Cucumis are very popular worldwide and have great market value. However, their fruit quality and yield are hindered by viral diseases. C. metuliferus is considered a wild species with resistance to viral diseases that is lacking in cultivated crops of the Cucumis genus, such as melon. The C. metuliferus line L37 shows extreme resistance against Papaya ringspot virus (PRSV-HA), whereas line L35 is a susceptible line. In this study, reciprocal grafting experiments between L35 and L37 were performed, and the PRSV-HA strain was pre-inoculated in the rootstock leaves. The results revealed that the resistance signal in the L37 rootstock could transmit and provide resistance to the L35 scion. Subsequently, double sandwich grafting was performed using the pre-inoculated L35 as the rootstock, which was then grafted onto the L37 intermediate and the L35 scion. The results showed that PRSV-HA RNA accumulated in the L35 rootstock leaf, petiole, and stem tissues, whereas PRSV-HA RNA accumulated in some intermediate and scion petiole and stem tissues. No HCPro RNA was detected in the L35 scion leaves. The results showed that the suppression of the virus occurred in the leaves, and the resistance effect spread from the rootstock in the scion direction. Hence, this study has demonstrated that RNA silencing of systemic signals is responsible for L37 resistance against PRSV. C. metuliferus L37 could provide a valuable resistance source for crops of the Cucumis species against viral diseases through grafting.Vegetable crops of the genus  Cucumis metuliferus (2n = 24) is a member of the Cucumis genus of the Cucurbitaceae family. C. metuliferus was grouped into the Melo subgenus of the Cucurbitaceae family based on the sequence similarity of six chloroplast loci and the nuclear ribosomal ITS1-5.8S-ITS2 regions [C. metuliferus is covered with stout, conical, blunt-based, and sharp stalk spines. Hence, it has several common names such as African horned melon, Kiwano, spiny cucumber, or recently, the commonly named devil fruit. It is consumed as a vegetable and is commercially grown for export due to its orange-reddish color and long shelf life. Widely distributed throughout the tropical and subtropical sub-Saharan regions of Africa [C. metuliferus is a monoecious annual species with climbing or sprawling vines [C. metuliferus could provide resistance against root-knot nematodes [Watermelon mosaic virus (WMV), Papaya ring spot virus (PRSV) [Cucurbit yellowing stunting disorder virus (CYSDV) [uliferus n = 24 isng vines . It has ematodes , and virs (PRSV) , and CucN gene encodes a nucleotide-binding site-leucine-rich repeat (NBS-LRR)-type protein that provides resistance against Tobacco mosaic virus (TMV) [Plants employ multiple defense mechanisms, such as the R (resistance) gene-mediated response and RNA silencing against pathogen infection. One of the common types of R-gene mediated defense responses is the induction of rapid localized cell death, which is highly recognizable by its necrotic lesions, called the hypersensitive response (HR). HR could be triggered by plant resistance proteins (R proteins), which recognize directly or indirectly virulence effectors from the virus, thereby activating the plant’s defense response. For example, the tobacco us (TMV) . Alternaus (TMV) .Both DNA and RNA viruses can trigger plant antiviral defense responses, and RNA is the target of RNA silencing, also called posttranscriptional gene silencing (PTGS) . In planGrafting is a traditional technique for asexual propagation and cultivation in tree and vegetable crops. It is accomplished most commonly by connecting two plant segments, the upper part known as the scion and the lower part called the rootstock. Early fruit agronomists noticed that certain rootstocks found in seedling populations could provide favorable characteristics for the scion, such as scion vigor, an improvement of fruit quality, climatic adaptability, and resistance to pests and diseases. To date, grafting has also been important for studying the function of proteins, RNAs, and hormones that act over long distances in plants. Transmissible signals from rootstock to scion or the opposite direction through phloem have been convincingly demonstrated ,16. For C. metuliferus line Acc. 2459 , including leaf chlorosis, a reduction of leaf size, and short internodes observed 7–10 dpi (days after inoculation) [PRSV-HA, a PRSV isolate from Hawaii, causes disease symptoms in the ulation) ,7,18. ImTo validate PRSV resistance and the influence of grafting on PRSV-HA transmission in L37 and L35 plants, self- and reciprocal-grafting experiments were performed. In nongrafting controls (L35 and L37), PRSV-HA was artificially inoculated on one-month-old seedlings (with two cotyledon leaves and the first true leaf). The PRSV-HA-infected L35 showed severe mosaic, malformation, and narrow leaf blades at 21 days post inoculation (dpi), whereas no symptoms were observed in the PRSV-HA-inoculated resistant control L37 A no. 7 aTo monitor the existence of PRSV-HA, reverse transcription PCR (RT-PCR) of PRSV-HA’s HCPro gene was performed on the systemic leaves of the rootstock and scion. As shown in HCPro gene in the L35 scion leaves at 7 dpi was evaluated by quantitative Real-Time PCR (qRT-PCR)  A,B no. 3qRT-PCR) B no. 7–9To further study the effect of the systemic resistance signals derived from the L37 rootstock through grafting in detail, double sandwich grafting experiments were performed. The pre-inoculated PRSV-HA L35 was used as the rootstock. The L37 intermediate and the L35 scion were grafted onto the PRSV-HA pre-inoculated L35 rootstock A no. 3. Furthermore, the PRSV-HA HCPro RNA in the stem, petiole, and leaf tissues of the plants in the double sandwich grafting was surveyed. The results showed that HCPro RNA was detected in the leaf, petiole, and stem tissues of the pre-inoculated L35 rootstock as expected  and RTM proteins against potyviruses in the host A. thaliana [In contrast to other plant pathogens, viruses are obligate parasitic organisms, and they enter host cells through mechanical inoculation or infection with the aid of vectors such as insects to overcome the cell wall barrier of plant hosts. After entry into plant cells, viruses face two major defense mechanisms, namely, the plant single dominant R gene-mediated response and the RNA silencing machinery, which may cooperate with each other in the early stages of the plant hosts against virus infection (as reviewed in ). First,thaliana .C. metuliferus resistance line L37  gene was identified as differentially expressed between susceptible L35 and L37 and could potentially be one of the candidates of Wmv [serine proteinase inhibitor (SPI), the disease symptoms were observed at 21 dpi after PRSV-HA infection. This suggests that SPI might be one of the components required for PRSV-HA resistance in L37 [SP1 gene in transgenic susceptible L35 has been reported, which supports the idea that the SP1 gene fragment from L37 could provide resistance for L35. Therefore, the single dominant gene Wmv remains unidentified, and little is known about the extreme resistance mechanism in L37.In this study, no symptoms or viral RNA were observed in the nongrafted control line L37  no. 7. Ts of Wmv . SubsequSecond, RNA silencing is another well-documented resistance mechanism in plant hosts against virus infection. It is well known that RNA silencing is a fast and efficient method of anti-viral resistance, including the suppression of virus replication and short- and long-distance movement. Several grafting studies have shown that RNA silencing signals could be transmitted and provide antiviral resistance from rootstock to scion in transgenic or nontransgenic plants ,23,24. TPotyvirus is a well-known viral proteinase essential for Potyvirus infection. Moreover, the HCPro protein also suppresses RNA silencing via directly binding to siRNAs and prevents virus RNA silencing by inhibiting RISC assembly [HCPro gene could overcome the immunity of L37, this further implies that RNA silencing would be the important mechanism responsible for L37 resistance against PRSV-HA.In addition, additional evidence of RNA silencing responsible for the L37 resistance mechanism is evident in studies of the PRSV-HA 5-1 strain. Interestingly, PRSV-HA 5-1, a nitrous acid-induced attenuated mutant of PRSV-HA, was shown to break down the extreme resistance, and it showed symptoms in L37, but it was reported to be symptomless or only cause a mild infection in L35 . The HCPassembly . Since tFurthermore, disease symptoms and PRSV-HA RNA were detected, and no recovery was consistently found in the L35 rootstock in the double sandwich grafted plants . This inNtTOM1/NtTOM3 transgenic tobacco (Sd1 line) showed resistance to Tomato mosaic virus (ToMV) and Tobacco mosaic virus (TMV) by silencing the genes of two host factors, NtTOM1 and NtTOM3, supporting tobamovirus multiplication. The nonsilenced tomato and tobacco scions grafted onto the Sd1 line showed 50% and 60% degrees of silencing in the scions, and siRNAs were detected in the Sd1 rootstock and the grafted scions. This result also indicated that systemic RNA silencing is not 100% efficient and that the presence of the siRNAs was consistent with silencing in the scions [PRSV-HA RNA accumulation in double sandwich grafted plants showed different types of patterns in this study . In genee scions . In summIn plants, viral dsRNA in the host triggers RNA silencing as a defense mechanism against viruses. These dsRNAs are rapidly processed into small RNAs (sRNAs) of 21 to 28 nucleotides (nt) in length, which then interact with RISC and ultimately give rise to secondary sRNAs for target gene RNA silencing. RNA silencing generates systemic signals that spread from the initiation cell to neighboring cells through plasmodesmata and systemically spread over long distances through the phloem tissue ,31. HencCucumis and could support virus-resistant breeding programs for cultivated melon or other Cucumis crops.In double sandwich grafted plants, pre-inoculated L35 was used as a rootstock to constantly provide PRSV-HA stimulus to L37. The results clearly showed that the L37 resistance signal did not restrict the systemic movement of PRSV-HA, which could be found in the stem and petiole tissues, but PRSV-HA was suppressed in the leaves of the L37 intermediate and the L35 scion . These rC. metuliferus is considered to be the most primitive species among the cultivated Cucumis species based on its external fruit characteristics and flesh bitterness taste [C. metuliferus has not been commonly used in cultivated melon improvement to date due to the difficulty of sexual hybridization between C. melo and C. metuliferus [C. metuliferus were reported to be grafting compatible with cultivated melons [C. metuliferus has been used as a rootstock for grafting to cultivated melons to improve root-knot nematode resistance. After being grafted onto C. metuliferus rootstocks, the susceptible melon lines showed the lowest root galling and nematode egg densities compared with the nongrafted lines. In addition, grafting showed no significant impact on the fruit quality of the tested lines. Hence, we propose that grafting susceptible scions onto L37 rootstock might help prevent virus infection without the need for transgenic plants; hence, C. metuliferus could be a valuable resistance resource for breeding resistant cucurbit crops.Wild relatives are commonly considered to be valuable resources for the genetic improvement of cultivated varieties. ss taste . Howeveruliferus . Alternad melons ,34. C. mC. metuliferus susceptible line Acc. 2459 (L35) and resistant line PI292190 (L37) seeds were germinated in pots and grown in a temperature-controlled greenhouse at 25 °C for one month (third true-leaf stage). Viruses were inoculated on two cotyledons and the first leaf with a PRSV-HA strain. The virus inoculum source was kindly provided by Dr. Fuh-Jyh Jan . The plants were mechanically inoculated with fresh inoculum sap diluted 1:50 (w/v) in sodium phosphate buffer . Inoculated plants were kept in the same greenhouse for subsequent symptom observations and virus detection.HC-Pro gene expression by RT-PCR and qRT-PCR analysis. The extraction of total RNA from 100 mg leaf tissues was carried out using TriPure isolation reagent  according to the manufacturer’s instructions. The RNA quantity and quality were determined by using spectrophotometry and gel electrophoresis. One microgram of total RNA was used for the first-strand cDNA synthesis reaction consisting of 1 × M-MLV buffer, 1 unit of M-MLV reverse transcriptase , 0.2 mM dNTPs, 0.1 mM primers, and 2.5 unit RNaseOUT . The reaction was performed in a thermal cycler under the following conditions: 60 min at 42 °C and then 10 min at 72 °C. PRSV HC-Pro-specific primers  and beta-actin gene primers of C. metuliferus  were adapted from previous studies [beta-actin gene was used as an internal control in each RT-PCR or qRT-PCR analysis. For RT-PCR, it consisted of 20 μL total volume PCRs, consisting of 1 × PCR buffer, 0.2 unit of ProTaq polymerase , 0.2 mM dNTPs, 0.15 mM primers, and 1 µL first strand cDNA templates. All amplifications were performed in a thermal cycler under the following conditions: 2 min at 94 °C, followed by 30 s at 94 °C, 30 s at 55 °C for annealing temperature, 1 min at 72 °C for 35 cycles, and 5 min at 72 °C for a final extension. The PCR products were electrophoresed and visualized on 1% agarose gels. For qRT-PCR, 10 μL total volume PCRs consisting of 1 × PCR buffer, 0.025 unit of WizPure™ Taq polymerase , 0.05 mM dNTPs, 0.6 μM primers, and 3 µL 1/5X first strand cDNA templates. The sequences of the qRT-PCR primers were as follows: PRSV HC-Pro-specific primers  and beta-actin gene primers of C. metuliferus  were adapted from previous studies [The presence of PRSV and its quantity were monitored using the expression level of  studies ,35. The Two grafting and virus inoculation methods were used. (i) Plants that were approximately one month old were used for grafting. The clef grafting method was employed . RootstoDouble sandwich grafts were performed as the first grafting and virus inoculation method. Graft inoculation involved the grafting of healthy scions onto the infected rootstock. Scion L35 and the intermediate L37 were grafted on the pre-inoculated L35 rootstock in turn. The presence of disease symptoms and viruses in the leaf, petiole, and stem tissues were determined 10 days after grafting by using RT-PCR analysis.


In this data, 75,320,390 raw reads were acquired from African bullfrog mRNA using Illumina paired-end sequencing platform. De novo assembly resulted in a total of 136,958 unigenes. In the obtained unigenes, 30,039 open reading frames (ORFs) were detected. This dataset provides basic information for molecular level analysis of this species, which undergoes a state of dormancy under dry conditions at ordinary temperatures called estivation.In this article, we report the first  This frog inhabits a savanna area of east Africa, south Africa, and the southern part of central Africa, where the air temperature ranges from 20–30˚C throughout the year, and there are pronounced dry and rainy seasons. During the dry season, the frogs burrow underground and form a tough cocoon to reduce evaporative water loss and decrease the respiration rate to less than 10% that during the active stage de novo transcriptome assembly of the African bullfrog Pyxicephalus adspersus. Total RNAs were purified from 11 tissues of young male. The mixed RNA was sequenced on the Illumina HiSeq 2500 platform. The properties of the reads and the assembled sequences are shown in Xenopus tropicalis proteins). The African bullfrog, a species belonging to the family of 22.1The Animal Care and Use Committee of Okayama University approved this work . This research was performed in strict accordance with the recommendation of the Fundamental Guidelines for Proper Conduct of Animal Experiment and Related Activities in Academic Research Institutions under the jurisdiction of the Ministry of Education, Culture, Sports, Science, and Technology in Japan, 2006.2.2Acheta domestica) or wax worms  for the first 3 weeks, and then fed every day with artificial diets  for 2 months.A captive bred African bullfrog was purchased from a specialty reptile and amphibian store . The purchased frog was maintained in a plastic container with coarse sand (5–7 mm diameter) and water. The frog was fed every day with house crickets (2.33 (0.1–0.3 g) and frozen in liquid nitrogen. The frozen samples were maintained at -80˚C. Total RNAs were extracted from the inner organs, intestines, muscles, and skin using the chaotropic extraction protocol for mouse pancreatic RNA, described by DeLisle 3, 0.1–0.3 g) were submerged in 10 ml of TRIZOL Reagent , an amount three times larger than that recommended by the supplier. The tissues were then homogenized at 14,000 rpm three times for 30 s each using the Polytron homogenizer . After incubation for 5 min at room temperature, 2 ml of chloroform was added and the mixture was vortexed for 15 s. The mixture was incubated for 3 min at room temperature and centrifuged at 12,000 g for 10 min at 4˚C. The upper aqueous phase (4 ml) was transferred to a fresh 50-ml tube and mixed with an equivalent amount of isopropyl alcohol. The sample was centrifuged at 12,000 g for 10 min and an RNA pellet was obtained. The RNA pellet was vortexed with 10 ml of 75% ethanol and centrifuged at 7500 g for 5 min at 4 ˚C. The RNA pellet was air-dried and dissolved in 200 µl of RNase-free water by incubating for 10 min at 55 ˚C. Further purification to remove contaminated genomic DNA was performed using a Monarch Total RNA Miniprep Kit , according to the supplier's protocol. The RNA was eluted from a column by 100 µl of RNase-free water and kept at −80 ˚C. The concentration and purity of the isolated RNA was determined using a spectrophotometer and RNA integrity was assessed by RNA ScreenTape assay. The concentrations of RNA isolated from 11 tissue segments of inner organs, intestines, muscle, skin, and head ranged from 0.52–3.34 µg/µl or 0.38–1.60 µg/mg tissue.The young adult frog (15 g body mass) was kept without feeding for 36 h. The frog was anesthetized by placing it into crushed ice for 10 min and then dissected on ice. The intestines were separated, the intestinal contents removed in phosphate buffered saline (pH7.4), and the intestines frozen in liquid nitrogen. The other tissues, including inner organs, muscle, and skin, were quickly excised to 3–5 mm2.4An equivalent amount of total RNAs (0.52–3.34 µg/µl) isolated from 11 tissue segments of inner organs (3 segments), intestines (2 segments), muscles (2 segments), skin (3 segments), and head (1 segment) were mixed to obtain 100 ng/µl total RNA. The mixed total RNA was analyzed by TapeStation  and determined to RIN (RNA integrity number) = 9.0. The poly (A)+ fraction was isolated from the total RNA, followed by its fragmentation. A strand-specific library with an insert size of 200 bp was prepared after conversion of the fragmented mRNA to cDNA and subjected to paired-end 2 × 100 bp sequencing on the HiSeq 2500 platform with v4 chemistry.2.5de novo assembled by Trinity 2.2.0 All analyses were performed mainly using the RNA Galaxy workbench 2.0 2.6https://www.uniprot.org/) . Homologous proteins found in the Uniprot database  with an E-value lower than 1E-5 were subjected to gene ontology (GO) analysis The detected ORFs were homology searched using local BLASTp  against the Uniprot database (The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


C. elegans to human. These domains are thought to mediate interactions between proteins but also between proteins and RNA in complex networks associated with mRNA splicing, chromatin structure, transcription, cell cycle and cell structure. A precise and tight regulation of their function is achieved through phosphorylation of a number of serine residues within the SR/RS motifs by the Serine-Arginine protein kinases (SRPKs) that lead to delicate structural alterations. Given that coronavirus N proteins also contain SR/RS domains, we formulate the hypothesis that the viruses exploit the properties of these motifs to promote unpacking of viral RNA and virion assembly.SR/RS domains are found in almost all eukaryotic genomes from  Coronaviruses (CoVs) have a single-stranded positive-sense RNA genome covered by an enveloped structure. The order of genes (5′–3′) contained in their RNA genome is as follows: replicase ORF1a/b, spike (S), envelope (E), membrane (M), and nucleocapsid (N) . The twoOf the four structural proteins, the nucleocapsid protein N is the one that is responsible for packaging the viral RNA . Many N The N protein of coronaviruses is structurally organized into two domains, the N-terminal domain (NTD) and C-terminal domain (CTD), which are separated by a flexible linker . Both NTThe flexible linkers of the different coronaviruses share very low sequence homology, yet they share some common properties: they are intrinsically disordered regions, they all contain a serine-arginine (SR)-rich domain  and theyViruses hijack cellular machineries in many ways to cause infection and to force the host cell to produce copies of the virus. In the context of this hijacking procedure the viruses take advantage of short linear motifs that have the capacity to encode a functional interaction interface and are found in intrinsically disordered regions of both cellular and viral proteins . These sFollowing entry, the coronavirus genome needs to be translated by the cellular translational machinery to yield initially polyproteins pp1a and pp1ab and then through proteolytic processing the non-structural proteins that will drive viral genome replication and subgenomic mRNA synthesis . For traCoronavirus RNA-dependent RNA synthesis includes two distinct processes: genome replication, during which the coronavirus replicase synthesizes full-length negative “antigenomic” RNA which in turn serves as a template for the synthesis of new genomic RNA and transcription during which the replicase-transciptase complex synthesizes a set of negative sub-genomic RNAs which are then used as templates for the synthesis of positive-sense sub-genomic RNAs encoding the viral structural and accessory proteins . Both thN protein is the most abundantly expressed protein during viral infection  and is sVirus assembly occurs in the ER-Golgi intermediate compartment (ERGIC) and is orchestrated by the M protein , 2019. AThe nucleocapsid is then incorporated into a viral particle. Finally, coronavirus particles budded into the ERGIC are transported in smooth-wall vesicles and trafficked via the secretory pathway for release by exocytosis .Although the N proteins of different coronaviruses share low sequence homology, they all contain an SR/RS domain. A SARS-CoV genetic construct lacking the SR motif from the N gene showed defective replication and significantly reduced levels of infectious virions , highligAll authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.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.


Flexibility in signal transmission is essential for high-level brain function. This flexibility is achieved through strict spatial and temporal control of gene expression in neurons. Given the key regulatory roles of a variety of noncoding RNAs (ncRNAs) in neurons, studying neuron-specific ncRNAs provides an important basis for understanding molecular principles of brain function. This approach will have wide use in understanding the pathogenesis of brain diseases and in the development of therapeutic agents in the future. Brain cytoplasmic RNAs (BC RNAs) are a leading paradigm for research on neuronal ncRNAs. Since the first confirmation of brain-specific expression of BC RNAs in 1982, their investigation has been an area of active research. In this review, we summarize key studies on the characteristics and functions of BC RNAs in neurons. Prior to completion of sequencing of the human genome, the complexity of living organisms had been assumed to derive from the diversity of protein-coding genes, which were estimated to number ≈120,000 in the human genome . The amoNoncoding RNAs (ncRNAs), commonly defined as transcripts that lack the ability to produce a protein, are classified according to size as long noncoding RNAs ) and small noncoding RNAs . About 60,000 lncRNAs have been identified in human cells; as an indication that ncRNA research is still in its infancy, ≈70% of these lncRNAs remain unnamed . AlthougThe brain, the most complex organ in the body, interprets a variety of stimuli from the outside world, generating the appropriate signal and passing it on to other organs. This process, carried out by 80 billion neurons spread throughout the central nervous system , owes muIn 1982, Sutcliffe and colleagues first discovered a polymerase III (pol III)-dependent ncRNA that was expressed only in the rat brain . While aBC1 RNA and BC200 RNA, collectively called brain cytoplasmic RNAs (BC RNAs), are specifically located in neuronal dendrites and control protein translation by inhibiting translation–initiation factors  32,33].,33.32,33Ala (tRNAAla) harboring 3′ unique sequence. Their subsequent study confirmed that BC1 RNA is conserved among rodents, including the guinea pig, Syrian golden hamster, and mouse [In 1984, Sutcliffe and colleagues extended their initial work, examining BC1 RNA expression in additional tissues of the rat, including the adrenal gland, spleen, testis, lung, heart, muscle, and gut. At this point, BC1 RNA had still been identified only in the brain , and wasnd mouse .In 1987, Watson and Sutcliffe identified a 200 nt-long RNA that exhibited brain-specific expression in primates and named it BC200 RNA. This gene is derived from a monomeric Alu element and the transcript has a poly A region similar to BC1 RNA, but the overall sequences are very different ,36. HoweIn 2007, Khanam et al. showed in transgenic mice that the BC200 RNA gene harboring only 250 bp upstream sequences expressed a stable amount of RNA comparable to a construct harboring about 2.3 kb upstream . In 2017Normally, BC RNAs are expressed only in neurons, but this regulated expression disappears completely during tumorigenesis. In 1997, Chen et al. confirmed that BC200 RNA was abnormally expressed in esophageal, lung, breast, colon, and cervical cancer tissues . Since tRecently, Sonawane and his colleagues published important results on tissue-specific gene expression . They coAnother characteristic of the biosynthesis of BC RNAs is that the level of expression is strongly affected by the development and activity of neurons. In 1998, Muslimov et al. observed that BC1 RNA remained low until synapses were fully formed and that its expression was suppressed by neuronal activity inhibitor, tetrodotoxin . ActivitRNA stability is another factor that determines the number of intracellular transcripts . Kim et BC RNAs are strong translational regulators. In 2002, using an in vitro system, Wang and colleagues first discovered that BC1 RNA exerts a translation-inhibitory effect . SubsequThe generation of BC1-knockout (BC1-KO) mice by Skryabin et al. was an important starting point for the physiological study of BC RNAs . In 2004Taken together, these observations indicate that BC RNAs regulate signal transmission by maintaining a balance between translational activation and inhibition in postsynaptic regions. This regulation allows neurons to exhibit experience- and context-dependent plasticity and ultimately perform higher-level brain functions, such as cognition and learning.Although BC RNAs can affect global expression in vitro, they may have a limited effect in vivo ,63. ThisIn 2019, a study by Muslimov et al. confirmed that the dendritic localization of BC200 RNA was severely impaired in the autoimmune disease systemic lupus erythematosus (SLE) . They fuIn 2003, Zalfa and colleagues reported that BC1 RNA binds to fragile X mental retardation protein (FMRP), suggesting that the BC1 RNA-FMRP complex might regulate the translation of specific mRNAs . HoweverBooy et al.’s work offers potential for new regulatory systems of BC RNA function in neurons . Among nBC RNAs are associated with several neuronal diseases . In 2007FMR1 gene on the X chromosome [FMR1 gene, FXS patients have over 200 CGG repeats, which reduces the expression of FMRP protein and causes brain dysfunction [Fragile X syndrome (FXS) is a congenital brain disease characterized by a wide range of neuropsychiatric manifestations, including mental retardation, hyperactivity, and autism . The mairomosome . Indepenromosome . Althougfunction . BC1-KO function ,79, suggFMR1 gene have been reported to directly affect the function of BC RNAs. People with 55-200 CGG repeats inserted into the FMR1 gene are classified as premutation [In addition, CGG repeats of the mutation . Young pmutation . Muslimomutation . They foMuslimov and colleagues recently reported that the mislocalization of BC RNAs is also associated with SLE . ResearcIn 1997, abnormal expression of BC200 RNA was first detected in several cancer tissues, including breast, esophagus, lung, ovary, parathyroid, and tongue . In 2004In 2016, Singh and colleagues constructed BC200 knock-out cell lines by the clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9 (CRISPR/Cas9) system . They obIn 2017, Shin et al. investigated the effect of BC200 RNA on genome-wide expression profiling of the cervical cancer cell line HeLa . As a reThe study of BC RNAs in cancer has a relatively shorter history. Although more detailed research on mechanisms is required, it is interesting to have the potential shown for BC200 RNA to act as a more diverse mechanism within cells compared to previous studies in neurons. Future studies need to confirm whether these mechanisms are also preserved in neurons. Although the environment of BC RNAs varies dramatically from cell to cell, neurons and cancer cells may share some of the same binding proteins and functions of BC RNAs. The BC RNAs of neurons have the potential to regulate gene expression through more complex mechanisms than have been identified to date.If a protein is the object that performs a physical function in a cell, ncRNAs are regulators that control this function so that it occurs at the right time and place . The proThe long history of BC RNA research in the neuron is divided into three stages . In the This flow of studies provides important inspiration for future research of BC RNAs and other ncRNAs in neurons. An enormous number of ncRNAs expressed in the central nervous system have still remained in the first stage ,27,90. M


Viral RNA exported from the coronavirus replication organelle is likely to be mediated by a crown-shaped molecular pore. et al. used cryo–electron microscopy to identify a molecular pore that spans the double membrane . Six copies of a large coronavirus transmembrane protein formed the core of this structure, which may constitute a viral RNA export channel and provide a target for future antiviral interventions.Coronaviruses transform host cell membranes into peculiar double-membrane vesicles that have long been thought to accommodate viral genome replication. However, because these compartments appeared to be completely sealed, it has remained unknown how the newly made viral RNA could be exported to the cytosol for translation and packaging into new virions. Wolff Science, this issue p. 1395; see also p. 1306 Coronavirus genome replication is associated with virus-induced cytosolic double-membrane vesicles, which may provide a tailored microenvironment for viral RNA synthesis in the infected cell. However, it is unclear how newly synthesized genomes and messenger RNAs can travel from these sealed replication compartments to the cytosol to ensure their translation and the assembly of progeny virions. In this study, we used cellular cryo–electron microscopy to visualize a molecular pore complex that spans both membranes of the double-membrane vesicle and would allow export of RNA to the cytosol. A hexameric assembly of a large viral transmembrane protein was found to form the core of the crown-shaped complex. This coronavirus-specific structure likely plays a key role in coronavirus replication and thus constitutes a potential drug target. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the third and most impactful example of a potentially lethal coronavirus infection in humans within the past 20 years , Middle East respiratory syndrome coronavirus (MERS-CoV), and SARS-CoV-2. One advantage of MHV over these class 3 agents is the absence of serious biosafety constraints, thus making MHV suitable for in situ cryo-EM studies. We performed electron tomography (ET) on cryo-lamellae prepared by focused ion beam milling of cells in the middle stage of MHV infection. The tomograms revealed abundant perinuclear DMVs with an average diameter of 257 ± 63 nm (±SD), occasionally interconnected or connected to the ER as part of the reticulovesicular network described in previous work  to analyze the structure of coronavirus-induced ROs in their native host cellular environment. The murine hepatitis coronavirus (MHV) is a well-studied model for the genus Reoviridae  , and engTo investigate whether nsp3 is a component of the DMV molecular pore, we imaged cells infected with a well-characterized engineered MHV expressing nsp3 with an enhanced green fluorescent protein (EGFP) moiety fused to the Ubl1 domain [MHV-Δ2-GFP3  revealedThe molecular pores frequently appeared to interact with other macromolecules on both the cytosolic and DMV luminal sides (fig. S8). In the subtomogram averages, these appeared as largely blurred out densities , which sThe interaction partners of the cytosolic nsp3 prong ranged from chain-like masses to larger assemblies . The subdomains of the long N-terminal nsp3 domain engage in a range of viral and virus-host interactions (Our findings suggest a pathway for newly made viral genomic RNA from the DMV interior, via the channel of the pore, to the cytosolic sites of encapsidation. In our model, specific replicase subunits may associate with the pore complex to guide the newly synthesized RNA toward it . As propThe double-membrane–spanning molecular pore revealed here may constitute the exit pathway for coronaviral RNA products from the DMV’s interior toward the cytosol, with the large and multifunctional nsp3 being its central component. Although the exact mode of function of this molecular pore remains to be elucidated, it seems to be a key structure in the viral replication cycle that is likely conserved among coronaviruses and thus may offer a coronavirus-specific drug target.


RNA-binding proteins (RBPs) play a crucial role in regulating RNA function and fate. However, the full complement of RBPs has only recently begun to be uncovered through proteome-wide approaches such as RNA interactome capture (RIC). RIC has been applied to various cell lines and organisms, including plants, greatly expanding the repertoire of RBPs. However, several technical challenges have limited the efficacy of RIC when applied to plant tissues. Here, we report an improved version of RIC that overcomes the difficulties imposed by leaf tissue. Using this improved RIC method in Arabidopsis leaves, we identified 717 RBPs, generating a deep RNA-binding proteome for leaf tissues. While 75% of these RBPs can be linked to RNA biology, the remaining 25% were previously not known to interact with RNA. Interestingly, we observed that a large number of proteins related to photosynthesis associate with RNA in vivo, including proteins from the four major photosynthetic supercomplexes. As has previously been reported for mammals, a large proportion of leaf RBPs lack known RNA-binding domains, suggesting unconventional modes of RNA binding. We anticipate that this improved RIC method will provide critical insights into RNA metabolism in plants, including how cellular RBPs respond to environmental, physiological and pathological cues. RNA-binding proteins (RBPs) interact with RNAs to form dynamic ribonucleoprotein (RNP) complexes that regulate the fate and function of RNA at virtually every step of its life cycle . TherefoIn the last decade, many efforts have been undertaken to identify RBPs in a comprehensive manner. For example, in vitro studies have employed protein arrays to determine proteins that capture fluorescently-labelled RNAs ,7, or imA recently developed approach termed RNA interactome capture (RIC) can systematically and comprehensively identify the proteins that interact with polyadenylated RNAs in living cells ,13,14. RSince 2012, RIC has been applied to multiple organisms including Trypanosoma brucei , LeishmaArabidopsis thaliana, comprising 717 RBPs. While 75% of the leaf RBPs were annotated to bind RNA, 25% of the RBPs were previously not known to interact with RNA, including metabolic enzymes and proteins from the photosynthetic apparatus. Our analysis revealed cellular RBPs harbouring classic RBDs as well as dozens of proteins lacking known RBDs, indicating unconventional modes of protein–RNA association. ptRIC offers an unprecedented opportunity to study the dynamics of RBPs under different developmental and cellular stages, and in response to changes in environmental conditions. These analyses will allow us to integrate RBP activity into multi-omic analyses to understand plant gene expression.In this study, we report an improved RIC protocol that allows the efficient application of RIC to plant leaves, which are broadly used to study plant biology and are critical for understanding plant physiology and functioning. Using this modified protocol, referred to here as ‘plant RNA interactome capture’ (ptRIC), we identified a comprehensive leaf RBPome for the model plant Arabidopsis thaliana plants (5–6 weeks old) of the Col-0 ecotype were used for all experiments. Plants were grown in soil at neutral day conditions  at 20 °C and a light intensity of approximately 100 μmol/m2/s.Mature A step-by-step protocol including all information about the reagents used is included in the 2 of UV light at 254 nm wavelength. We allowed 30 s pause in between irradiations. For the non-crosslinked (NoCL) negative control, leaves were placed on ice for approximately the same time the crosslinked (CL) samples were maintained on the ice pads during irradiation (~3 min). After irradiation, the leaves were immediately frozen in liquid nitrogen to preserve the molecular interactions and sample integrity. Both CL and NoCL samples were processed in parallel following the same protocol.For UV crosslinking, leaves of mature Arabidopsis plants were excised and placed on a plastic sheet on top of ice pads to prevent sample overheating. The leaves were crosslinked three times with 150 mJ/cmLeaf tissue was ground to a fine powder in liquid nitrogen using a mortar and pestle. Next, 1.2 g of tissue was mixed with 12 mL of lysis buffer in a 50 mL tube. The lysates were kept on ice to minimise RNA degradation. To further homogenise the lysates, a Potter–Elvehjem homogeniser  was used. Samples were homogenised for 1 min, while they were kept on ice to avoid sample overheating. The lysates were cleared by centrifugation  and filtration of the supernatant through Miracloth . To shear the gDNA, lysates were passed through a narrow needle (27 G) five times. The lysates were cleared again by centrifugation  and filtration of the supernatant through Miracloth.wt/v), 0.02% IGEPAL,2.5% PVP40 (wt/v), 1% B-ME (v/v), 5mM DTT, protease inhibitor and RNase inhibitor), using a magnet to trap the magnetic beads followed by supernatant removal. Next, 250 μL of beads was added to each of the lysates and incubated at 4 °C for 1 h in a rotator (10 rpm) to allow hybridisation of the oligo(dT) to the poly(A) tail of the RNAs. The beads were captured on a magnet for ~20–30 min at 4 °C, making sure the supernatant was cleared. The supernatant was collected and stored on ice for a second round of capture. The beads were then washed with 1.5 mL of different buffers with an incubation of 5 min on ice, inverting the tube every 30–60 s, followed by magnet capture and supernatant removal. First, the beads were washed with lysis buffer, which is a stringent buffer that allows the removal of unspecific binders sticking to the oligo(dT) beads or bound to the RNAs non-covalently or via protein–protein interactions. The beads were then washed with harsh buffer (20 mM Tris-HCl (pH 7.5), 2 M LiCl, 1 mM EDTA, 1% LiDS (wt/v), 0.02% IGEPAL (v/v), 5 mM DTT) at room temperature to increase the removal of unspecific binders and avoid precipitation of the LiDS. The harsh buffer contained higher concentrations of the ionic detergent LiDS and LiCL to further increase the stringency of the capture. The beads were then washed two times with buffer I (20 mM Tris-HCl (pH 7.5), 500 mM LiCl, 1 mM EDTA, 0.1% LiDS (wt/v), 0.02% IGEPAL (v/v), 5 mM DTT), two times with buffer II (20 mM Tris-HCl (pH 7.5), 500 mM LiCl, 1 mM EDTA, 0.02% IGEPAL (v/v), 5 mM DTT), one time with buffer III with detergent (20 mM Tris-HCl (pH 7.5), 200 mM LiCl, 1 mM EDTA, 0.02% IGEPAL (v/v), 5 mM DTT), and one time with buffer III without detergent (20 mM Tris-HCl (pH 7.5), 200 mM LiCl, 1 mM EDTA, 5 mM DTT), as this detergent interferes with downstream mass spectrometry analyses. To elute the RNA–protein complexes, beads were resuspended and incubated with 300 μL of elution buffer (20 mM Tris-HCl (pH 7.5), 1 mM EDTA) for 3 min at 55 °C. The beads were pelleted using a magnet and the supernatant containing the RNA–protein complexes was transferred to a new tube and stored at −80 °C.Before starting the oligo(dT) capture, aliquots of 200–500 μL of the input samples  were taken and stored at −80 °C. Oligo(dT) beads (250 μL/sample) were activated by washing them three times with lysis buffer (20 mM Tris-HCl (pH 7.5), 500 mM LiCl, 1 mM EDTA, 0.5% LiDS .For each of the samples, the two eluates from different rounds of capture were pooled and quantified using a NanoDrop spectrophotometer  Elution buffer was used to adjust the volume of the samples so that the same amount of RNA was present in each sample.After isolation of the RNA–protein complexes, we treated each of the eluates with 4 μL of RNase A and T1 mix  for 1 h at 37 °C followed by incubation for 15 min at 50 °C. Samples were then analysed as follows: (i) Western blotting with specific antibodies; (ii) silver staining for total protein analysis; and (iii) quantitative mass spectrometry to identify and quantify the isolated proteins. Out of the 600 μL of the eluate samples, 50 μL was used for Western blotting and silver staining analyses, and the remaining material (550 μL) was used for mass spectrometry (stored at −80 °C).v/v) and incubated for 4 min at 95 °C. Proteins were separated in a 10–12% acrylamide gel followed by western blot or silver staining. Western blots were performed following standard procedures  > 1.5 and an adjusted p-value (FDR) < 0.01 were defined as ‘RBPs’, whereas the remaining proteins (‘non-enriched in +UV/−UV’) were discarded to avoid a high incidence of false positives. During the process of optimising ptRIC, we analysed a pilot experiment by mass spectrometry. For this early experiment, we analysed one CL and one NoCL sample. Therefore, in this pilot experiment we classified a protein as RBP if log2FC [CL/NoCL] ≥ 2.The identified proteins were separated into two groups and analysed using different methods as follows. The proteins with values in the CL samples, which were not detected in any of the NoCL samples (negative control) were analysed using a modified version of the semi-quantitative approach described by Sysoev and colleagues . We clasge limma . The resHochberg  to calcuThe proteins defined as RBPs by either semi-quantitative or quantitative methods were pooled to define a high-confidence Arabidopsis leaf RBPome. Bioinformatic analyses of the Arabidopsis leaf RBPome were performed as described in the https://www.ebi.ac.uk/pride/archive/) with the dataset identifier PXD018141.The mass spectrometry proteomics data were deposited into the ProteomeXchange Consortium via the PRIDE  partner Arabidopsis thaliana. In brief, leaves were detached from the plants and irradiated three times with UV-C light at 150 mJ/cm2 (CL) to induce covalent bonds between RNAs and proteins that were in intimate contact (≤2Å). Next, 1.5 g of tissue was disrupted and cells were lysed, followed by capture of poly(A) RNAs and covalently linked proteins using oligo(dT) magnetic beads. This isolation was performed under stringent denaturing conditions using ionic detergents and high LiCl concentrations to exclusively isolate direct RNA binders, as described by Reichel [2FC [CL/NoCL] ≥ 2). More importantly, 59% were not enriched over the non−UV irradiated negative control, representing a set of unspecific binders that co-purified with RNA in a UV-independent manner  and determined that the optimal crosslinking protocol was three irradiations at 150 mJ/cm2 spaced by a 30 s pause, as previously proposed by Reichel and colleagues [To circumvent this problem, we optimised multiple parameters that we suspected were affecting RBP recovery. A sufficient UV dose is critical for efficient protein–RNA crosslinking; however, excessive UV exposure can lead to RNA damage and, therefore, reduction of RBP yield ,46. Leaflleagues . MoreoveWe suspected that some of the non-enriched unspecific binders A,B couldwt/v) and of LiCl (2M). The inclusion of this wash step was expected to fully dissociate non-covalent interactions that might remain attached to the beads or oligo(dT) probe throughout the RNA capture process. By applying these modifications, we increased the proportion of proteins enriched in UV-irradiated samples over controls to 89% and, as a consequence, the proportion of +UV/−UV non-enriched proteins dropped to 11%  and the chaperone HEAT SHOCK COGNATE PROTEIN 70-3 (HSC70-3), which were classified as RBPs by ptRIC, were enriched in eluates in a UV-dependent manner B. Severa2 fold change between UV crosslinked and non-crosslinked samples (log2FC (CL/NoCL)) for each protein and estimated the false discovery rate (FDR) of each change using data from four biological replicates. We classified proteins as high-confidence RBPs when the log2FC (CL/NoCL) > 1.5 and the adjusted p-value (FDR) < 0.01. Following this method, 427 proteins were classified as high-confidence Arabidopsis leaf RBPs. However, a large number of proteins identified had missing values  in all the NoCL controls  annotations. Importantly, approximately 56% of the leaf RBPs were annotated as ‘RNA binding’, and an additional 19% had annotations linked to RNA biology A, indicaThe leaf RBPs were annotated to be localised not only in the nucleus (219 RBPs) or cytoplasm (303 RBPs), but also in the chloroplast (310 RBPs) and mitochondria (86 RBPs), indicating that ptRIC allows deep analyses of the Arabidopsis RBPome, including discovery of RBPs from different subcellular compartments and organelles C. When cInterestingly, we identified larger numbers of RBPs localising to the chloroplasts than anticipated C. AccordWe expected a strong enrichment in previously characterised RNA-binding domains (RBDs) if our RBPome was enriched in bona fide RBPs. In agreement, based on Pfam annotations about 60% of the leaf RBPs harboured known RBDs , whereas the remaining 40% harboured no recognisable RBDs A. These We identified 280 RBPs of which the RNA-binding activity could be explained by non-classical RBDs. These included ribosomal, pentatricopeptide repeat (PPR), helicase C-terminal and zf-CCHC as the most prominent domains C. The faThe PPR-containing protein family has expanded in plants as compared to metazoans  ,58 and mA large proportion of the identified RBPs did not possess any recognisable RBD  domain. Moreover, proteins containing the DUF1296/GIP1 domain were also identified in the RBPome of etiolated Arabidopsis seedlings . Hence, bind RNA ,66, to obind RNA ,68,69. Abind RNA , their RHence, domains that are enriched in RBPs but lack previous links to RNA biology could represent novel RBDs. However, further experiments have to be undertaken to determine if these domains are endowed with RNA-binding activity. Alternatively, RBPs that do not possess known RBDs could also bind RNA through disordered regions in analogy to other eukaryotic systems . Recent Next, we compared our leaf RBPome with those previously published for Arabidopsis ,35,37. WWe also tested whether the large number of ptRIC-specific RBPs was due to a more efficient capture of RBPs from subcellular compartments. To test this, we constructed a superset of the Arabidopsis RBPs identified in previous studies  and evalTo obtain deeper insights into the 409 plant RBPs exclusively identified here, we analysed their domain distribution across known and newly discovered RBPs . We discbona fide RBPs.Importantly, 42% of the leaf ptRIC-specific RBPs are annotated as ‘RNA binding’ and an additional 25% had previously been linked in some extent to RNA biology . Hence, Moonlighting functions have been described for a number of metabolic enzymes across eukaryotes, including plants ,23,35,71Additional moonlighting enzymes were identified in the leaf RBPome, such as COBALAMIN-INDEPENDENT METHIONINE SYNTHASE, (S)-2-HYDROXY-ACID OXIDASE, GLYCINE DEHYDROGENASE and GLUTAMATE-GLYOXYLATE AMINOTRANSFERASE, which, to our knowledge, have not previously been linked to RNA biology . MoreoveWe observed that about 8% of the leaf RBPs (56 proteins) had annotations related to photosynthesis or photosystems , and of Chlamydomonas reinhardtii contains an RRM domain at the N-terminus that becomes exposed under oxidising conditions and triggers its capacity to bind RNA [Chlamydomonas reinhardtii regulates its own RNA translation by binding the 5′UTR via the C-termini of the protein [A priori this may seem surprising, since photosynthesis has not been extensively linked to RNA metabolism. However, there are multiple lines of evidence that support the hypothesis that components of the photosynthetic apparatus can associate with RNA. Firstly, the intensity of most of these photosynthetic proteins in ptRIC was relatively high, suggesting that these were bona fide RBPs . Secondlbind RNA ,78,79. T protein .Given the evidence that photosynthetic proteins may associate with RNA, it is tempting to speculate that components of the photosynthetic apparatus may act as RNA regulators similarly to moonlighting metabolic enzymes . AlternaWe improved RIC to perform efficiently in plant leaves. This modified protocol, referred to here as ‘plant RNA interactome capture’ (ptRIC), has generated a comprehensive Arabidopsis leaf RBPome, which includes 717 proteins. While 75% of the RBPs identified by ptRIC had known or predicted links to RNA biology, 25% were previously unknown to interact with RNA, offering new avenues of research to the plant community. Notably, ptRIC captured several metabolic enzymes interacting with RNA, and showed that the photosynthetic apparatus engages with RNA through several of its components. Moreover, 39% of the identified RBPs lacked known RBDs, suggesting the existence of novel modes of RNA binding that remain to be discovered. The existence of still uncharacterised RBDs spans from yeast to human and plants, reinforcing the importance of this phenomenon . Because


RNAs adopt specific structures in order to perform their biological activities. The structure of RNA is an important layer of gene expression regulation, and can impact a plethora of cellular processes, starting with transcription, RNA processing, and translation, and ending with RNA turnover. The development of high-throughput technologies has enabled a deeper insight into the sophisticated interplay between the structure of the cellular transcriptome and the living cells environment. In this review, we present the current view on the RNA structure in vivo resulting from the most recent transcriptome-wide studies in different organisms, including mammalians, yeast, plants, and bacteria. We focus on the relationship between the mRNA structure and translation, mRNA stability and degradation, protein binding, and RNA posttranscriptional modifications. RNA molecules fulfill a number of important functions. They take part in not only gene expression, as the intermediate between the DNA and protein, but also in many other cellular processes. It has emerged that the RNA structure, which single stranded RNA can fold into , adds anIn the last decade, the development of new, sophisticated methods for the measurement of RNA structures in living cells has revolutionized the field of structural and functional RNA studies. Coupling RNA chemical probing in vivo with next generation sequencing and advanced bioinformatics tools has allowed for shifting the perspective from low-throughput studies to thousands of heterogeneous RNAs, and even whole transcriptomes, in complex cellular environments . The interplay between the translation and RNA structure constitutes the subject of discussion, and it remains an open question whether the RNA structure guides translation or whether translation guides the RNA structure in cells . In geneE. coli transcriptome complemented with ribosome profiling has indicated that the unstructured sequence upstream of the start codon is a general feature of E. coli genes and the secondary structure in this region is negatively correlated with gene expression [E. coli cells that showed that the translation efficiency is regulated by unfolding kinetics of structures overlapping the ribosome binding site (RBS) [E. coli transcriptome in vivo suggests that TE is only weakly correlated with local RBS structure and is rather regulated by structure of the entire coding sequence (CDS) [Numerous studies have shown that the stable structures in the 5′ UTR and near the start codon of mRNA can repress the effective initiation of translation in bacteria and eukaryotes ,55. The pression . Consistte (RBS) . Accordice (CDS)  . Nevertheless, the RNA stability and translation efficiency are undeniably inseparable and very often, factors, including 3′ UTR binding proteins that control transcript stability and decay, are also engaged in TE regulation ,62,63.E. coli transcripts, especially the coding regions of mRNA tend to be less structured in vivo [Arabidopsis thaliana and yeast, the average in vivo structure of CDSs was higher or not distinguishable from the UTRs, respectively [Oryza sativa), the structure of the CDS regions is higher than 3′ UTR, but lower than 5′ UTR [In general, mRNAs have a lower structure in cells than in vitro, but different RNA structural patterns between species have been found. Across  in vivo ,43. A st in vivo . In addi in vivo . In contectively ,9. Additn 5′ UTR ,42.E. coli transcriptome in vitro [E. coli showed a strong negative correlation between the mRNA structure and TE [The correlation between CDS structure and ribosome density was not found in the PARS study of in vitro , but thee and TE ,43. In ce and TE . FurtherA strong correlation between the RNA structure and TE was also observed in transcriptome-wide DMS-seq analysis of zebrafish embryos during maternal to zygotic transition (MZT) . A decreIn contrast to the above studies, the early genome-wide DMS-seq RNA structure analysis in yeast suggested the global unfolding of RNA in vivo, which is not correlated with translation machinery . In thisA. thaliana transcriptome [Furthermore, a recent study of mammalian RNA structures across subcellular compartments has shown that the majority of the transcripts preserve their structure as they transfer from chromatin to the nucleoplasm and cytoplasm . This sucriptome ,41.Arabidopsis thaliana, and rice (Oryza sativa), is a three-nucleotide periodicity in the mRNA secondary structure [E. coli transcriptome-wide study [An intriguing feature of the coding region detected in diverse organisms in vivo, including, mouse, tructure ,10,42,66de study .The stability and fate of mRNA molecules in the cell are under strict control, and at the end of their life cycle, RNAs undergo a carefully regulated degradation. The cap at the 5′-end and the poly(A) tail at the 3′-end are considered as major determinants of mRNA stability, but it can be also regulated by the other intrinsic features of transcripts, such as the sequence, length, and structure of UTRs and RNA modifications in UTRs ,49,67,68In human and yeast, the structure of 5′ UTRs is negatively correlated with mRNA stability, whereas the secondary structure in 3′ UTRs is associated with longer half-lives and higher abundance of mRNAs ,50,68. TIn contrast to human mRNAs, in yeast, RNA structure content in 3′-ends is similar to other mRNA regions . HoweverAn important role of the 3′-end structure for mRNA stability was also observed in zebrafish in vivo. The 3′ UTRs of zebrafish mRNAs are structurally dynamic and changes in their structure can regulate the stability of mRNAs during MZT by modulating miRNA activity . In addiE. coli [D. melanogaster [Moreover, genome-wide RNA decay analysis indicates that codon optimality is also critical for mRNA stability in many organisms, including E. coli , yeast [E. coli , zebrafiE. coli , D. melanogaster , and humnogaster . The staModern technologies combining high-throughput sequencing with in vivo UV crosslinking and RNA immunoprecipitation , together with new computational approaches provided new insights into the landscape of the RNA binding proteome . To dateS. cerevisiae transcripts with their interacting proteins revealed that many RBPs recognize evolutionary conserved RNA structures in CDS, possibly formed by the degenerated codons [Many RBPs need to recognize a specific RNA sequence or structure for their function in a cell ,86,87, bd codons . These iA recent investigation of human transcriptome focused on the interplay between the structure of RNA and its ability to facilitate protein binding revealed a relationship called the RNA structure-driven protein interactivity, which has an important functional role . Accordi6A modification, facilitates HNRNPC-binding in the chromatin RNA fraction [In contrast, other genome-wide studies in mammalian cells point to the contribution of RBPs to the RNA structural rearrangements that are distinct from ribosome—or ATP-helicase induced RNA unwinding ,37. As dfraction , seems t6-methyladenosine (m6A). Thus, it is not surprising that m6A modification is of particular interest in research. Development of an m6A RNA immunoprecipitation approach followed by high-throughput sequencing (MeRIP–seq) allowed to study m6A modification landscape in a transcriptome-wide manner [6A sites, mainly in the context of the sequence GGm6ACU, in more than 7000 human transcripts. The sites of m6A modification are highly conserved between humans and mice and preferentially appear in internal long exons, around the stop codons, and in the 3′ UTRs. However, the exact functions and, in particular, the effects on RNA folding are still not completely understood. The regulatory function of m6A mRNA modification has been shown in transcription, splicing, mRNA export and stability, and translation [As the RNA, in its life cycle, undergoes numerous post-transcriptional modifications, adding various chemical groups to their bases can significantly influence the RNA folding, stability, and interactions with cellular factors ,95,96. Te manner ,99. Thesnslation ,101,102.nslation , mammalinslation , and planslation ,106.6A influences the RNA structure because of the rotation of the methlyloamino group, from syn to anti conformation, with a higher energy, thus destabilizing the RNA duplexes by 0.5–1.7 kcal/mol [6A can contribute to increasing the stability of the RNA molecule, probably by base stacking. Ex vivo studies of human transcriptome confirmed the structural RNA changes at the m6A modification sites, with a strong tendency for unwinding RNA secondary structure [6A in RNA has been proposed to work as a “molecular switch” for the RNA structure [6A-modified vs. unmodified transcripts, and showed that in the cell RNA regions, both surrounding and including the modified A residues, tend to be unpaired [6A destabilizing effect on RNA duplexes or the modification machinery preferentially methylate adenosine at single-stranded sites of RNA, they performed genetic knockout of N6-adenosine-methyltransferase (Mettl3) in mouse ES cells. The depletion of Mettl3 led to a transcriptome-wide reduction of the SHAPE signal at the m6A modification sites, confirming the RNA destabilizing properties of m6A modification in vivo.Thermodynamic study has shown that mkcal/mol . The opptructure . Therefotructure . Spitaleunpaired . To chec6A enriched regions in transcripts are far less structured than the same, but unmethylated, sites of RNAs, and the patterns of m6A-induced structural destabilization are similar in chromatin, nucleoplasm, and cytoplasm [METTL3-METTL14 complex’s localization to specific nuclear loci [6A local destabilization of RNA for their binding. For example, in the chromatin fraction, m6A modification can facilitate the binding of HNRNPC by the disruption of the local RNA secondary structure in close proximity to the binding sites [6A can work as an “RNA molecular switch” in plants [6A in rice has been observed only in 3′ UTR and not in the CDS or 5′ UTR regions.Recent transcriptome-wide studies across the three different cellular compartments in mammalian cells further supported that mytoplasm . Howeverear loci ,108. Furng sites ,107. In n plants . HoweverRecent transcriptome-wide studies have significantly increased the knowledge about the interplay between the RNA secondary structure and RNA functions in cells. They allow for considering the influence of various cellular factors on RNA folding in vivo, and vice versa, as well as the impact of the RNA structure on critical biological processes. However, there are still many unanswered questions and challenges. For example, it remains unclear whether the RNA folding pattern is species-specific or observed differences result from application of various RNA structure probing methods. An important area for advancement is further development of methods for more accurate studying of the coexisting RNA conformers, RNA co-transcriptional folding, and differentiation of intramolecular RNA interactions and intermolecular RNA–protein or RNA–RNA bindings. Together, with continuous experimental/technical development, there is also a need to advance the computational tools not only for high-throughput data analysis, but also for the experimentally supported accurate modeling of the RNA structure in the native in vivo form.


Rationale: Long extracellular RNAs (exRNAs) in plasma can be profiled by new sequencing technologies, even with low abundance. However, cancer-related exRNAs and their variations remain understudied.Methods: We investigated different variations  in diverse long exRNA species (e.g. long noncoding RNAs and circular RNAs) using 79 plasma exosomal RNA-seq (exoRNA-seq) datasets of multiple cancer types. We then integrated 53 exoRNA-seq datasets and 65 self-profiled cell-free RNA-seq (cfRNA-seq) datasets to identify recurrent variations in liver cancer patients. We further combined TCGA tissue RNA-seq datasets and validated biomarker candidates by RT-qPCR in an individual cohort of more than 100 plasma samples. Finally, we used machine learning models to identify a signature of 3 noncoding RNAs for the detection of liver cancer.Results: We found that different types of RNA variations identified from exoRNA-seq data were enriched in pathways related to tumorigenesis and metastasis, immune, and metabolism, suggesting that cancer signals can be detected from long exRNAs. Subsequently, we identified more than 100 recurrent variations in plasma from liver cancer patients by integrating exoRNA-seq and cfRNA-seq datasets. From these datasets, 5 significantly up-regulated long exRNAs were confirmed by TCGA data and validated by RT-qPCR in an independent cohort. When using machine learning models to combine two of these validated circular and structured RNAs  with a miRNA (miR-122) as a panel to classify liver cancer patients from healthy donors, the average AUROC of the cross-validation was 89.4%. The selected 3-RNA panel successfully detected 79.2% AFP-negative samples and 77.1% early-stage liver cancer samples in the testing and validation sets.Conclusions: Our study revealed that different types of RNA variations related to cancer can be detected in plasma and identified a 3-RNA detection panel for liver cancer, especially for AFP-negative and early-stage patients. Recent studies have shown that circulating cell-free RNAs (cfRNAs) in liquid biopsies are promising biomarkers PD-L1, an mRNA transcript associated with the response to treatment for non-small lung cancer showed reliable clinical performance in plasma PCA3, was identified as a prostate cancer biomarker in urine RN7SL1, a signal recognition particle RNA (srp RNA), was found to be correlated with breast cancer progression circMYBL2, regulates FLT3 translation by recruiting PTBP1 to promote FLT3-ITD AML progression Previous exRNA biomarker studies have primarily focused on miRNAs In addition to change in abundance of RNAs, post-transcriptional RNA isoform variations are also associated with cancer development and progression, and such variations could serve as cancer biomarkers In comparison with small RNA-seq, long RNA-seq can provide more information than abundant level. For instance, long RNA-seq can be used to detect various types of RNAs, including circRNA and lncRNA, as well as post-transcriptional variations like alternative splicing and editing of mRNAs. Therefore, in this study, we investigated the expression profiles of long exRNAs in multiple cancer types in order to find cancer-related long exRNA species and their variations in plasma samples. Moreover, we validated selected liver cancer variations with multiple cohorts and various methods, including exosomal RNA-seq (exoRNA-seq), TCGA tissue RNA-seq, cell-free RNA-seq (cfRNA-seq), exosome enrichment assays, and RT-qPCR. Our findings revealed a multi-RNA panel that could be used for the diagnosis of liver cancer, especially for alpha feto-protein (AFP)-negative and early stage patients.We first analyzed exosomal RNA-seq (exoRNA-seq) data from patients with three types of cancer and healthy donors (HDs) in 79 datasets that were curated by exoRBase 2FC > 0 or < 0, △PSI > 0 or < 0 and △editing ratio > 0 or < 0) among the CRC, HCC, and PAAD patients  among the RNA variations identified in CRC, HCC, and PAAD patients Figure C, suggess Figure D.In addition to the variations found in CRC, HCC, and PAAD patients, we also evaluated the functional relevance of variations specific to each cancer type. We curated known cancer genes (positive controls), as well as non-cancer genes (negative controls) We further investigated functional coherence by calculating enrichment in curated pathways from the Kyoto Encyclopedia of Genes and Genomes (KEGG) We used liver cancer as an application example and identified recurrent variations in multiple datasets Figure . First, We previously identified 1,168 differentially expressed genes in exoRNA-seq data Figure  to identUGT2B7 and CAMK4), 2 circRNAs (circ-0073052 and circ-0080695), 2 lncRNAs (HULC and LINC01226), and 1 snoRNA (SNORD3B-1). Additionally, we collected 50 tumor and paired normal tissue RNA-seq datasets of liver cancer from the TCGA database and evaluated the expression levels of these genes in tissues. The results of these experiments revealed that 6 of the 7 selected genes had similar expression patterns in the TCGA data and the tissue samples , we selected 7 of 122 recurrently differentially expressed genes for further assays, including 2 mRNAs  (see Methods in detail) for further validation. However, none of these 3 genes had the same AS pattern in multiple samples from the TCGA or exoRNA-seq data  as potential exRNA biomarkers for further analysis and validation.Finally, we validated 10 candidate events  using RT-qPCR in the 26 samples that were previously sequenced . ActuallHULC has already been established as a diagnostic biomarker of HCC HULC and novel biomarker candidates LINC01226, circ-0073052, circ-0080695, and SNORD3B-1.The 5 selected exRNAs include various types of RNAs, including lncRNA, circRNA, and snoRNA. Among them, the lncRNA SNORD3B-1, we collected the structure profile from the Rfam database and RBP binding hotspots from the POSTAR2 database SNORD3B-1 was enriched with RBP binding sites and RNA secondary structures . The shape and size of exosomes were typical of extracellular vesicles (EVs)  of the 6 miRNAs Previous studies identified miRNAs as HCC diagnosis biomarkers, so 6 known miRNA biomarkers SNORD3B-1, circ-0080695, and miR-122) obtained the highest average AUROC (89.4%) in comparison with a set of 5 long exRNAs (76.9%), a set of 6 miRNAs (73.4%) SNORD3B-1, circ-0080695, and miR-122 as a 3-RNA detection panel for liver cancer  data were downloaded from exoRBase Cell free RNAs (cfRNAs) were isolated from ~1 ml plasma using the Plasma/Serum Circulating RNA and Exosomal Purification kit  according to the manufacturer's protocol. The DNA residuals in the cfRNAs were digested using Recombinant DNase I (RNase-free). We then cleaned up the cfRNAs using RNA Clean and Concentrator-5 kit (Zymo), and eluted them with 7 µL elution buffer.We added 1µL External RNA Controls Consortium (ERCC) spike-in (with suitable dilution) to the 7 µL eluted cfRNAs as input to prepare the cfRNA sequencing libraries, with SMARTer Stranded Total RNAseq-Pico Input Mammalian kit (Clontech) according to the manufacturer's instructions. Finally, the RNA libraries were sequenced on Illumina XTen (2 × 150 bp) platform with depth of >10 million reads per sample.We used 32 plasma samples  for the In miRNA RT reactions and single-primer qPCR experiments, we used the miRcute Plus miRNA First-Strand cDNA Synthesis Kit and miRcute Plus miRNA qPCR Detection Kit . In long RNA RT reactions, we used TIANScript II First-Strand cDNA Synthesis Kit and FastFire qPCR PreMix (SYBR Green) Kit . Each Ct value was the average of 3 replicates, and the detection limit was lower than 0.1 pg. We list primer sequences for the RT-qPCR of long RNAs in HULC as a positive control for lncRNAs in liver cancer We used We used Cutadapt to remove adapters and read pairs with an average quality score below 30 in either read of the pair. Paired reads were first mapped to the rRNA database (from NCBI RefSeq database). The unmapped reads were then mapped to the human genome sequences (UCSC genome build hg38). A genome index was generated using STAR with splicing junction annotations from GENCODE V27. Remaining reads after the first two steps were mapped to the circular RNAs from circBase. We only considered a paired reads (from 5'-end in read 1 to 3'-end in read 2) that overlapped with the back-splicing junction as circular RNA reads. Only read pairs mapped to the human genome or circular RNAs were included in further analyses. We removed duplicated read pairs using picard MarkDuplicates because the dataset contained a large proportion of duplicated reads due to PCR amplification in library construction. Finally, a gene count matrix was generated using featureCounts with options “-M -p -s 1 -t exon -g gene_id”.We used DESeq2 When selecting candidate events recurrently identified in both exoRNA-seq and cfRNA-seq Figure A, we ranIn order to obtain differentially edited sites, we applied RNAEditor The alternative splicing analysis was performed by rMATS When selecting recurrent events Figure C, we staTo discover alternative polyadenylation events, we used DaPars We downloaded the cancer gene sets as positive controls from the following resources: the Network of Cancer Genes (NCG5) We downloaded the gene sets unrelated to cancers as negative controls from the following resources: a conservative version of the negative AGO list (NegAGOClean) Finally, differential expression, alternative splicing, alternative polyadenylation, and differential RNA editing gene sets were overlapped with above gene sets. The ratios of the overlapped genes over the size of each gene set were calculated.P-value were annotated as tumorigenesis and metastasis, immune or metabolism-related pathways.Pathway analyses were carried out using Metascape P-values of less than 0.05 were considered to be statistically significant.All tests for comparing the expression levels in different groups were Wilcoxon rank sum tests. Machine learning methods were applied to evaluate the classification performance of the selected features. We used cross-validation for model selection. Samples were subjected to 5-fold cross-validation with 10 repeats (RepeatedStratifiedKFold in the scikit-learn package), yielding 50 class-balanced independent training and testing sets in an 80% - 20% manner. AUROC (Area Under the Receiver Operating Characteristic curve) was utilized as the performance metric. We applied a grid search strategy (GridSearchCV in scikit-learn package) to select hyper-parameters that maximize the classification score (score method of classifiers in scikit-learn package) . The impTo compare and select an optimal feature set among 11 features (5 ncRNAs and 6 miRNAs) in the RT-qPCR data sets, we traversed feature numbers of 3, 5, 7, 9, and all of the corresponding feature combinations . The combination with the highest average AUC among all possible combinations was selected. Due to the instability of random partition and small sample size, we averaged the results of 50 cross-validations to obtain a ROC curve and measured the prediction performance of different feature combinations.Finally, a Random Forest model-based panel with the selected 3 features  was trained on the AFP-positive samples, tested and validated on the AFP-negative samples. The original RT-qPCR values were scaled to 0-1 in the machine learning model-based panel.Supplementary figures and tables.Click here for additional data file.


Many of the currently used methods rely on the particular reverse transcription signatures left by RNA modifications in cDNA; these signatures may be naturally present or induced by an appropriate enzymatic or chemical treatment. The newest approaches also include labeling at RNA abasic sites that result from the selective removal of RNA modification or the enhanced cleavage of the RNA ribose-phosphate chain , followed by specific adapter ligation. Classical affinity/immunoprecipitation-based protocols use either antibodies against modified RNA bases or proteins/enzymes, recognizing RNA modifications. In this survey, we review the most recent achievements in this highly dynamic field, including promising attempts to map RNA modifications by the direct single-molecule sequencing of RNA by nanopores.The precise mapping and quantification of the numerous RNA modifications that are present in tRNAs, rRNAs, ncRNAs/miRNAs, and mRNAs remain a major challenge and a top priority of the epitranscriptomics field. After the keystone discoveries of massive m Post-transcriptional RNA modifications  can be detected in RNA while using various methods and approaches exploiting the chemical and physico-chemical properties of these non-canonical RNA nucleotides. In addition to classical RNA techniques, such as 5’/3’ and specific internal labeling as well as nucleoside/RNA oligonucleotide analysis by Liquid Chromatography coupled to Mass Spectrometry (LC-MS) or tandem Mass Spectrometry (LC-MS/MS), methods that are based on second- (abbreviated as NGS for Next Generation Sequencing) and third-  generation sequencing become increasingly popular. These approaches aim to provide single-nucleotide resolution for the identification of the modified RNA position, but they may be less accurate in the exact nature of the modified residue due to a rather generic treatment used during the library preparation step. The most popular and reliable methods using NGS analysis rely on various specific chemical treatments that are applied to specifically alter RNA-modified residues to make them detectable either as RT-stop or as a mis-incorporation of nucleotides into cDNA. Actually, NNGS approaches mostly use ion-current profiles through the nanopore or kinetics of deoxynucleotide triphosphates (dNTP) incorporation in PacBio chips to deduce the presence of unusually modified nucleotides.6–107 nucleotides), even methods with an extremely good False Discovery Rate (FDR )<0.001 will still provide thousands of false positive hits. Thus, extreme care should be taken in the interpretation of large transcriptome-wide datasets claiming the presence of hundreds, or even thousands, of detected RNA modified nucleotides  and NNGS (single-molecule sequencing) the nature of the signal may be only indirectly related to the chemical nature of the RNA modification, thus mis-identifications are not only possible, but actually rather frequent. In addition, if the analysis is performed at the whole-transcriptome scale (→10The mapping of RNA modifications by NGS approaches is mainly based on: (1) altered base pairing during a reverse transcriptase (RT)-driven primer extension step, (2) altered chemical reactivity of the base due to a specific reagent (3) associated cleavage of the ribose-phosphate chain, and (4) a differential recognition of modified RNA nucleotides by specific antibodies (Ab) or proteins. In some instances, an enzymatic treatment or in vivo metabolic labeling can be used to exacerbate the chemical reactivity of a given modified nucleotide. Altered base-pairing is typically exploited for RNA modifications bearing extra chemical groups at the Watson–Crick (WC) edge of the base  or for ‘RT-silent’ modifications after chemical derivatization affecting their WC edge. Methods that are based on the specific cleavage of the phosphodiester bond either rely on altered recognition by a specific enzyme or on the formation of an RNA abasic site, followed by a specific (and highly selective) ligation step. Antibody (protein enrichment) protocols exploit differential noncovalent or covalent binding to the modified site using UltraViolet light (UV) or chemical cross-linking steps.Current approaches using NNGS (single-molecule sequencing) are mostly based on the use of direct nanopore RNA sequencing. Indeed, the profile of ion current registered for modified nucleotides passing through the nanopore is substantially altered when compared to the unmodified counterparts. While the experimental setup is generally rather straightforward and the RNA treatment is not different from classical RNA-Seq analysis, the extraction and analysis of raw nanopore sequencing data require complex and time-consuming bioinformatics treatment. Because of these limitations, NNGS methods are only at the emerging stage and cannot be considered to be an alternative to replace established NGS protocols. This will certainly evolve in the nearest future, and experience that accumulated in now-routine NGS analysis will be extremely helpful in the development of NNGS single-molecule analysis.The analysis of RNA modifications by NGS is still a very recent topic (the first publications in the field are from 2012); however, a number of excellent review articles have already covered previous achievements ,4,5,6,7.Different principles are currently employed for the detection of RNA modifications in the epitranscriptome using NGS and NNGS. We classify them in: (1) an analysis of RNA signatures that are visible in sequencing profiles , (2) the treatment-induced cleavage of the RNA phosphodiester chain followed by a selective ligation of sequencing adapters, and 3) affinity-based enrichment protocols exploiting the specificity of polyclonal or monoclonal antibodies and specific enzymes installing modifications in RNA. In many instances, the developed protocols use a combination of different principles .Natural RT signatures consist of the altered reading of the modified nucleotide during primer extension by an RNA-dependent RNA polymerase . Depending on the nature of the nucleotide, such a signature may represent a “mutation” as compared to the expected reference sequence or constitute a more complex profile composed of mis-incorporations at different proportions in addition to the abortive RT products that end at the modified nucleotide. Such RT signatures can be manipulated by the pre-treatment of the RNA template , by the choice of a particular condition of the primer extension, or by the use of non-natural dNTP substrate(s) and mutants of the RT active site. The in vivo metabolic labeling described later in this review also allows for the incorporation of reactive chemical groups at positions of certain modifications (methyl groups) and, thus, also helps to alter the RT signature of the modified RNA template D.The adenosine-to-inosine (A-to-I) deamination editing events in RNAs are catalyzed by enzymes from the ADAR/ADAT family. Because inosine base pairs with C and not with U (T) anymore, such events show up as A to G substitutions when the sequences of the genomic DNA and the cDNA are compared A. Such “3C, m1A, m1I, m22G, and m1G sites in human tRNAs, of which a selection of predicted m3C sites was experimentally validated. Later, the application of a specific RT-signature was developed for m1A mapping in low-complexity RNA (such as rRNA and tRNAs)  can also affect the properties of RTs. This was explored for the detection of m1A, m22G, m1G, and m3C by replacing Mg2+ with Mn2+ in the reaction buffer of four different RTs. The arrest rate and mis-incorporation profiles are both strongly modulated under these altered conditions, with an increase in nucleotide skipping (deletions). Every RT polymerase shows an individual sensitivity to increased [Mn2+] and it depends on the nature of the RNA modification tested, allowing for a more reliable detection [The modulation of divalent metal ions [Mgetection .6A due to the restricted base pairing and, thus, was used to generate a specific m6A signature in RNA [Finally, the detection of RT-silent RNA modifications can be achieved by the use of synthetic base-modified dNTP. Selenium substituted thymidine (4Se-dTTP) was found to induce RT stops at me in RNA .Thermus thermophilus in the original study) show a relative selectivity during the incorporation of nucleotides opposite m6A in the RNA template [6A-modified residues at selected loci.Native DNA polymerases with RT activity . The selected mutant carrying six amino acid replacements shows both a robust RT primer extension activity and a well-defined RT signature at the modified position, suitable for the mapping and quantitative assessment of m1A modification [A similar approach was also applied for the selection of HIV-1 RT variants showing a particular sensitivity towards N1-methyladenosine (mfication .A proof of concept for metabolic RNA labelling while using the S-adenosyl-L-methionine (SAM) analog was first demonstrated by the enzyme-catalyzed transfer of alkynyl moiety onto RNA . However6A mapping in cellular RNAs by the combination of SAM analog bearing the biorthogonal propargyl group and the “click-chemistry”-driven biotin labeling of the modified RNA molecules. For in vivo labeling, the SAM analog was replaced by propargyl-L-selenohomocysteine  that is used in SLAM-seq [4U, which causes s4U to be retrotranscribed as a C, increasing the apparent transition rate to near-quantitative. While this method was mostly applied to trace the incorporation of s4U nucleoside into a nascent RNA in metabolic labeling experiments, it is certainly suited to trace posttranscriptional s4U modifications in bacterial tRNAs.Thio-substitued uridine at position 4 is a natural modified nucleotide that is found at position 8 in bacterial tRNAs, but it is rather known as an artificially introduced random RNA modification widely used in Photoactivatable Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation (PAR-CLIP) experiments . It was SLAM-seq  alters t4U was first introduced by metabolic labeling followed by conversion to cytidine residues by osmium (OsO4) treatment, thus abolishing the RT signature and allowing for a direct assessment of s4U-labeled RNA [In another sequencing approach (named TUC-Seq), seled RNA .5C mapping and quantification by Schaefer and Lyko [The conversion of C-to-U residues in DNA and RNA by bisulfite is probably the most used chemical reaction in nucleic acid chemistry. Bisulfite RNA sequencing, which was adapted from 5mC detection in DNA, was described for mand Lyko . The metand Lyko ,49,50,51and Lyko ,53,54,55and Lyko ,57,58.Optimized and presumably more reliable bisulfite conversion protocols have been proposed with improved C-to-U conversion rates in the presence of formamide  and with5C in RNA were suggested based on DNA-related protocols [5C by NaBH4, and hm5C in RNA is detected by its resistance to deamination, similarly to m5C. Alternatively, f5C is first protected by O-ethylhydroxylamine (fCAB-Seq for RNA), and this adduct becomes resistant to subsequent deamination [As a further extension of RNA bisulfite deamination chemistry, approaches for the detection of frotocols ,60. Thismination .5C/hm5C in RNA was also suggested. It uses the selective oxidation of hm5C to trihydroxylated-thymine (thT) that is mediated by peroxotungstate. The thT residue base pairs with A during the RT step and, thus, becomes T in sequencing. If combined with the TET-mediated oxidation of m5C to hm5C, this method is also suitable for base-resolution m5C mapping [A bisulfite-free single-base-resolution mapping protocol for m mapping .5C by potassium oxoruthenates (K2RuO4), followed by an aldol-type addition-elimination-cyclization sequence [5C detection in RNA.Of note, in DNA 5hmC can be oxidized to fsequence , and the6A mapping and quantification. The protocol, termed NOSeq [6A to chemical deamination under conditions where all other RNA nucleotides are efficiently converted. The protocol allows for targeted sequencing for the confirmation of m6A’s presence and the relative quantification of the modified residue.Recently, the global deamination of all RNA bases by nitrous acid treatment was used for med NOSeq , exploitN-Cyclohexyl-N’-(2-morpholinoethyl)carbodiimide metho-p-toluenesulfonate, usually abbreviated as CMCT) [Classical approaches for RT-silent pseudouridine (Ψ) detection are based on the use of soluble carbodiimide (as CMCT)  and theyas CMCT) ,70,71,72as CMCT) ,74. Openas CMCT) , potentias CMCT) ). Carbodas CMCT) .2+ ions, the Ψ-monobisulfite adduct undergoes heat-induced ribose ring opening and Mg2+-assisted reorientation, causing base skipping during cDNA synthesis. These 1–2 base deletions in the read sequence can be detected and used for precise Ψ mapping by NGS [Alternative chemistry for Ψ detection in RNA was proposed in RBS-Seq, the protocol that uses RNA bisulfite deamination under optimized conditions. In the presence of Mgg by NGS .1A, which causes mis-incorporation and abortive cDNA synthesis [6A. This reaction was essentially used for validation by observing the disappearance of m1A RT-signatures in RNA incubated under alkaline conditions at high temperatures [Under alkaline conditions, mynthesis , undergoeratures ,78.4C), which is present in tRNAs and rRNAs, a chemical reduction with sodium borohydride (NaBH4) leading to the saturation of the 5,6 double-bond in acetylated cytidines was employed. Reduced ac4C, in turn, produced ~20–30% of mis-incorporation signals in cDNA, as determined by Sanger sequencing [3) under acidic conditions. This new mapping approach did not confirm previously reported mRNA ac4C data, but revealed a temperature-dependent modulation of rRNA/ncRNA and mRNA acetylation from hyperthermophilic archaea [For the 4-acetylcytidine , allows for the high-throughput detection of m7G modifications at a single-nucleotide resolution [Samples were treated with NaBHsolution .4 reduction of m7G was used for its profiling in human miRNAs, but the resulting RNA abasic site was used for Schiff base formation with Biotin-ARP (Aldehyde Reactive Probe) reagent containing the reactive NH2 group for the selective enrichment of the modified sites  [7G in miRNA is still under debate [The same NaBHRed-seq)  , led to the scission of the tRNA chain at the modified nucleotide and the position of the cleavage can be followed as RT-stop in RNA-Seq data in a combination termed TRAC-seq [The reduction of mTRAC-seq ,101. Dem7G detection, named AlkAnilineSeq [7G . Aniline cleavage results in downstream RNA fragments with 5’-phosphate, and a selective 5’-primer ligation ultimately led to a positive selection of fragments and, thus, to an exquisite sensitivity  are also sensitive to hydrazine, a comparison between native and AlkB-treated (to remove m3C residues) samples is mandatory.The same chemical reagent, hydrazine, was also proposed for the specific mapping of m in RNAs . The tec4) oxidation. RNA is first randomly fragmented either chemically (Zn2+) through cleavage at 95 °C (Nm-Seq) or enzymatically by benzonase (RibOxi-Seq), (de-phosphorylated if necessary) and subjected to 3’-terminal ribose oxidations by NaIO4. 3’-terminal Nm residues resist cleavage and remain competent for the ligation of 3’-adapter, while unmodified riboses are converted to a non-ligatable dialdehyde structure (4 oxidation/aniline treatment/de-phosphorylation is strictly required. Better enrichment is achieved when multiple cycles (up to eight cycles used in Nm-Seq protocol) of 3’-terminal nucleotide removal are performed. In comparison, Nm-Seq provides a better sensitivity in Nm detection, but it requires substantial amounts of input RNA, due to the inevitable loss occurring during NaIO4-oxidation/aniline treatment/de-phosphorylation steps.Two conceptually similar methods, Nm-Seq  and RibOtructure E. Becaus6A at a single-nucleotide resolution was achieved by use of MazF endonuclease, which is sensitive to the m6A methylation status of ACA motifs, overlapping with the classical DRACH  motif for m6A modification by METTL3 [6A-related protection, some ACA sites are absent from the library and this proportion can be quantified. The systematic quantitative profiling of my METTL3 . RNA is The use of specific antibodies for the detection of RNA modifications was proposed and successfully implemented in the late 1970s ,109,110,MeRIP-Seq is the most straightforward application of specific antibodies for RNA modification analysis. RNA is fragmented into pieces (100–150 nt in length), immunoprecipitated with RNA modification-specific antibody, and the enriched fragments are converted to libraries and sequenced A. A comp6A detection [1A mapping [5C [4C [7G [The applications of MeRIP-Seq for RNA modification detection are rather numerous, starting with cornerstone publications on metection ,119. A s mapping , as revi mapping . More reping [5C , ac4C [1 [5C [4C , and m7G [4C [7G .6A and m6Am [6Am detection by miCLIP, differential maps were generated using PCIF1 KO [4U) and cross-linking by mild UV-light irradiation at 365 nm [Further improvements in MeRIP-Seq specificity and resolution were brought about by the inclusion of a UV-crosslinking step in addition to Ab-driven immunoprecipitation B. Specifand m6Am ,125, as and m6Am . For spePCIF1 KO . PAR-CLIt 365 nm .5C-MTases, including two distinct steps: the methylation of the target base and the recycling of the covalent intermediate to release the enzyme from the covalent complex with RNA [The idea to use an RNA modification enzyme  for covalent attachment to the target sequence in RNA is based on a two-step mechanism of mwith RNA ,130,131.E. coli radical SAM m8A-MTase RlmN by miCLIP-MaPseq [A high-throughput implementation was developed for human NSUN2 catalytic mutant (C271A) and it was called individual-nucleotide-resolution crosslinking and immunoprecipitation (iCLIP) protocol C. This aP-MaPseq ; for a rP-MaPseq .5C-MTase in vivo [An alternative cross-linking protocol, called Aza-IP (AzaIP-Seq), uses 5-azacytidine-mediated RNA immunoprecipitation to form a covalent bond between an RNA substrate and native m in vivo . Crossli in vivo . The use in vivo . 5U54-MTase RUMT [The formation of covalent adducts with RNA substrate was also known for mase RUMT , and thease RUMT . Thus, tase RUMT   is an attractive alternative to classical cluster sequencing protocols. Indeed, cluster sequencing involves an amplification step, providing only an average picture of modifications in a population of RNA molecules. Single-molecule analysis should be performed to obtain information regarding the exact combinations of modifications in a given RNA chain  .6A) by single-molecule sequencing was established seven years ago by using the PacBio single-molecule, real-time (SMRT) technology. HIV-1 and AMV RT were loaded to a zero-mode waveguide (ZMW) chip and the extension of DNA primer on the RNA template was monitored [The proof of principle for the analysis of RNA modifications  as well as pseudouridine modifications [More recent examples of single-molecule RNA sequencing for the detection of RNA modifications concern direct RNA sequencing by nanopores (Oxford Nanopores). Using direct RNA sequencing, it was demonstrated that mualities . With apications . 6A Identification using Nanopore Sequencing), which assigns m6A methylation status to more than 13,000 previously unannotated DRACH  sites in endogenous HEK293T transcripts and identifies more than 40,000 sites with isoform-level resolution in a human mammary epithelial cell line [It is clear that the major challenge in the field of direct RNA sequencing and RNA modification mapping by nanopores consists of the use of appropriate data analysis software and algorithms. Analysis can be either conducted by standard base calling and the identification of “sequencing signatures” or by the extremely laborious, but direct, analysis of ion current traces. The first solution is implemented in the software MINES , was trained on various types of synthetic modified RNA and applied to rRNA and mRNA sequencing. ELIGOS is able to accurately predict known classes of RNA methylation sites (AUC > 0.93) in rRNAs from an cells . Model-ban cells . Anotheran cells . 5C modification in SARS-Cov-2 [In the context of the COVID-19 pandemic, the direct RNA nanopore sequencing of full-length coronavirus genomic RNA allowed for us to predict multiple sites of mRS-Cov-2 . HoweverRS-Cov-2 . In conclusion, direct RNA modification analysis by nanopore sequencing is rapidly developing and improving in reliability, but it has still not reached maturity for routine application in RNA epitranscriptomics. Thus, classical approaches are still widely used in the routine analysis of RNA modifications, and the use of nanopores is only envisaged as an alternative validation technique.