Drosophila melanogaster to systematically study the LD (r2) between editing sites and infer its impact on the adaptive signals of RNA editing. Pairs of editing sites (PESs) were identified from the transcriptome. For CDS PESs of two consecutive editing sites, their occurrence was significantly biased to type-3 PES (Syn-Nonsyn). The haplotype frequency of type-3 PES exhibited a significantly higher abundance of AG than GA, indicating that the rear Nonsyn site is the driver that promotes the editing of the front Syn site (passenger). The exclusion of passenger Syn sites dramatically amplifies the adaptive signal of Nonsyn RNA editing. Our study for the first time quantitatively demonstrates that the linkage between RNA editing events comes from hitchhiking effects and leads to the underestimation of adaptive signals for Nonsyn editing. Our work provides novel insights for studying the evolutionary significance of RNA editing events.Adenosine-to-inosine (A-to-I) RNA editing leads to a similar effect to A-to-G mutations. RNA editing provides a temporo-spatial flexibility for organisms. Nonsynonymous (Nonsyn) RNA editing in insects is over-represented compared with synonymous (Syn) editing, suggesting adaptive signals of positive selection on Nonsyn editing during evolution. We utilized the brain RNA editome of  AG motif, where H denotes non-G nucleotides, is prone to being targeted by ADAR , s, sADAR, Drosophila still needs to be consolidated by new evidence and data.However, the discovery of this adaptive signal requires (1) meticulous identification of RNA editing events to exclude the wide-spread synonymous SNPs (single nucleotide polymorphisms) in the genome, and (2) a strict comparison between the RNA editing sites and the unedited adenosines in the same set of genes. Any technical or methodological biases would introduce false-positive synonymous editing sites that reduce the observed Nonsyn/Syn ratio for RNA editing and dampen the adaptive signals. The confidence in the conclusion of adaptive RNA editing in AAC (1st codon position) will be annotated as Asn > Asp (AAC > GAC), while the A-to-G mutation in AAC (2nd codon position) will be annotated as Asn>Ser (AAC > AGC)  records each variation site. Each line of VCF contains the functional annotation of one variant. For example, an A-to-G mutation in C > AGC) D. This aC > AGC) D. The inr2) and p values of each pair of editing sites (PES). The r2 ranges from 0 to 1 and a higher r2 represents a stronger LD. We indeed found wide-spread linkage between RNA editing events and proposed that the annotation of editing sites should be updated. However, we did not make further implications on how the linkage between editing events will impact our understanding on the adaptive signals of RNA editing sites.To fill the gap between independent annotation and the potential interaction between RNA editing sites, we previously developed an algorithm to measure the linkage between RNA editing sites . BrieflyD. melanogaster to systematically study the LD (r2) between editing sites and infer its impact on the adaptive signals of RNA editing. Totally, 1518 PESs were identified. CDS PES had the strongest LD, suggesting potential epistasis between CDS editing sites. For CDS PESs of two consecutive editing sites, including Nonsyn-Nonsyn (type-1), Nonsyn-Syn (type-2), and Syn-Nonsyn (type-3), their occurrence was significantly biased to type-3. The haplotype frequency of type-3 PES exhibited a significantly higher abundance of AG than GA, indicating that the editing of the rear Nonsyn site drives the editing of the front Syn site. Therefore, the Nonsyn sites in type-3 PES act as drivers and the paired Syn sites are passengers. The exclusion of these passenger Syn sites dramatically amplifies the adaptive signal of RNA editing by increasing the observed Nonsyn/Syn ratio for editing sites. Our study for the first time quantitatively demonstrates that the linkage between RNA editing events comes from hitchhiking effects and leads to the underestimation of adaptive signals for Nonsyn editing. Our work provides novel insights for studying the evolutionary significance of RNA editing events.In this work, we utilized the brain RNA editome of D. melanogaster (Dipteran: Drosophilidae) from FlyBase . The transcriptome (RNA-Seq) data of Drosophila brains were downloaded from NCBI  under accession SRP074828. The 2114 brain RNA editing sites were downloaded via link , and their linkage information was downloaded via link . The phyloP score across the D. melanogaster genome were downloaded from the UCSC genome browser .We retrieved the genome of r2 and p values of each pair of RNA editing sites (PES). Due to the limitation of sequencing coverage and read length, only a limited number of RNA editing sites could be covered by the same reads. Not all editing sites in the same gene have a pair-wise LD with each other.We followed the pipeline of our previous study , which whttps://github.com/lindenb/jvarkit.git, accessed on 2 June 2023). Consequently, a total of 1518 PESs were identified from the sequencing reads. Notably, each PES should cover at least one editing event. For example, if two nearby editing sites both have 20% editing level, but the “edited reads” at one site did not cover another site (and the reads covering both sites are unedited at both positions), then this pair of editing sites could not be counted as a PES in our results. We only consider the PESs for which we could find a read that contains at least one editing event at one of the two positions. For these PESs, we would count the numbers of the four haplotypes AA, AG, GA, and GG, and the corresponding haplotype frequencies were AAf, AGf, GAf, and GGf. The calculation of r2 is based on these haplotype frequencies. The 1518 PESs had AAf < 1. A PES with AAf = 1 suggests no editing events were detected at both positions and was not considered as we explained above (but the editing event might be included in other reads that did not cover both positions).We mapped the RNA-Seq reads to the reference genome using STAR v2.4.2a  with defThe calculation of LD parameters, statistical tests, and graphical works were conducted in Rstudio version 3.6.3.r2 between two RNA editing sites that could be covered by the same sequencing reads. With 135.6 M mapped reads from brains of D. melanogaster, a total of 1518 pairs of editing sites (PESs) were identified, including 331 PESs in CDS, 412 PESs in UTR, 521 PESs in intron, and 254 PESs in other regions (r2) of CDS PESs was remarkably higher than the r2 of PESs in UTR and intron (r2) decreases with distance . For PESs in CDS, the distance between two editing sites, together with the frame of the first site, will determine whether the two sites are annotated as type-1 (Nonsyn-Nonsyn), type-2 (Nonsyn-Syn), type-3 (Syn-Nonsyn), or type-4 (Syn-Syn) , an essential difference between CDS PESs and non-coding PESs is the existence of tri-nucleotide periodicity (reading-frame) in CDS. For PESs in non-coding regions, the distance between two sites has no direct effect on the functional consequence of the two editing sites. However, in CDS, A-to-I(G) RNA editing at the 1st and 2nd codon positions will leads to nonsynonymous (Nonsyn) mutations and the editing at the 3rd codon position leads to synonymous (Syn) mutations, except for one case (ATSyn-Syn) A. First,Syn-Syn) A.We found that for most groups of PESs, the numbers of type-1 PES (Nonsyn-Nonsyn) were much higher than the numbers of other types of PES  B. This sD. melanogaster genome. Using the genome-wide data as a control, we found that the expected numbers and fractions of types 1, 2, and 3 were 754,666 (50.1%), 412,912 (27.4%), and 338,895 (22.5%) . The over-represented type-3 (Syn-Nonsyn) PESs in group1 might reflect a non-random process between two editing sites. Next, we investigated the group1 PESs and studied the possible interactions between two neighboring editing sites.To test how unexpected it is to see such a high proportion of type-3 PESs among group1, we needed to find a negative control. We calculated the numbers of two consecutive (unedited) adenosines in the CDS regions of the  (22.5%) C, respecr2) of those PESs  had an extraordinarily high fraction of type-3 PESs (Syn-Nonsyn), we set out to explain this unique phenomenon. We first looked at the strength of LD , we could clearly see that the editing levels in type-1 and type-2 PESs were much lower than the editing levels in type-3 PESs because the GG haplotype had a high frequency only in type-3 PESs  Whe-3 PESs B,C. For e-3 PESs B. This sInterestingly, according to the inferred editing process of type-3 PESs C, the reDrosophila brain editome recorded 678 Nonsyn editing sites and 144 Syn editing sites. The Nonsyn/Syn ratio is 4.71 (p = 0.067 by Fisher’s exact test); although, the expected Nonsyn/Syn could be slightly reduced when only the edited genes were considered. Here, if the 26 passenger Syn editing sites in type-3 PESs were removed, the observed Nonsyn/Syn ratio will be 678/118 = 5.75, which is significantly higher than the random expectation of 3.97 (p = 1.40 × 10−4), regardless of whether the edited genes or all genes were used. Therefore, studying the linkage between CDS editing sites would help us to clarify which editing sites are the main target of natural selection and which sites are just byproducts. This information would also deepen our understanding of the adaptive nature of the RNA editing mechanism in Drosophila as well as other insects and metazoans.The  is 4.71 A, which Drosophila editome come from hitchhiking, which means that they are byproducts of Nonsyn editing events, we wondered whether we could find more evidence and data to enlarge the adaptive signals of RNA editing events. Auxiliary evidence for adaptive editing is that the Nonsyn editing sites are usually  more conserved than the Syn editing sites [D. melanogaster, the conservation level of each genomic site could be quantitatively measured using the phyloP score . A higher phyloP score represents a higher conservation level of a site across the phylogeny. We found that the 26 Syn editing sites in type-3 PESs had significantly higher conservation levels than the remaining Syn editing sites  that showed strong linkage and a biased profile of haplotype frequency. We argue that the synonymous editing site within this PES is the passenger produced by the editing of adjacent nonsynonymous site. Then, these passenger synonymous sites should be excluded in the evolutionary analyses involving Nonsyn/Syn ratios. This is a key conclusion based on our observations.By systematic identification of the linked RNA editing sites in the The measurement of linkage between RNA editing sites is a novel field with technical challenges. The idea came from the LD measurement in population genetics ,38. Withr2) between RNA editing sites decreases with distance, which is similar to what should be observed for DNA mutations, the mechanisms are largely different. The linkages between DNA mutations are eroded by the recombination of chromatids: the farther apart two mutations are located, the higher probability they will be separated by recombination. For RNA editing sites, the linkage between two closely related sites is simply caused by the “batch production” of ADAR proteins: the farther apart the two editing sites are located, the less likely they will be edited by ADARs at the same time. This is the essential difference between RNA editing and DNA mutation.Notably, in our analyses, although we observed that the LD . The exclusion of passenger Syn sites dramatically amplifies the adaptive signal of Nonsyn RNA editing (by elevating the Nonsyn/Syn ratio). The linkage information should be considered when studying the functional consequence and evolutionary significance of RNA editing sites.Our study for the first time quantitatively demonstrates that the linkage between RNA editing events comes from hitchhiking effects and leads to the underestimation of adaptive signals for Nonsyn editing. Our work provides novel insights for studying the evolutionary significance of RNA editing events.


Despite the popularity of modern medicine, medicinal plants remain a cornerstone of treatment for numerous diseases, particularly among ethnic groups and tribal communities around the globe. Ethnomedicine offers advantages such as ease of use, convenience, and economic benefits. Medicinal plant knowledge within Bulang ethnic community of southwest China is a valuable complement to Chinese ethnomedicine systems. Accumulated medical knowledge is due to the extensive length of occupation by Bulang People, considered the earliest inhabitants of Xishuangbanna; this has resulted in the development of various traditional treatment methods with local characteristics and unique curative effects. Therefore, there is exceeding value in exploring the medical knowledge of Bulang.A total of 175 local informants participated in the interviews and distribution of questionnaires in 10 Bulang villages in Menghai County, Xishuangbanna Prefecture, Yunnan Province, China. We documented the community of Bulang's use of medicinal herbs, and we used both the informant consensus factor (ICF) and use value (UV) methodologies to analyze the data. Furthermore, we conducted a comparative study to explore the potential of Bulang traditional medicine by comparing it to traditional Dai medicine.Camellia sinensis (0.94) and Zingiber officinale (0.89) showed the highest UV values, while the function of Phyllanthus emblica L. and Houttuynia cordata Thunb. were also noted. The ICF revealed digestive system related diseases were the most commonly treated, with conditions of the motor system using the highest number of plant species. Finally, a comparison with traditional Dai medicine determined that 22 plants (36.67%) of the 60 surveyed had higher medicinal value in Bulang medicine.The study recorded 60 medicinal plant species belonging to 41 families and 59 genera, including 22 species of herb, 22 species of shrub, nine species of trees, and seven species of liana. Araceae, Compositae, Lamiaceae and Leguminosae were found to have the highest number of species. The affordability and cultural heritage of Bulang medicine make it advantageous, Investigated Informants report that increased usage of Western medicine (88%), less availability of herbal medicine (95.43%), and the reduction in medicinal plant resources (80.57%) pose significant threats to Bulang medicine. All Bulang medicinal plants are naturally grown, with only 22 per cent being cultivated. Bulang communities primarily source medicinal plants from the wild. Should environmental damage lead to the extinction of these medicinal plants, it could result in a shift toward modern Western medicine as a preferred medical treatment. Bulang ethnomedicine is a vital supplement to China's traditional medicine, particularly aspects of ethnic medicine relevant to daily life. Future research should emphasize inter-ethnic medical studies to reveal the untapped potential of medicinal plants. International traditional medicine comprises various forms, such as Indian, Arabic, and Chinese; connecting different genres is essential to transmitting medicinal civilization and maintaining social relationships. Ayurveda, the predominant school of traditional Indian medicine, boasts a lengthy history in the Ganges Valley and has notably influenced South Asian traditional medicine. Traditional medicine in South Asia represents a traditional medicine system with Indian medicine serving as its core. During the Arabian era between the seventh and fourteenth centuries AD considered a romantic period in the history of Western medicine, the culture of therapy underwent significant development, playing a pivotal role in fostering later medicinal advancements. Traditional Chinese Medicine (TCM) maintains an inseparable connection with Arabic and Indian medicine, with these three primary therapeutic systems playing an integral role in transmitting human medicinal civilization and sustaining social relations , 2. And In contrast with Traditional Dai Medicine (TDM), TDM is an ancient ethnic medicine system in China that has more than 2500 years of experience and has been collecting and organizing for years , 9. Its Some scholars have made statistics on the development status and drug resources of Bulang Traditional medicine, but there is little relevant literature. Li et al. applied and analyzed the medicinal plant resources of six major ethnic minorities living in Xishuangbanna and recorded 49 kinds of Bulang medicinal plants, and 95 In summary, despite a few investigations into Bulang medicinal knowledge, the detailed records of Bulang medicinal plants and the comparative research with TDM still need to be included. The paper aims to record the therapeutic differences between the Bulang medicinal plants and compare the medicinal properties of the Dai and Bulang.(Camellia sinensis (Linn.) var. assamica (Masters) Kitamura) in the Yunnan province of China, has become increasingly popular in Southeast Asia may be due to its multiple health benefits. The unique sensory characteristics of Pu’er tea arise from the multitudinous chemical changes and transformations of the chemical constituents of the sun-dried green tea leaves that occur during the post-fermentation process[Bulang people primarily inhabit complex terrain characterized by high mountains and deep valleys, situated on both sides of the middle and lower reaches of the Lancang and Nujiang rivers, mainly south of 25°N latitude, ranging between 1500 and 2300 m above sea level during the post-fermentation process. Bulang people are primarily engaged in mountain agriculture, in sharp contrast to Dai people living in the flatland area  to examine variability in data obtained from ethnobotanical field surveys. The ICF is defined as the degree of variation in the number of medicinal plant species utilized by doctors in treating a particular type of disease. formants , 21.Use Value (UV) is a valuable tool for assessing the significance of local species. It reflects the frequency of use for a given species among the informants, with Ui representing the number of uses reported by each informant and Ut being the total number of informants:  culture , this ap culture , 24.The study comprised 175 informants  the declining number of Bulang herbalists (95.43%); (2) the limited range of diseases that Bulang medicine can treat compared to Western medicine (88%); (3) the declining availability of medicinal plants due to environmental degradation (80.57%); (4) the unpalatable taste and difficulty in swallowing Bulang herbal medicine in contrast to Western medicine tablets (76.57%).A total of 60 species belonging to 41 families and 59 genera of medicinal plants are identified, for which scientific name, Chinese name, Bulang name, family, habit, use value, habitat, parts used, medicinal use, and disease treatment for each species are all provided Tables , Fig. 8.Phyllanthus emblica L. and Houttuynia cordata Thunb. were both found to be 0.97, indicating their significance in local practice. Additionally, Camellia sinensis var. assamica (J. W. Masters) Kitamura (0.94) and Zingiber officinale Roscoe (0.89) were also among the plants with high UV values. In contrast, Tetrastigma hemsleyanum Diels et Gilg (0.09), Trachycarpus fortunei (Hook.) H. Wendl. (0.04), and Saurauia napaulensis DC. (0.03) had the lowest recognition for their medicinal properties.We utilized the use report (UR) to calculate the use value (UV) of the reported medicinal plants, providing a means to assess their relative importance in the study area and shed light on the preferred medicinal plants used by Bulang people Table . The UV Table Six primary methods are used in the preparation of traditional medicinal plants by Bulang people. The Decoction is The most common method, accounting for 80% of all preparations. This method involves medicinal components in fresh, sugar or alcohol. Following decoction, the most prevalent methods include pounding (16.67%) and infusing (15%), typically involving steeping in water or alcohol. The remaining methods include powdering (6.67%), cooking (6.67%), and chewing (1.67%). Oral application was the most commonly utilized , followed by external application  and medicinal washing .We systematically categorized distinct symptoms based on human body systems disease systems, identified ten unique groupings of symptoms and subsequently determined the number of illnesses that fell within each classification Table . The typAs per the results of the informants' interview, the majority of individuals familiar with Bulang traditional medicine fell between the ages of 30 and 60 (83.43%), with this age group demonstrating a higher level of definitive knowledge regarding medicinal plants than other age cohorts –28. InteIn addition, the survey results revealed that a significant proportion (88%) of participants perceived modern medicine as more efficacious in treating diseases than traditional medicine. As China's education rate has increased in recent decades, individuals may increasingly value modern medicine's scientific underpinnings and express concerns regarding the potential adverse side effects of herbal medicine. Moreover, 80.57% of interviewees expressed the availability of medicinal plants is declining. While not all Bulang people may be practicing herbalists, they are generally knowledgeable about the flora of their surroundings since they need to differentiate between edible and poisonous plants. Unfortunately, the number of medicinal plants used by Bulang people is limited, and there is no active cultivation of these plants. The rapid changes in the environment and habitat destruction make it increasingly difficult for herbalists to find medicinal plants in the wild, which could lead to the discontinuation of their use or a reduction in their efficacy, ultimately causing patients to turn to Western medicine. This lack of sustained access to medicinal plants represents a significant challenge to the continuity of Bulang medicine.Socioeconomic changes could result in losing or reducing medicinal plants and related indigenous knowledge . ResearcMore than 80% of the survey participants emphasized the cultural significance of Bulang medicine, viewing it as a crucial aspect of Bulang ethnic identity. The development and evolution of traditional medicinal knowledge among ethnic minorities have been significantly shaped by the interplay of cultural, historical, environmental, and belief systems. These communities highly value traditional medicine knowledge, considering it a significant cultural heritage with deep cultural roots . In ethnPhyllanthus emblica and Houttuynia cordata, ascertaining their significant ethnobotanical value (0.97). Phyllanthus emblica belongs to the Phyllanthus genus of the Phyllanthaceae family and is extensively distributed across subtropical and tropical regions in countries such as China, India, Indonesia, and Malaysia. Its fruits are known to have high concentrations of vitamin C and superoxide dismutase, exhibiting hepatoprotective, antibacterial, anticancer, and anti-inflammatory properties [Phyllanthus emblica has been documented in traditional Bulang and Dai medicine for treating various ailments. Bulang medicine employs Phyllanthus emblica to treat liver and gallbladder diseases, pharyngitis, abdominal distension, abdominal pain, cough, scurvy, heat clearing and detoxification, liver and gallbladder disorders, pharyngitis, abdominal distension, abdominal pain, cough, scurvy, stopping itching, sores, fever, cough etc.Upon analyzing the dataset for Use Value, the two botanical specimens with the greatest reported usage were operties –13. PhylHouttuynia cordata is a widely distributed and highly esteemed edible plant in southwestern China, highly regarded and consumed by Dai, Bulang, Lahu, Hani, Yao, and Dong ethnic groups [Houttuynia cordata into modern food systems may significantly improve human health and well-being. Saurauia napaulensis, with the lowest Use value, is primarily distributed in southeastern and southwestern Yunnan, southwestern and northwestern Guang Xi, Gui Zhou, as well as in India, Nepal, Myanmar, Laos, Thailand, Vietnam, and Malaysia. It thrives in mountainous areas, sparse forests, and thickets situated at an altitude range of approximately 500-1500 m. Despite its extensive distribution, there needs to be more research on this plant, domestically or internationally, with only a few studies examining its chemical composition [c groups , 48. Usic groups . Therefoposition . It is uCamellia sinensis var. Assamica scores a high Use value of 0.94. Herbal beverages are consumed for recreational or therapeutic purposes [Camellia sinensis var. assamica, endemic to Yunnan, China. The characteristic brown hue of the tea leaves is a result of microbial fermentation by Aspergillus niger during processing, in conjunction with the action of leaf oxidase [With 165 individuals reporting its medicinal value, purposes –55. Tea purposes . Pu'er t oxidase . Researc oxidase –61. Nota oxidase .Bulang people use Pu'er tea as both food and medicine. Ubiquitous are Paste Rice Tea and Ming Zi Tea. Paste Rice Tea is prepared by baking glutinous rice in an earthen teapot until it turns yellow and then adding tea leaves, boiled water, sliced ginger, and brown sugar. It is believed to have therapeutic properties that help alleviate colds, coughs, sore throats, heat, dry lungs, and other ailments. In addition, Ming Zi tea is made similarly to paste rice tea but with pine resin, a sticky substance secreted by pine trees, a combination of natural oils from pine and wood fibers. The different parts of the pine and cypress trees have varying oil content, with the roots containing the highest levels and the higher branches having lower levels. This tea is believed to help alleviate gastrointestinal discomfort, constipation, and other related conditions. Another unique tea consumption method is Sour Tea, which involves fermented tea leaves. Rather than being brewed with boiling water, Sour Tea is chewed directly, allowing its flavor and aroma to fill the mouth while promoting digestion, quenching thirst, and generating fluids.Before comparing the two ethnomedicines, understanding the difference in living altitude created a boundary between the two groups is essential. Dai, who inhabit the plains, historically referred to Bulang people living in the mountains as Man or Ka, meaning 'mountain-dweller' and 'slave', respectively. Xishuangbanna has traditionally been more economically advantageous for the Dai than the Bulang. This advantage was based on the pattern of Dai occupying the more agriculturally accessible lowlands. Nevertheless, Bulang people practiced subsistence cultivation in mountainous areas, trading tea and other substances in local periodic markets. However, inter-ethnic relations have undergone significant changes with the establishment of modern market systems and the focus on market economics. In particular, the combined efforts of foreign capital and the local resources of Pu'er tea have led to a change in the relationship between the Bulang and Dai , 64.Current ethnic medicine narratives emphasize TDM's formal acknowledgement as one of China's four traditional medicines, but Bulang medicine has yet to be thoroughly investigated and structured. When Theravada Buddhism first appeared in Xishuangbanna in 1437, the Dai written language was primarily intended to preserve and transmit Buddhist teachings. Bulang people, who lacked written language, occasionally adopted the Dai script through their conversion to Buddhism. Consequently, research on Bulang medicine is still in its nascent stage due to the absence of written records; with scarce ancient literature dedicated to the subject matter and few references to Bulang medicine in other historical texts, oral transmission remains the primary mode of preserving and transmitting the existing traditional medicine knowledge among the Bulang ethnic group –67.Psidium guajava, for example, is commonly employed in TDM for heat clearing, detoxification, and skin conditions. In contrast, Bulang medicine primarily treats gastrointestinal ailments like enteritis, dysentery, and hemostasis. This highlights medicinal plants' unique approaches and applications in the two ethnic therapies. This plant finds applications for treating diarrhea, dysentery, diabetes, cardiovascular disease, cancer, parasitic infections, gastroenteritis, hypertension, diabetes, caries, pain relief and improvement in locomotor coordination. Previous research indicates that Psidium guajava is commonly used to produce essential oils with antibacterial, anti-inflammatory, mosquito-repellent, and wound-healing properties[After analyzing the research data, we find 31 medicinal plants possess a greater therapeutic spectrum in TDM compared to Bulang, seven plants exhibit an equivalent therapeutic range in both ethnic groups Table , while troperties–70. ThesEntada phaseoloides is a plant commonly used in traditional Bulang and Dai medicine to treat soreness, fever, and amygdalitis. Recent studies have revealed its use in Chinese Yao ethnic medicine to treat rheumatism, as a nutritional supplement, and to promote blood circulation [Callerya cinerea and Argyreia synesis var. cinerea, both included in Bulang medicine, are purported to have therapeutic effects on gynecological ailments. However, this curative property needs to be mentioned in TDM or widely acknowledged in current research on these plants in China and abroad. Eclipta prostrata is recognized for its medicinal value in treating abdominal pain in Bulang medicine. However, in TDM, this plant is also used to treat detumescence, analgesia, dysentery, soreness, and irregular menstruation, as well as for liver protection, immunity regulation, and detoxification. These therapeutic effects have been verified through relevant studies [In addition, there are several other plants worth discussing. culation . Another studies .Tetrastigma hemsleyanum is only known in TDM for its ability to treat detumescence, while Bulang medicine recognizes its potential to promote hemostasis, stimulate blood circulation, and alleviate swelling. Further research demonstrates that Tetrastigma hemsleyanum, particularly its root tuber and whole herb, possesses additional pharmacological activities such as heat clearing and detoxification, blood circulation activation, pain relief, wind and phlegm dispelling, and efficacy against conditions like poisonous snakebites, whooping cough, bronchitis, pneumonia, pharyngitis, hepatitis, pediatric hyperthermia, and tumors [Conversely, d tumors –75. CompThe study investigated the ethnobotanical knowledge of medicinal plants among Bulang people, evaluating the current status of research and utilization of their medicinal knowledge. A total of 60 species, 41 families and 59 genera of medicinal plants were utilized by Bulang people. Environmental changes are increasingly leading to the extinction of medicinal plants, which could contribute to people preferring modern Western medicine over traditional medicine. As the disappearance of these plants has the potential to reduce the availability of medicinal materials and limit the development of treatments, it also risks hindering the progress of scientific and medical research. It is, therefore, crucial to preserve these plants and their use by fostering sustainable harvesting practices, protecting habitats, and supporting research on their potential benefits. Notably, all medicinal plants used were mainly distributed in the wild, with the root being the most used part and the primary preparation method being decoction. Results of the study revealed that 41 diseases were treated with medicinal plants, with illnesses related to the digestive system being the most common. The most used plant species were those related to the motor system category.A comparison between Bulang and Dai medicine revealed that 22 (36.67%) of the 60 plants investigated had more curative potential in Bulang medicine than Dai medicine. To further investigate the significant significance of medicinal plants, it is imperative to prioritize collaborative research efforts focused on the interplay between traditional ethnic remedies. The study also highlighted that the most significant medicinal values were in ethnomedicine closest to daily life, such as the therapeutic values of tea, ginger, and other staples. However, the medicinal values of some plants are gradually declining with environmental changes, and there is a growing concern that they may be forgotten or replaced by increasingly convenient western medicines. The decrease in the number of Bulang traditional herbalists was identified as the most significant threat to the development of Bulang medicine. In conclusion, the study provides essential insights into the rich ethnobotanical knowledge of Bulang people, highlighting the potential for further research to explore their medicinal plants' therapeutic values and safeguard their traditional medicinal knowledge.


Saccharomyces cerevisiae. It also focuses on how TOR regulates transcription depending on external cues. Finally, it discusses the simultaneous coordination of the three RNA pols through common factors regulated by TOR and summarizes the most important similarities and differences between S. cerevisiae and mammals.Ribosomes are the basis for protein production, whose biogenesis is essential for cells to drive growth and proliferation. Ribosome biogenesis is highly regulated in accordance with cellular energy status and stress signals. In eukaryotic cells, response to stress signals and the production of newly-synthesized ribosomes require elements to be transcribed by the three RNA polymerases (RNA pols). Thus, cells need the tight coordination of RNA pols to adjust adequate components production for ribosome biogenesis which depends on environmental cues. This complex coordination probably occurs through a signaling pathway that links nutrient availability with transcription. Several pieces of evidence strongly support that the Target of Rapamycin (TOR) pathway, conserved among eukaryotes, influences the transcription of RNA pols through different mechanisms to ensure proper ribosome components production. This review summarizes the connection between TOR and regulatory elements for the transcription of each RNA pol in the budding yeast  Ribosomes are macromolecular complexes that act as ‘factories’ to carry out the neosynthesis of virtually all peptides in cells. There are about 200,000 ribosomes in an exponentially growing yeast cell, whose production rate has been estimated at ~2000 ribosomes per minute ,2. This S. cerevisiae. We also discuss the possible factors implicated in the simultaneous coordination of RNA pols through the TOR pathway.The biogenesis of each yeast ribosome requires the production of the precursor of ribosomal RNA (rRNA) 35S by RNA polymerase I (RNA pol), which generates mature 18S, 5.8S and 25S rRNAs, the synthesis of 5S rRNA by RNA pol III and the expression of mRNAs from 138 genes) by RNA pol II to generate 79 different ribosomal proteins (RPs) ,7,8,9. T8 genes bIt is essential for cells to have mechanisms that allow their metabolism and growth to be adjusted depending on external conditions. Cells respond to a wide variety of environmental cues by modulating gene expression. The TOR signaling pathway, conserved from yeast to higher eukaryotes, is one of the most important mechanisms by which cells sense nutrient availability and regulate their protein biosynthetic capacity and the ribosome production rate ,19,22,23S. cerevisiae -like family [TOR gene, in S. cerevisiae, a paralog exists, TOR2, which encodes Tor2, a PI3K-like kinase with ~70% sequence homology to Tor1 [TOR was initially described in the budding yeast iewed in ). In S. e family ,8,22. Al to Tor1 . Two dis to Tor1 . However to Tor1 . TORC1 i to Tor1 ,25,26. I to Tor1 ,28,29, b to Tor1 ,31. TORC to Tor1 . TORC2 i to Tor1 . As the TORC1 is sensitive to a wide variety of environmental cues, such as nutrient starvation , redox stress, rapamycin, among others ,28. The The signaling downstream of TOR has been classically considered on two main branches termed the Sch9 branch and the Tap42-PPases branch. Some of the important cited roles of TOR, especially in RNA pols transcription regulation, are driven by AGC family member kinase Sch9, one of the major TORC1 substrates . The othrRNA production by RNA pol I represents almost 60% of overall transcription in yeast and is a limiting step for ribosome biogenesis . TOR inhRRN3 and RPA43 are essential genes but can be deleted in a strain that overexpresses fusion protein Rrn3-Rpa43 (termed CARA protein) [TOR has been proposed to control mainly RNA pol I transcription via transcription factor Rrn3 ,41. Thisprotein) . In CARAprotein) . Anotherprotein) . It can RNA pol I recruitment to rDNA promoters is primarily mediated by Sch9 , one of the TORC1 major targets, a process that is inhibited by rapamycin . HoweverHMO1 and RPA49, which encodes Rpa49 [HMO1 expression is regulated in a TOR-dependent manner [HMO1 gene [The high-mobility group B (HMG) family member Hmo1 was identified as a factor involved in rDNA transcription by genetic interactions between es Rpa49 . Hmo1 dies Rpa49 , and HMOt manner , with thMO1 gene . Taken tThe Ccr4-Not complex is a well-known element involved in several RNA pol II-dependent gene expression steps (reviewed in ). Ccr4-Npaf1Δ cells after histidine starvation [paf1Δ cells compared to the wild-type [The RNA polymerase-associated factor 1 complex (Paf1C) is a well-known transcription factor involved in RNA pol II transcription . A role arvation , and rapild-type . These fild-type .S. cerevisiae is ~150, half of which are transcriptionally active in exponentially growing cells [It has been estimated that the number of chromosomal rDNA copies in ng cells . Growth ng cells ,65, and ng cells . Althougng cells . TOR posng cells . Furtherng cells .asf1Δ and rtt109Δ mutants show hypersensitivity to rapamycin [Several histone H3 modifications are positively regulated by TOR . Of themapamycin , TOR couapamycin .Tor1 kinase shuttles between the nucleus and the cytoplasm . NutrienTaken together, these data support that TOR acts at different levels to control RNA pol I activity and, hence, ribosome biogenesis. The roles of the TOR pathway in RNA pol I transcription are summarized in About 50% of all RNA pol II-mediated transcription events are devoted to the production of RPs . The TORTranscription factor Rap1 is a key player for RPs and RiBi gene expression. It binds most RP gene promoters and, hence, accounts for ~50% of total RNA pol II-mediated transcription ,73,74,75Split zinc (Zn)-finger protein Sfp1 participates in RP and RiBi gene expression in a TOR-dependent manner ,80,81. ISfp1 rapidly translocates from the nucleus to the cytoplasm following the stress conditions that lead to the downregulation of RP and RiBi gene expression ,80,81. SGeneral transcription factor Abf1 has a well-known role as an activator of RP and RiBi gene expression under optimal nutrient conditions by binding a small group of RP and ~50% of RiBi gene promoters ,85. HoweHMO1 deletion does not lead to the overall downregulation of RP genes compared to wild-type cells [Hmo1 binds to the rDNA gene body and plays a well-known role in RNA pol I transcription see . Interespe cells . Shortlype cells . Moreovepe cells .hmo1Δ cells compared to the wild-type, which indicates that Hmo1 is also required for the repression of RP genes in a TOR-dependent manner [As with RNA pol I, Hmo1 dissociates from RP gene promoters after rapamycin treatment, which correlates with the downregulation of RP genes . Notablyt manner .The downstream kinase effector of TOR, Sch9, positively regulates RiBi and RP gene expression in a TORC1-mediated manner ,79. Sch9INO80, which renders ino80Δ cells resistant to rapamycin, partially mimics the ribosome biogenesis genes’ transcriptional profile observed in rapamycin-treated cells [As with RNA pol I, TOR plays a role in chromatin remodeling during RNA pol II-mediated transcription. The RSC (Remodeling the Structure of Chromatin) complex is an essential ubiquitous and ATP-dependent chromatin remodeler that performs nucleosome repositioning to activate or repress transcription . After Ted cells .As previously indicated, TOR seems to play a role in histone modification. TOR regulates the recruitment of Esa1  to RP gene promoters. This allows histone H4 acetylation, which activates the transcription of these genes . AccordiThe roles of TOR in RNA pol II regulation for ribosome biogenesis are schematized in RNA pol III is specialized in the high-rate production of short RNAs through several rounds of reinitiation over the DNA template. It synthesizes rRNA 5S, which is needed for ribosome biogenesis and all the tRNAs required for translation. To synthesize these transcripts, the RNA pol III holoenzyme needs the participation of two different basal transcription factors, termed TFIIIB , TFIIIC , among other proteins (reviewed in ). In 5S As with RNA pols I and II, rapamycin treatment disturbs RNA pol III transcription and causes the downregulation of RNA pol III-dependent genes ,89. How maf1Δ cells are defective in the downregulation of RNA pol III transcription under different growth conditions, including TOR inhibition [Maf1 is the master regulator of RNA pol III-mediated transcription, conserved from yeast to humans (reviewed in ). Indeedhibition . Co-immuhibition ,101,102 hibition . Notablymsn5Δ cells, Maf1 is constitutively located in the nucleus but does not cause the downregulation of RNA pol III transcription [Maf1 activity and cellular localization are regulated by phosphorylation (reviewed in ). Under cription . This incription ,102,105.cription . The kinMAF1 in sch9Δ cells restores transcriptional defects [SCH9 deletion renders dephosphorylated Maf1 and causes its nuclear localization [SCH9 (termed DESCH9), which uncouples Sch9 activity from the upstream regulation by TORC1, blocks the repression of RNA pol III transcription [7E, a phosphomimetic version of native Maf1. This led the authors to suggest that Sch9 could target additional factors to promote RNA pol III transcription [Many studies have pointed out that Sch9 is the major kinase of Maf1 under optimal growth conditions , but crSCH9 deletion leads to Bcy1 (the regulatory PKA subunit) hyperphosphorylation, which attenuates PKA activity [Some observations suggest that Sch9 can phosphorylate the PKA consensus sites of Maf1, even when PKA activity is lacking, but this redundant effect is not bidirectional ,107. Theactivity . Finallyactivity .The overall data suggest that TORC1 acts on Sch9 to regulate Maf1 phosphorylation under favorable growth conditions, while the Ras/PKA pathway probably provides an additional mechanism to control Maf1 activity through sensing glucose availability. Whether PKA has a stronger impact on Maf1 phosphoregulation under other growth conditions cannot be ruled out.The CK2 enzyme is an essential serine/threonine kinase involved in numerous important cellular processes . CK2 actCka2-Maf1 interaction has been reported in a carbon source-dependent manner, and CK2 phosphorylates Maf1 both in vitro and in vivo . NotablyTOR inhibition causes the rapid dephosphorylation of Maf1, which leads to Maf1-RNA pol III interaction and transcription repression ,102. In S. cerevisiae, the PP4 core complex is composed of three subunits: Pph3, the catalytic subunit; Psy2, the regulatory one; Psy4, the regulatory/scaffold subunit [PPH3 or PSY2 genes leads to the impairment of both Maf1 dephosphorylation and RNA pol III repression after TOR inhibition, which falls in line with the role of PP4 in Maf1 regulation  phosphorylation is TOR-dependent . Two kinpression . Kns1 ispression . Mck1 isrocesses .maf1Δ cells causes similar RNA pol III repression to the triple mutant maf1Δ kns1Δ mck1Δ, which indicates that Kns1 and Mck1 kinases do not seem to regulate RNA pol III independently of Maf1 [Interestingly, adding rapamycin to  of Maf1 . Therefo of Maf1 .Bdp1 is an essential subunit of the TFIIIB complex and is required for RNA pol III recruitment to promoters . NotablyBdp1 phosphorylation  seems to be TOR-dependent . Indeed,The roles of TOR in the regulation of RNA pol III are schematized in Although crosstalk among the three RNA pols has been proposed, whether RNA pols are temporally and spatially regulated through the TOR pathway remains unclear. Sch9 indirectly influences not only the transcription of RNA pol III by phosphorylating Maf1 . HoweveRPH1 and TOR1 genetically interact, and rph1Δ cells exhibit rapamycin resistance and defects in RNA pol I and II repression, which is consistent with a negative role of Rph1 under TOR-inhibiting conditions [Another possibility is that TOR could control the transcription of the three RNA pols via a common transcription factor. TBP is a universal transcription factor that mediates the transcription of the three RNA pols . A phospnditions . Under onditions . Thus, Tnditions . Yet wheHMO1 in CARA cells largely alleviates the upregulation of most RP genes upon rapamycin treatment [Cells can adjust the transcription of rDNA in response to the downregulation of RP genes . In CARAreatment . This fireatment . HoweverAnother intriguing hypothesis is that TOR could simultaneously regulate the activity of the three RNA pols for optimal ribosome biogenesis through chromatin remodeling. As mentioned in The TOR signaling pathway is highly conserved throughout eukaryotes. Unlike yeast, all other eukaryotes have only one Tor protein , which forms two TOR complexes: mTORC1 and mTORC2 . As in yrDNA transcription regulation by mTORC1 is not fully understood, although the global process seems conserved. Like yeast, mTORC1 inhibition leads to a rapid decrease in rRNA production ,147, andS. cerevisiae, RPs, and RiBi gene expression are regulated by TOR via different mechanisms that directly or indirectly influence RNA pol II activity. In contrast, mammalian mTORC1 regulates RPs production mainly at the translational level [In al level . mTORC1 al level ). These al level .mTORC1 inhibition causes RNA pol III transcription attenuation . This meThe transcriptional coordination of the three RNA pols by mTORC1 has also been proposed in mammals, but the mechanisms governing this process are still poorly understood . One of Ribosome biogenesis regulation at the transcriptional level has become an interesting and exciting research field. There is growing evidence that cells require the tight regulation of ribosome production to properly respond to fluctuating environmental cues, and signaling pathways play a critical role in this process. This review overviews the transcriptional regulation of the three RNA pols by the highly conserved TOR signaling pathway. To date, some transcription factors have been identified as effectors of TOR to modulate the activity of each RNA pol. Understanding the mechanisms by which TOR regulates these factors has been an important step to further comprehend RNA pol transcription regulation, ribosome biogenesis and cell growth. Interestingly, recent advances in this field have led to the hypothesis about the factors that could simultaneously coordinate RNA pols under the control of TOR. These mechanisms could include modifications in the common subunits of RNA pols, the factors that target these subunits, general transcription factors, global epigenetic modifications and chromatin remodeling. Further research is needed to examine the mechanisms that govern these processes. Furthermore, crosstalk between RNA pols must exist, or even between RNA processing and transcription. Future exploratory perspectives may include the existence of feedback loops to finely tune and regulate RNA homeostasis and environmental fluctuations.


RNA modifications are dynamic and reversible chemical modifications on substrate RNA that are regulated by specific modifying enzymes. They play important roles in the regulation of many biological processes in various diseases, such as the development of cancer and other diseases. With the help of advanced sequencing technologies, the role of RNA modifications has caught increasing attention in human diseases in scientific research. In this review, we briefly summarized the basic mechanisms of several common RNA modifications, including m6A, m5C, m1A, m7G, Ψ, A-to-I editing and ac4C. Importantly, we discussed their potential functions in human diseases, including cancer, neurological disorders, cardiovascular diseases, metabolic diseases, genetic and developmental diseases, as well as immune disorders. Through the “writing-erasing-reading” mechanisms, RNA modifications regulate the stability, translation, and localization of pivotal disease-related mRNAs to manipulate disease development. Moreover, we also highlighted in this review all currently available RNA-modifier-targeting small molecular inhibitors or activators, most of which are designed against m6A-related enzymes, such as METTL3, FTO and ALKBH5. This review provides clues for potential clinical therapy as well as future study directions in the RNA modification field. More in-depth studies on RNA modifications, their roles in human diseases and further development of their inhibitors or activators are needed for a thorough understanding of epitranscriptomics as well as diagnosis, treatment, and prognosis of human diseases. RNA molecules like DNA and proteins are also chemically modified through RNA modifying enzymes. With the fast development of detecting technologies, such as chemical labeling, mass spectrometry and high-throughput sequencing, more than 170 different types of post-transcriptional modifications on RNA have been identified, dynamically regulating RNA functions and stability . RNA modRNA modifying enzymes can be classified as “writers”, “erasers” and “readers”. “Writers” are usually modifying enzyme complexes that install RNA modifications on RNA substrates . DiffereRecent studies have revealed the important roles of RNA modifying enzymes in human diseases, including cancer, neurological disorders, cardiovascular diseases, metabolic diseases, as well as developmental and genetic disorders. For example, the m5C methyltransferase NOP2/Sun RNA methyltransferase 2 (NSUN2) is overexpressed in breast cancer and is correlated with cancer development and progression . In contThe increasing understanding of RNA modification and its role in cellular processes has provided new potential in the diagnosis, treatment, and prevention of a variety of diseases. Therefore, in order to help researchers to thoroughly understand the roles of RNA modifications in diseases and ways to target these modifications for clinical purposes, we summarize in this review the functional mechanisms of seven of the better studied RNA modifications, including N6-methyladenosine, 5-methylcytosine, N1-methyladenosine, internal 7-methylguanosine, pseudouridine, adenosine-to-inosine editing, and N4-acetylcytidine. We also discuss currently available small molecules targeting these modification pathways as well as their applications in human diseases.To date, the methylation of internal adenosines at the N6 position (m6A) has been found in messenger RNA (mRNA) and non-coding RNAs  in eukaryotic cells Fig. a-b 27]..27]. In The methyltransferase complex consists of METTL3/METTL14/Wilm’s-tumor-1-associated protein (WTAP) triplet as well as several co-factors including RNA-binding motif protein 15 (RBM15)/15B, VIRMA and KIAA1429  technologies have laid the foundation for studying the role of m6A mRNA modification in different diseases.m5C was first discovered in 1925, known to occur on rRNA, tRNA, ncRNA and mRNA  and 5-formyl-2’-O-methylcytidine (f5Cm34), suggesting that ALKBH1 mediates demethylation of m5C modification . . 109]. AIt has been indicated that YTH domain family  can interact with m1A-carrying RNA, and are thereby jargonized as “readers”  modification refers to adding a methyl group at the 7ion Fig. a-b . A. A148]. Saccharomyces cerevisiae homolog of human NAT10, was uncovered to catalyze the ac4C-1773 of 18S rRNA, promoting the formation of pre-18S rRNA . Al. Al15]. 18S rRNA . Subsequ18S rRNA . NAT10-m18S rRNA . Althoug18S rRNA . SIRT7 w18S rRNA . Therefo18S rRNA . More fuDysregulation in RNA modifying enzymes has been reported in various disease models, including multiple cancer types Fig. , Table 1Cancer is a major public health problem that affects people’s life all over the world. It is a complex disease with many subtypes and variations and can be further classified based on specific characteristics, such as the stage and grade. Treatment approaches and outcomes vary depending on the type and stage of cancer. Many studies have provided evidence suggesting that dysregulation of RNA modifications or RNA modifying enzymes contributes to human cancers. The list of important RNA modifying enzymes that play essential roles in cancer keeps expanding as research progresses in this field Table .m6A dysregulation has been implicated in various aspects of cancer development. Dysregulation of m6A may lead to altered RNA stability, disrupted splicing patterns, disrupted RNA processing and maturation, as well as altered efficiency and accuracy of translation, resulting in aberrant expression of oncogenic or the tumor-suppressive genes, thus contributing to cancer initiation and progression . m6A dysDepending on the different m6A-modified RNA targets, the role of the METTL3/14 complex may be contradictory in some cancer models, as opposite effects have been reported in different studies .Most research studies on the m6A writer METTL3 or METTL14 indicate their oncogenic roles in cancer. For example, METTL3 is abundantly expressed in Acute myelogenous leukemia (AML) and has been identified as a crucial gene for AML cell proliferation by a whole-genome CRISPR dropout screening approach , 166. MEMETTL3 expression was also elevated in glioblastoma stem-like cells (GSCs) and was attenuated during differentiation . This elBesides AML and glioblastoma, oncogenic roles were also reported for METTL3 in multiple other cancer types. Elevated METTL3 expression was observed in human lung adenocarcinoma, where METTL3 played an essential role in promoting cancer cell survival, proliferation and invasion . CytoplaTumor suppressor functions of METTL3-METTL14 complex have also been reported the in several cancer models. For example, Cui et al.showed that the METTL3-METTL14 complex inhibited GSC self-renewal and tumorigenesis by regulating mRNA m6A enrichment and expression of genes with critical oncogenic functions, such as a disintegrin and metalloproteinase domain-containing protein 19 (ADAM19)  domain . This doInsulin-like growth factor 2 messenger RNA binding proteins (IGF2BPs) specifically recognize m6A-modified RNAs through their KH domains , althougm5C is another RNA modification that has been implicated in cancer. Similar to m6A, dysregulation of m5C modification can lead to altered RNA stability, disrupted splicing patterns, altered epigenetic patterns . DysreguLike m6A, most m5C modifiers are known for their oncogenic roles. NSUN1 was first identified as a proliferation nuclear antigen . NSUN1 wBy analyzing 382 tumors and 362 normal specimens from pancreatic cancer (PC) patients, Yang and colleagues characterized NSUN6 as an important factor regulating PC cell proliferation and suppressing PC development .m1A is a relatively less studied RNA modification that has only gained attention in recent years. Although its role in cancer is less extensively studied compared to m6A and m5C, emerging evidence suggests that m1A dysregulation may also contribute to cancer development and progression. Dysregulation of m1A in cancer may affect the stability of specific RNA molecules, impact the translation of specific mRNA transcripts, disrupt RNA processing and maturation, perturb RNA–protein interactions, as well as disturb the balance between m1A deposition and removal, leading to cancer development and progression . BioinfoMembers of the m1A methyltransferase complex, TRMT6 and TRMT61A, were overexpressed in advanced HCC tissue and correlated negatively with HCC survival . TRMT61AWAF1/Cip1 and p27Kip1 in lung adenocarcinoma cells . . 225]. PBy analyzing the TCGA cohort, Yan et al. found significantly elevated expression of the rRNA m7G methyltransferase WBSCR22 in human CRC tissue, which led to oxaliplatin resistance . SilenciWBSCR22 and its cofactor TRMT112 synergistically suppressed tumorigenesis and progression of pancreatic cancer by transcriptionally regulating interferon‑stimulated gene 15 (ISG15), a ubiquitin‑like modifier enzyme involved in metabolism and proteasome degradation .RNA methyltransferase (RNMT) recruitment to the promoters of genes in the canonical Wnt/β-catenin signaling pathway promoted by MYC-mediated Ser 5 phosphorylation of RNA Polymerase II enhanced mRNA cap methylation and increased translational capacity and elevating protein expression in cancer cells Fig. d 231]..231].In summary, the aberrant regulation of RNA methylation is associated with cancer development . The fouPseudouridine (Ψ) is one of the most abundant modifications found in various RNA species, including mRNA, tRNA, rRNA, and ncRNAs . Ψ playsOne of the best characterized PUS1 targets is the steroid receptor RNA activator 1 (SRA1) ncRNA . PUS1-meDKC1, a member of a snoRNP complex, contains TruB Ψ synthase motifs and requires an RNA guide for its catalytic activity on rRNA, snRNA, snoRNA and TERC . DKC1 plDKC1 overexpression in HCC patients was correlated with MYC and MKI67 expression, thus may be an unfavorable prognostic factor predicting advanced clinical stage and poor patient prognosis . In lungTP53 gene were responsible for the inactivation of p53 function as tumor suppressor in over half of human cancers . . 225]. MRNA modifications have the potential to serve as biomarkers for cancer diagnosis, prognosis, and treatment response. Identifying and validating specific modification patterns or signatures that correlate with different cancer types or disease states would aid in early detection and personalized treatment strategies. Studying the regulatory mechanisms that control RNA modifications in each specific cancer type and disease stage will provide valuable insights into the underlying molecular processes. Further exploring the roles of RNA modifiers in cancer cells may uncover novel therapeutic targets and inform the development of targeted interventions. Elucidating the dynamics of RNA modifications in cancer is an important research direction. Investigating how modifications change in response to therapeutic interventions, disease progression, or specific cellular contexts will provide insights into their functional roles and potential as therapeutic targets. Developing strategies to target and manipulate RNA modifications for therapeutic purposes is also an exciting area of research. This includes the development of small molecules, antibodies, or gene editing tools to modulate specific modifications and investigate their therapeutic potential in cancer treatment. Exploring the feasibility and implications of RNA modification editing, similar to DNA base editing, is also an important emerging area of interest. Developing precise editing tools to manipulate specific RNA modifications in cancer cells would enable the exploration of their functional consequences and therapeutic potential.Addressing these gaps will require collaborative efforts among researchers, technological advancements, larger and well-characterized patient cohorts, and multidisciplinary approaches. As research progresses, we will gain deeper insights into the roles of RNA modifications in cancer and potentially uncover novel therapeutic targets and biomarkers for improved cancer diagnosis and treatment.Neurological disorders are a diverse group of conditions that affect the structure and function of the nervous system, including the brain, spinal cord and peripheral nerves. Dysregulation of RNA modifications are involved in multiple neurological disorders, such as dysregulated brain development, stroke, neurodegenerative diseases and traumatic injuries.Among all organs, m6A methylation of RNAs is highest in the brain and is known to promote cell survival after adverse conditions .Drosophila nervous system, inhibited translation of key transcripts involved in axonal growth regulation via its interaction with Fmr1, the fly homolog of Fragile X mental retardation RNA binding protein (FMRP) . M. M35]. MConsidering the structure and activity of Rhein and MA, a novel scaffold N-phenyl-1H-indol-2-amine was used as a base to design FTO inhibitors . Among aBased on MA structure, Huang et al.synthesized FB23 (IC50 = 0.06 μM) and FB23-2, which directly bound to FTO and selectively inhibited the m6A demethylase activity of FTO  and pyridine-2,4-dicarboxylate , occupied the 2OG binding pocket of FTO by forming electrostatic and hydrogen bond Fig. b 416]. . 416]. SThrough structure based virtual screening, Chen et al. recently found two robust FTO inhibitors CS1 and CS2 with IC50 at nanomolar range in AML cells Fig. b 419]. . 419]. CUsing structural and biochemical studies, Peng et al.identified entacapone, an FDA-approved drug for treating Parkinson’s disease , as an FThrough virtual screening, structural optimization and bioassay, Xie et al.developed a novel small-molecule FTO inhibitor, 18,097, which selectively bound to FTO active site and significantly inhibited breast cancer cell proliferation and metastasis both in vitro and in vivo Fig. b 422]. . 422]. MUsing in silico-based rational target-tailored development methods, Selberg et al. developed two small-molecule FTO inhibitors with potent inhibition, 4-amino-8-chloroquinoline-3-carboxylic acid  and 8-aminoquinoline-3-carboxylic acid , which supported the survival of growth factor-deprived primary dopamine neurons in culture , originally reported as an oncometabolite highly produced by mutant isocitrate dehydrogenase 1/2 (IDH1/2) enzymes, suppressed leukemia cell proliferation/viability and promoted cell-cycle arrest and apoptosis by inhibiting FTO demethylase activity and manipulating the expression levels of CCAAT/enhancer-binding protein alpha (CEBPA) and MYC  Fig. c. CitratNakao et al.designed and synthesized a series of 1-aryl-3,4-substituted-1H-pyrazol-5-ol derivatives as ALKBH3 inhibitors, among which 1-(1H-5-methylbenzimidazol-2-yl)-4-benzyl-3-methyl-1H-pyrazol-5-ol (HUHS015) demonstrated the highest potency IC50 = 0.67 μM) against ALKBH3  inhibited the ALKBH enzymes and sensitized IDH mutant cells to alkylating agents, such as procarbazine and lomustine (CCNU) Fig. c 435]. . 435]. CResearch on ALKB homologs is ongoing, as their protein structures are resolved, their functions have been better understood and corresponding drugs have been designed. Several ALKB homolog inhibitors can regulate iron concentrations and subsequently affect ALKB homolog activity as iron chelators. Although not identical in terms of amino acid sequence, the ALKBH family proteins show similar structures over the active site Fig. b 438]. . 438]. HThe YTH-domain containing proteins are a group of proteins that possess a conserved RNA-binding domain known as the YTH (YT521-B homology) domain, which enables proteins such as YTHDF1-3 and YTHDC1-2 to act as readers of m6A-containing transcripts  family consists of three members, IGF2BP1 , IGF2BP2 (IMP-2 or IMP2/Hum2/HuR-BP2), and IGF2BP3 (IMP-3 or KOC) Fig. d 443, 44. They aBy binding to specific mRNA targets, IGF2BPs regulate multiple aspects of RNA metabolism, including stability, localization, translation, and alternative splicing, thus influencing several important aspects of cell function, such as cell polarization, morphology, proliferation, migration, metabolism and differentiation . DysreguUsing a fluorescence anisotropy-based assay, Mahapatra et al. screened 160,000 small molecules and identified a potent and selective IGF2BP1 inhibitor, BTYNB, which inhibited melanoma and ovarian cancer cell proliferation by downregulating several IGF2BP1-mediated mRNA transcripts, including c-Myc, β-TrCP1, NF-κB and eEF2 Fig. a 445]. . 445]. TAccumulating evidence has shown that m5C modulates the stability, nuclear export, translation, and cleavage of RNAs to mediate cell differentiation, proliferation, stress responses, apoptosis, and other biological functions . In humaConsidering the chemical similarity between DNA 5mC and RNA m5C, drugs designed to interfere with DNA methylation may target RNA methylation as well. For example, the RNA m5C methyltransferases DNMT2 and NSUN3 have been suggested to be important targets of the DNA methylation inhibitor 5-Azacytidine (5-AZA) in AML, while the interaction between NSUN1 and the transcriptional co-activator BRD4 induced resistance to 5-AZA Fig. b 236]. . 236]. NThe TRMTs (tRNA Methyltransferases) are responsible for adding methyl groups to specific nucleotides within tRNA molecules –453. TheBy screening potential inhibitors blocking TRMT6/TRMT61A interaction from an FDA-approved drug bank, Wang et al.identified a potent inhibitor Thiram against TRMT6/TRMT61A complex that remarkably suppressed self-renewal of liver CSCs and liver tumor growth Fig. a. ThiramSince Ψ modifications play roles in various cellular processes and diseases, targeting specific PUS family members could therefore potentially affect these processes and have therapeutic benefits.Escherichia coli Ψ synthase TruB containing critical eukaryotic homologs was not inhibited, but rather handled the RNA containing 5-FU as a substrate . . 462]. Pubstrate . The E. ing 5-FU . Uridineing 5-FU , 471. Ining 5-FU . They deing 5-FU . 5'-monoing 5-FU .Other than 5FU-containing tRNAs, studies have also been conducted searching for potential inhibitors targeting other factors involved in pseudouridinylation. For example, Rocchi et al.sought to identify small molecules able to inhibit the catalytic activity of human dyskerin (DKC1). Using various in silico techniques, they selected compounds and analyzed the binding modes and the interaction patterns of ligands in the human dyskerin catalytic site. They identified four molecules  that significantly inhibited dyskerin pseudouridylation activity, among which only compound 1 (pyrazofurin) showed a significant cytotoxic activity in MCF7 breast cancer cells  and 4,086 FDA-approved drugs, Cui et al.identified C17 as a potent inhibitor against PUS7 . In cellResearch into Ψ related modifiers as therapeutic targets is still in its early stages, and much more work is needed to fully understand their functions, regulation, and potential for therapeutic intervention. Nonetheless, the emerging understanding of Ψ modifications and their roles in diseases holds promise for future therapeutic strategies.ADARs are enzymes catalyzing the chemical conversion of adenosines to inosines in dsRNA substrates . ADAR faADAR-mediated A-to-I RNA editing has significant consequences for RNA molecules since inosine is later recognized as guanosine during translation, leading to potential changes in RNA sequences and subsequent protein coding. ADAR-mediated RNA editing is particularly prominent in the nervous system, where it contributes to the diversification of neuronal transcriptomes and fine-tuning of neurotransmitter receptors. ADARs are involved in many physiological and pathological processes, including neuronal development, synaptic plasticity, and neurological disorders. Dysregulation of ADAR activity or RNA editing has been associated with various diseases, including cancer, neurological disorders , and autoimmune diseases, suggesting that ADARs may be recognized as potential therapeutic targets. There are ongoing efforts to develop small molecule inhibitors and nucleic acid-based approaches to modulate ADAR activity. These strategies aim to specifically target ADAR enzymes or their binding domains, interfere with RNA editing events, or restore proper RNA editing in disease-associated contexts.In 2006, Maydanovych synthesized C6-substituted derivatives of 8-azanebularine and showed that C6-methyl derivative was incorporated into an RNA substrate for ADAR2 via the phosphoramidite and showed a strong inhibitory effect on editing enzyme binding, suggesting that methylation at C6 must be considered in the design of new ADAR inhibitors based on this ring system Fig. d 477]. . 477]. APrimary cultured cortical cells treated with the ADAR inhibitor erythro-9-(2-hydroxy-3-nonyl) adenine hydrochloride (EHNA) significantly reduced the editing efficacy of 5-HT2CR mRNA in a dose-dependent manner Fig. d 480]..480].Through high-throughput screening on 2115 FDA-approved compounds for possible repurposing in inhibition of the Zα domain, Choudhry et al. selected three compounds, alendronate, etidronate, and zoledronate based on their XP Gscore Fig. d 481]. . 481]. AADAR1p110-to-p150 splice isoform switching induced by inflammatory microenvironment drives cancer stem cell generation and therapeutic resistance in multiple malignancies. Crews et al. developed Rebecsinib, a selective ADAR1 inhibitor that reversed ADAR1 splice isoform switching, thus attenuating LSC self-renewal and extending survival of humanized LSC mice Fig. d 482]..482].Failure of GluA2 RNA editing resulting from ADAR2 loss often occurs in ALS cases and causes motor neuron death. Recovering ADAR2 level in the ADAR2-depleted mechanistic sporadic ALS mouse model by intravenously injecting adeno-associated virus serotype 9 (AAV9)-ADAR2 rescued mice from motor neuron death by normalizing TDP-43 expression, suggesting a possibility of using AAV9-mediated ADAR2 gene delivery as a gene therapy for ALS .The development of therapies targeting ADARs is still in its early stages, and it requires a comprehensive understanding of the complex regulatory mechanisms and consequences of RNA editing. Off-target effects and unintended consequences of modulating ADAR activity need to be carefully considered. ADARs have multiple RNA targets, and altering their activity could have broader effects on RNA processing and gene expression. Therefore, while ADARs hold potential as therapeutic targets, further research is needed to fully understand the mechanisms, functions, and consequences of ADAR-mediated RNA editing in various diseases. Overcoming the challenges of achieving target selectivity and minimizing off-target effects will be crucial for the development of effective and safe therapies targeting ADARs.The functional significance of Ac4C modifications in RNA is still being actively researched, and their specific roles and implications in cellular processes and disease conditions are not yet fully understood. However, dysregulation of ac4C has been reported in multiple cancer types as well as a few other disease models. Therefore, it is important to screen for inhibitors targeting ac4C modifiers.The only NAT10 inhibitor reported so far, Remodelin (~ 40 μM), effectively suppressed NAT10 protein expression as well as its activity by binding to its Acetyl-CoA binding site Fig. e 21, 48. RomedelRecently, Dalhat screened a library of FDA-approved drugs aiming to identify novel inhibitors of NAT10 activity. They selected four drugs, namely fosaprepitant, leucal, fludarabine and dantrolene that bound to NAT10 with a better binding capability, indicating that these four drugs might serve as potential NAT10 inhibitors, although further cellular testing and evaluation are needed to verify their functions against NAT10 related diseases Fig. e 491]..491].At present, the research on ac4C modifications in RNA is still in its early stages, and further investigations are needed to elucidate their functional significance and therapeutic potential. Continued research in this area will contribute to a better understanding of the roles of ac4C in RNA biology and its potential as a therapeutic target.Although more and more inhibitors against RNA modifiers have been developed, targeting RNA modifications still face a great challenge.First, many RNA modifications occur at low abundance, making it challenging to selectively target and manipulate the modified RNA molecules without affecting the unmodified ones. Achieving specificity in targeting a particular modification while minimizing off-target effects is a significant hurdle. RNA modifications may need to be targeted in specific cells or organs. Efficient delivery of therapeutic agents or editing tools specifically to the desired cell types or tissues is also difficult. RNA modifications are often dynamically regulated and vary in response to different physiological or pathological conditions. Targeting and manipulating modifications in real-time or in a specific context may require precise temporal and spatial control, which can be technically demanding. Some RNA modifications may locate in regions of the RNA molecule that are structurally or functionally inaccessible. For example, modifications within highly structured regions or bound by RNA-binding proteins may be difficult to target or modify using traditional approaches.Second, our knowledge regarding the structures of some RNA modifiers is still quite limited. For example, the domain structures of the NSUN family and the TRMT family members have not been thoroughly reported yet, it is thus more challenging to design inhibitors against these proteins.Third, the development of effective tools and technologies for targeting and manipulating specific RNA modifications is an ongoing challenge. Techniques such as CRISPR-based systems or small molecules that specifically modulate RNA modifications need to be refined and optimized for different modifications. The functional consequences and biological roles of many RNA modifications are still not fully understood. Targeting specific modifications without comprehensive knowledge of their functional implications may lead to unpredictable side effects or limited therapeutic outcomes. Any therapeutic intervention targeting RNA modifications must consider potential safety concerns and minimize off-target effects.Addressing these difficulties requires continued research and development of innovative technologies, improved delivery systems, a deeper understanding of modification functions, and rigorous safety assessment. Overcoming these challenges will pave the way for the development of targeted therapies and interventions that leverage the potential of RNA modifications in various biological and disease contexts.The study field of RNA modifications is rapidly evolving with ongoing research and discoveries. Despite the discovery of more than 170 different types of RNA modifications, there could still be undiscovered modifications. Identifying novel RNA modifications requires innovative technologies that can accurately detect and characterize these modifications. Mass spectrometry (MS) is a traditional powerful analytical tool for identifying and quantifying RNA modifications. Advanced MS techniques, such as liquid chromatography-mass spectrometry (LC–MS) and RIP-LC–MS, have been instrumental in discovering and characterizing new RNA modifications , 493. AnIt is important to note that there are differences in the identification of RNA modifications among different RNA species such as mRNA, tRNA, rRNA, and ncRNA. These differences arise due to variations in the abundance, structure, modification patterns, and techniques employed for each RNA species. mRNA is generally more abundant compared to tRNA, rRNA, and ncRNA, making it easier to obtain sufficient quantities for analyses. Considering these differences, it is important to tailor the experimental approaches and techniques for the identification and analysis of RNA modifications based on the specific RNA species of interest. Researchers often employ a combination of techniques and methodologies to comprehensively study modifications across different RNA types and unravel their functional significance.RNA modifications are increasingly recognized for their roles in human diseases, including cancer, neurological disorders, cardiovascular diseases, metabolic disorders and genetic diseases. However, there is still much to learn about the functional consequences of RNA modifications in disease contexts and their potential as therapeutic targets. So far, only a few RNA modifications have been well-characterized, and a great majority of the 170 currently discovered RNA modification types are still poorly understood regarding their biological functions, not to mention their clinical relevance and therapeutic potential. The enzymes responsible for RNA modification are not yet well characterized except for m6A, much work is needed to fully understand the specific mechanisms by which they catalyze the modification of RNAs. Understanding the functional consequences and regulatory mechanisms of RNA modifications is crucial for deciphering their roles in cellular processes. This includes investigating how RNA modifications influence RNA structure, stability, localization, translation, and interactions with other molecules, as well as elucidating the enzymes responsible for modifying RNA and their regulation. Researchers are actively exploring novel modifications, such as ac4C, m1A and m6Am, to understand their functional roles, regulatory mechanisms, and implications in various biological processes, including development, disease, and epigenetic regulation.It is necessary to mention that RNA modifications do not function in isolation but often interact with and influence each other. Investigating the interplay and cross-talk between different RNA modifications will provide insights into their synergistic effects, functional consequences, and potential regulatory networks. Further investigations into the epitranscriptomic landscape of disease samples, such as patient tissues or biofluids, may lead to the identification of disease-specific RNA modifications, potential diagnostic markers, and therapeutic targets. Addressing these gaps will advance our understanding of RNA modifications and their significance in biological processes and disease pathogenesis and pave the way for the development of novel therapies and diagnostic tools targeting RNA modifications.With the growing appreciation of the functional importance of RNA modifications, there is interest in developing targeted editing technologies to precisely modulate specific RNA modifications. Such technologies, including base editors or chemical modification tools, can be used to investigate the functional consequences of specific modifications and potentially correct disease-associated dysregulation. CRISPR-Cas systems have also been adapted to develop RNA-targeting tools, such as RNA-targeting Cas proteins and RNA-targeting guide RNAs, which can be used to specifically enrich or manipulate RNA modifications for downstream analysis. For instance, the RNA-targeting Cas13 enzyme has been utilized for the detection and characterization of RNA modifications . TargetiTaken together, RNA modifiers can be promising targets for clinical therapies. Targeting RNA modifiers may potentially transform the way we diagnose, treat, and prevent a variety of diseases. RNA modification has been shown to play a role in the development and progression of various types of cancer, neurological disorders, cardiovascular diseases, metabolic disorders and genetic disorders. Targeting RNA modification has the potential to be an effective therapeutic strategy for the treatment of these human disease by modulating RNA stability and translation of important proteins. Continued research in the field of RNA modification can certainly improve our understanding of the biological roles of RNA modifications, improve existing therapies, as well as develop new therapies. As our understanding of RNA modification continues to deepen, it may lead to new breakthroughs in the diagnosis, treatment, and prevention of human diseases.