Cherry virus A (CVA) is a member of the genus Capillovirus in the family Betaflexiviridae. It has a positive single-stranded RNA genome of ~ 7400 nucleotides (nt). The length of the CVA 5′-UTR is ~ 100 nt; however, the function of this long UTR has not yet been reported.A variety of FLuc (FLuc), and in vitro translation of the corresponding transcripts was evaluated using wheat germ extract (WGE). Then, in-line structure probing was performed to reveal the conserved RNA structures in CVA-5′UTR.Molecular and phylogenetic analyses were performed on 75 CVA sequences, which could be divided into four groups, and the RNA secondary structure was predicted in four CVA 5′-UTR types. These four CVA 5′-UTR types were then inserted upstream of the firefly luciferase reporter gene FLuc construct containing these four CVA 5′-UTR types increased the translation of FLuc by 2–3 folds, suggesting weak translation enhancement activity. Mutations in CVA 5′-UTR suppressed translation, suggesting that the conserved RNA structure was important for function.The four CVA 5′-UTR types appeared to have a conserved RNA structure, and the The conserved RNA secondary structure was identified by structural evolution analysis of different CVA isolates and was found to regulate translation. In viruses, the biological functions of RNA based on their spatial structures and various cis-acting RNA elements are translation, RNA synthesis, and genome packaging –4. EfficCapillovirus in the family Betaflexiviridae, was first reported in 1995  ATP, which was purified by electrophoresis through 5% denaturing polyacrylamide gels. End-labeled RNA denatured at 75 °C, followed by slowly cooling to 25 °C. For in-line probing, 5 pmol of end-labeled RNA was incubated at 25 °C in 50 mM Tris–HCl (pH 8.5) and 20 mM MgCl2 for 12 h. Samples were separated by 8% polyacrylamide gel electrophoresis (8 M urea) alongside a hydroxide-generated RNA cleavage ladder and RNase T1 digestion product on labeled RNA fragments. Then, the gels were dried and exposed to a phosphorimager screen, followed by detection with the Typhoon FLA 7000 .In-line probing was performed as previously described . BrieflyTo effectively investigate the CVA RNA structure, a population that was neither genetically highly structured nor interrelated was selected. Using SNPs , principal component analysis (PCA) was performed and a phylogenetic tree was constructed to quantify the population structure of the 75 variants. On the basis of the phylogenetic tree and PCA analyses, five clades were classified—types 1–4 and Admix  of the four types and all CVAs showed great variation along the whole genome Fig. . The π vGene expression was possibility controlled by the 5′-UTR structure determined by nucleotide sequence. The MEME program was used to predict the motifs of the 5′-UTR sequences in the four types Fig. . Almost CUUUACAGAGCUUCCCAACUGUAAAG, that forms a stem-loop in which the under lined bases are paired. And its loop is particularly rich in pyrimidine. Thus we hypothesized that the polypyrimidine sequences could interact with a region of 18S rRNA to enhance translation activity, as was previously found for Tomato bushy stunt virus, Barley yellow dwarf virus, Turnip crinkle virus and Triticum Mosaic Virus [The RNA secondary structures were predicted through the conserved motif sequences using the Mfold software Fig. . Type 1,ic Virus –23.Fig. FLuc gene  reported the genetic diversity of CVA isolates from China by analyzing three genomic regions that corresponded to the coat protein, RNA-dependent RNA polymerase, and the core region, which resulted in at least seven major clusters [. (2017), Phylogenetic analysis resulted in six major groupings [Sweet cherry is a major fruit crop of increasing economic importance. CVA is among the most common viruses infecting sweet cherry , 25. In clusters . Moreoveroupings . In our . 207 reporteConserved stem-loop structures that function as translational enhancers have been previously identified in viruses –29. To dFLuc construct containing these CVA 5′-UTR types increased the translation of FLuc by 2–3 folds, suggesting weak translation enhancement activity. We identified the conserved RNA structure by structural evolution analysis of different CVA isolates and found that it presents IRES activity to regulate translation.Molecular and phylogenetic analyses were performed on 75 CVA sequences, which could be divided into four groups. The four CVA 5′-UTR types appeared to have a conserved RNA structure, and the


RNA plays an important role in biology, and more than 170 RNA modifications have been identified so far. Post-transcriptional modification of RNA in cells plays a crucial role in the regulation of its stability, transport, processing, and gene expression. So far, the research on RNA modification and the exact role of its enzymes is becoming more and more comprehensive. Human immunodeficiency virus 1 (HIV-1) is an RNA virus and the causative agent of acquired immunodeficiency syndrome (AIDS), which is one of the most devastating viral pandemics in history. More and more studies have shown that HIV has RNA modifications and regulation of its gene expression during infection and replication. This review focuses on several RNA modifications and their regulatory roles as well as the roles that different RNA modifications play during HIV-1 infection, in order to find new approaches for the development of anti-HIV-1 therapeutics. Post-transcriptional modification of RNA is a common phenomenon in biological processes . Since tN1-methyladenosine (m1A), 5-methylcytosine (m5C), N6-methyladenosine (m6A), N7-methylguanosine (m7G) and N6,2′-O-dimethyladenosine (m6Am), etc. [RNA methylation is the most well-characterized modification of RNA modifications. According to the site of methylation, RNA methylation can be divided into various forms, including m), etc. ,6. The h6A, m1A, m5C, m7G, m6Am, pseudouridine (ψ), 2′-O-methylation (2′-O-Me or Nm) and N4-acetylcytidine (ac4C) modifications  and methyltransferase-like 14 (METTL14). METTL3 is the is a functional active site as a catalytic subunit, and METTL14 is an essential site that promotes RNA binding and is a scaffolding structure [6A methyltransferase-related  [6A [6A includes the fat mass and obesity-related protein (FTO) and the alkylated DNA repair protein ALKB5 (ALKBH5), which are mainly present in the cytoplasm or nucleus, respectively, and can selectively demethylate RNA [6A in mRNA, but also shows demethylase activity of m6A m and m1A of specific tRNA, snRNA or mRNA [6A methylation and generate functional signals include the YTH-RNA-binding domain family  [6A-binding YTH domain and a variable N-terminal effector domain with unclear function [6A modification adds a new layer of post-transcriptional gene expression regulation that is involved in T cell response to HIV infection, production of type I interferons, and T cell differentiation and homeostasis  , METTL16IAA1429) , RNA binIAA1429) , zinc fiIAA1429)  and the IAA1429)  are esselate RNA ,31,32. F or mRNA . “ReaderTDHC1-2) ,34, the TDHC1-2) ,36, IGF2TDHC1-2) , eukaryoTDHC1-2) , HUR [39TDHC1-2)  and leucTDHC1-2) . All threostasis .1A is a reversible modification of mRNA and tRNA and is a positively electrostatically methylated nucleotide under physiological conditions [1A is high in tRNA and rRNA but low in mRNA. The mRNA containing m1A is 10 times less than that containing m6A [1A was first identified in tRNAs, usually located at positions 9, 14, and 58 of tRNAs [1A is critical for tRNA folding, stability, and tRNA-protein interactions [1A is prevalent in mitochondrial-encoded transcripts, and through the study of manipulating m1A level by a mitochondrial located m1A methyltransferase trmt61b, it was found that m1A interferes with the translation of mitochondrial mRNA [1A58 modification of cytoplasmic tRNAs [1A at positions 9 and 58 in mitochondrial tRNA [1A is methylated at position 1309 by the nucleolar protein RRP8 , and this methylation is required for rRNA biogenesis [1A, TRMT61B installs m1A in mt-mRNA transcripts, and TRMT10C methylates position 1374 of ND5 mt-mRNA [1A can be catalyzed by the demethylases FTO, ALKBH1 and ALKBH3 [1A [mnditions . Methylanditions . The conning m6A ,85. m1A of tRNAs . m1A is ractions ,88. m1A ial mRNA . Methylaial mRNA . TRMT61Aic tRNAs . In addiial tRNA ,46; in hogenesis ,91. In m mt-mRNA ,47. m1A d ALKBH3 ,49,85. YKBH3 [1A ,51.5C) of DNA dates back to the 1950s and is essential for gene expression and epigenetic regulation [5C in RNA, and high-throughput detection methods found that m5C is an RNA modification widely present in mRNA and lncRNA, including transfer RNA (tRNA), ribosomal RNA (rRNA), Long non-coding RNA (lncRNA), small nuclear RNA (snRNA), microRNA (miRNA), and enhancer RNA (eRNA) [5C in mRNA show different regulatory activities, they can promote or inhibit translation; in tRNA, m5C can stabilize secondary structure and affect anticodon stem-loop conformation; in rRNA, m5C can affect translation preservation Authenticity [5C methyltransferase usually uses S-adenosylmethionine (SAM) as the methyl donor to transfer the methyl group to the fifth carbon position of the RNA cytosine base [5C methyltransferases have been identified, including enzymes of the NOL1/NOP2/SUN domain (NSUN) family, DNA methyltransferase (DNMT) homolog DNMT2, and tRNA specific methyltransferase (TRDMT) family members [5C are somewhat controversial and have yet to be determined. In recent years, some reports indicate that the ten-eleven translocator (Tet) family has the potential to act as an RNA demethylase. In mRNA, enzymes of TET can oxidize m5C to generate 5-hydroxymethylcytosine (hm5C) [5C34 to f5Cm34 (5-formyl-2′-O-methylcytidine) and hm5Cm34 (5-hydroxymethyl-2′-O-methylcytidine) along mt-tRNAMet and anticodon cytoplasmic tRNALeu [5C reader proteins ALYREF (RNA and export factor-binding protein 2) and YBX1 (Y-box-binding protein 1) exert biological effects by recognizing and binding m5C sites [The discovery of 5-methylcytosine  . Modificenticity . The m5C members . The erae (hm5C) . Further tRNALeu ,98. RNA 5C sites ,55,56.7G, one of the ubiquitous RNA modifications in various species, is a positively charged RNA modification produced by the addition of a methyl group to the N7 position of nucleoguanosine [7G also regulates miRNA biosynthesis and biological function, tRNA stability, 18S rRNA processing and maturation [7G -containing tRNAs are present in eukaryotes, they are primarily found in yeast and more recently in mice, and this modification is catalyzed by the Trm8/Trm82 complex in yeast and the METTL1/WDR4 complex in humans [muanosine ,100. Eukuanosine . In addituration . m7G -con humans . Studiesn humans , but its6Am [7G cap can be methylated on the ribose to form 2′-O-methyladenosine (Am), which can then be further methylated at its N6 position to generate m6Am [6Am is the most prevalent RNA modification in many species, including mammals, present in the 5′-UTR, 3′-UTR and stop codons, and in the first transcription nucleus in about 30% of cellular mRNAs nucleotides can have a significant impact on gene expression in the transcriptome [6Am [An abundant RNA modification close to the mRNA cap structure is dimethylated adenosine, m6Am . The firate m6Am ,102. m6Acriptome . Recent ome [6Am ,62.Ψ, the C5 glycoside isomer of guanosine, was the first post-transcriptional modification discovered, the fifth nucleotide of RNA, and the most abundant modification in RNA . Ψ is wiO-methylation  is an abundant and highly conserved modification. Nm is modified to add a methyl group to the 2′ hydroxyl group of the ribose moiety, any of the four nucleotides  may have ribose methyl groups at different stages of the ribosome biogenesis process. In addition, Nm is abundant in tRNA, rRNA and SnRNA, which is important for the biogenesis, metabolism, and function of these molecules [O-methylation of ribose in RNA is formed by two cellular mechanisms: through independent methyltransferases or through complex assembly of proteins associated with snoRNA guides (snoRNPs) [O-methyltransferase FTSJ1, also known as MRX9, TRMT7, JM23, can target the C32 and N34 positions in the anticodons of tRNAPhe and tRNATrp [O-methyltransferase Sbp1 (human homolog: FTSJ3), which does not involve an antisense guide RNA [2′-olecules ,70,71. Nolecules ,105,106.olecules ,107. ThesnoRNPs) . Most mRsnoRNPs) . The hum tRNATrp . FTSJ1 h tRNATrp ,75. In e tRNATrp . The G29uide RNA .4C is an ancient and conserved RNA modification that was first described in the E. coli tRNA met anticodon [4C is widely distributed in the human transcriptome, with most sites occurring in the coding sequence (CDS), and acetylation can promote the expression of target genes by improving mRNA stability and translation [4C in eukaryotic RNA occur in the central nucleotide of the 5′-CCG-3′ consensus sequence [4C modification contributes to the correct recognition of codons, stabilizes the tertiary structure of the tRNA molecule, increases the high fidelity of protein translation, and plays a key role in cellular thermotolerance [4C modification on rRNA is a characteristic of thermophilic organisms [4C modification can also increase mRNA stability, enhance substrate translation, and promote mRNA translation efficiency [N-acetyltransferase 10 (NAT10) is involved in catalyzing the deposition of ac4C on tRNA, rRNA and mRNA, and acetyl coenzyme A (CoA) acts as an acetyl donor [4C catalyzed by NAT10 on tRNA and rRNA requires the assistance of the cofactors THUMPD1 and snoRNA, respectively [acnticodon ,111, andnticodon ,113. ac4nslation . All thesequence . For tRNolerance ,80; The rganisms , it alsorganisms . In addificiency . N-acetyyl donor . Furtherectively . WhetherHuman Immunodeficiency Virus (HIV) is a virus that attacks the body’s immune system. HIV was first isolated and identified in 1983 from the lymph nodes of a patient with lymphadenopathy ,117. TheIn terms of virus taxonomy, HIV belongs to the genus Lentivirus of the Retroviridae family, and is currently classified into type 1 and type 2  according to their genetic characteristics and differences in viral antigens . CompareThe HIV genome consists of two identical single-stranded RNA molecules enclosed within the core of the viral particle. The HIV genome, also called previral DNA, is produced by reverse transcription of viral RNA genomic RNA into DNA, degradation of RNA, and integration of double-stranded HIV DNA into the human genome .The replication cycle of HIV can be divided into early and late stages. In the early stage, the virus binds to the prime cell surface receptor, then enters the cell, the viral RNA is reverse transcribed into DNA, the viral capsid is uncoated, the viral DNA is imported into the nucleus, and then the DNA is integrated, and the viral DNA is integrated into the host cell genome. Mature HIV-1 surface glycoprotein gp120 binds to CD4 receptors on host cells . After tThe HIV-1 genome encodes nine open reading frames, 15 different proteins. Both ends of the genome have long terminal repeats (LTRs) . There a6A, m5C RNA modification [6A modification of HIV-1 RNA plays an important role in viral infection and HIV-1 protein synthesis [Replication and infection of HIV-1 virus are also regulated by RNA modifications. Once HIV-1 proviral DNA is integrated into the host cell chromosome, viral latency is affected by post-transcriptional modifications (acetylation/deacetylation) . For virfication . The m6Aynthesis . The eff6A modifications were also found in viral RNA decades ago in Rous sarcoma virus [6A modification exists in HIV-1 RNA and is abundant in the entire viral genome, present in regulatory regions, coding sequences and structural regions. The results suggest that HIV-1 infection of T cells promotes methylation of viral and host RNAs, and that RNA methylation status is positively correlated with viral replication. Silencing of ALKBH5 increases RRE RNA methylation, which promotes REV binding and increases nuclear export of viral RNA, which promotes viral replication; In contrast, silencing of METTL3 and/or METTL14 reduced RRE RNA methylation and subsequent recruitment of Rev to the RRE, thereby reducing viral RNA export and inhibiting viral replication. In vitro experiments determined that efficient Rev binding requires A7883 in the RRE structure, one that flips out from the RRE stem region and interacts directly with REV’s W45 via van der Waals contacts [Internal mma virus , influenma virus  and SV40ma virus . Howevercontacts . Furthercontacts .6A modification sites on the HIV-1 RNA genome were precisely located, mainly located in the 3′ untranslated regions (3′ UTRs) of various HIV-1 mRNAs. Studies have shown that m6A residues in the 3′ non-coding region can promote the expression and replication of HIV-1 gene by increasing the stable expression level of viral mRNA. When YTHDF1-3 was overexpressed, HIV-1 gene expression was affected in 293T cells, and increased levels of Gag, Nef, Tat, and Rev mRNAs were observed, as well as increased protein levels of Gag and Nef. Furthermore, HIV-1 is highly sensitive to m6A “reader” YTHDF2 expression levels in infected T cells, and the expression level of YTHDF2 is positively correlated with its replication [In the study of Kennedy et al., the mlication .6A “readers” led to a decrease in the level of HIV-1 infection. In primary CD4+ T cells, HIV-1 RNA is modified by m6A in infected cells, and “readers” YTHDF1-3 protein inhibits viral reverse transcription and gene expression by binding to m6A -modified HIV-1 RNA, thereby inhibiting HIV-1 infection. Knockdown of m6A “writers” reduced HIV-1 Gag synthesis and virion release in virus-producing cells, whereas knockdown of “erasers” had the opposite effect [In the study of Tirumuru et al., it was found that in 293T cells, the overexpression of me effect .6A “writers” and “erasers” in HIV-1 gene expression, the above experiments demonstrated that knockdown of m6A “writers” METTL3 and METTL14 resulted in decreased Cap24 and Gag protein levels and total gp120 mRNA levels in cells and supernatants decreased, whereas knockdown of “erasers” FTO and/or had the opposite effect. Although the roles of m6A “writers” and “erasers” in HIV-1 gene expression are clear, the role of m6A “readers” remains controversial. Using different methods, cell lines, and other reagents may have contributed to different results when studying the relationship between YTHDF protein and HIV-1 replication and expression, so these results need to be clarified by continued research.Regarding the role of mA new study shows that in human CD4+ T cells, the YTHDF3 protein is incorporated into HIV particles in a nucleocapsid-dependent manner and is able to reduce viral infectivity during the next infection cycle, while HIV restricts the viral activity of YTHDF3 by cleaving virion-bound YTHDF3 at different sites by HIV protease .6A methylation in HIV-1 RNA to increase viral replication [6A-related RMPs to reduce m6A in HIV-1 RNA and inhibit virus replication.Selberg et al. showed that activation of the METTL3/METTL14/WTAP RNA methyltransferase complex can promote mlication . Therefo1A profiling and deep sequencing methods revealed that HIV-1 virion RNA contains a large amount of 1-methyladenosine, but m1A is present in tRNA not present in the HIV-1 genome [1A 58 modification in the early and late stages of HIV-1 replication and in cellular tRNA modification networks. TRMT61A and TRMT6 are responsible for m1A58 modification of cytoplasmic tRNAs. HIV-1 is a positive-strand RNA virus. HIV-1 is a positive-strand RNA virus. After entering the host cell, cytoplasmic tRNA3Lys is used as a primer, and then most of the viral RNA is degraded by RNaseH, leaving two short viral RNA’s polypurine tracts, which are used as primers for positive-strand DNA synthesis [1A 58 is required for strong positive-strand blockade of retroviruses in vitro. In their study, integration of the HIV-1 genome into host cells was greatly reduced when the virus was generated from TRMT6 mutant cells. In the late stage of HIV-1 replication, the protein levels of HIV-1 capsid p24 and its precursors GagPol pr160 and Gag pr55 were reduced in TRMT6 mutant cell lines, and HIV-1 integrase and its precursor GagPol Pr160 and processing intermediates were greatly reduced, Tat protein decreased. Thus TRMT6 is required for the early steps of HIV-1 replication and for efficient late accumulation of HIV-1 protein and virus production [1A58 modification plays an important role in the early and late stages of HIV-1 replication and in the tRNA modification network. tRNA m1A58 is required for reverse transcription to produce strong positive-strand termination in vitro.Liquid chromatography-mass spectrometry (LC/MS) analysis of HIV-1 virion RNA combined with m1 genome . HIV-1 iynthesis . ‘Plus-synthesis .After thynthesis . In expeoduction . Collect5C promotes viral mRNA translation. In the study of Courtney, a highly prevalent modification of m5C was detected on HIV-1 gRNA in infected cells, with m5C levels about 14 times higher than cellular mRNA. In their study, they identified NSUN2 as the primary m5C “writer” on HIV-1 gRNA and showed that loss of NSUN2 reduced HIV-1 protein expression but did not affect viral RNA levels. Furthermore, NSUN2 deletion disrupts ribosome recruitment, affecting HIV-1 alternative splicing [5C RNA methyltransferase NOP2/NSUN1 is an HIV-1 restriction factor. Proteomic studies have found that NOP2 protein is an RNA-binding protein associated with the 5′UTR of HIV-1 [5C methylation of the TAR, blocking HIV-1 transcriptional elongation. The competition of NOP2 with Tat and binding to TAR are mediated by the catalytic domain (MTD) of RNA MTase [5C methylation of HIV-1 RNA genome, which is beneficial to viral protein synthesis, while NOP2 mediates m5C methylation of HIV-1 TAR and interferes with pre-transcription. Addition of m5C to HIV-1 RNA affects alternative splicing of its transcripts. HIV-1 has high levels of epitranscriptional m5C modifications, which in turn promote viral gene expression by regulating viral RNA splicing and promoting viral mRNA translation. The role of m5C in viral replication is an understudied area that deserves further attention.msplicing . Kong etof HIV-1 . ThroughNA MTase . Therefo7G cap to the 5′ end of nascent RNA [7G cap helps initiate translation in mammalian cells, whereas uncapped pre-mRNA is degraded by 5′ exonuclease to accelerate decay [7G cap [7G- or trimethylguanosine-caps (TMG-caps) to varying degrees. In experiments they found that the HIV-1 transcript initially transcribed was m7G-capped. Unspliced or partially spliced HIV-1 RANs containing RREs bind to Rev and then recruit PIMTs to these RNAs. In this way, PIMT is introduced into viral RNA, which then hypermethylates the m7G cap to TMG-CAPS. PIMT-mediated TMG-caps can promote RNA expression. In addition, experiments have also shown that PIMT can regulate the expression of Rev- and CRM-1–dependent Gag-Pol-RRE RNA, and TMG cap is a hallmark of the CRM-1 pathway [7G-cap significantly promotes the production of infectious virus. In addition, hypermethylation of viral mRNA leads to cap-binding complex exchange to heterodimeric CBP80/NCBP3, thereby expanding the functional capacity of HIV-1 in immune cells [RNA polymerase II (RNAP II) can add a mcent RNA . The m7Gte decay ,154. Whi [7G cap . There i [7G cap ,157. Yed pathway . What’s ne cells .6A methylation on 5′-terminal 2′-O-methylated A on mRNA is catalyzed by methyltransferase phosphorylation of CTD-interacting factor 1 (PCIF1) [6Am RNA methylome through viral Vpr protein-mediated PCIF1 degradation. In their study they found that HIV genomic RNA did not contain any m6Am modifications. In HIV infection, m6Am modification is reduced in many cells, which is mediated by Vpr-induced PCIF1 degradation. In addition, they found that PCIF1 is a broad HIV inhibitor that inhibits viral replication and limits HIV infection through methylation of ETS1 mRNA. The binding of ETS1 to the HIV promoter can reduce HIV transcription. However, PCIF1 does not methylate HIV genomic RNA. Briefly, PCIF1 inhibits HIV infection by enhancing the stability of the host m6Am gene, but HIV relieves this restriction by Vpr-induced PCIF1 degradation during pathogenesis, PCIF1 affects the replication phase of HIV [The m (PCIF1) ,62. The e of HIV . PCIF1 m7SK RNA is a highly conserved non-coding nuclear RNA in vertebrates . StudiesO-methylation. When there is RNA without 2′-O-methylation modification in the cell, the cytoplasmic sensors Mda5 and RIG-I in the cytoplasm will recognize this type of exogenous RNA and further activate the expression of type I interferon (IFN) to exert antiviral function [O-MTase, whose encoded product can methylate adenine in the viral genome, evading the innate immune response of the immune system [O-methyltransferase that is recruited to HIV RNA via TRBP. The transfection of HIV RNA expressed in FTSJ3 knockdown cells caused a significant increase in IFN-α/IFN-β compared with the control group. Later, the researchers used the knockdown of the RNA cytoplasmic receptors Mda5 and RIG-I cells to conduct experiments and proved that FTSJ3-TRBP completes immune escape through the Mda5 pathway through the 2′-O-methylation modification of HIV RNA by FTSJ3-TRBP. The FTSJ3-TRBP system can be hijacked to complete the 2′O-methylation modification of viral RNA to reduce the sensitivity of the MDA5-IFN pathway, reduce immune system recognition, and increase HIV-1 replication [In higher eukaryotes, the cellular innate immune system can distinguish endogenous mRNA from exogenous mRNA through the molecular signature of RNA 2′-function . The gene system . In theilication . Furtherlication .4C residue on HIV-1 RNA to 11 distinct sites. Their studies identified that the cellular acetyltransferase NAT10 adds ac4C to HIV-1 transcripts at multiple discrete locations, which promote HIV-1 gene expression by increasing the stability of viral RNA while viral mRNA translation was not affected [The study by Tsai et al. mapped the acaffected .In the decades since RNA modifications were discovered, scientists have discovered multiple roles for dynamic RNA modifications. RNA modification can dynamically reprogram gene expression and regulate RNA stability, metabolism, splicing, translation, localization, transport, binding to other RNAs or binding to RNA-binding proteins. RNA modifications are also regulated by regulatory proteins. As the virus that causes AIDS, HIV-1 is also regulated by RNA modifications in the process of infecting the human body. Current studies have shown that several different RNA modifications can indirectly promote viral replication either by directly increasing viral RNA stability or translation, or by allowing the virus to evade innate immune recognition of viral transcripts. Therefore, the exploration of inhibitors that block viral RNA modification may inhibit the target of viral genes and promote the antiviral immune response of the host. In addition, this type of drug targets proteins associated with RNA modifications, so there is little chance of resistance or viral mutation. However, much remains unknown about the role of RNA modifications in HIV-1 infection. So, exploring how viruses modify, avoid or use RNA modification processes to regulate the expression of their own viral genes, and have a better understanding of the role of RNA modifications in HIV-1 infection may help in the search for new targets for antiviral therapy.


Nlgn2), Cdc42 guanine nucleotide exchange factor 9 (Arhgef9) BLCAP apoptosis inducing factor (Blcap), and cytoplasmic FMR1 interacting protein 2 (Cyfip2), as well as two noncoding RNA sites in small nucleolar RNA host gene 11 (Snhg11) and the maternally expressed 3 (Meg3) gene. Moreover, significant changes were observed in gene functions and pathways enriched by genes with A-to-I RNA editing in losers and especially winners compared to controls. Our results demonstrate that repeated winning and losing experiences in chronic social conflicts are linked to A-to-I RNA editing pattern difference, underlining its role in the molecular mechanism of agonistic interactions between conspecifics.Winner-loser effects influence subsequent agonistic interactions between conspecifics. Previous winning experiences could strengthen future aggression and increase the chance of winning the next agonistic interaction, while previous losing experiences could have the opposite effect. Although the role of A-to-I RNA editing has been recently implicated in chronic social defeat stress and aggressive behavior, it remains to be further elucidated in chronic social conflicts in agonistic interactions, especially in the repeated aggression (winners) and repeated defeat (losers) resulted from these conflicts. In the current study, transcriptome-wide A-to-I RNA editing in the dorsal striatum was investigated in a mouse model of chronic social conflicts, and compared between mice repeatedly winning and losing daily agonistic interactions. Our analysis identified 622 A-to-I RNA editing sites in the mouse dorsal striatum, with 23 to be differentially edited in 22 genes, most of which had been previously associated with neurological, psychiatric, or immune disorders. Among these differential RNA editing (DRE) sites four missense variants were observed in neuroligin 2 ( Agonistic behavior during social conflicts between conspecifics results in winners and losers. Winner-loser effects could influence future agonistic behavior in such conflicts . PreviouHtr2c)   was implHtr2c) . RNA ediHtr2c) . HoweverThe striatum is a subcortical region integrating social information into coding of reward and social action and is involved in agonistic behavior . The strTherefore, the current study conducted a comprehensive investigation of A-to-I RNA editing in the dorsal striatal transcriptome of male mice with alternative social experiences of agonistic interactions  and showhttps://www.ebi.ac.uk/ena). The dataset (PRJEB36194) included the dorsal striatum from three groups (N = 3 per group) of adult C57BL/6J male mice with alternative social experiences  formed in agonistic interactions (Raw data of RNA-Seq were downloaded from the European Nucleotide Archive (ENA) of the European Molecular Biology Laboratory  by using VarScan (version 2.4.3) , filterehttps://david.ncifcrf.gov/tools.jsp) (https://maayanlab.cloud/Enrichr/). Items with a false discovery rate (FDR) < 0.05 were considered significant.Enrichment analysis of differentially edited genes using DAVID online prediction tools (ols.jsp)  and Enriols.jsp)   analysis was conducted and further visualized using R (version 3.6.3).The levels of RNA editing among groups were compared using the ANOVA test, and In our RNA-Seq data analysis, a total of 662 high-confidence A-to-I RNA editing sites were observed in 342 genes in the mouse striatum . These sWe then looked into the A-to-I RNA editing sites and edited genes in individual groups. 540, 531, and 577 sites were found in controls, winners, and losers, respectively . Among tpost-hoc analysis showed that the DRE sites in ten genes including LUC7 Like (Luc7l), Fas activated serine/threonine kinase (Fastk), CDP-diacylglycerol synthase 2 (Cds2), vesicle associated membrane protein 4 (Vamp4), BLCAP apoptosis inducing factor (Blcap), Rab-like protein 6 (Rabl6), SRC kinase signaling inhibitor 1 (Srcin1), small integral membrane protein 14 (Smim14), tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein beta (Ywhab), and Neuregulin 1 (Nrg1) had significantly different editing levels between winners and losers with opposite change directions compared to controls  sites in 22 genes were found among the three groups . DAVID acontrols .Nlgn2) gene  and p.Arg380Gly in the Cdc42 guanine nucleotide exchange factor 9 (Arhgef9) gene . Compared to controls, winners had an increased editing level of Nlgn2:chr11:69834033, and losers had the highest editing level of Nlgn2:chr11:69834033. The editing of Arhgef9 p.Arg380Gly was observed only in controls but not in winners or losers. Another two missense DRE sites included p.Gln5Arg in Blcap  and p.Lys320Glu in the cytoplasmic FMR1 interacting protein 2 (Cyfip2) gene . The editing level of Blcap p.Gln5Arg was increased in winners but decreased in losers compared to controls. In contrast, the editing level of Cyfip2 p.Lys320Glu was only significantly increased in losers.It was noted that among the four missense DRE sites, two ranked the top two among all DRE sites, including p.Asn136Asp in the neuroligin 2 (Snhg11)  and the Maternally Expressed 3 (Meg3) gene .In addition to protein-coding genes, two long noncoding RNA (lncRNA) genes were also differentially edited, including Small Nucleolar RNA Host Gene 11 (Cxcl14), Cds2, Arhgef9, Fastk, Srcin1, Cyfip2, kelch like family member 2 (Klhl2), kinesin family member 5C (Kif5c), muskelin 1 (Mkln1), Nrg1, Nlgn2, replication protein A1 (Rpa1), and Ywhab  (Our previous study showed enrichment of DRE genes with multiple G-to-A RNA editing. Our results showed that the top 10 genes with multiple RNA editing  included= 0.002) .Adult mice experiencing alternative social conflicts provide a useful model to study repeated winning and losing experiences in chronic social conflicts. Recently, RNA editing has been reported to be involved in defeated stress as well as in aggressive behaviors , 8, 9. TSRCIN1 gene encoding p140Cap/SNIP, a scaffolding protein localized in dendritic spines, was recently reported as a hub for postsynaptic proteins involved in neuropsychiatric disorders  was found to be the most differentially edited among mice with alternative social experiences. Winners had an increased editing level of the variant compared to controls, and losers had the highest editing level. Nlgn2 encoded encodes a transmembrane scaffolding protein that plays a role in cell-cell interactions in both neurons and other cells types and the formation and remodeling of GABAergic synapses by recruiting and clustering synaptic proteins  and Cyfip2 (p.Lys320Glu), which were also reported in humans  remains unclear. Meg3 is a maternally expressed imprinted gene that functions as a lncRNA tumor suppressor  genes, the RNA of which were also the most intensively edited targets in the mouse dorsal striatum. An intronic variant c.207-1847A>G in l injury . Neverthppressor . Recent  surface . The expperidone . A study pathway . A recen by Meg3 . The larMoreover, in line with our previous report, genes with multiple editing sites showed a significantly higher possibility of being differentially edited among alternative social experiences. Among the top ten genes with the most editing sites, four exhibited DRE , 3. SuchHtr2c encoding serotonin receptor 2C was reported to be increased in isolation-induced aggressive behavior of BALB/c mice  can be found in the article/Ethical review and approval was not required for the animal study because ethical review and approval are not applicable for this study.F-XR and FK performed the bioinformatic analysis. Z-YW improved the data analysis pipeline. C-YR, Y-SH, S-YX, Y-NL, Y-PL, and J-JF participated in the data interpretation and discussion. J-HC conceived the project and planned the study. All authors contributed to the final manuscript.This study was supported in part by grants from the National Natural Science Foundation of China (No. 31671311), the National first-class discipline program of Light Industry Technology and Engineering (LITE2018-14), the Six Talent Peak Plan of Jiangsu Province (No. SWYY-127), the Innovative and Entrepreneurial Talents of Jiangsu Province, the Program for High-Level Entrepreneurial and Innovative Talents of Jiangsu Province, Natural Science Foundation of Guangdong Province/Guangdong Basic and Applied Basic Research Foundation (2019A1515012062), Taihu Lake Talent Plan, and Fundamental Research Funds for the Central Universities (JUSRP51712B and JUSRP1901XNC), and Postgraduate Research and Practice Innovation Program of Jiangsu Province (KYCX20_1946).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Our work alsoreveals a negative cooperativity effect between the effects of Mg2+ and Na+ on polyrA condensation. Finally, we observethat polyrA sequences can act as promoters of phase separation inmixed sequences. These results demonstrate the potential for noncanonicalinteractions to act as networking stickers, leading to specific condensationproperties inherent to polyrA composition and structure, with implicationsfor the fundamental physical chemistry of the system and functionof polyA RNA in biology.RNA–RNA interactionshave increasingly been recognizedfortheir potential to shape the mesoscale properties of biomolecularcondensates, influencing morphology, organization, and material statethrough networking interactions. While most studies have focused onnetworking via Watson–Crick base pairing interactions, previouswork has suggested a potential for noncanonical RNA–RNA interactionsto also give rise to condensation and alter overall material state.Here, we test the phase separation of short polyA RNA (polyrA) homopolymers.We discover and characterize the potential for short polyrA sequencesto form RNA condensates at lower Mg These includethe inhibition25 and induction26 of conformational changes in protein bindingpartners, the formation of Watson–Crick interactions in repeatsequences,15 electrostatic interactionswith polycation partners,27 and potential structure-inducedeffects.28RNA andRNA-binding proteins are among the most abundant moleculesin biomolecular condensates. Several cellular ribonucleoprotein (RNP)granules are enriched in these molecules, making them targets of interestin understanding how and why condensates form. There is much evidencethat multivalent interactions between RNA and proteins can lead tophase separation. These interactions can include specific interactionswith RNA-binding proteins or nonspecific interactions with chargedintrinsically disordered proteins (IDPs). While many phase separationstudies have focused on IDPs and the necessity of key proteins inRNP granules, the significance of RNA in BMCs is increasingly beingrecognized.31 While several studies have focused on the role ofWatson–Crick-based interactions,29in vitro studies have also shownthat noncanonical RNA interactions have the capacity to drive phaseseparation and tune the material and compositional properties of RNAcondensates.31 Previous work has demostratedthat even RNA homopolymers can phase separate in the presence of highconcentrations of salt, likely mediated by noncanonical RNA–RNAinteractions.30 In such homopolymer-based systems, the identityof the homopolymers has a dramatic influence on the phase behavior.In the presence of PEG and high concentrations of NaCl, long RNA polymersof polyrU, polyrC, and polyrA condense to form droplets with distinctproperties, while polyrG forms aggregates, presumably due to its tendencyto form stable G-quadruplex structures in the presence of monovalentsalts.30 Other studies have also shownthat polyrU can phase separate in the absence of PEG when mixed withsufficient concentrations of divalent cations17 and that differences in scaffold identity influence partitioningand organization.23 These findings andothers in the field have suggested that noncanonical RNA–RNAinteractions and the chemical properties of constituent polymers influencethe phase behavior and physical properties of RNA condensates.Amongthese studies is a growing body of evidence that RNA–RNAinteractions can drive condensate formation and influence the mesoscaleproperties of complex systems.33 From a chemical perspective, polyrA, despite its simplicity of sequence,has a complex array of structural forms. Due to its potential forself-interaction, polyrA has the potential to transition between randomcoils, single-stranded helices, and double-stranded structures dependingon environmental conditions.40 Furthermore, recent studies indicate that polyrA interacts withMg2+ in a manner distinct from polyrU and polydA with respectto both hydration and binding preference compared to Na+.42Of these homopolymers, polyrA is especially interesting from bothbiological and chemical perspectives. Physiologically, long tractsof polyadenylated tails are highly conserved, making it among themost universally conserved homopolymer present in cells. The lengthand presence of these sequences have been functionally associatedwith mRNA half-life, degradation machinery, and transcriptional processes.35 Recent work has shown that these precipitates can be liquid-likecondensates with long polyrA sequences in the presence of crowders.30 Other studies have also highlighted the unique role of Mg2+ in interacting with polyrA to form aggregates with both long polyrA41 and short polyrA.42 Despite the potential for interesting features in polyrA phase separation,Mg2+-mediated polyrA droplet formation has not yet beencharacterized for short polyrA sequences. This presents an opportunityto explore the limits of RNA phase separation and to further probethe implications of polyrA–Mg2+ interactions inthe context of phase separation.In this paper, we explore the waysin which short polyrA sequencescan also exhibit interesting phase behavior. Work as far back as 50years ago showed that polyrA sequences can form helical structuresand precipitate in the presence of monovalent salt.20) to characterizethe phase separation properties of shorter homopolymeric polyrA. Wefound that rA20 phase separates into condensates at substantiallylower Mg2+ concentrations than previously observed in longerpolyrU sequences. Our results revealed that these condensates displayproperties consistent with kinetically arrested states with slow internalrearrangement. We also discovered an unexpected cooperativity effecton rA20 condensation between Mg2+ and Na+. Additionally, given the importance of polyrA tails in biologyand the unique phase properties we observed of rA20, wehypothesized and tested its ability to act as a phase separation promoterwhen tagged onto a generic mixed RNA 20-mer construct (rN20), comparing 40-mers with tails of rA20 to those withtails of rU20 and the mixed sequence rN20.Here we use 20-mer polyrA oligomers were ordered from Sigma-Aldrich  with HPLC purification.These were resolubilized in diethyl pyrocarbonate (DEPC) treated water(Santa Cruz Biotech) and diluted into buffer . Solidsof MgCl2 and NaCl purchased from Sigma-Aldrich  as well as polyethylene glycol  were dissolvedin DEPC treated water.All oligomers  unless otherwisespecified. Concentrations of the master mix of nucleic acid and bufferbefore condensation were checked by a 5- or 10-fold dilution usingA260. The 40-mer mimic constructs were made so that the total amountof bases in solution was comparable to the 20-mer experiments(∼38μM). For experiments with PEG or monovalent salt, a 4X nucleicacid master mix was made and aliquoted. The resultant mixtures werethen diluted 2-fold with the respective additive and mixed. All nucleicacid master mixes were allowed to equilibrate to room temperaturebefore a final step of MgCl2 addition, which brought concentrationsto the final values recorded.To solutionsof nucleic acidsin buffer , varying concentrations of 2 × MgCl2 and nucleic acid master mixes were remade,with error bars depicting the standard deviation of values betweenthe runs.UV–vis absorption spectrawere recorded with a Nanodrop 2000c. Absorbance at 350 nm was usedto report on the formation of aggregates. Data shown represents triplicateruns where MgCl20 labeled condensates, 0.3% of 488nm laser power was applied, while for Cy3-rA20 labeledcondensates, laser power was 0.8% at 514 nm for standard readings.In readings where time series were applied over a period of time greaterthan 10 min, reflection autofocus was turned on and was applied every5 min.All differential interference contrast(DIC) and fluorescence microscopy images were recorded on a ZeissLSM 780 laser scanning confocal microscope. Samples were imaged atroom temperature using a 100× oil immersion objective (Plan-Apochromat100×/1.40 Oil DIC M27) with a scan speed of 314 ms/pixel anda pixel size of between 0.17 and 0.08 μm. For DIC images, 40%of 488 nm laser power was applied, and averaging was set to 4. InFRAP and fusion-based studies, averaging was turned off and lowerlaser power settings were applied to mitigate the effect of photobleachingover time. For 6FAM-rA2, samples were mixedand subsequently transferred to Lab-Tek chambered no. 1.5 borosilicatecover glass which had been treated with 10% TWEEN-20 for 35 min, rinsedwith DEPC treated water, and dried overnight prior to storage anduse. Droplets were formed separately within a minute of each otherand then sequentially added to the coverslip after about 10–30min of incubation on the cover glass after droplet formation as specifiedbelow.Fluorescently labeled oligomers and dyes were added priorto condensate formation such that their final concentrations were1 μM. After the addition of MgClAll images and videos were analyzed with Fiji software.20 oligomer used as a marker. Dropletswere partially bleached (region of interest (ROI) area ∼ 0.35μm2 in droplets with radii approximately 5-fold thebleached radii). Experimental droplets were bleached using four tosix iterative pulses of 100% 488 and 405 laser power, after whichthe bleached area was below 50% of its initial signal . Images were corrected for xy driftby use of the Correct 3D drift option on the Fiji Registration plug-in.43 For a few images with more xy drift, the StackReg plug-in was first applied with the Rigid Bodycorrection44 and Correct 3D drift was appliedafter.For fluorescence recovery after photobleaching(FRAP) experiments, samples were prepared in the same manner as describedabove, with the 6FAM-rA20 + rA20 constructs,normalized curves were plotted and fit to an exponential curve usingGraphPad Prism, y = y0 + A(1 – et/τ), from which the immobile fraction was calculated with the equation A corresponds tothe empirical fit parameter. Multiple droplets (n = 3) were used for each condition. For a detailed description ofFRAP conditions, please see the Supporting Information.Fluorescence recovery of the bleached area was referencedto an area inside an unbleached droplet of a comparable size to accountfor photobleaching and to calculate a normalized intensity:20 or Cy3-rA20 oligomer as a marker. Droplets wereformed with either the Cy3 or FAM labeled oligomers nearly simultaneously.The Cy3-rA20 was then transferred to the cover glass andused to focus the confocal microscope on the z-dimensionof interest. An image was collected each minute starting from 15 minafter formation and incubation of Cy3-rA20 for a totalof 60 min. After 5 min, FAM-rA20 was then added to thetop of the mixture. Fusion of these droplets just above the coverglass was observed. Three-dimensional images were rendered with the3D Viewer Plugin on Fiji/ImageJ.To visualize droplet fusionand mixing, two sets of samples were separately prepared in the samemanner as described above, with the use of either the 6FAM-rA30 the extent to which these homopolymers could phase separate givenshorter, fixed lengths was yet unknown. To test the potential forshort, nucleic acid oligomer phase separation, we compared the phaseseparation of 20-mer RNA constructs  and corresponding DNA counterparts  in the presence ofMg2+ with increasing divalent cation concentrations upto 1 M Mg2+. Using DNA and RNA forms of equal length allowedfor comparison across different polymers given variations in bothnucleobase and backbone sugar structures. Although they would havebeen a useful data point, polyG oligomers were not tested here givendifficulties in acquiring oligomers on this length scale.Whileprevious studies have shown the ability of long RNA homopolymersto phase separate in the presence of high concentrations of cationsand, in some cases, crowder,2+ absorptionwas observed, were used as a proxy for condensate formation as previouslyreported.17 Droplet formation and morphology were confirmedwith DIC imaging for select points in the data set.Tomeasure condensate formation, we used a combination of absorptionspectroscopy and confocal microscopy. Changes in absorbance at 350nm, a region where no native nucleic acid or Mg20 begins phaseseparating at 30 mM Mg2+, which we throughout this paperrefer to using the notation, “≥30 mM Mg2+”, marking the lowest Mg2+ concentration testedwhere absorbance at 350 nm increases above background levels (2+ concentration than thepreviously published 250 mM Mg2+ for long polyrU (800–1000kDa) in the absence of a crowder.17 Itis also significantly lower than the concentration of monovalent saltnoted in the phase separation observed with long polyrA in the presenceof 750 mM NaCl with a crowder.30 Interestingly, thetransition appears to show nonmonotonic behavior, where the curveincreases from about 30 mM Mg2+, passes through a maximumaround 80 mM, and then subsequently decreases. Previous examples ofnonmonotonic, reentrant behavior in multicomponent systems have beenobserved for example in charge-based systems,45 where a balanced ratio of charged species tends to favor separationand strongly unbalanced systems do not due to charge repulsion andscreening of networking interactions. In this way, the presence ofreentrant behavior could be indicative of multiple competing forcesinvolved in the phase separation of polyrA and Mg2+ whichinclude both charge-based networking interactions and the more hydrophobicrA–rA interactions that are not screened by salt. Just as inthe reentrant systems of charged polymer species, we hypothesize thatit is possible for rA–Mg2+ systems to have an optimalbalance of these interactions based on the concentrations of rA andMg2+. However, it is also possible that rather than thequantity of material phase separated, the size and shape of the dropletprofile are driving the nonmonotonic behavior. Either of these possibilitiesis interesting and could warrant future exploration.Our resultsindicated that short polyrAd levels 1A. This 20 did form sparsely populated irregular aggregates, while all samples at 1.0, 0.8, and 0.6 mg/mL phase separatedat ≥30 mM Mg2+, with the intensities of absorbanceat 350 nm increasing with concentration and likely suggesting an earlieronset of condensation , further reducing Mg2+ thresholds.Predictably, we observed a shift in the observed phase boundarytoward lower Mgensation 1C. Thus,34 We expected a similar relationship to occur with MgCl2 addition with the possibility for such a reduced length to not phaseseparate under the conditions established. To test this, we examinedthe phase separation potential of rA10, rA15, rA20, and rA30 where the final concentrationof each species was adjusted to approximately 0.5 mg/mL, the equivalentconcentration used for rA20 in the RNA/DNA base comparisons.Consistent with previous studies which demonstrated the importanceof multivalency in phase separation,47 our data showedthat short polyrA condensation is also a length dependent process,with increasing length favoring phase separation and shifting thephase boundary toward lower Mg2+ concentrations. The shortestlength polyrA to phase separate under these conditions was rA15, which phase separated around 100 mM Mg2+, followedby rA20 (≥30 mM Mg2+) and rA30 (≥25 mM Mg2+) . This is consistent with the idea that, below acritical length, RNA no longer forms liquid droplets29 under these conditions and shows that even as few as 15nucleotides of polyrA are sufficient to observe phase separation withinthe polyrA/Mg2+ concentration regime tested.In addition to investigating the effectsof RNA concentration onthe phase separation of the system, we were also interested in determiningthe effects of length on the phase boundary and observing whetherthere was a lower bound to phase separation given a lower valency.Previous fractionation of long polyrA showed a positive relationshipbetween polymer length and phase separation propensity given phaseseparation with NaCl and temperature.mM Mg2+) 1D with r2+-mediated polyrA condensation does appear to followthese expectations makes it more striking that rA20 comparativelyphase separates at such low concentrations of Mg2+ in theabsence of crowders and at lower lengths and concentrations comparedto previously demonstrated long homopolymer work.30 That thisis unique to polyrA of the nucleic acids tested is also interestingin part because of the aforementioned differences in how it interactswith Mg2+ when compared to both polyrU and its DNA analogue42 as well as its potential for self-interaction.40 Networking and phase transitions rely upon favorable interactionswith other macromolecules.7 In this sense, the sameπ–π and cation−π interactions thatenable the formation and destabilization of secondary structural elementswith itself given changes in environment both with cations and withpH49 could be related to polyrA’s ability to phase separate underrelatively low Mg2+.Theseobservations of length, concentration, and crowding are consistentwith the current understanding of the importance of valency, concentration,and crowding in tuning phase boundaries and condensation. The factthat Mg50 However, unlike typical liquid-like condensates,these rA20 droplets seem to exhibit very slow mixing andinternal exchange while still fusing into spherical droplets. Here,we use “fusion” to refer to the process of dropletscombining to form a single droplet separate from the process of internalcontent mixing.Like other liquid-like condensates, polyrA dropletsmerge upon contact and form spherical droplets 2A. In pr20 dropletsseparately labeled with two different dyes, 6FAM-rA20 andCy3-rA20. The fluorescent markers were added to the sampleprior to droplet formation to a final concentration of about 1 μMsuch that the ratio of labeled to unlabeled rA20 was around1:77. Images were taken every minute for 1 h. Upon initial fusion,the droplets do not appear to mix, instead forming spheres but maintainingtheir segregation  on the phase behavior of the rA20/Mg2+ system. If nonspecific charge screening was the predominant roleof the divalent cation, we expected that the addition of significantconcentrations of monovalent salt would lower the phase boundary dueto the extra ions available to screen backbone charges.In previous work focused on thehomotypic separation of polyrU in the absence of crowder,2+ andmonovalent cations, our results revealed opposing cooperativity effectsin polyrA condensates compared to those in long polyrU. Where theaddition of NaCl was additive in polyrU, shifting phase separationto lower concentrations of MgCl2 appended to a “generic” 20 nt sequence  of unstructured RNA frequentlyused in deadenylation studies,61 to create three constructs: rN20 + rN20, rN20 + rA20, and rN20 + rU20.Giventhe ability of polyrA to undergo phase separating at such low lengthand concentration thresholds, one question that emerges is the extentto which such an RNA sequence could serve a functionally similar purposeas IDPs or other low complexity domains in promoting or tuning phaseseparation properties of slightly longer mixed sequences. We expectedthat if this were true, sections of homopolymer RNA may be able totune condensates given the properties of their observed pure, shortforms. To test this idea, we used a mixed construct containing selectoligomer tails . This was a slightlyhigher value relative to the rA20 construct alone, whichseparated around 30 mM ([rA20] = 76.6 μM), but lower than thephase separation propensity of the 0.2 mg/mL rA20 (approximately31 μM) separation, which occurred around 100 mM Mg2+. Through the promotion of phase separation enabled by the rA20 tail, we show that the phase separation properties of noncanonical,polyrA interactions can promote phase separation of a mixed sequencethat otherwise would not have phase separated at the same lengthsand concentrations.Our data from turbidity and microscopy show that the polyrAtailed constructs phase separated more readily, while the mixed 40-merand the polyrU tailed construct did not 4A. Phase20 + rA20 constructsdemonstrated very slow recovery over the course of 30 min  assess the limits of polyrAas a condensation tag, (b) determine whether these same arrested dynamicsoccur in more complex and more physiologically relevant systems ,and (c) establish whether adenylation or deadenylation significantlyalters phase behavior of longer, more complex sequences.Finally,the role of polyrA tails in condensates could be an intriguingavenue of research. While our system uses a higher Mg


As a semi-dominant inheritance mouse model, the heterozygous Myo6C442Y/+ mice recapitulated the progressive SNHL in human.2 Xiao et al. used RNA base editing system, basically composed of base editors and guide RNA (gRNA) to treat this mouse model. To screen optimal RNA base editors and gRNAs for Myo6C442Y correction, Xiao et al. overexpressed Myo6C442Y RNA in the human embryonic kidney (HEK) 293T cells, cotransfected with different RNA base editors (mini dCas13X or Cas13b was fused with ADAR2dd v1 or ADAR2dd v2) and gRNAs targeting C442Y mutation. The mini dCas13X fused with ADARdd-v1(mxABE-v1) showed high editing efficiency with low off-target.Many mutations of human Myo6C442Y/+ mice at postnatal day 0 (P0) to P2. Significant base editing can be detected 2 weeks after the injection, and the AAV-mxABE-T-treatment resulted in the preservation of electrophysiological property, embodied by partial recovery of decreased Ca2+ current amplitude in inner hair cells (IHCs). Importantly, compared to Myo6C442Y/+ mice with substantial degeneration of outer hair cells (OHCs) and substantial disorganization of surviving IHCs and OHCs, the Myo6C442Y/+ mice treated with AAV-mxABE-T showed more OHCs in the middle and basal turns of the cochlea, and more organized hair bundles in OHCs and IHCs. Furthermore, AAV-mxABE-T treatment led to significantly decreased ABR and DPOAE thresholds, indicating an improvement in hearing function. Together, AAV-mxABE-T treatment attenuates some typical microscopic lesions of Myo6C442Y/+ mice in vivo, which may rescue the auditory function of the diseased mice.Next, Xiao et al. packaged mxABE-v1 and gRNA targeting C442Y mutation into adeno-associated virus (AAV)-PHP.eB (hereafter referred to as AAV-mxABE-T) and injected it into the inner ear of 3 Although Cas13a to c have shown varying dependence on a protospacer flanking sequence (PFS) for efficient RNA targeting, Cas13d and Cas13X have no PFS bias. Coupled with its compact size, the mini base editor mxABE provides possibility to treat genetic diseases in vivo via a single AAV vector. By packing mxABE-v1 and gRNA into an AAV, Xiao et al. directly corrected Myo6C442Y/+ mutation in vivo without breaking double-strand DNA and bringing in eternal genomic changes.SNHL is a common disease with no effective treatment. However, the use of drugs, stem cells and gene therapy is struggling to develop as potential strategies for improving hearing cells or auditory function in SNHL patients. Since genetic factors contribute to approximately half of the congenital SNHL cases, gene therapy is potentially important means to inhibit the disease progression and improve the symptom. CRISPR-Cas systems are the most widely used gene editing tools, and base editing is a newer application of these systems to generate precise point mutations in DNA or RNA. The permanent genomic alterations and requirement of a protospacer adjacent motif (PAM) at the editing site may limit the use of DNA base editors, such as Cas9 and Cas12. Since RNA editing is reversible and non-heritable, CRISPR-Cas13 system, specifically targets and edits RNA, should better meet the requirements of ethics, especially in clinical application.4 Since RNA editing is temporary and reversible, repeated AAV injection may be needed in some process of treatment, an optimal highly infectious serotype, an appropriate time, dosage, and method to transfer the virus need to be selected. Besides, large preclinical data about effective methods of controlling the AAVs immunogenicity are required before clinical translation, and methods to reduce the anti-AAV humoral response are required, such as inhibiting B cell activation, modifying AAV surface, and transiently reducing IgG levels. In addition, alternative strategies for AAV methods are being exploited. Several studies have shown that Lipid nanoparticles (LNPs) play an important role in mRNA vaccines against COVID-19 and other nucleic acid-based therapies, which hold promises for their potential to deliver molecules of mxABE and gRNA to the target cells. Moreover, to cure the patients suffering from Myo6,C442Y/+ extended preclinical and clinical studies are required to identify most suitable delivery methods with high efficiency and specificity, and low safety risks.The delivery method is one of the most important issues with the use of mxABE. AAV is considered a promising virus to deliver gene therapy vectors in vivo, but the results of three children’s death in a clinical trial for X-linked myotubular myopathy caused great worry about the safety of recombinant AAVs.5 We expect this variant and other high-fidelity Cas endonucleases to be used to optimize the therapy strategy. Furthermore, it is our opinion that for the foreseeable future, many new anti-CRISPR proteins or small molecule drugs will be found and improved. Either of them, acts as a failsafe or an adjuvant to reduce off-target effects in patients, will make it possible to block the activities of mini dCas13X when it causes side-effects or after required gene editing has achieved to result in precise RNA editing.Because of the off-target effects, strategies for improving the efficiency and fidelity of mxABE are required. An appropriate gRNA is important, even the best-designed gRNA sometimes needs engineering by chemical modifications or other methods to target more specifically. On the other hand, Cas endonucleases are equally important and have been developed rapidly. Tong et al. reported a high-fidelity Cas13X variant exhibiting efficient on-target activity but markedly reduced collateral activity.Lastly, widely recognized international laws and regulations on acceptable therapeutic interventions in humans are required, and tougher measures should be introduced to avoid ethical issues for the clinical application of CRISPR-Cas system.Myo6C442Y mutation and partially repair phenotypic traits in a mouse model. It provides a potential utility of RNA editing tools as a possible therapeutic approach to cure sensorineural hearing loss and other genetic diseases.Taken together, this result from Xiao et al. demonstrates that mxABE has the ability to correct the


RNA–protein complexes regulate a variety of biological functions. Thus, it is essential to explore and visualize RNA–protein structural interaction features, especially pocket interactions. In this work, we develop an easy-to-use bioinformatics resource: RPpocket. This database provides RNA–protein complex interactions based on sequence, secondary structure, and pocket topology analysis. We extracted 793 pockets from 74 non-redundant RNA–protein structures. Then, we calculated the binding- and non-binding pocket topological properties and analyzed the binding mechanism of the RNA–protein complex. The results showed that the binding pockets were more extended than the non-binding pockets. We also found that long-range forces were the main interaction for RNA–protein recognition, while short-range forces strengthened and optimized the binding. RPpocket could facilitate RNA–protein engineering for biological or medical applications. RNA–protein complexes play irreplaceable roles in biological processes, including gene regulation, protein synthesis, and virus replication ,2,3. ForCurrently, the available RNA–protein databases can be divided into three categories. (1) The structural RNA–protein database. The Nucleic Acid Database (NDB) and Protein Data Bank (PDB) provide experimentally determined RNA–protein complexes with sequences and structures ,13. In aWe propose a pocket-based interaction database, RPpocket, for RNA–protein complexes. We first identify the RNA–protein interaction interface and binding sites. Then, we systematically analyze the secondary structures and topological features for the interface interaction. The results show that the protein loop prefers to bind in the RNA stem region. The electrostatic interactions are the main force for long-range recognition, while short-range forces optimize and strengthen the interactions. RPpocket provides a straightforward framework to reveal RNA–protein recognition principles for RNA-related drug development or medical applications. There are 74 non-redundant RNA–protein structures and 793 pockets in the RPpocket database. The RNA length is 20~266 nucleotides, and the protein length is 22~477 amino acids. According to the different functions, we divided these 74 structures into the following categories : tRNA  were more extended than the non-binding pockets .The pocket geometrical volume and surface area information are some of the most critical characteristics of RNA–protein interactions. We analyzed the topological features of the RNA pockets, including 60 binding and 32 non-binding pockets. The volume, surface area, effective radius, sphericity, and pocket centroid were calculated and recorded in our database. We performed a statistical pocket geometrical analysis for different RNA categories . The resTo understand RNA–protein interaction patterns, we calculated the binding frequency between the nucleotides and amino acids of the representative structures in the RPpocket database. There were more positive amino acids  involved in the interactions (31.48%) than negative amino acids . Moreover, the proportions of hydrophobic amino acids , polar amino acids , and other amino acids (glycine and proline) were 26.94%, 23.21%, and 10.80%, respectively a. We furIn general, RPpocket has the following characteristics and advantages: (1) containing 132 RNA pockets and 661 protein pockets extracted from non-redundant 74 RNA–protein complexes; (2) providing the sequence and tertiary structure of all complexes in the dataset; (3) showing the interaction patterns of binding sites and secondary structures between RNA and protein; (4) analyzing the topological pocket features ; (5) allowing the users to rotate and translate the structure in the visualization module; (6) supplying tools for RNA complex prediction.RPpocket has eight modules: Home, Search, Visualization, Download, Links, Tutorial, Statistics, and Contacts. The Home module mainly introduces the RPpcoket database and the navigation to other modules . The seaThe NDB (Nucleic Acid Database) and PDB (Protein Data Bank) are widely used databases for biologists ,13. The Currently, several databases focus on RNA–protein complexes . NPInterSeveral existing protein-related databases also provide sequence, structure, or interaction information . For exaTo construct the RNA–protein dataset , we usedAn RNA–protein interface was defined if the heavy atom distance between nucleotide and amino acid was less than 4 Å. We extracted the secondary structure in dots and brackets format from the RNA FRABASE 2.0 . The secV and A represent the volume and area of the pocket [The RNA pockets were detected using a rolling probe method with the 3V program ,43,44,45e pocket .The protein pockets were detected by DoGSiteScorer, which is widely used for detecting protein surface pockets and subpockets ,48,49,50We statistically analyzed RNA–protein complex sequences, secondary structures, and topological pocket features in this work. The results show that protein loops prefer to bind to the RNA stem region. Long-range electrostatic interactions bring RNA and protein together. Short-range interactions optimize the complex. We also developed one easy-to-use database, RPpocket, to provide pocket-based topological interaction information. We hope that RPpocket will facilitate RNA-related study and inhibitor design.


Previously, interferon-α was the only recommended therapy for HDV infection. In recent years, some new therapies targeting these regions, such as Bulevirtide, Lonafarnib, Nucleic acid polymers have appeared, with better curative effects and fewer adverse reactions.Hepatitis Delta Virus (HDV) is the smallest mammalian single-stranded RNA virus. It requires host cells and hepatitis B virus (HBV) to complete its unique life cycle. The present review summarizes the specific regions on hepatitis D antigen (HDAg) and hepatitis B surface antigen (HBsAg) that drive HDV to utilize host cell machinery system to produce three types of RNA and two forms of HDAg, and hijack HBsAg for its secretion and  Hepatitis delta virus is a defective virus that requires HBV to proliferate in human hepatocytes. A recent meta-analysis estimated that 292.0 (251.5–341.1) million individuals were HBsAg positive, corresponding to a global prevalence of 3.9% (3.4–4.6) ; anothervia two ways: coinfection and super-infection. HBV and HDV simultaneously co-infect healthy people, causing extensive hepatic necrosis and severe fulminant hepatitis with a high fatality rate. Most adults can eliminate both viruses and recover from coinfection, and < 5% of cases develop chronic hepatitis D (CHD) (Hepatitis delta virus infection occurs  D (CHD) . Compare D (CHD) .Hepatitis B virus vaccine protects healthy individuals from HBV and HDV infections. However, there is currently no HDV vaccine available protecting chronic hepatitis B patients from HDV infection. Before the European Medicines Agency approved the conditional marketing authorization of Bulevirtide in July 2020, interferon (IFN)-α was the only therapy for CHD recommended by professional and societal guidelines. However, IFN-α can lead to common and sometimes severe adverse reactions, with a high recurrence rate .We summarize the specific regions on HDAg and HBsAg that drive HDV proliferation, and the curative effect of some therapies targeting these regions. We further list some mechanisms underlying the HDV life cycle that are yet ill-understood, and some regions that are potential therapeutic targets. Investigating HDV proliferation mechanisms and the functions of specific regions on HDAg and HBsAg will help us to develop new treatment strategies associated with better therapeutic effects, fewer adverse reactions, and lower recurrence rates.Hepatitis delta virus is 35–37 nm in diameter and composed of HDV ribonucleoprotein (RNP) complex and HBsAg-containing envelope . HDV RNPThere are currently eight HDV genotypes (HDV-1–8), the ribonucleotide number of HDV gRNA is between 1,672 and 1,697 (genotype-dependent) . HDV gRNGenomic strand ribozyme and antigenome strand ribozyme are the only viral enzymes possessed by HDV, display related sequences and are similar in the predicted secondary structure, and both are crucial for HDV RNA replication . HDV gRNTwo forms of HDAg that are translated from HDV mRNA have been identified, including small-HDAg  and large-HDAg . HDV mRNA structure is similar to that of the host mRNA, containing a 5′ cap, a 3′ poly-A tail, and a coding region . A variaThe three critical regions on the S-HDAg and L-HDAg N-terminal identity region (1–195 aa) are as follows: (i) RNA-binding domain , binding to HDV RNA to form HDV RNP ; (ii) Covia CCS  trvo entry ; (iii) cvo entry ; (iv) anvo entry ; (v) Tryvo entry .de novo entry of HDV relies on the HBsAg-containing envelope, and the mechanism underlying de novo entry is also similar to that in HBV infection. HDV primarily targets human hepatocytes, and no evidence has shown that HDV naturally infects other cells , which is expressed on the outer face of the hepatocyte membrane ] . Throughnfection . Primarynfection .via clathrin-mediated endocytosis pathway , antigenomic RNA , and mRNA (∼800 nt). The copy number of these RNA is approximately 300,000, 60,000, and 600/cell, respectively . MoreoveIn nuclear, the parental gRNP migrates to Pol-II-containing speckles . Pol-II After enough mRNA are transcribed, parental gRNP migrates to the nucleolus for agRNA replication. Although the migration machinery is not yet clear, it seems to be related to the newly synthesized S-HDAg . SubsequThe structure of circular agRNA is similar to gRNA, it wraps around the newly synthesized HDAg multimers to form agRNP, and migrates to Pol-II-containing nuclear speckles. Pol-II uses agRNP as a template, through the rolling-circle mechanism, to replicate linear gRNA polymers. HDV genome strand ribozyme cleaves linear gRNA monomers from linear gRNA polymers. The linear gRNA monomers are self-ligated into circular gRNA molecules and wrap around the HDAg multimers to become the progeny gRNP.According to the amplification process of HDV RNA, the progeny gRNP works as parental gRNP to amplify more mRNA or agRNA, or leaves the nucleus to form progeny virus.As mentioned above, the amplification of HDV RNA is accompanied by the production of S-HDAg. Notably, after translation from mRNA, S-HDAg requires posttranslational modification, which is critical for its function. These critical modifications include: (i) Arginine methyltransferase catalyzes methylation at Arg-13, which is essential for RNA replication, affecting HDAg subcellular localization ; (ii) AcAfter binding to HDV RNA, S-HDAg further interacts with several components of the host cell: (i) Pol-II. S-HDAg interacts with 9/12 subunits of Pol-II , the comIn the Pol-II-dependent mRNA and gRNA amplification, S-HDAg mainly activates RNA amplification and promotes RNA elongation. In the Pol-I dependent agRNA replication, S-HDAg stabilizes the agRNA, protects it from nuclease decomposition, and promotes its cleavage and self-ligation .Hepatitis delta virus RNA amplification requires S-HDAg, while L-HDAg prevents HDV RNA amplification and is essential for virus assembly. After accumulating some S-HDAg in cells, HDV RNA will be edited to produce L-HDAg. The newly synthesized L-HDAg is also posttranslational modified.A family of proteins called adenosine deaminases acting on RNA (ADAR) catalyzes the adenosine (A) to inosine (I) editing reaction on dsRNA . HDV RNAWhen Pol-II uses the edited-agRNA as a template, it replicates an ACC triplet on edited-gRNA. Pol-II further uses the edited-gRNA as a template to transcribe a W codon (UGG) on edited-mRNA. The edited-gRNA is defective for edited-agRNA replication .As mentioned above, there is a variable amber/W site in mRNA. For original mRNA, the site contains an amber codon  and the translation stops here to produce an S-HDAg; For edited-mRNA, the site contains a W codon , the translation doesn’t stop here, keeps adding the C-terminal 19 amino acids of L-HDAg until the next termination codon .Adenosine deaminases acting on RNA 1 regulates the HDV life cycle by converting the early stage of HDV RNA amplification (mediated by S-HDAg) into the later stage of virus assembly (mediated by L-HDAg). Some genotypes of HDV  do not display an A-C mismatch pair at the editing site, thereby use alternative transient conformation to increase the binding efficiency of ADAR1 .Phosphorylation, acetylation, methylation, and sumoylation are posttranslational modifications of S-HDAg that also occur in the N-terminal identity region (1–195 aa) of L-HDAg. Additionally, the C-terminal extension region (196–214 aa) of L-HDAg requires farnesylation. The last four amino acids at the C-terminal (211–214 aa) of L-HDAg are Cys-Arg/Thr-Pro/Gln-Gln (C-R/T-P/Q-Q), which constitute a farnesylation signal . The farNuclear localization signal drives both farnesylated and non-farnesylated L-HDAg to enter into nucleus through the nuclear pore complex and the positively charged RBD drive them bind to HDV RNA. Nonetheless, the farnesyl group on L-HDAg is necessary for the combination between HDV RNP and HBsAg-containing envelope , and is After the start of RNA editing, the amount of L-HDAg in the nucleus increases in a time-dependent manner until the balance between the two HDAg is achieved. A small amount of L-HDAg is sufficient to inhibit the replication of gRNA. When L-HDAg exceeds S-HDAg, it inhibits the amplification of agRNA and mRNA .As the amount of L-HDAg increases in the nucleus, the proportion of L-HDAg monomers on the newly synthesized HDV gRNP is also increased. Considering the number of S-HDAg and L-HDAg are equivalent in circulating HDV , we specExperiments have shown that despite the binding to HDAg, agRNA cannot leave the nucleus, most of the agRNA remains in the nucleus, but the amount of HDV gRNA in the cytoplasm and nucleus is almost equal . Howevervia HBV budding mechanism. As helper virus, HBV only mediates HDV de novo entry and secretion.After assembling with HBsAg-containing envelope, HDV is secreted Via ESCRT/MVB pathway, Some HBsAg self-assembles to form filament-SVPs, which contain a much larger amount of the L-HBsAg compared to sphere-SVPs, and are secreted through ESCRT/MVB pathway like infectious viral particles (12–1013/mL) in serum of chronic hepatitis B patients is much higher than infectious HBV particle (108–109/mL)  , which i10–1011/mL serum  envelope protein) into HCV host cells, HDV replicates and binds to E1E2, then is secreted and enters into other cells via E1E2 . But clivia E1E2 . These fvia E1E2 .Without HBsAg expression, HDV persists in humanized mice for at least 6 weeks, leading to host cell detachment and death due to rapid virus replication, or be converted by HBV infection to develop coinfection . Cell dede novo entry, RNA amplification, HDAg production, assembly, and secretion) and the regions that drive each step are illustrated in The entire life cycle of HDV  and injection site reactions (Bulevirtide (formerly known as Myrcludex B) is a myristoylated synthetic lipopeptide, with the same amino acids sequence as the N-terminal 47 aa in L-HBsAg RBS. eactions .Lonafarnib is a farnesyl transferase inhibitor that prevents the farnesylation of L-HDAg, thus blocking the combination between HDV RNP and HBsAg. Lonafarnib should be administered with other antiviral drugs to achieve the best effect. When Lonafarnib and Ritonavir (RTV) are administered in combination for 24 weeks, 38.5% of CHD patients had undetectable HDV RNA . When LoNucleic acid polymers (NAPs) are phosphorothioated oligonucleotides, which means in every phosphodiester linkage of the oligonucleotides, one of non-bridging oxygen atom is replaced by a sulfur atom. In local hydrophobic microenvironment, the free electron in the non-bridging sulfur atom can transit from the unshared (charged) state to a shared (uncharged) state with the central phosphorous atom. The transition ability of the sulfur atom allows NAPs to adopt a localized hydrophobic property along their long axis and form a hydrophobic surface, the rest part of the NAPs forms a hydrophilic surface, altogether confer NAPs amphipathic property .via folding together of the hydrophobic surfaces, amphipathic NAPs bind amphipathic α-helices in class I viral glycoproteins, which are present on the surface of many enveloped viruses  RNA amplification. S-HDAg activates the amplification of mRNA and gRNA, stabilizes agRNA and promotes its cleavage and self-ligation; (iii) HDAg production. After modification, mediated by CCS and NLS, HDAg oligomerizes and re-enters the nucleus; (iv) Assembly. HDV RNA wraps around HDAg multimers to form HDV gRNP via RBD, NES drives the egress of HDV gRNP from the nucleus. After leaving the nucleus, HDV gRNP anchors to the endoplasmic reticulum membrane via farnesyl group, and binds to HBsAg-containing envelope via the interaction between TRM and farnesyl group to form HDV; (v) Secretion. Driven by HBsAg-containing envelope, HDV secretes via the Golgi apparatus classic constitutive secretory pathway.Hepatitis delta virus is a robust pathogen that spreads widely. Since an ideal therapy is yet unavailable, in-depth exploration is essential. The present review summarizes the effects of the specific regions of HDAg and HBsAg on HDV life cycle: (i) Some regions have been applicated for HDV therapy: (i) RBS: Bulevirtide is an L-HBsAg RBS analog, competing with RBS for specific receptor hNTCP to block HDV entry; (ii) Farnesyl group: Lonafarnib is a farnesyl transferase inhibitor, preventing the farnesylation of L-HDAg, thus blocking the combination between HDV RNP and HBsAg; (iii) HBsAg: NAPs are phosphorothioated oligonucleotides that bind to amphipathic α-helices of HBsAg, preventing the secretion of HBsAg. Except these three regions, several other regions are potential therapeutic targets. For example, blocking NLS or NES of HDAg will prevent HDAg nucleocytoplasmic shuttling, blocking CCS or RBD of HDAg will prevent the assembly among HDAg or between HDAg and RNA, and blocking TRM of HBsAg will prevent the assembly between HDV RNP and HBsAg-containing envelope.de novo entry; key factors driving HDV to remove the HBsAg-containing envelope and expose HDV RNP; method to change the HDV RNP dominant signal from farnesyl group (anchor to endoplasmic reticulum) in assembly stage to NLS (enter into the nucleus) in new round of the de novo entry; whether and how Pol-III participates in the amplification of HDV RNA; the domains or signals of S-HDAg involved in the migration of gRNP from the nucleoplasm to the nucleolus during RNA amplification; enzymes catalyzing the connection of HDV RNA linear monomers to form circular monomers. Investigating HDV proliferation mechanisms and the functions of these specific regions on HDAg and HBsAg will help us to discover new treatment strategies and develop new drugs associated with better therapeutic effects, fewer adverse reactions, and lower recurrence rates.There are still several mechanisms underlying the HDV life cycle are yet ill-understood, including but not limited to the following: the assembly structure of HDV RNA and HDAg; similarities and differences between HBV and HDV during the JZ: data curation, formal analysis, writing-original draft, and writing-review and editing. XG: data curation, writing-original draft, and writing-review and editing. JD: writing-review and editing. HX: formal analysis, validation, and writing-review and editing. JN: funding acquisition, supervision, and writing-review and editing. XC: data curation, supervision, validation, and writing-review and editing. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The development of novel target therapies based on the use of RNA interference (RNAi) and antisense oligonucleotides (ASOs) is growing in an exponential way, challenging the chance for the treatment of the genetic diseases and cancer by hitting selectively targeted RNA in a sequence-dependent manner. Multiple opportunities are taking shape, able to remove defective protein by silencing RNA , by arresting mRNA translation , or by reactivating modified functional protein  or a not very functional protein. In this last case, the use of ASOs permits modifying the expression of specific proteins by modulating splicing of specific pre-RNAs  or by downregulation of transcript levels  in order to restore the biochemical/physiological condition and ameliorate quality of life. In the era of precision medicine, recently, an experimental splice-modulating antisense oligonucleotide, Milasen, was designed and used to treat an 8-year-old girl affected by a rare, fatal, progressive form of neurodegenerative disease leading to death during adolescence. In this review, we summarize the main transcriptional therapeutic drugs approved to date for the treatment of genetic diseases by principal regulatory government agencies and recent clinical trials aimed at the treatment of cancer. Their mechanism of action, chemical structure, administration, and biomedical performance are predominantly discussed. The his-A “RNAse-dependent mechanism” that takes place in the nucleus and is mediated by the enzyme “RNAse H”, able to degrade the RNA strand of RNA/DNA duplex .-a.One is dependent from the interaction between ASO and transcript, thus preventing RNA loading on the ribosome and arresting mRNA translation  9];;9];b.Another mechanism is operating through the splicing process —that is,“RNAse-independent mechanisms” that take place in the cytoplasm; in this case, the ASO can act through different paths:“Antisense Oligonucleotides” (ASOs) are short-length single sequences of deoxynucleotides, 12–28 bases, that are synthesized to be complementary to a sequence of mRNA or pre-mRNA to generate a DNA/RNA heteroduplex; in this way, they can regulate the target expression. For the first time, Zamecnik and Stephenson used ASO to inhibit Rous sarcoma virus (RSV) cycle ..24].those acting through regulation of transcript levels (1) and those modifying the splicing of specific pre-RNAs (2).The first transcript-targeted therapies authorized for use in clinical practice are focused against genetic diseases. These therapies, based on RNAi or ASOs, can be distinguished between (1). Downregulation of transcript levels by RNAi-based drugs  by the ASO-based drug (Inotersen) and second-generation ASO-based drug (Volanesorsen/Waylivra).(A) Hereditary Transthyretin Amyloidosis (hATTR) is a rare autosomal dominant, multisystemic, progressive disease caused by more than 120 mutations in the gene encoding transthyretin (TTR) located on chromosome 18q12.1. The protein TTR binds and transports thyroxine and retinol. The most common mutation is a single-nucleotide substitution that causes the substitution of Valine in position 30 with Methionine   . The assAn alternative therapeutic target is represented by the reduction in the circulating level of TTR, thus decreasing the amount of proteins that can form the amyloid fibrils. Two drugs have been designed based on this mechanism of action: “inotersen”, an ASO-based drug, and “patisiran”, a RNAi-based drug. These drugs are useful alternatives to other classical treatment such as liver transplantation and TTR stabilizers.ClinicalTrials.gov number: NCT01737398, https://clinicaltrials.gov/ct2/show/NCT01737398, accessed on 30 April 2022), inotersen was administered by subcutaneous injections, three times a week for the first week followed by once a week administration for 64 weeks. Patients that received inotersen obtained an improvement of their life’s quality. However, they showed different ADRs such as glomerulonephritis, thrombocytopenia, and death . I. ISpheriThe use of ASO- and siRNA-based therapeutics for the treatment of a range of genetic diseases is an established fact. It is likely that the improvement of delivery vectors and chemical formulations will translate into an improvement in clinical efficacy. On the contrary, the use of this kind of therapeutics is still an area of active investigation for cancer treatment. The achievement of clinical utility will probably require several efforts in order to define molecular targets for specific tumor subtypes and to design selective delivery procedures . In part