 was considered highly statistically significant.All data are presented as the mean ± S.D. of three independent experiments. PiRNA is involved in transposon silencing, spermatogenesis, genome rearrangement, epigenetic regulation, protein regulation and reproductive stem cell maintenance . HoweverResearch on snoRNA has developed rapidly, and recent studies have shown that snoRNA is also involved in genetic diseases, human variation, hematopoiesis, metabolism and cancer . Small RSnRNA includes a large group of non-coding RNAs with large differences, and these non-coding RNAs are mainly involved in the selective splicing of mRNA precursors and the processing of ribosomal RNA . We analMiRNAs are involved in the maintenance of endothelial cell function , 31. SmaAll RNAs have a tendency to spontaneously fold into secondary or tertiary structures. Although RNA has only four types of nucleotides, they can form a variety of structures, thus affecting cell function. Aberrantly expanded, repeated RNA sequences can exhibit gain-of-function abnormalities and become pathogenic, giving rise to many diseases . The obtThe relationship between RNA structural and functional diversity in different organisms is the frontier of major basic research in life science. Especially with the proposal of genome and proteome projects, RNA omics research has become a new growth point of molecular biology research. In this study, we found the relationship between small RNAs and ox-LDL-induced ED by small RNA sequencing and qPCR. PiRNA is a type of small RNA isolated from mammalian germ cells, and piRNA can play its regulatory role only when combined with members of the piwi protein family . CurrentSnoRNA is a type of non-coding RNA with a length of 60–300 nucleotides that widely exists in eukaryotic cells . In vertSnRNA is the main component of the RNA spliceosome in the posttranscriptional processing of eukaryotes . There aMiRNAs are the most studied sncRNAs, and their function is involved in almost all aspects of biological processes. An increasing number of studies have shown that miRNAs play an important regulatory role in cardiovascular diseases . SeveralThere are relatively few reports on the relationship between repeat-associated RNA and disease. Repeat-associated RNA was reported to be associated with myotonic dystrophy (DM), Fuch’s endothelial corneal dystrophy (FECD) and amyotrophic lateral sclerosis (ALS) . We firsUsing small RNA high-throughput sequencing and qPCR detection, we found that the expression of piRNAs, snoRNAs, snRNAs, miRNAs and repeat-associated RNAs significantly changed during ox-LDL-induced endothelial cell injury. Our results preliminarily show that these sncRNAs are significantly upregulated or downregulated during ox-LDL treatment. PiRNA, snoRNA, snRNA, miRNA and repeat-associated RNA may be biomarkers of endothelial cell injury and could be useful in the future as therapeutic targets. Further research is still needed to reveal the cellular pathways regulated by sncRNAs that are involved in ox-LDL-induced endothelial cell injury.The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in this article/This animal study was reviewed and approved by the Animal Ethics Committee of Shidong Hospital.YW wrote the manuscript, conducted the data analysis, and participated in the conception and conduction of the study. TL participated in the conception and conduction of the study and extensively engaged in the preparation and revision of the manuscript drafts. WX participated in the conception of the study, data analysis, and revision of the manuscript drafts. YB, DY, and LF participated in the conception of the study, supervised the conduction and data analysis, and extensively engaged in preparation and revision of the manuscript drafts. All authors contributed to the article and approved the submitted version.


Drug transporters play an important role in the maintenance of chemical balance and homeostasis in different tissues. In addition to their physiological functions, they are crucial for the absorption, distribution, and elimination of many clinically important drugs, thereby impacting therapeutic efficacy and toxicity. Increasing evidence has demonstrated that infectious, metabolic, inflammatory, and neurodegenerative diseases alter the expression and function of drug transporters. However, the current knowledge on transporter regulation in critical protective barriers, such as the brain and placenta, is still limited and requires more research. For instance, while many studies have examined P-glycoprotein, it is evident that research on the regulation of highly expressed transporters in the blood–brain barrier and blood–placental barrier are lacking. The aim of this review is to summarize the currently available literature in order to better understand transporter regulation in these critical barriers. Critical barriers surrounding the brain and fetus, respectively termed the blood–brain barrier (BBB) and the blood–placental barrier (BPB), form functional barriers between these vulnerable tissues and the systemic circulation, thereby playing a key role in protection against potentially toxic environmental and endogenous compounds. Furthermore, transporter systems within specialized cells of the barriers serve as mechanisms to provide adequate exchange of essential substances, gasses, and wastes. A number of these transport systems are also involved in the exchange of drugs and are highly expressed in the apical and/or basolateral membranes of the BBB and BPB . The proAs excellently summarized by Daneman and Prat , the terIn this review, we aim to focus on the mechanisms shown to regulate transporters in the endothelial cells of the BBB. The ABC-transporter P-glycoprotein (P-gp), which is encoded by ABCB1/MDR1, is the best-studied transporter in the context of pathologies. In addition to P-gp, multiple other transporters are assumed to contribute to the functionality of the BBB. Ranking transporters for their abundance as detected in isolated human brain microvessels by mass spectrometry suggests higher levels for the glucose transporter 1  as compared to all other transporters observed in the currently published mass proteomic studies. Of note, there are a limited number of studies that have quantified the abundance of transporters in isolated human brain microvessels and there are inconsistencies in the reported data ,6,7,8. HIn addition to abundance, the role of a transporter is also impacted by its localization in the polarized endothelial cells. Determination of ultrastructural localization within endothelial cells of the human BBB allows for prediction of their contribution towards the net transcellular transport of their substrates. Indeed, confocal and electron microscopy analysis has confirmed luminal localization of BCRP and P-gp ,12, whilThe placenta forms a barrier that serves not only as a physical barrier between maternal and fetal circulation (blood), but also plays a critical role in regulating nutrient and waste exchange between mother and fetus. Thus, the placental barrier protects the developing fetus and later embryo from toxicants and xenobiotics, and it ensures its proper development. The placenta is the largest fetal organ, and its main components include the chorionic villi and the fetal vascular endothelium ,25. In dThe ABC transporters, BCRP and P-gp, are highly abundant in the syncytiotrophoblasts throughout gestation ,28,29. DSLC transporters also play a crucial role in biodistribution and fetal exposure of their substrates as they facilitate the cellular entry of several drugs, thereby impacting either the maternal-to-fetal or fetal-to-maternal net transport, depending on localization and directionality of transport. It is believed that many of the SLC transporters are found within the basolateral membrane of placenta, thereby influencing cellular entry at the fetal side of syncytiotrophoblasts. Indeed, immunofluorescence studies have confirmed localization of OCT3, OATP2B1, and OAT4 within the basolateral membrane of syncytiotrophoblasts ,35,36,37Placental expression data of several other drug transporters has been reported, including for ENTs, concentrative nucleoside transporters (CNTs), and additional members of the MRP family. Their expression, physiological role, and implications in maternal-drug transfer are much less characterized and there are contradictory findings in the literature ,39. For In summary, there are multiple transport systems expressed in the syncytiotrophoblast; however, the current knowledge on transporter-protein abundance and functional implication of alterations in this critical protective barrier is still limited and requires further research.Major neurological diseases, such as multiple sclerosis, stroke, epilepsy, Alzheimer’s disease, and Parkinson’s disease, are linked to structural and functional changes in the BBB . The imptau protein. Aβ derives from amyloid precursor protein (APP), a single-pass transmembrane protein highly expressed in the brain, which is metabolized through sequential proteolysis by β- and γ-secretase enzymes -verapamil PET study results, where AD patients exhibited higher binding potential of this P-gp substrate in most areas of the AD brain and lower extraction ratios compared to age-matched, healthy volunteers .,98.95,98As recently summarized by Ghafouri-Fard et al., there are several miRNAs that are differentially expressed in animal models of epilepsy and in the brains of patients with epilepsy . Some ofTaken together, there is some evidence linking epilepsy to an upregulation of P-gp with evidence from rodent models indicating that overexpression may contribute to drug resistance. It appears noteworthy at this point that there are rodent in vivo and in vitro data that antiepileptic drugs such as phenobarbital, phenytoin, carbamazepine, and valproic acid increase P-gp expression and function in the brain ,129. NevParkinson’s disease (PD) is one of the neurodegenerative conditions where expression of P-gp has been investigated as a representative of the efflux transporter family contributing to barrier function. In general, PD is characterized by the progressive loss of dopaminergic neurons within the substantia nigra and the intracytoplasmic accumulation of abnormal aggregates of misfolded α-synuclein protein (Lewy bodies) ,134. TheOne feature of the PD brain is the structural and functional alteration in the BBB’s integrity. Indeed, vascular degeneration has been shown histologically  and func11C]-verapamil. In 2005, Kortekaas et al. found enhanced signals (18%) in the midbrain region of five PD patients compared to five healthy controls measured with [11C]-verapamil, thereby suggesting a reduction of P-gp function associated to the disease [11C]-(R)-verapamil as the PET tracer, the same group did not observe these changes in the midbrain even if they reported a larger variation in the volume of distribution of verapamil in PD patients compared to healthy controls [11C]-verapamil PET data of an extended study population showing enhanced [11C]-verapamil uptake in frontal white matter regions of advanced PD patients compared to controls, suggesting a regional downregulation of P-gp function [In terms of estimating the impact of PD on the expression and activity of P-gp in humans, direct measurements are rather limited . Therefo disease . Howevercontrols . Finallyfunction . From thFor the BBB, we have so far focused on neurodegenerative diseases and their impact on the regulation of drug transporter expression and function. However, these diseases are commonly linked to neuroinflammation, with respective changes in cytokine profiles and various regulatory pathways. Neuroinflammation, as an underlying mechanism of changes in transporters expression and function in the BBB, is also assumed for infectious diseases. One major disease which should be considered to be of high interest would be the Human Immunodeficiency Virus (HIV) infection. Here, the CNS is considered a sanctuary site, an anatomical compartment where HIV replication can take place in the presence of Highly Active Antiretroviral Therapy (HAART) due to poor drug exposure . The BBBFor P-gp, there are data showing higher amounts of this efflux transporter in patients with HIV-encephalitis  with sigThroughout pregnancy, the placenta undergoes morphological transformations along with gestational-dependent changes in transporter expression. Although there are discrepancies in earlier reports, recent proteomic analysis has shown a gestational decline in protein expression of BCRP and P-gp, decreasing nearly two-fold from the first trimester to term. On the other hand, levels of OAT4 and OCT3 were found to increase from the first or second trimester to term . VariousProgesterone and estradiol, steroid hormones which physiologically fluctuate during pregnancy, have been shown to increase placental expression of BCRP and P-gp, while maternal administration of glucocorticoids decrease levels of OATP2B1 and BCRP ,185,186.In addition to these physiological factors, several maternal diseases such as preeclampsia, gestational diabetes, and bacterial or viral infection have shown an overall dysregulation of drug transporters ,176,177 Many of these diseases or conditions are associated with elevated immune responses and induction of TNF-α, IL-1β, IL-6, and IL-17. Involvement of inflammation in regulatory control of placental transporters is highly plausible as treatment of primary cultures of placental explants or trophoblast with proinflammatory cytokines have been reported to alter expression of several transporters ,188. DruPreeclampsia is a highly prevalent worldwide disorder of pregnancy which is characterized by new-onset maternal hypertension and proteinuria along with other biochemical signs of kidney or organ damage. It remains as one of the most common causes of maternal and fetal morbidity . Its etiIn line with this, Dunk et al. (2018) reported a significant downregulation of P-gp in villous and extravillous trophoblast from preeclamptic patients and further conducted a series of siRNA knockdown and functional experiments in primary and continuous cultures of cytotrophoblasts and trophoblasts. The authors demonstrated a role of P-gp in trophoblast migration, invasion, and differentiation, suggesting that P-gp downregulation is involved in the pathogenesis of the disease, rather than a consequence .In the same way, placental expression of BCRP and its association with preeclampsia has been investigated. A downregulation of BCRP was seen in placenta obtained from preeclamptic women complicated with hemolysis-elevated liver enzymes and low platelets (HELLP) syndrome as compared to healthy or preeclamptic patients . A subseMDR1 (P-gp) in placenta obtained from insulin-managed GDM patients as compared to gestation-matched controls. Although significant changes in the expression of other ABC transporters such as BCRP were not seen between groups, a significant positive correlation between BCRP expression (mRNA and protein) and levels of hA1c  was observed in diabetic patients [One of the most common complications during pregnancy is gestational diabetes mellitus (GDM), which is characterized by a persistent hyperglycemia that develops during pregnancy and resolves after birth . Numeroupatients . Kozlowspatients . InteresIn addition to regulation of the drug transporters in placenta, there are data linking gestational diabetes to aberrant adenosine transport in placental vascular cells. Indeed, there are preclinical data indicating that nucleoside transporter expression is affected in this metabolic condition. In this context, different groups have evaluated the role of ENT1 and ENT2. In human umbilical vein endothelial cells (HUVECs) exposed to high levels of D-glucose, a reduced adenosine transport was linked to decreased mRNA expression of ENT1/2 . SimilarOther factors associated with metabolic disease, such as obesity, may potentially impact transporter regulation; however, there are relatively few studies in the literature. Wang et al.  recentlyIn the same way, the persistent increase of maternal blood cholesterol in pregnancy, a condition known as maternal supraphysiological hypercholesterolemia (MSPH), has been shown to impact placental ABC transporter expression. The research of Fuenzalida et al. revealed that this condition can cause an increase in the expression of cholesterol efflux regulatory protein or CERP (ABCA1) in placental tissue and primary human trophoblasts from women with MSPH, whereas the expression of ABCG1 (ABC8) was decreased . These rThe impact of HIV on expression of transporters has also been investigated in the context of pregnancy. Several preclinical and clinical studies have revealed that changes in placental transporters that have been reported in HIV can be related to the pathogenesis of the disease itself or can be induced by pharmacological treatment of the disease. Immune dysregulation is a hallmark of chronic HIV infection. Studies in HIV-1 transgenic (HIV-Tg) rats found that, while basal expression of transporters in placenta were similar to that of wild-types, exposure to low levels of endotoxin resulted in augmented inflammatory responses and amplified dysregulation of transporters in the HIV-Tg rats . In the Although the effect of hepatitis infection on transporter regulation during pregnancy has not been extensively explored, a few studies suggest that hepatitis may have an impact. The use of PCR array screening in pregnant woman with Hepatitis C Virus (HCV) has revealed the increased expression of P-gp and BCRP in comparison to uninfected/healthy controls, while the nucleoside transporters ENT1 and ENT2 remained unchanged. This was corroborated with qRT-PCR and Western blot experiments .Transport proteins play a critical role in the maintenance of homeostasis in different organs. While there is clear evidence that transporters play a pivotal role in controlling access and accumulation of substrates in brain and placenta, there is increasing preclinical and clinical evidence demonstrating disease-mediated regulation within these protective barriers. Maintaining the integrity and functionality of these membrane barriers are essential to the proper functioning of the brain and placenta. More importantly, a large number of drugs used for therapeutic management are substrates of these transporters; hence, modulation of their expression and function may have an impact on their distribution, efficacy, and toxicity.As we have discussed throughout the review, changes in expression for certain transporters can not only be considered a consequence of a pathological condition but may also be involved in the pathogenesis or even progression of certain diseases. This is especially true for Alzheimer’s Disease in the brain. In neurological diseases, P-gp is the most-studied BBB transporter. Since P-gp is assumed to be involved in the clearance of Aβ peptides, multiple studies suggest a role of this efflux transporter in the development of AD. Various molecular mechanisms and regulatory pathways are associated with downregulation of P-gp, such as NF-κB activation, P-gp ubiquitination, and suppression of the Wnt/β-catenin pathway. Nevertheless, whether these findings can link decreased expression and activity of P-gp to the pathogenesis of AD remains to be determined. Contrarily, P-gp levels are increased in epilepsy. One mechanism that might be involved includes glutamate-mediated activation of the NMDA receptor and COX-2 that together with TLR-4 lead to NF-κB activation, which plays a key role in the development of neuroinflammation. Likewise, neuroinflammation caused by HIV infection may alter BBB functionality and lead to P-gp upregulation. On the other hand, experimental data regarding P-gp are rather contrasting and inconclusive in PD, but highlight changes in vascular morphology, integrity, and permeability of the BBB.Finally, for most of the diseases mentioned in this review, there is a lack of clinical data concerning transporters’ regulation at the BBB, since data are mainly obtained from in vitro and animal studies. Although this information is relevant, clinical in vivo studies are needed to confirm these findings. In addition, reported data is often conflicting and inconclusive. Therefore, deeper investigations into transporter regulation, with a particular focus on human studies, is needed to optimize therapeutic dosing regimens in pathological conditions. For brain-targeted therapies, the poor penetration of drugs into the CNS is one of the biggest issues limiting the efficacy of current treatments, although efflux at the BBB is crucial to the safety profile for many other drugs. For example, ivermectin, loperamide, non-sedating antihistamines, and antimuscarinics would likely cause more CNS side effects and would have less applications without their efflux at the BBB. This pharmaco-resistance phenomenon involves either reduced uptake or increased efflux of drugs to and from the brain tissue; consequently, alterations in the levels of drugs transporters expressed at the BBB have a crucial implication in therapeutic efficacy or failure. A better understanding on the regulation of transporters at this localization will help to improve the current therapeutic interventions for neurological disorders.Likewise, the regulatory factors contributing to modulation of placental transporters in health and disease are fairly unknown. To date, P-gp and BCRP have been best characterized in placenta with regards to their dysregulation in the context of underlying maternal diseases and pregnancy complications. However, there are important data on the regulation of some SLCs such as OCT3, OAT4, and OATP2B1 which have potential implications in disease progression and therapeutic intervention. Nucleoside transporters such as ENT1/2 have also been studied because of their impact on the distribution and fetal exposure to antiviral agents. Although crucial information on the possible regulatory mechanisms of these transporters has been obtained through in vitro studies using choriocarcinoma cell lines, primary cultures of syncytiotrophoblasts, or human placental explants, the ultimate proof of functional significance requires in vivo studies. Animal models provide important data on the expression and regulation of drug transporters and their impact in drug distribution; however, due to interspecies differences in placental structure and transporter expression/function, models based on human placental tissues or in vivo human studies are essential to draw final conclusions applicable to clinical practice. A better understanding of the regulation of placental drug transporters in normal and pathological pregnancies will aid in predicting fetal drug exposure and in the development of more targeted medication for mothers.For both the BBB and BPB, there are still inconsistencies regarding the abundance as well as the cellular and subcellular localization for many transporters. There are also discrepancies between research groups and studies reporting expression or localization of drug transporters in these critical protective barriers. However, in order to properly investigate the net contribution of specific transporters at barriers possessing multiple transport systems, we need information on their abundance and polarized localization as well as function. Considering that most regulatory pathways modify numerous transporters at the same time, investigations would certainly profit from more specific substrates. Moreover, improvements are needed in the analytical or imaging methods to assess the functionality of these transporters in human tissues. If possible, these should be analyses via mathematical modeling of such in vitro data, with the aim to establish in vitro-to-in vivo extrapolation options for a set of compounds, since this will allow integration of such data into existing physiologically based pharmacokinetic/pharmacodynamic models. However, as multicell models are generally required, the complexity to translate such assays to the in vivo situation will be challenging. To date, more information has been generated from placental studies as compared to brain. This is mainly due to the limitations to obtain adequate brain tissue samples from healthy or disease populations. Apart from cultured cell models, most information on the regulation of transporters in human brain comes from postmortem studies or genetic screening of tumor biopsies. More recently, valuable information has also been generated through noninvasive imaging studies. Development of more specific, labeled substrates will improve current knowledge on transporter expression in the brain and will thus allow more in vivo insights into the functional regulation of these transporters.While we have identified many gaps in our knowledge, we are beginning to understand the underlying mechanistic pathways and connectivity that regulate transporter expression and activity in critical barriers such as in brain and placenta. Understanding transporter regulation in those barriers will be instrumental in gaining access to new medication for widespread diseases.


The universal dihydrouridine (D) epitranscriptomic mark results from a reduction of uridine by the Dus family of NADPH-dependent reductases and is typically found within the eponym D-loop of tRNAs. Despite its apparent simplicity, D is structurally unique, with the potential to deeply affect the RNA backbone and many, if not all, RNA-connected processes. The first landscape of its occupancy within the tRNAome was reported 20 years ago. Its potential biological significance was highlighted by observations ranging from a strong bias in its ecological distribution to the predictive nature of Dus enzymes overexpression for worse cancer patient outcomes. The exquisite specificity of the Dus enzymes revealed by a structure-function analyses and accumulating clues that the D distribution may expand beyond tRNAs recently led to the development of new high-resolution mapping methods, including Rho-seq that established the presence of D within mRNAs and led to the demonstration of its critical physiological relevance. The ribinhydrin ,32. The xtension . Both trreatment ,35. Thes3H-labelled D on a cellulose membrane after oxidation with an inorganic salt and reduction with borohydride .,95.−), w in vivo .T. maritima revealed an oxidoreductase enzyme with two domains; an N-terminal TIM barrel and a C-terminal helical domain [T. thermophilus DusA crystal highlighted the same general structure [Phe complex revealed that DusA interacts with the D-stem loop, the anticodon stem loop and the T-stem loop of tRNA and that the D-loop but not the D-stem is strongly distorted when DusA is bound. The third published bacterial Dus structure was DusC from E. coli that also displays a two-domain conformation with an N-terminal catalytic domain and a C-terminal RNA binding domain [T. thermophilus DusA and E. coli DusC led to the hypothesis that they share the same catalytic mechanism. Remarkably, notable structural dissimilarities were discovered by comparing the bacterial DusA (targeting tRNA-U20 and 20a) and DusC (targeting tRNA-U16). Both enzymes adopted the same general fold – while having different substrate specificities – but bound and recognized the tRNA in different orientations. The tRNA binding differed by a 160° rotation that resulted in the proper integration of the targeted uridine in the catalytic pocket. This trademark way of catalyzing a reaction is unique in RNA enzymology and is achieved through specific binding signatures. According to its target, each Dus enzyme has a cluster of amino acids – that is phylogenetically conserved in Bacteria – that defines the docking of tRNA to allow the reduction of a specific uridine [E. coli DusB adopted the same overall structure with an N-terminal TIM barrel fold carrying the catalytic function and a C-terminal helical domain. The tRNA docking in DusB was similar to the one of DusC, which makes sense since DusB and C modify neighboring nucleotides (17 and 16). However, a major difference between DusB and C relied on the positioning of the nucleobase into the catalytic center. Reversed polar and nonpolar amino acids in the catalytic pocket of DusB led to the 180 degrees rotation of the nucleobase that is targeted for reduction. In conclusion, diversification of bacterial Dus specificities was made possible through two astonishing strategies; nucleobase rotation or tRNA docking rotation  . The seql domain . Using al domain ,103.6.4 or OH− treatments result in an abasic site (nucleobase-free ribose) leading to the cleavage of the RNA chain with aniline treatment [7G. This is likely due to the incomplete formation of abasic sites at D position following limited alkaline hydrolysis.As indicated above, NaBHreatment ,35. Thisreatment ,105. Whi+ RNAs, raising the possibility of mRNAs hDUS3-specific modification in this specialized cell type [On a completely different basis, RNA-mediated activity-based protein profiling (RNABPP) relies on metabolic RNA labelling, mRNA interactome capture and quantitative proteomics. In this approach, a 5-halopyrimidine  is incorporated in RNA and form stable RNA-protein adducts with the modifying enzyme, allowing RNA interactome capture . These eell type .4 and then covalently bound by the rhodamine fluorophore (Rho). From there, the Rho-seq protocol unfolds as follows; (I) RNA extraction from WT and ∆4 strains (lacking the four dus genes), (II) Rho+ and mock-treatments, (III) ribodepletion, (IV) cDNA synthesis and library preparation, (V) strand-specific deep-sequencing, (VI) data analysis by implementation of a multifactorial analysis. The detailed protocol is available in a separate publication [We recently introduced Rho-seq to globally detect the presence of D residues on RNA . In a twlication .Rho-seq provided the first transcriptome-wide Dus1 to 4-specific repertoire of the distribution of D and highlighted that D is an integral component of yeast and human mRNAs, though at low level. These data also provided a framework for the previous findings that DUS1 and DUS3 cross-link to mRNAs in both yeast and human ,111.The absence of detected D-sites on the bacterial coding transcriptome suggests that mRNA dihydrouridylation may be a eukaryotic-specific mechanism although this requires additional support .in silico tools were developed to predict the presence of D on various RNA types. These computational predictions need however to consider a larger set of proven D-containing sequences to build more objective tools [A set of ve tools .E. coli tRNAs through the nanoscale pore [Finally, the detection of modified nucleotides with the Oxford Nanopore Technologies is still in its infancy but further developments will likely confirm the seminal detection of D on ale pore .7.dus3 in fission yeast specifically affects meiotic chromosome segregation and the translation of a set of proteins including tubulins while the mitotic cell cycle is barely affected [Similarly to other mRNA modifications, the detected D-sites could be relevant in a specific physiological context. Two previously reported cases are illustrated in the literature: induced pseudouridylation upon heat shock and widespread methylation during the meiotic programme ,118. We affected .in vitro coding properties of D-containing oligoribonucleotides. Despite the technical boundaries encountered at that time, they concluded that the presence of D resulted in the loss of coding ability. Rottman and Cerutti showed that a ribopolymer carrying 4.2% of D (and 95.8% of U) lost up to 60% of Phe(UUU) residues incorporation into the protein [Ribosome profiling and proteomics revealed that a dihydrouridylated mRNA can be translated but that the modification affects this process, which may result in modulation of the translational speed and ribosome stalling . In the  protein . Another protein . The con protein ,122. TheThe mRNAs encoding several subunits of the CCT (chaperonin-containing tailless complex polypeptide 1) complex, which is required for the folding of newly synthesized tubulin and actin proteins , containAs stated above, the alterations of the expression level of some Dus enzymes and the D landscape in cancers will be the topic of additional investigations to understand if translation is affected and to test if this is mediated by modified mRNAs and/or tRNAs.It took almost 40 years from the first detection of dihydrouridine within tRNAs to the discovery of Dus enzymes, and 20 more years to expand the D landscape to the coding RNA world. Recent advances open up several interesting research directions.E. coli is the 23S-D2449 deposition is independent of the dusA-B-C genes [non-canonical dihydrouridine synthase. In Bacteria, uracil can be reduced to dihydrouracil by the dihydropyrimidine dehydrogenase complex formed by PreA and PreT [H. sapiens) leads to a severe sensitivity to the administration of 5-fluorouracil, an agent used widely to treat cancer [A peculiarity of -C genes . To our and PreT . Noticeat cancer .The catalytic activity of the DPYD enzyme is reversible, suggesting the immediate possibility that Dus enzymes may also function backward as D eraser to restore uracil from D in some circumstances. A proof of concept supporting this possibility was already reported .A. thaliana where the DUS orthologs are expressed at low level in rosette leaves and apex tissues, unlike other RNA modifying enzyme genes [It will also be of interest to explore the D landscape in plants where it is predicted to be present on mRNAs as well . Interesme genes .3D) from D in T. brucei is another important perspective to unravel the biology of this complex modification [Deciphering the synthesis of a carboxypropylated dihydrouridine  to interact with D . InteresThe abundant, simple yet fascinating D modification is only about to reveal its importance.Click here for additional data file.


Despite the early promise of RNA therapeutics as a magic bullet to modulate aberrant signaling in cancer, this field remains a work-in-progress. Nevertheless, RNA therapeutics is now a reality for the treatment of viral diseases (COVID-19) and offers great promise for cancer. This review paper specifically investigates RNAi as a therapeutic option for HCC and discusses a range of RNAi technology including anti-sense oligonucleotides (ASOs), Aptamers, small interfering RNA (siRNA), ribozymes, riboswitches and CRISPR/Cas9 technology. The use of these RNAi based interventions is specifically outlined in three primary strategies, namely, repressing angiogenesis, the suppression of cell proliferation and the promotion of apoptosis. We also discuss some of the inherent chemical and delivery problems, as well as targeting issues and immunogenic reaction to RNAi interventions. Hepatocellular carcinoma (HCC) therapeutics remain an intractable global problem against a background of rising incidence and changing etiology . A wide This review paper specifically investigates RNAi as a therapeutic option for HCC. RNAi therapeutics involves the deployment of RNA molecules in sequence-specific modulation to repress gene specific expression . In thisSmall interfering RNA (siRNA) are double stranded RNA (dsRNA) that load a guide strand into some form of Argonaut (AGO) based RNA-induced silencing complex (RISC) after separation from its passenger strand see Figure 1in vivo but challenges remain due to off-target effects, as well as unintended immunogenic reaction . Alternatively, manufactured siRNA that are miRNA mimics can be utilized for RNAi. Manufactured siRNA-based therapeutics use siRNAs that have been chemically stabilized to increase their potency and reduce RNase degradation reaction . The stain vivo  are short single stranded (ss) DNA/RNA that exist in nature as small RNA molecules. RNA based ASOs bind to a complementary nucleotide sequence site in mRNA causing splicing, degradation (R Nase H) or the repression of mRNA translation , 12 and Aptamers are short, single-stranded DNA or RNA (ssDNA or ssRNA) molecules that can selectively bind to a specific target. Aptamers can fold into a multi-functional state to bind to a target DNA/RNA in a similar fashion to an antibody to suppress protein expression. Aptamers, often labeled as chemical antibodies, can bind to a wide range of molecules including toxins, viruses, proteins and peptides and its binding facility is not based on complementarity but rather its ability to fold into a 3-dimensional structure to enfold a target molecule in the same way as an antibody , 6. RNA Ribozymes are ribonucleic acid enzymes that play a catalytic silencing role to promote the cleaving of target mRNA by hybridizing with. This is a process where two ssRNA molecules combine into a single dsRNA molecule. There are three predominant forms of ribozyme, namely, hammerhead (HH), hepatitis delta virus (HDV) and hairpin (HP) ribozymes  but the O-Me, 2′-deoxy-2′-C-allyl uridine, and terminal inverted 3′-3′ deoxyabasic nucleotides (Ribozymes can be directed to target the 5’ end of RNA to induce RNaseP and cleavage in a sequence specific manner. Liposome mediated ribozymes can be delivered exogenously or transcribed endogenously to cleave target mRNA and they can be used repeatedly because they are not consumed in the cleavage process. Ribozyme therapy can be used to target most non-coding RNA (ncRNA) including siRNA, microRNA (miRNA), long noncoding (lncRNA), piwi-interacting RNA (piRNA) and small nucleolar RNA (snoRNA) that all play a regulatory role in carcinogenesis , 18. Traleotides .Riboswitches are a group of dynamic ncRNA motifs found upstream, in the 5′ untranslated region of mRNAs, where they regulate translation through binding of their cognate ligand to their aptamer domain . A ribosin vivo due to speedy degradation via nuclease activity  molecules. Modifications to the RNA backbone can also increase nuclease resistance and involve substituting non bridging phosphate oxygen for sulfur, as well as boranophosphate substituted with the BH3 group . These mA wide variety of nanoparticles (NPs) have been developed and tested over 40 years. Delivery of RNAi based drugs can utilize lipid-based (cholesterol and α-tocopherol) NPs, polymer-based (polyethylene-PEG) NPs, inorganic NPs and a range of exosome-mimetic nanovesicles , 28.Lipid-based NPs (LNPs) include liposomes, micelles, emulsions and solid lipid nanoparticles (SLNPs). LNPs also include stable nucleic acid lipid particles (SNALPs) , lipidoiin vivo, as well as enhances the targeting of its RNA based cargo (Polymer-based NPs like Polyethylene (PEG) can be complexed with Polyvinylimine (PEI) to form a PEG-PEI based RNA conjugate that increases stability ed cargo . PEG caned cargo . Upon deed cargo .in vivo and also specific to a chosen target (NPs can use a ligand to target delivery where these conjugates promote improved binding and reduced immunogenicity. Upon cell entry the NPs can be separated (cleaved) to facilitate RNA loading into an AGO based RISC. For example, a Ga1NAc-siRNA conjugate readily binds to ASGPR thus providing siRNA entry access to hepatocytes , 33. Antn target . The deln target  includinn target .in vitro and in vivo, including high efficacy by natural cell transduction, sustained gene expression, and long-term gene silencing. The use of an adenovirus vector encoding a siRNA, for example, can deliver RNA  suppressor networks, the Wingless-related integration site/beta-catenin (WNT/β-Catenin) pathway, and the phosphoinositide 3-kinase/mitogen-activated protein kinase (PI3K/MAPK) and Janus kinase/signal transducer (JAK/STAT) pathways and transforming growth factor-beta (TGF-β) signaling . Typicalreceptor . In addireceptor .Three primary strategies are interrogated in this section, namely, anti-angiogenesis, anti-cell proliferation and pro-apoptosis. In each therapeutic strategy, aberrantly expressed mRNA in HCC pathogenesis have been identified and targeted by RNAi therapy see Figure 2HCC pathogenesis including hypervascularity and marked vascular abnormalities are common feature of disease progression in HCC . VasculaOther growth factors driving angiogenesis are hepatocyte growth factor (HGF) and its receptor c-Met, angiopoietins (ANG1/2 and Tie2) and endoglin (CD105) , 120–123The development of RNAi drugs to repress angiogenesis in HCC has been promoted by the moderate efficacy of the Sorafenib family of drugs. RNAi therapeutics to repress angiogenesis include the deployment of siRNA, aptamers, riboswitches, ribozymes, ASOs and CRISPR/Cas9 See Figure 2Small interfering RNA: This branch of therapeutics has largely focused on siRNA repression of the drivers of angiogenesis in HCC including VEGF, FGF, PDGF, HGF, angiopoietins (ANG1/2), endoglin (CD105) and their respective receptors. A preponderance of siRNA therapeutic models targeting angiogenesis in HCC have targeted vascular endothelial growth factor (VEGF) expression , Notch, JAK/STAT and Hippo pathways  also plays a role in the promotion of proliferation (The Hedgehog (Hh) pathway also plays an important role in cell proliferation and invasion , as wellferation . Finallyferation  by a ranferation .siRNA: RNAi based therapeutics to modulate cell proliferation in HCC pathogenesis has deployed a range of polymer and lipid-based NPs, Stable nucleic acid lipo-particles (SNALPs), and chemically stabilized siRNA  was disrupted to reduce cell proliferation and highlight the key role of methylation of DNA in HCC  expressing HCC cell-line via the suppression of oncogene expression in c-jun and c-fos. This aptamer can also targeted nucleolin  model targeting PKM2, induced apoptosis and growth arrest by downregulating PKM2 in HCC  in vivo . In anotyclin D1 . SNALP byclin D1 , 101 whiyclin D1 . Anotherpoptosis . In anotferation . Cyclin feration . Another in vivo . CXCL1 e in vivo . In anot in vivo .A multifunctional polymeric NP, that has progressed to clinical trial stage, has been developed to transport shRNA survivin and VEGF siRNA to trigger apoptosis and inhibit angiogenesis in HCC patients . This woCRISPR/Cas9: In one study using a mouse xenograft model, a CRISPR/Cas9 model used Cas9-based genome editing to introduce the prodrug-converting enzyme herpes simplex virus type 1 thymidine kinase (HSV1-tk) into the genomes of HCC cells resulting in the precipitation of apoptosis  was tested in three HCC cell lines, HepG2, HuH7 and JHH6. When delivered by liposomes. GT75 was able to effectively reduce HCC cell viability and promote apoptosis. No effects on cell cycle were observed and the effect of this aptamer was likely due to the interference with eEF1A activity  because of its role in promoting cell survival  drug in 1998, first aptamer drug in 2004, the first RNAi clinical trial in 2010 and the first siRNA drug in 2018. These RNAi drugs were based on the synthetic development of complementary RNA sequences that can bind to mRNA or a protein to repress (silence) expression. Six FDA approved RNA based drugs have been approved from 1998 to 2019 with the majority developed for muscular dystrophy/atrophy .To date, a few clinical trials have been registered for siRNA RNAi based therapy in HCC. These trials include an LNP to target MYC mRNA to repress proliferation, a SNALP to target PLK1 mRNA  to retarin vivo, as well as delivery and host immunogenic issues. RNAi targeting is often problematic because of the cross-talk between genes and some RNAi studies have demonstrated that repressing one oncoprotein can result in stimulating another (Despite the early promise of RNA therapeutics as a magic bullet to modulate aberrant signaling in cancer, this field remains a work-in-progress largely due to the inherent instability of RNA  another . Neverth another . A wide  another .KS conceptualized, developed first draft and responded to reviewer edits and comments to develop final draft. SA reviewed and revised drafts, AC reviewed and revised drafts, AK reviewed and revised drafts. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.