{"paper_id":"ed28e01a-eed8-4bb2-81fa-d328fecd423c","body_text":"Natural antisense transcripts (NATs) transcribed from eukaryotic genes are primarily long (>200 nucleotides) transcripts that do not encode proteins, that is, long non-coding RNAs (lncRNAs). NATs are transcribed in the opposite direction to mRNA from the same gene and are distributed in the nucleus and cytoplasm. NATs are highly abundant in the human genome and are found at many gene loci ( Krappinger et al., 2021 ;  Seal et al., 2023 ).\nAlthough NATs are heterogeneous and are mostly expressed at low levels, short-read sequencing (RNA sequencing) has enabled the identification of many lncRNAs, including NATs, as well as other regulatory RNAs.\nBased on their relative positions, NATs are classified into three types ( Latgé et al., 2018 ;  Krappinger et al., 2021 ): (1)  intergenic , a NAT (lncRNA) located between two genes; (2)  intronic , a NAT located in an intron; and (3)  overlapping , a NAT overlapping with an mRNA and involving mutual interactions ( Figure 1A ). Standardized nomenclature was assigned to lncRNAs, including overlapping and intronic NATs ( Seal et al., 2023 ). For example, the overlapping antisense transcript of the human interferon ( IFN ) gene is designated  IFN-AS  ( Kimura et al., 2015 ).\nMessenger RNA (mRNA) and regulatory RNAs in cells.  (A)  Transcription of mRNA and NAT: An mRNA is transcribed from a gene, and then spliced, capped, and polyadenylated. NATs (shown as  AS ) synthesized from corresponding genes are >200 nucleotides in the length and do not code for proteins, that is, lncRNAs. Three types of NATs are intergenic, intronic, and overlapping NATs ( Latgé et al., 2018 ;  Krappinger et al., 2021 ).  (B)  RNA duplex formation between mRNA and NAT: An overlapping NAT hybridizes with an mRNA at a single-stranded region, such as a loop or bulge in the secondary structure to form a short mRNA–NAT duplex, which is then bound to RNP(s). Resultant complex stabilizes (sometimes destabilizes) the mRNA ( Nishizawa et al., 2015 ;  Nishizawa et al., 2022 ).  (C)  ceRNAs: miRNA is synthesized as a precursor from a given gene and then undergoes processing by Drosha and Dicer ( Virtue et al., 2012 ). circRNA is also synthesized. MREs are located in mRNAs, NATs, and circRNA. Binding of miRNA to an MRE(s) of mRNA is competitively inhibited by NATs and circRNAs if they share the common MRE(s) ( Karreth and Pandolfi, 2013 ;  Tay et al., 2014 ;  Kimura et al., 2015 ).\nNATs play diverse roles in regulating gene expression at the epigenetic, transcriptional, and post-transcriptional levels in the nucleus and cytoplasm ( Wahlestedt, 2006 ;  Nishizawa et al., 2015 ;  Werner et al., 2024 ). NATs are regulatory RNA molecules implicated in epigenetic modifications, transcriptional interference, modulation of alternative splicing to mediate translational efficiency, mRNA stability, masking of microRNA response elements (MREs) to sponge microRNAs (miRNAs), and other mechanisms ( Faghihi and Wahlestedt, 2009 ;  Nishizawa et al., 2015 ;  Krappinger et al., 2021 ). miRNAs modulate protein expression through mRNA degradation and translational repression ( Virtue et al., 2012 ).\nTwo well-studied mechanisms of NAT actions are (1) an antisense transcript (overlapping NAT) and its relevant mRNA form an mRNA–NAT duplex at loops in the secondary structure, to which RNA-binding proteins (ribonucleoproteins, RNPs) bind to stabilize the mRNA ( Matsui et al., 2008 ) ( Figure 1B ); and (2) common MRE(s) shared between antisense transcript and its mRNA. The NATs act as a competing endogenous (ce) RNA by sequestering MRE-shared miRNAs, which dowenregulate their expression ( Karreth and Pandolfi, 2013 ;  Tay et al., 2014 ) ( Figure 1C ). Consequently, NATs in the ceRNA network are involved in various pathological processes, including immune responses, neurodegenerative disorders, tumorigenesis and oncogenic progression ( Krappinger et al., 2021 ).\nHere, we focus on several examples of NAT-mediated mechanisms and provide an overview of the regulatory networks involving mRNAs, NATs, and other regulatory RNAs. We also discuss the biological functions and potential applications of NATs in the treatment of NAT-associated diseases.\n\nInterferon-α1 is a type I interferon family member that is induced in response to viral infection as a key part of the innate immune response. The interferon-α1 ( IFNA1 ) mRNA levels are post-transcriptionally regulated by its antisense transcript  IFNA1-AS , which stabilizes  IFNA1  mRNA through cytoplasmic  IFNA1  mRNA– AS  duplex formation at a single-stranded loop domain and enhances the accessibility of RNPs ( Kimura et al., 2013 ) ( Figure 1B ). Another possible mechanism may involve this  IFNA1-AS , as well as the mRNAs and antisense transcripts of other members of the  IFNA  multigene family, acting as ceRNAs via common MREs ( Kimura et al., 2015 ) ( Figure 1C ). Therefore,  IFNA  mRNAs and  IFNA-AS  determine post-transcriptional  IFNA1  mRNA levels.\nIFNA-AS  represses the growth of human influenza A virus in a guinea pig model, harboring MX dynamin-like GTPase 1. Antisense oligoribonucleotides, which represent the functional domains of  IFNA1-AS,  inhibit influenza A virus proliferation in the respiratory tracts of virus-infected guinea pigs ( Sakamoto et al., 2019 ). These antisense oligoribonucleotides mimicked  IFNA1-AS  and exhibited antiviral activity.\nSeveral lncRNAs, including NATs, have been implicated in breast cancer development.  Latgé et al. (2018)  summarized these studies and discussed the molecular mechanisms underlying their functions. EPHA2 is a receptor tyrosine kinase that is overexpressed in breast and other cancers.  EPHA2  expression is thought to be causally related to tumorigenesis. Two splice variants of the NATs of  EPHA2  gene ( EPHA2-AS1/2 ) overlap with  EPHA2  mRNA and may form  EPHA2  mRNA– AS  duplexes. They modulate  EPHA2  mRNA levels in human breast adenocarcinoma cell lines and patient samples, with the highest levels detected in triple-negative breast cancer cells, such as MDA-MB-231 cells ( Okuyama et al., 2020 ).\nEPHA2-AS1/2  modulates cytoplasmic  EPHA2  mRNA levels by interacting with a complementary single-stranded region of the mRNA specific to  AS1/2  in MDA-MB-231 cells. This result was confirmed by  EPHA2-AS1/2  silencing using a sense oligonucleotide or by overexpression of an antisense oligoribonucleotide, in which both the sense and antisense oligonucleotides were derived from the functional region of  EPHA2  mRNA targeted by  EPHA2-AS1/2  ( Okuyama et al., 2020 ). These antisense transcripts promote the proliferation and migration of cells through EPHA2-dependent Ras signaling pathways. These findings suggest that  EPHA2-AS1/2  is a potential target for the triple-negative breast cancer treatment ( Odaka et al., 2024 ).\nThus, NATs may play a crucial role in the pathophysiology of inflammatory diseases. iNOS (also known as NOS2) catalyzes the production of the proinflammatory mediator nitric oxide (NO). In sepsis, excessive NO production in hepatocytes and macrophages has been implicated in tissue injury ( Nishizawa et al., 2015 ;  Nishizawa et al., 2022 ). Similar to  EPHA2-AS1/2 , the overlapping antisense transcripts of  iNOS  gene ( iNOS-AS ) interact with and stabilize  iNOS  mRNA with RNPs ( Matsui et al., 2008 ). When an oligonucleotide representing the loop of  iNOS  mRNA (i NOS  sense oligonucleotide) targeted by  iNOS-AS  was introduced into rat hepatocytes, it decreased both  iNOS  mRNA and protein levels by interfering with mRNA– AS  interactions in the cytoplasm ( Nishizawa et al., 2015 ).\nTo target the mRNA–NAT duplex mechanism, NAT-targeted regulation technology (NATRE) using sense oligonucleotides may be applied in the treatment of animal disease models. A sepsis/endotoxemia model was established by administering D-galactosamine and bacterial lipopolysaccharide to rats. When an  iNOS  sense oligonucleotide was simultaneously administered with D-galactosamine and lipopolysaccharide to rats, the survival rate significantly increased. The  iNOS  and tumor necrosis factor-α mRNA levels were also decreased in the livers of the sense oligonucleotide-administered rats ( Okuyama et al., 2018 ;  Nishizawa et al., 2022 ).\nRecombinant soluble thrombomodulin (rTM), an endothelial-type anticoagulation cofactor that inhibits intravascular coagulation by binding to thrombin, is used to treat disseminated intravascular coagulopathy (DIC) by suppressing coagulation, inflammation, and apoptosis. Because DIC, also known as sepsis-associated coagulopathy, is a frequent complication of sepsis, combination therapy with an  iNOS  sense oligonucleotide and rTM was evaluated for hepatoprotection in a rat model of septic shock after partial hepatectomy. The combination of the anti-DIC drug rTM and the  iNOS  sense oligonucleotide improved the survival of sepsis model rats compared with the sense oligonucleotide alone and reduced hepatic  iNOS  mRNA levels and serum NO concentrations; however, it remains unclear whether rTM affects  iNOS  mRNA –AS  interactions ( Nakatake et al., 2023 ). Collectively, sense oligonucleotides may act as nucleic acid drugs that suppress inflammatory responses, and their efficacy may be enhanced by other drugs with different mechanisms of action.\n\nTwo typical NAT-mediated mechanisms that target mRNA–NAT duplexes and ceRNAs through MRE(s) are reviewed above. They function in the cytoplasm to regulate mRNA levels, whereas NATs also regulate gene expression in the nucleus. Other NAT-mediated mechanisms are discussed below.\nNATs regulates the alternative splicing. The leukocyte common antigen  CD45  encodes protein tyrosine phosphatase receptor type C, which is expressed in all nucleated hematopoietic cells. The primary  CD45  transcript uses three alternative exons to produce four protein isoforms. The antisense transcript ( CD45-AS ,  PEBP1P3 ) was transcribed in the opposite direction of the  CD45  gene (intron 2). Intronic  CD45-AS  regulates alternative splicing of  CD45  mRNA, possibly by regulating histone H3 modification (acetylation and methylation) and DNA methylation of  CD45  gene intron 2.  CD45-AS  also decreases the binding of the chromatin conformation organizer CCCTC-binding factor (CTCF) to intron 2 and alternative splicing exons of  CD45  gene. The expression levels of  CD45  splicing isoforms affect lymphocyte function and development and thus, immune system activity. Therefore, changes in the expression levels of  CD45  alternative splicing isoforms caused by  CD45-AS  may contribute to autoimmune diseases or immune deficiencies ( Su et al., 2021 ).\nCircRNAs are single-stranded, covalently closed RNA molecules that are ubiquitous across species ranging from viruses to mammals. CircRNAs classified as novel ncRNA species were identified by transcriptome analysis using RNA sequencing. Several circRNAs have been identified. Lariat RNAs, which are covalently closed and spliced from precursor mRNAs, are well-known intronic circRNAs. Due to their covalent ring structure and lack of free ends, circRNAs are highly stable ncRNAs in cells. circRNAs are expressed in mammalian cells in a tissue- or cell-specific manner and primarily function as miRNA sponges ( Zhou et al., 2020 ). Because circRNAs compete with NATs when they share common MRE(s), circRNAs and NATs are ceRNAs.\ncircRNAs are involved in the development and progression of cancer, cardiovascular diseases, diabetes, and neurological disorders ( Zhou et al., 2020 ;  Karimi et al., 2025 ). NATs and their corresponding regulatory RNAs play crucial roles in the pathogenesis of neuropathic pain. Peripheral nerve injury or noxious stimuli can induce extensive changes in the expression of miRNAs, NATs, lncRNAs, and circRNAs. miRNAs regulate neuroinflammation during the progression of neuropathic pain. Antisense transcripts of early growth response 2 ( Egr2-AS ) and potassium voltage-gated channel subfamily A member 2 ( Kcna2-AS ) were upregulated in Schwann cells and dorsal root gangla, respectively. LncRNAs and NATs, such as X-inactive specific transcript ( XIST ) and nuclear paraspeckle assembly transcript 1 ( NEAT1 ), are upregulated in the spinal cord, and  XIST  and  NEAT1  act as miRNA sponges. The circRNA circHIPK3 was abnormally expressed in the dorsal root ganglia, whereas ciRS-7, cirZNF609, and circ_0005075 were upregulated in the spinal cord. Sequestration of miRNAs by NATs and circRNAs leads to the expression of pain-related molecules or modulation of miRNA processing ( Jiang et al., 2022 ).\nEpigenetic modifications such as  N \n 6 -methyladenosine (m 6 A) and 5-methylcytosine (m 5 C) are frequently observed in various RNA transcripts. These modifications alter RNA structure and properties, thereby modulating lncRNA functions and interactions ( Cusenza et al., 2023 ). The m 6 A modification of lncRNAs facilitates transcriptional activation ( Lee et al., 2021 ). The binding of YTH domain-containing family protein 2 (YTHDF2), an m 6 A reader, to m 6 A leads to the degradation of m 6 A-containing mRNA ( Wang et al., 2014 ). The functions of m 5 C are less clear, although its biological effects include regulation of RNA localization, stability, and transcription efficiency ( Cusenza et al., 2023 ).\nThe m 6 A modification affects the transcription of many genes and has been implicated in cancer ( Cusenza et al., 2023 ), diabetic nephropathy ( Huang et al., 2024 ), and Alzheimer’s disease ( Zhang et al., 2022 ). The antisense transcript of the  STEAP3  gene ( STEAP3-AS1 ) is highly expressed in human colorectal cancer tissues.  Zhou et al. (2022)  reported that  STEAP3-AS1  is induced by hypoxia-inducible factor-1α (HIF-1α) and affects  STEAP3  mRNA stability by binding to the m 6 A reader YTHDF2.  STEAP3-AS1  competitively interacts with YTHDF2 to dissociate YTHDF2 from  STEAP3  mRNA and prevent m 6 A-mediated  STEAP3  mRNA degradation. High-level expression of the STEAP3 protein activates the Wnt/β-catenin signaling pathway and promotes colorectal cancer progression.\nEVs have been studied extensively over the past decade. EVs, including exosomes (endosome-derived particles with plasma membranes, classified as small EV), are secreted by cells into the extracellular space, i.e., their microenvironment and systemic circulation such as blood and urine ( Jeppesen et al., 2023 ). EVs carry cell-derived proteins and RNAs (mRNAs, NATs, and miRNAs) and transport them to other cells ( Miceli et al., 2024 ). The EV cargo detected by liquid biopsy may seerve as disease biomarkers. EV uptake generally occurs in the liver, kidneys, spleen, lungs, colon, and bones ( Jeppesen et al., 2023 ). EVs are involved in intercellular communication with other cells. The incorporation of NATs from cancer cell-derived EVs into other (acceptor) cells may promote cell growth and metastasis.\nAbnormal NAT expression of the actin filament-associated protein 1 gene ( AFAP1-AS ) and lncRNAs, including  H19  and metastasis-associated lung adenocarcinoma transcript 1 ( MALAT1 ), is observed in endometriosis, a common gynecological disease. Additionally, the antisense transcript of  HIF1A  gene ( HIF-AS ,  aHIF ) is highly expressed in the serum EVs of patients with endometriosis. These NATs can be transported through small EVs and subsequently introduced into other cells. These transferred endometriosis-related NATs can sponge miRNAs, promoting the invasion and metastasis of endometrial stromal cells and leading to endometriosis progression ( Liu et al., 2023 ).\nSolute carrier family 16 member 1 (SLC16A1) accelerates lactate influx and induces M2 macrophage polarization. Small EVs from hepatocellular carcinoma (HCC) cells carry NAT ( SLC16A1-AS1 ), which stabilizes  SLC16A1  mRNA by facilitating its interaction with heterogeneous nuclear ribonucleoprotein A1 (HNRNPA1). IL-6 secreted by M2 macrophages activates signal transducer and activator of transcription 3 (STAT3) to induce methyltransferase 3 ( METTL3 ) transcription in HCC cells. Increased METTL3 enhances m 6 A methylation of  SLC16A1-AS1 , and m 6 A-methylated  SLC16A1-AS1  increases the stability of  SLC16A1  mRNA. When  SLC16A1-AS1  in EVs is incorporated into other HCC cells, it promotes progression toward malignancy ( Hu et al., 2024 ).\nFinally, nodal growth differentiation factor (NODAL) is a transforming growth factor-β superfamily member and required during early embryonic development. NODAL is also involved in tumor progression and metastasis.  NODAL-AS  ( LADON ), a NAT that overlaps with exon 2 of  NODAL  gene, is highly expressed in metastatic melanoma cell lines.  NODAL-AS  may interact with  NODAL  mRNA, resulting in the upregulation of oncogenes and the downregulation of metastasis suppressor genes.  NODAL-AS  is enriched in small EVs derived from melanoma cells;  NODAL-AS  are incorporated to other melanoma cells, promoting tumor progression and metastasis. Collectively,  NODAL-AS  regulates melanoma progression ( Dutriaux et al., 2023 ).\n\nSeveral reports have demonstrated the therapeutic potential of targeting mRNA–NAT duplex formation ( Figure 1B ). Oligonucleotides that hybridize with mRNA and/or NAT are good drug candidates. As hybridization is highly specific to genes, these oligonucleotides (including DNA, RNA, and synthetic nucleic acids) may be used as drugs to treat diseases. Nucleic acid-based therapeutics belong to the third major drug class after low-molecular-weight (LMW) drugs and antibody-based therapeutics; therefore, their specificity and efficacy should be examined and compared.\nNucleic acid-based therapeutics are expected to affect complex RNA–protein networks among mRNA and regulatory RNAs, including NATs. Several difficulties must be overcome to develop new drugs, including optimizing their nucleotide sequences to avoid off-target effects, chemically modifying them to increase nuclease resistance, and incorporating them into cells. These points have been discussed in detail using  iNOS  sense oligonucleotides ( Yoshigai et al., 2013 ).\nRNPs such as HuR and hnRNPs contribute to the interactions among mRNAs, NATs, circRNAs, and miRNAs. NATs and other RNAs regulate mRNA levels in concert with RNPs. Therefore, the involvement of RNPs should be considered when investigating the NAT-mediated mechanisms.\nAnother approach involves controlling the NAT-mediated network by targeting mRNA–NAT duplexes using LMW molecules. For example, dexamethasone, a synthetic glucocorticoid, destabilizes  iNOS  mRNA, possibly by modulating the interactions among mRNA, NAT, and RNP ( Ozaki et al., 2010 ). Pharmacologically active LMW compounds underlie the effects of traditional Japanese (Kampo) medicines and functional foods. Similar to sense oligonucleotides, LMW compounds may also recognize RNA structures (stem-loop structures and mRNA– AS  duplexes) that affect mRNA stability ( Nishizawa et al., 2022 ). AHCC® (a standardized extract of cultured  Lentinula edodes  mycelia) downregulated both  EPHA2-AS1/2  and  EPHA2  mRNA, possibly by reducing  EPHA2  mRNA– AS  duplex ( Odaka et al., 2024 ).\nThe ceRNA mechanism is another potential target for disease treatment.  IFNA1-AS , mRNAs, and NATs of the other  IFNA  multigene family members serve as ceRNAs ( Kimura et al., 2015 ). Pseudogenes, lncRNAs, and circRNAs also serve as ceRNAs. Crosstalk between ceRNAs and mRNAs via shared MREs plays an important role in the pathophysiology of various diseases. RNA pharmaceuticals based on pain-related NATs and circRNAs may serve as novel analgesics for treating neuropathic pain by sequestering miRNAs.\nEVs can be used to deliver nucleic acid-based drugs. As endometriosis-related NATs promote the invasion and metastasis of endometrial stromal cells by sponging miRNAs ( Liu et al., 2023 ), endometriosis progression may be suppressed by lowering NATs. Thus, cancer-related NATs may be suitable targets for cancer treatment.\nRecent RNA sequencing data have indicated the presence of many NATs and circRNAs in cells ( Zhou et al., 2020 ). These findings raise complex questions: how do NATs interact with circRNAs, and why are so many circRNAs involved in gene regulation and disease pathophysiology? However, the functions and biological roles of NATs and circRNAs require further investigation.\nCell-based studies cannot address problems related to  in vivo  efficacy, off-target effects, and organ delivery. Therefore, animal experiments are essential for evaluating nucleic acid-based drugs. Rat models of sepsis/endotoxemia can be used to assess the effects of nucleic acid-based therapeutics; for example to evaluate an  iNOS  sense oligonucleotide targeting  iNOS-AS  ( Okuyama et al., 2018 ).\nIFNA1  antisense oligoribonucleotides exhibited antiviral effects in influenza A virus-infected guinea pigs ( Sakamoto et al., 2019 ). When the functional domains of a NAT are known, as in the case of  IFNA1-AS , antisense oligoribonucleotides act as NATs and manifest their effects. Because dysfunction of  IFNA1-AS  may contribute to autoimmunity by failing to sustain  IFNA1  gene expression and function, targeting  IFNA1-AS  could modulate overstimulated innate immune pathways to treat inflammatory autoimmune disorders, such as autoimmune cutaneous diseases ( Muntyanu et al., 2022 ).\nKhorkova et al. (2022)  reviewed commercially available nucleic acid-based therapeutics to humans, including antisense oligonucleotides and short-interfering RNAs. Nucleic acid-based and/or LMW drugs can be loaded onto EVs and lipid nanoparticles (LNPs) to target specific genes and control their expression. EVs and LNPs are potent carriers of synthetic oligonucleotides and mRNAs in cells ( Jeppesen et al., 2023 ) ( Figure 2 ). These nanotechnologies offer robust platforms for precison-drug delivery to various cancers, including HCC ( Shi et al., 2026 ).\nIntercellular communication mediated by extracellular vesicles and introduction of nucleic acids and drugs. In the cells, mRNA, NATs ( AS ), miRNA, and circRNA are present and mutually interacted. MRE(s) may exist in mRNA and regulatory RNAs. EVs, which include RNA and protein are secreted from the donor cell and transported to the acceptor cell. When the donor cell is a cancer cell, features of the acceptor cell may be affected by the EV transport. RNA therapeutics (synthetic oligonucleotides, mRNA, and LMW drugs, as well as plasmids and viral vectors) can be directly introduced to the acceptor cell using transfection or infection. Alternatively, they can be loaded onto EVs or LNPs and are transported to the acceptor cell, such as cancer cells. Thin arrows represent endogenous transport pathways of EVs; thick arrows represent introduction of exogenous molecules using EVs, LNPs, or viral particles to alter endogenous mRNA levels in the acceptor cells. These nanotechnologies may be applied to the administration of RNA therapeutics to humans.\nRapidly emerging findings on the intercellular communication of NATs should also be considered. NATs and miRNAs are transported in EVs and alter signal transduction and gene expression in the acceptor cell, leading to malignant effects such as promotion of cell growth and metastasis. The application of LNP-encapsulated small-interfering Ythdf1 significantly improves the efficacy of anti-PD-1 therapy in metabolic dysfunction-associated steatohepatitis (MASH)-HCC allograft models ( Wang et al., 2023 ).\nOverall, NAT-based regulatory networks play crucial roles in immune response, inflammation, and tumorigenesis. Various approaches are essential to better understand NAT-mediated networks and to develop nucleic acid-based drugs for disease.","source_license":"CC-BY-4.0","license_restricted":false}