Along with the development of targeted inhibitors of epitranscriptomic modulators  now entering clinical trials, the field holds significant promise for treating these abundant cancers. Here, we present the most current summary of this work, while also highlighting the therapeutic potential of these discoveries.Dynamic modifications on RNA, frequently termed both, “RNA epigenetics” and “epitranscriptomics”, offer one of the most exciting emerging areas of gene regulation and biomedicine. Similar to chromatin-based epigenetic mechanisms, writers, readers, and erasers regulate both the presence and interpretation of these modifications, thereby adding further nuance to the control of gene expression. In particular, the most abundant modification on mRNAs, N These f970s [6A , led to 970s [6A ,5. 6A has by far been the most studied of the RNA modifications, and thus, will be the major focus of this review. Readers are referred to the following recent excellent reviews for further information on other modifications [These diverse RNA modifications now are thought to form an “epitranscriptomic code”, which may enable or enhance RNA-dependent reactions, and change RNA structure-function relationships. Collectively, RNA epigenetics now offers an entirely new layer of regulation to control gene expression in a spatiotemporal manner. As discussed below, given the extensive changes in gene expression, which occur during the self-renewal of epithelial tissues, they provide a great model in which to study the role of epitranscriptomics in both homeostasis and disease states. Here, we summarize the most recent findings at the intersection of RNA epigenetics and epithelial biology. As the most abundant modification on mRNAs, mications ,6,7,8.6A is conserved across multicellular eukaryotes including plants and vertebrates, single-cell organisms such as archaea, bacteria, and yeast, as well as among viral RNAs. In vertebrates, approximately 25% of mRNAs contain at least one m6A modification [6A is catalyzed in the nucleus typically at DRACH  motifs, and is enriched transcriptome-wide in the 3’ untranslated region (UTR), as well as within internal exons and near stop codons [6A and ultimately reduced efficiency of translation, suggesting that m6A deposition relies on the dynamics of the transcribing RNA polymerase II [6A catalytic complex can be recruited to DNA by both transcription factors  [6A methylation occurs co-transcriptionally. For example, H3K36me3 was shown to be recognized and bound directly by methyltransferase-like 14 (METTL14), a part of the m6A catalytic complex, which in turn, binds to an adjacent RNA polymerase II to deposit m6A co-transcriptionally [6A itself was recently shown to be important for recruiting the histone demethylase, KDM3B, to chromatin in order to demethylate the repressive histone modification, H3K9me2 [mfication . In mammp codons . Notablyerase II . Consist, CEBPZ) , as well, CEBPZ) . Collecttionally . Intrigu H3K9me2 .6A methylation. For example, m6A has been shown to either promote mRNA degradation or alternatively its translation by increasing its stability, depending upon the cellular context [6A, the amount of m6A can also impact the response as highly m6A modified transcripts display a slightly lower translational efficiency than unmodified transcripts, suggesting that m6A impacts transcript turnover [6A may affect mRNA splicing, though evidence in mammalian systems is limited [6A methylation on an array of chromosome-associated regulatory RNAs (carRNAs) [6A methylation led to increased chromatin accessibility and nascent transcription in embryonic stem (ES) cells.As detailed further below, diverse aspects of mRNA metabolism have been shown to have been affected by m context . Beyond turnover . In addi limited ,14. BeyocarRNAs) . Inhibit6A in mediating DNA damage and repair processes. For example, it has been shown that m6A is deposited upon mRNA rapidly (within ~2 min) at sites of DNA damage following exposure to ultraviolet (UVC) radiation [6A on DNA damage-associated mRNAs, where it ultimately recruits RAD51 and BRCA1 to perform homologous recombination (HR)-mediated repair [Beyond affecting gene expression, some other recent studies have uncovered a novel role for madiation , and thaadiation . Anotheradiation . This phd repair .6A, m6A -modified regions are significantly enriched for the presence of single nucleotide polymorphisms (SNPs). Indeed, the number of SNPs decreases as the distance from the m6A sites increases [6A -associated quantitative trait loci (QTLs) can significantly alter the binding sites of RNA-binding proteins and have heterogeneous downstream effects, supporting a role for m6A in human disease heritability [6A, and suggest that much remains to be revealed regarding this critical modification. Underscoring the potential disease relevance of mncreases . Additiotability . Togethe6A in mRNA is deposited by a large multicomponent writer complex within the nucleus  as well as on methionine adenosyltransferase 2A (MAT2A) [RNA methylation, similar to the known DNA or histone epigenetic modifications, is regulated by “writers” (methyltransferases), “readers” (RNA binding proteins), and “erasers” (demethylases). m nucleus . METTL3  on Mrna ,20. Whil on Mrna ,22,23. T on Mrna . METTL3 nsferase . Beyond  (MAT2A) . Similar (MAT2A) .6A methyltransferase associated), is critical for the deposition of m6A at 3’ untranslated regions (UTRs) [The METTL3/14 heterodimer also binds a number of different adaptors which function in unique ways to facilitate the functions of the complex. For example, Wilms tumor-associated protein (WTAP), though it does not possess methyltransferase activity, binds to METTL3/14 and is required for optimal substrate recruitment and heterodimer localization by binding to chromatin and transcription factors at specific promoters ,28. Anots (UTRs) . The ZC3s (UTRs) , while Rs (UTRs) . Collect6A was thought to potentially be an essential part of m6A function, more recent findings suggest that it may occur on a limited basis and in a limited number of tissues under physiological conditions. As mentioned above, the first discovered m6A eraser was FTO (fat mass and obesity-associated protein) [6A in the nucleus , which is found in the 5’UTR [6Am likely occurs on snRNAs and not mRNAs [6A eraser ALKBH5 has no activity towards m6Am [Alkbh5-knockout mice appear generally normal [6A as compared to FTO, expression levels of ALKBH5 are more highly negatively correlated with m6A levels in comparison to FTO [While initially, the active demethylation of mprotein) , which r nucleus . FTO’s she 5’UTR . Furtherot mRNAs . In contrds m6Am . ALKBH5 rds m6Am . Surprisy normal , though y normal . Consistn to FTO . As discn to FTO .6A methylation on mRNA are attributed to the different readers that bind to it. The first discovered m6A readers were members of the YTH domain-containing proteins, as the ~150aa YTH domain was demonstrated to bind RNA in a m6A-dependent manner. In the mammalian genome, there are five YTH domain-containing proteins, the YTHDCs (YTH domain-containing) and the YTHDFs (YTH domain family) readers. The nuclear m6A readers are the YTHDCs. YTHDC1 regulates mRNA splicing by interacting with a variety of splicing regulators such as SRSF3 [The unique outcomes of mas SRSF3 , and alsas SRSF3  , as well as methylated RNA immunoprecipitation sequencing (MeRIP-seq) [6A-seq is unable to differentiate between m6A and N6,2′-O-dimethyladenosine (m6Am). Second the resolution of these approaches is in the hundreds of base pairs. These limitations have driven the development of methods that are antibody-free and limited to a single nucleotide resolution. Fundamental to all the recent advances in the epitranscriptomics field has been the discovery of new methods to detect and study these modifications genome-wide. The earliest and most widespread of these for mRIP-seq) . However6A-modified transcripts and the m6A antibody to induce mutations during reverse transcription. The more precise localization of m6A peaks called miCLIP or m6A-CLIP [6A-seq or the MeRIP-seq. For single nucleotide resolution studies, significant improvement is obtained by adding a UV crosslinking step after the formation of non-covalent complexes between mm6A-CLIP . It has 6A residues that are then detected using RNA-seq, using as little material as 10 ng of total RNA. Endoribonuclease-based approaches include the MAZTER-seq method which relies on the ability of the bacterial RNase MazF to cleave RNA upstream of an “ACA” sequence, but it does not cleave upstream of “m6A-CA” and can map m6A at single nucleotide resolution [6A-sensitive RNA-endoribonuclease–facilitated sequencing (m6A-REF-seq) uses the m6A-sensitive endoribonuclease ChpBK to identify and quantify transcriptomic m6A sites at specific motifs at single base resolution [Antibody free approaches include DART-seq (deamination adjacent to RNA modification targets), which uses APOBEC1 (a cytidine deaminase) fused to the YTH domain of YTHDF2 . This fusolution . In addisolution .6A [Finally, complementing these sequencing-based methodologies are biophysical methods, such as thin layer chromatography and mass spectrometry that can serve as powerful tools for both validation and the identification and quantification of m6A .Dynamic, self-renewing tissues like epithelia undergo dramatic alterations in gene expression as they differentiate, suggesting that RNA epigenetic mechanisms may play important roles in their physiology. Epithelia are found lining internal and external surfaces, such as the lining of the intestinal track and the surface of the skin, and form the major barriers between the internal and external environments. The different types of epithelia are characterized as being stratified, transitional, or glandular, and depending on the region of the body, the cells can vary in shape and amount of stratification . These tIn stratified epithelia, basal stem-like progenitor cells adhere to the basement membrane and through a continuous regenerative process, go on to replace the “suprabasal” cells above them . With fuAny dysregulation of this highly coordinated differentiation process can lead to hyperplasia, abnormal differentiation, and diseases ranging from the neoplastic (squamous cell carcinoma) to the inflammatory . Indeed, while significant progress has been made in unraveling many of the pathways and players involved in driving these processes across epithelial tissues, such as WNT, Notch, Sonic Hedgehog (Shh), and BMP signaling ,50,51, a6A to promote either mRNA degradation or translation, perhaps it is not surprising that altering METTL3-m6A may lead disparate outcomes on differentiation depending upon the timing of Mettl3 deletion during development. Notably, a complete absence of m6A due to Mettl3 deletion is early embryonic lethal. Knockout of METTL3 from both human and mouse ES cells results in increased transcript half-life of key pluripotency regulators such Nanog, Sox2, and Klf4, improves self-renewal and proliferative abilities, and prevents differentiation from naïve pluripotency into downstream lineages [6A is thought to impair differentiation and promote carcinogenesis in a variety of cancer contexts [As suggested by the ability of METTL3-mediated mlineages ,55. In ccontexts , such ascontexts  and cutacontexts . 6A’s roles during epithelial homeostasis and differentiation, studies are very limited. One recent examination utilizing a Krt14-Cre recombinase to delete Mettl3 in epidermis and oral epithelium demonstrated broad developmental defects including a significant failure of hair morphogenesis, premature interfollicular differentiation, and a loss of filiform papillae in the tongue. The authors attributed these effects to altered Wnt signaling [Mettl14 in the murine epidermis impaired the m6A-dependent association between the long non-coding RNA (lncRNA), Pvt1, and MYC. This interaction was shown to be critical for the promotion of epidermal stemness and wound healing capabilities [6A in epithelial homeostasis, as well as the need for future studies to answer all of the outstanding questions at the intersection of the epitranscriptome and normal epithelial functions.In terms of our understanding of mignaling . Anotherbilities . These s6A in particular have been implicated in numerous aspects of epithelial cancer biology. Similar to the way in which SCCs share numerous common biological underpinnings [6A function can promote similar broad effects across the diverse forms of SCC  secondary to metastatic cSCC , numbersA damage , the majHead and neck squamous cell carcinoma (HNSCC) is the sixth most common of all human cancers worldwide, and despite advances in cancer therapies, it continues to have a relatively poor prognosis and high rate of morbidity and mortality . HNSCC c6A regulatory genes and the survival rate of HNSCC patients [6A regulatory gene, followed by YTHDF3, METTL3 and YTHDF1. The increased expression of VIRMA was associated with higher cancer stages, tumor grade, and nodal metastasis, suggesting that its dysfunction may play an important role in the initiation and progression of HNSCC [6A enhanced the stability of LNCAROD, which then protected the oncogenic protein YBX1 from proteasomal degradation to promote HNSCC cell proliferation and mobility in vitro and tumorigenic ability in vivo [6A was shown to reduce the expression of ZNF750, an important pro-differentiation tumor suppressor [Seeking to identify potential biomarkers in HNSCC, one recent study examined data from The Cancer Genome Atlas (TCGA) to explore relationships between expression levels of mpatients . This stpatients . Anotherof HNSCC . Anotherof HNSCC . Mechani in vivo . Additioppressor .6A methylation in a YTHDF1-dependent manner [6A methylation of BMI1 promoted its translation in conjunction with IGF2BP1 [Mettl3 in vivo inhibited the development of OSCC upon carcinogen exposure [6A can clearly play an oncogenic role in OSCC pathogenesis, and suggests that targeting METTL3 pharmacologically may be a therapeutic approach worth testing. Oral squamous cell carcinoma (OSCC), a form of HNSCC, is the major subtype of oral cancer, responsible for approximately 90% of oral neoplasms . It is ct manner . Depletit manner . Another IGF2BP1 . They shexposure . Future 6A and subsequently increase the expression levels of β-catenin mRNA and protein in CESC cells. The increased β-catenin resulted in increased levels of the nucleotide excision repair factor (ERCC1) in CESC. By knocking down β-catenin, ERCC1 levels were reduced, and the cells (and particularly those with FTO overexpression) were sensitized to chemoradiotherapy [CESC is the fourth most diagnosed cancer and the fourth leading cause of cancer-associated mortality in women worldwide . Two grootherapy . 6A regulatory gene expression and patient prognosis utilizing TCGA data. This analysis revealed that higher FTO expression was associated with a poor prognosis in LUSC patients  that together could promote malignant transformation and the progression of BLCA in vitro and in vivo [6A regulation by METTL3 and ALKBH5. Their results revealed that depletion of METTL3 or overexpression of ALKBH5 in BLCA cells resulted in reduced ITGA6 translation and decreased cell adhesion in a m6A-dependent manner [6A in the 3′UTR of ITGA6 promotes its translation in conjunction with YTHDF1 and YTHDF3, and collectively this promoted the growth and metastatic capacity of BLCA cells [6A on MYC, AFF4, RELA and IKBKB to promote their activation [Urothelial carcinoma of the bladder is currently the fourth most common malignancy in men . A recen in vivo . Anothert manner . Their dCA cells . SimilarCA cells . This grtivation .YTHDC2 gene were associated with ESCC risk, and that knocking down YTHDC2 impaired the proliferation rate of ESCC cells [6A regulatory genes displayed increased expression in esophageal cancer tissue in comparison to normal tissue [6A reader, HNRNPA2B1, correlated with tumor diameter and lymphatic metastasis and promoted disease by upregulating fatty acid synthesis enzymes, ACLY and ACC1. Consistent with this, knockdown of HNRNPA2B1 could block proliferation, migration, and invasion in esophageal cancer models [Esophageal carcinoma is one of the most common malignant tumors worldwide, in which 90% of these are esophageal squamous cell carcinomas (ESCC) ,92,93. TCC cells . Anotherl tissue . In partr models . 6A epitranscriptome may contribute to carcinogenesis. In addition to all of the above examples from specific epithelial cancers, many other studies have highlighted other broad mechanisms that m6A may affect to promote cancer. For example, m6A has been shown to directly impact interferon signaling by leading to the decay of interferon gene mRNAs [6A could promote tumorigenesis. Additionally, METTL3- m6A has been shown to promote epithelial to mesenchymal transition (EMT) in several studies [6A, as well as the need to further understand all of the underlying mechanisms of its roles in cancer, to improve therapeutic outcomes. In summary, these findings demonstrate the numerous and frequently context-dependent manner by which the dynamic METTL3- mne mRNAs ,95. As a studies ,97,98,996A, many of which have been implicated in the balance between differentiation and cancer [5C) has been detected not only on rRNAs and tRNAs, but also on mRNAs by transcriptome-wide mapping approaches. This m5C on mRNAs has been shown to be catalyzed by NOP2/Sun RNA methyltransferase family member 2 (NSUN2). NSUN2 is overexpressed in a variety of human cancers, including cSCC, colon, and breast cancers, and its inhibition impaired MYC-dependent proliferation in keratinocytes and cSCC xenograft studies [5C promoting their translation and the invasiveness and metastasis of BLCA. The authors found that NSUN2 and the m5C reader YBX1 promoted the expression of the oncogene, HDGF, in a m5C dependent manner, and that higher expression of this NSUN2-YBX1-HDGF axis correlated with poor prognosis [As alluded to previously, there are numerous RNA modifications beyond md cancer ,100. For studies . In cSCC studies . A recenrognosis . NSUN2 wrognosis .More than 150 RNA post-transcriptional modifications have been identified to date, which are widely distributed on various types of RNA, including mRNA, tRNA, rRNA, small non-coding RNA (sncRNA), and lncRNA. The variety and breadth of these numerous modifications and numerous types of RNA underscores the vast potential they have to regulate biology and disease. On this basis, RNA epigenetic (epitranscriptomic) modifications have now been shown to play important roles in numerous critical physiological aspects of tissue development and disease. Despite this, there are many unknowns in this relatively nascent field. While the initial insights into the role of the epitranscriptome on epithelial biology and disease have been exciting, much remains to be discovered.RNA epigenetics provides for an entirely new layer of gene regulation, offering impressive nuance and complexity to the regulation of dynamic tissues, such as self-renewing epithelia. Indeed, given the ability to target the enzymes that write and erase these modifications pharmacologically, there is significant promise for the promise of epitranscriptome-targeting drugs to treat disease, as attested to by the significant number of companies that have sprung up to develop these compounds. However, as our summary above suggests, a great deal remains to be done, in order to better understand the mechanisms through which RNA modifiers impact epithelial disease first, so until then it is clear that the RNA epigenetics/epitranscriptomics field will remain one of the most exciting areas of biomedical discovery for years to come.


Young people living with HIV(YPLWH) in low-and middle-income countries are entering adolescence and young adulthood in significant numbers. The majority of the HIV-related research on these young people has focused on clinical outcomes with less emphasis on their sexual and reproductive health (SRH). There is an increasing awareness of the importance of understanding and addressing their SRH needs, as many are at elevated risk of transmitting HIV to their sexual partners and young women, in particular, are at significant risk for transmitting HIV to their infants. The purpose of this scoping review is to synthesize research investigating the SRH needs of young people living with HIV in low- and middle-income countries.We searched electronic databases for studies focusing on young people aged 10–24 years and 27 studies met inclusion criteria.This review identified four themes characterizing research on SRH among young people living with HIV: knowledge of SRH, access to SRH services, sexual practices, and future family planning and childrearing.Our findings suggest a need for additional research on comprehensive sexuality education to equip YPLWH with knowledge to facilitate desirable SRH outcomes, interventions on sero-status disclosure and condom use, and health provider capacity to provide SRH services in their pre-existing HIV clinical care. Young people living with HIV(YPLWH) in low-and middle-income countries are entering adolescence and young adulthood in large numbers. The majority of the HIV-related research on these young people has focused on clinical outcomes with less emphasis on their sexual and reproductive health. It is important to understand and address their sexual and reproductive health (SRH) needs, as many are at a high risk of passing on HIV to their sexual partners and young women, in particular, are at significant risk for passing on HIV to their infants. The purpose of this scoping review is to summarize research examining the SRH needs of young people living with HIV in low- and middle-income countries. We searched online databases for studies focusing on young aged 10–24 years and 27 studies were included in the review. This review identified four themes characterizing research on SRH among young people living with HIV: knowledge of SRH, access to SRH services, sexual practices, and future family planning and childrearing. Our findings suggest a need for additional research on comprehensive sexuality education to equip YPLWH with knowledge to facilitate desirable SRH outcomes, interventions on sero-status disclosure and condom use, and health provider capacity to provide SRH services in their pre-existing HIV clinical care. The widespread success of highly active antiretroviral therapy (HAART) has improved the outlook and life expectancy of the nearly 3.4 million young people living with HIV worldwide . While tConsidering that adolescence is characterized by a strong desire for autonomy and a rise in sexual expression and exploration , many YPThough SRH has been identified as a research and health priority for young people, globally, many countries, including low and middle income countries (LMICs), have difficulty promoting SRH outcomes among young people , 9. StruWe followed the Preferred Reporting Items for Systematic Reviews and Meta-Analyses Guidelines (PRISMA). An electronic search was conducted in March 2020 of the following databases: PubMed, Scopus, and Web of Science. Search terms included ‘adolescent’, ‘youth’, ‘HIV’, ‘AIDS’ ‘sexual and reproductive health’, ‘sexual behavior’, ‘reproductive health’, ‘family planning’, and ‘maternal and child health’. Eligibility criteria included the following: (1) published in a peer-reviewed journal; (2) written in English; (3) focused on YPLWH aged 10–24 years; (4) reported results from a LMIC; and (5) explicitly discussed the sexual and reproductive health of YPLWH. We manually identified countries as LMIC based on the World Bank classifications in February 2020.All three databases were searched by three independent reviewers . Titles and abstracts were read and evaluated based on the above inclusion criteria. Articles that met inclusion criteria based on the first review were included in a spreadsheet and uploaded to a shared reference manager. Articles that were identified by at least two independent reviewers automatically moved to the full text review. Articles were included in this scoping review if all three reviewers agreed on its inclusion. Discrepancies amongst reviewers were discussed with the research group until an agreement was achieved. This scoping review focused on primary sources; however, other types of reviews  that were identified during article extraction were reviewed manually for any mention of articles that did not appear in our electronic search.Data from articles were abstracted and charted detailing the author(s), year of publication, country setting, aims and purpose of the research, methodology, study population, and key findings. A qualitative content analysis was conducted on the key findings to identify common themes across studies.Figure Table Our review identified four themes characterizing SRH needs among YPLWH: knowledge of SRH, access to SRH services, sexual practices, and future family planning and childrearing. While the resulting studies were conducted in different countries and communities, the themes were consistent across geographical regions. We describe each theme in detail below.Thirteen studies reported that YPLWH either had low SRH knowledge or limited access to information about SRH. Five studies showed that YPLWH lacked an understanding of how HIV affected their bodies, ways in which HIV and other STIs were transmitted, or had limited SRH knowledge , 15–18. Additionally, YPLWH reported that they did not know where to go to obtain information about SRH such as contraception , 20. In Four studies assessed maternal health and pregnancy among adolescent girls living with HIV –26. ThesProvider characteristics were identified as barriers to accessing SRH services among YPLWH. We identified nine studies of healthcare providers and other health service delivery stakeholders that assessed their comfort, readiness, and/or competence to provide SRH to YPLWH. In four studies, providers reported low self-efficacy in their ability to provide SRH services such as contraception to YPLWH , 27, 28.Collectively, findings from reviewed studies suggested that YPLWH engaged in sexual activities at rates that were similar to their peers without HIV . MoreoveYPLWH also had the added burden of disclosing their HIV status to their sexual partners, which was often accompanied with fear of HIV-related stigma and rejection by sexual partners , 32. OurLastly, in four studies, concerns about future relationships and childbearing emerged as an SRH theme for YPLWH in LMICs. YPLWH reported desires to have children and be married in the future, but also expressed concerns with realizing these desires , 37, 38.This scoping review synthesized literature elucidating the sexual and reproductive health needs of YPLWH in LMICs. The review yielded 27 studies from 11 countries in sub-Saharan Africa and Asia. We identified four main sexual and reproductive health themes: knowledge of sexual and reproductive health, access to sexual and reproductive health services, sexual practices, and future relationships and childbearing. The findings from this review highlight specific gaps to be addressed in future research, policy, and programs in LMICs. First, there is a need for youth-friendly, sexual and reproductive health services for YPLWH in LMICs that integrate comprehensive sexuality education, which is characterized by open-mindedness, non-judgmental and positive attitudes by health providers. Second, there is a need for interventions that encourage young people to adopt safe sex behaviors, such as condom use, and prepare young people to disclose their HIV status to sexual partners.Inadequate knowledge of sexual and reproductive health, contraceptive options, and family planning coupled with limited access to sexual reproductive health services was a common finding across the studies. This finding has important implications for the health outcomes of YPLWH, particularly as it relates to SRH for adolescent girls living with HIV. Generally, adolescent girls and young women in LMICs have an increased risk of acquiring HIV and having unintended pregnancies, unsafe abortions, and birth complications . These rOur findings showed that YPLWH engage in sexual encounters at similar rates to young people not living with HIV, and YPLWH desire to be in relationships and have families. Interestingly, in two studies YPLWH reported a preference for romantic and/or sexual partners without HIV , 33. ThiLastly, findings from this review highlight the gap in health provider capacity to provide SRH services to YPLWH. Providers preferred abstinence-based SRH education and counseling. However, given that YPLWH continue to be sexually active despite receiving abstinence-based counseling and messaging, this demonstrates that health providers are not responding adequately to the needs of YPLWH. Lack of training in adolescent psychosocial development and the unique SRH needs of YPLWH has been linked to poor quality of care and low motivation to engage in SRH services by YPLWH , 27, 28.This review indicated that there are a few published interventions aimed at improving SRH among YPLWH in LMICs . We idenThis study is not without limitations. First, we limited our search to the three most well-utilized databases in public health and HIV research. As such, it is possible that some studies may have been missed and not included in this review. Nonetheless, a scoping review is not meant to be a systematic assessment of the literature on a specific topic. Instead, they are best utilized when topics are constantly evolving, as is the case of SRH among YPLWH in LMICs. Second, results were limited to studies published in English and may have led to a language bias. Third, the studies focused on YPLWH in SSA countries and a few countries in Asia. As a result, it is possible that these findings may not be generalizable for YPLWH outside of these settings. Despite these limitations, the findings in this review add to the growing body of knowledge on the unique SRH challenges of YPLWH. To the best of our knowledge, this is the first scoping review that has documented the SRH needs of YPLWH in LMICs –46.As YPLWH begin to transition to adulthood, they face the compounded burden of navigating a stigmatized, chronic condition while also experiencing the rapid physiological and sexual development characteristic of adolescence and young adulthood. While sexual and reproductive health is a key component of this developmental stage, additional research was needed to synthesize the unique SRH needs of YPLWH in LMICs. Our findings suggest a need for additional research on comprehensive sexuality education to equip YPLWH with knowledge to facilitate desirable SRH outcomes, interventions on sero-status disclosure and condom use, and health provider capacity to provide SRH services in their pre-existing HIV clinical care.


Scientific Reports 10.1038/s41598-021-81056-3, published online 18 January 2021.Correction to: In the original version of this Article, Sen Li was incorrectly listed as a corresponding author. The correct corresponding author for this Article is Jun Wang. Correspondence and request for materials should be addressed to wangjun@xinhuamed.com.cn.In addition, Jun Wang was incorrectly affiliated with ‘Department of Pediatric Surgery, Shanghai Children’s Hospital, Shanghai Jiao Tong University, No. 355, Luding Road, Shanghai, 200000, China’.Finally, the original version of this Article omitted an affiliation for Sen Li. The correct affiliations are listed below.Department of Pediatric Surgery, Xinhua hospital affiliated to Shanghai Jiao Tong University School of Medicine, No. 1665, Kongjiang Road, 200092, Shanghai, ChinaSen Li & Jun WangDepartment of Pediatric Surgery, Shanghai Children’s Hospital, Shanghai Jiao Tong University, No. 355, Luding Road, 200000, Shanghai, ChinaSen LiThe original Article has been corrected.


Proper development and plasticity of hippocampal neurons require specific RNA isoforms to be expressed in the right place at the right time. Precise spatiotemporal transcript regulation requires the incorporation of essential regulatory RNA sequences into expressed isoforms. In this review, we describe several RNA processing strategies utilized by hippocampal neurons to regulate the spatiotemporal expression of genes critical to development and plasticity. The works described here demonstrate how the hippocampus is an ideal investigative model for uncovering alternate isoform-specific mechanisms that restrict the expression of transcripts in space and time. These include RNA binding proteins (RBPs) and microRNAs, that then recruit other post-transcriptional regulators, such as motor proteins or translational regulators  and Long-term depression (LTD), respectively  reduces HCN1 expression, and (1b-2) results in the sequestering of HCN1 to intracellular puncta in hippocampal CA1 , a channel responsible for the IDlg3, also known as synapse-associated protein 102 or Sap102) regulates the localization of ion channel subunits during development, specifically glutamate ionotropic N-methyl-d-aspartate receptors (NMDARs) that mediate synaptic plasticity  expresses splice variants whose RNAs localize differently. Known more widely as G protein-gated inwardly rectifying K+ channel 2 (Girk2), it is a G-protein dependent hyperpolarizing potassium channel that maintains resting membrane potentials and has been shown to depotentiate LTP in hippocampal cultures , can undergo two modes of RNA regulation that affect its activity and localization- splicing and editing  and dendritic (stratum radiatum) layers, while Gria2flop mRNA is expressed exclusively in the cell soma  serve important isoform-specific roles by regulating many aspects of mRNA metabolism, including RNA localization, stability, and translation. Utilization of alternate UTRs leads to the incorporation of different Npas4 from an upstream promoter results in a variant with a longer 5′UTR that preferentially localizes to hippocampal CA1 dendrites  is another example with transcript isoforms that differ only in the UTRs. BDNF is a critical neurotrophin that regulates hippocampal plasticity  and 18 DIV, respectively  and methyl-CpG binding protein 2 (MECP2). Calcium influx upon neuronal activation causes the nuclear translocation of γCAMKII, which leads to phosphorylation of CREB1  is a Rho-family GTPase that is required for plasticity at CA1 spines  is a well-characterized gene involved in memory formation that encodes alterative isoforms with different length 3′UTRs due to alternative polyadenylation  RNA, the catalytic subunit of AMP-activated protein kinase (AMPK), a regulator of plasticity  data in order to identify the locations of RBPs along target RNA transcripts. Cell-specific RNA-RBP prediction networks that modulate local transcriptomes can be generated by combining compartment-specific transcriptome data with RBP-CLIP data, or when technically feasible, performing compartment-specific RBP-CLIP in different contexts. Fully elucidating UTR-mediated local RNA regulation by unraveling the RNA localization code may be beyond our technological reach at this time. However, advances in highly-multiplexed and iterative single-molecule imaging techniques to map RBP-RNA interactions in situ provide much optimism.As evidenced by Gria2, but the exact molecular players remain unidentified. RNA editing can occur co-transcriptionally like splicing, and some RNA editing enzymes have been implicated in splicing  to repress translation of target transcripts such as the long 3′UTR neurite-localized It remains an open question as to how alternate isoforms impact local translational capacity. Genome-wide measures of isoform-specific transcription and translation in mature neuronal cultures from differentiated human embryonic stem cells demonstrate that regulatory sequences in long 5′ and 3′UTRs generally repress translation, with the strongest effect from 3′UTRs  is almost exclusively present in the cytoplasm, while circStau2 localizes to synapses in both mouse and human neurons, suggesting a synaptic role for some circRNAs  that are bound by RBPs  are non-linear transcripts without a distinct 3′ or 5′ end that are produced as a result of back-splicing (Gokool et al., Developmental and activity-regulated gene expression programs can utilize cell- and context-specific RNA codes necessary to support compartment-specific functions. Despite all the elegant work described in this review, we have only scratched the surface of transcript isoform-dependent local RNA regulation. Recent work has cataloged cell- and compartment-specific isoform expression, but many questions remain with regards to the functional roles that specific sequences and their interactors play in regulating local transcriptomes. Advances in long-read technologies, such as nanopore sequencing, have illuminated the extent of previously underestimated levels of RNA isoform diversity. Nanopore sequencing allows for the direct sequencing of full-length transcripts, instead of the shorter reads utilized by RNAseq. This technology was able to identify 33,984 isoforms from 10,793 genes in human cells, of which 52.6% had an unannotated splice junction (Workman et al., 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.


Risdiplam is the first approved small-molecule splicing modulator for the treatment of spinal muscular atrophy (SMA). Previous studies demonstrated that risdiplam analogues have two separate binding sites in exon 7 of the SMN2 pre-mRNA: (i) the 5′-splice site and (ii) an upstream purine (GA)-rich binding site. Importantly, the sequence of this GA-rich binding site significantly enhanced the potency of risdiplam analogues. In this report, we unambiguously determined that a known risdiplam analogue, SMN-C2, binds to single-stranded GA-rich RNA in a sequence-specific manner. The minimum required binding sequence for SMN-C2 was identified as GAAGGAAGG. We performed all-atom simulations using a robust Gaussian accelerated molecular dynamics (GaMD) method, which captured spontaneous binding of a risdiplam analogue to the target nucleic acids. We uncovered, for the first time, a ligand-binding pocket formed by two sequential GAAG loop-like structures. The simulation findings were highly consistent with experimental data obtained from saturation transfer difference (STD) NMR and structure-affinity-relationship studies of the risdiplam analogues. Together, these studies illuminate us to understand the molecular basis of single-stranded purine-rich RNA recognition by small-molecule splicing modulators with an unprecedented binding mode.  Spinal muscular atrophy (SMA) is one of the most common lethal genetic diseases in new-borns ,2. In thA promising therapeutic strategy is to restore proper splicing of the SMN2 exon 7 to compensate for the loss of the SMN1 gene in SMA patients . NusinerAt binding site 1, one of the risdiplam analogues, SMN-C5 Figure , was fou50) of SMN-C5 significantly increases in ΔESE2 minigene compared to the wildtype sequences, and SMN-C5 failed to induce the complete inclusion of exon7 even at a high concentration (10 μM)  RNA that contains the GA-rich sequence selectively binds to SMN-C2 . Binding (10 μM) . However (10 μM) . These f (10 μM) ,16. The  (10 μM) .In view of this, it was hypothesized that the 5′ splice site is the primary target for the modulatory activity, and that ESE2 facilitates the modulator binding through a cooperative tertiary RNA structure with the 5′ splice site . HoweverIn this study, we interrogated the affinity and selectivity of risdiplam analogues that bind to the GA-rich sequence (binding site 2) in SMN2 exon 7 using a range of biochemical and biophysical tools. Instead of deleting the whole ESE2 sequence, we made single-point mutations within the GA-rich sequence that could disrupt the binding between risdiplam analogues and this GA-rich sequence. Molecular dynamics (MD) has proven useful in simulations of the structural dynamics of nucleic acid ,21, notahttp://www.oligoevaluator.com) using Lambert–Beer's Law. The risdiplam analogues (1 mM in DMSO) were diluted in 2× assay buffer  to make a 1 μM 2× working solution.All synthetic DNA and RNA oligomers were purchased from Integrated DNA Technologies and reconstituted in nuclease-free water (Invitrogen #AM9932) at 1 mM. The concentration of the oligonucleotides at ∼0.1 mM was calibrated with the A260 value measured by a NanoDrop ND-1000  and the predicted extinction coefficient  and DNA Seq6 (25 μM) in 1× assay buffer was prepared and incubated at room temperature for 10 min. 3 μl of SMN-C5 solution at each concentration was added to a 42 μl SMN-C2/DNA Seq6 mixture and incubated at room temperature for 10 minutes. The mixture was then transferred into a microplate (20 μl each well) in duplicates for fluorescence polarization readout. The experimental data were analysed using Prism 8 software . The dissociation constant (Kd) was calculated with 95% confidence interval after nonlinear curve fitting .A 1:2 dilution series (8 points) of each oligonucleotide was prepared in 20–30 μl water to desired concentrations (i.e. 1–128 μM). 20–30 μl 2× working solution containing the 2× assay buffer and the small-molecule ligand was added to each oligonucleotide sample in 1:1 (v/v) and mixed by pipetting for 4 times. For fluorescence polarization measurement, 20 μl of the above 1× working solution was transferred into a 384-well, black, flat-bottom microplates (Greiner Bio-One) in duplicates or triplicates. The plate was equilibrated at room temperature for 5 min before being read using a microplate reader  at 25°C. For compound SMN-C5, a competitive binding assay was employed to measure an apparent 1 was placed randomly at >15 Å away from the nucleic acid. Each simulation system was then prepared using the solution builder plug-in with the CHARMM-GUI web server  harmonic position restraints to DNA/RNA and ligand heavy atoms with a constant number, volume and temperature (NVT) ensemble. The system was further equilibrated for 125 ps at 298.15 K using constant number, pressure and temperature (NPT) ensemble with the same restraints as in the NVT run. Then cMD without any constrains was performed to further relax the system for 1 ns at 1 atm pressure and 298.15 K temperature.Initial energy minimization, equilibration, and conventional molecular dynamics (cMD) of compound max (–1.The simulations proceeded with GaMD (see method details in SI), which was implemented in the GPU version of AMBER18 . A shortmax ,27. One (Rg) were calculated using CPPTRAJ. They were also used as reaction coordinates for calculating a 2D PMF free energy profile by reweighting all five GaMD simulations combined (see method details for energetic reweighting of GaMD simulations in SI). The PyReweighting toolkit , followed by 20 μl 10× PBS pH 5.0 buffer, 10 μl D2O, 2 μl DMSO-d6 and H2O. The solution was then heated at 60°C for 1 min before 1 mM RNA Seq6 or DNA Seq4 solution was added to form a 200 μl small molecule-nucleic acid solution. The amount of the RNA Seq6 or DNA Seq4 solution and H2O were varied to form solutions of different ratios of RNA Seq6 or DNA Seq4 to compound 1. The series of solutions with 5 , 10, 20 mol% RNA Seq6, or 2.5, 5, 10, 20, 50, 100, 200, 400, 1000, 2000, 5000 and 8200 mol% DNA Seq4 were then transferred to 3 mm NMR tubes for analysis. 1H NMR spectra were acquired at 25°C with excitation sculpting (ES) or presaturation to suppress the water signals. The STD experiment was performed with saturation at 5.80 ppm for DNA and at 5.70 ppm for RNA . A train of selective gaussian pulses with a bandwidth of 125 Hz and 2 s mixing time were utilized, with 16k scans acquired for DNA and 32k scans for RNA samples.NMR spectra were acquired on a Bruker 600 MHz Avance III HD spectrometer equipped with a TCI cryoprobe, and a Varian INOVA 600 MHz spectrometer equipped with a cryogenic HCN probe. To a 1 ml Eppendorf tube was added 500 μM compound We previously reported that a risdiplam analogue, SMN-C2 Figure , binds tKd) of SMN-C2–RNA Seq6 (binding site 2) is 3.4-fold stronger than that observed with RNA Seq18 . They showed a 1:1 binding stoichiometry with a calculated M Figure  and B, way Table . In the ay Table . The resay Table . In RNA,ay Table . Importaay Table . The co-ay Table , reconfi1 . During five independent 500 ns GaMD production simulations, compound 1 appeared to spontaneously bind to both RNA (Rg) reduced to ∼8.0 Å  of compound 1 intercalated between the second and fourth bases of the first GAAG motif, whereas in RNA, the intercalation occurred between the second and fourth bases of the second GAAG motif  are below the detection limit when 20 mol% of the RNA Seq6 is present, whereas the aliphatic signals are still observed  of the doublet for 3-CH3 of compound 1 (∼1.4 ppm) only increases from 9.1 Hz at 0 mol% RNA to 11.8 Hz at 20 mol% RNA  also showed small STD signals compared to those observed with the aromatic protons. Thus, both the 1H NMR titration and the STD experiments strongly indicated that the interaction between compound 1 and the DNA Seq4 is mainly driven by the aromatic moieties, specifically rings B, C, and D, of this small molecule. This result is also consistent with the fact that SMN-C2 prefers to bind with purine-rich sequences since the latter engage in π-interactions more effectively than pyrimidine-rich sequences  experiments were carried out to further interrogate the interaction between compound 4 Figure  39). Th. Th1 andmeasured . The STDequences .1, ring A) does not substantially alter the binding affinity with RNA and DNA Seq6 (Table 2) did not reduce the binding affinity to DNA Seq6 significantly , an, an41), 3–5). Changing the Cl position from C4" into C2" reduces the binding affinity by more than 12-fold . Unlike these compounds with a monocyclic ring D, the new risdiplam analogues with a bicyclic ring D/E (compounds 10 and 11) both showed high binding affinity to the GA-rich sequence RNA and DNA Seq6 when the ring D is unchanged from SMN-C2  in cells . The binin cells . The TSLhnRNP A1  and Tra2hnRNP A1 . During 1. As an analogy of GNRA tetraloop in RNA, GNNA and GNAB  tetraloops can also stably form in DNA . Since the dipole moments of aromatic rings are important for π-stacking  were observed in the GaMD simulations. Nevertheless, relatively low-energy conformational states of each system could be identified from the simulations, which uncovered a folded double loop-like conformation induced by small-molecule binding in both DNA and RNA. The induction of the conformational change is consistent with the observation of peak shifts in circular dichroism (CD) spectra of RNA or DNA Seq6 in the presence of compound 1 .We also discovered that the binding to the GA-rich sequence is not sufficient to correct SMN2 splicing in cellular assays. Several risdiplam analogues retain the ssDNA or ssRNA-binding ability without showing any observable splicing modulation in cells  for the binding of the small molecules to either the GA-rich sequence in this study or to the 5′ splice site–U1 snRNP complex in the previous report  repeats ). To ourhttp://ambermd.org/), CPPTRAJ (https://amber-md.github.io/cpptraj/CPPTRAJ.xhtml), and PyReweighting (https://github.com/MiaoLab20/PyReweighting). Representative bound conformations generated by GaMD simulations are available in PDB files in Model Archive repository (https://modelarchive.org): RNA–compound 1 complex, Bound/Folded state (https://modelarchive.org/doi/10.5452/ma-fxj31), Bound/Intermediate state (https://modelarchive.org/doi/10.5452/ma-l2fj8); DNA–compound 1 complex, Bound/Folded state (https://modelarchive.org/doi/10.5452/ma-aghox), Bound/Intermediate state (https://modelarchive.org/doi/10.5452/ma-ut0vm), Bound/Unfolded state (https://modelarchive.org/doi/10.5452/ma-royyf).All software packages used in this report are available in public repositories, including AMBER18 (gkab602_Supplemental_FilesClick here for additional data file.


Protection of genome integrity is vital for all living organisms, particularly when DNA double-strand breaks (DSBs) occur. Eukaryotes have developed two main pathways, namely Non-Homologous End Joining (NHEJ) and Homologous Recombination (HR), to repair DSBs. While most of the current research is focused on the role of key protein players in the functional regulation of DSB repair pathways, accumulating evidence has uncovered a novel class of regulating factors termed non-coding RNAs. Non-coding RNAs have been found to hold a pivotal role in the activation of DSB repair mechanisms, thereby safeguarding genomic stability. In particular, long non-coding RNAs (lncRNAs) have begun to emerge as new players with vast therapeutic potential. This review summarizes important advances in the field of lncRNAs, including characterization of recently identified lncRNAs, and their implication in DSB repair pathways in the context of tumorigenesis. An intact genome is vital for the survival of living organisms as it ensures normal homeostasis . To safeMost of the known factors that build these regulatory circuits are identified as proteins. However, mounting evidence has revealed an unexpected new class of players to participate in genome surveillance pathways. These new factors constitute a subset of RNA molecules defined as non-coding RNAs (ncRNAs) ,3. NcRNAIn the current review, we focus on the role that long non-coding RNAs (lncRNAs) play as versatile tools in the various DDRR pathways, and specifically in the detection and repair of DNA double-strand breaks (DSBs), the most deleterious type of DNA damage ,4. PrincWhile a very large proportion of the human genome is transcribed ,6,7, onlNotably, while large numbers of curated human lncRNA entries are available in public repositories, starting from 16.000 up to 270.044, only a relatively small proportion of them have been functionally annotated ,12. LncRThe expression levels of lncRNA genes exhibit high cell-to-cell variation , while tMost lncRNA species are transcribed by RNA Pol II, and they usually undergo 5′- m7G capping, splicing, nonsense-mediated decay (NMD) and possess 3′-end poly(A) tails, thus resembling mRNA biogenesis ,17. A ca(i) Genomic location of lncRNAs. This classification involves: (a) intergenic lncRNAs (lincRNAs)—loci encoding such transcripts are located in the intervening regions among protein coding genes; (b) intronic lncRNAs—these transcripts are derived from introns that separate the coding exons of a gene.(ii) Location context. In this respect, lncRNAs relate to sense- or antisense-derived transcripts: (a) the former are transcribed from the sense strand and may overlap partly or completely with the entire sequence of a protein coding gene; (b) the antisense-derived transcripts originate from the antisense strand and can emerge from three routes: (1) antisense strand-derived transcripts may overlap with an exon of a sense gene, (2) transcripts may overlap with the intron of a sense gene, and (3) transcripts may cover the entire protein-coding sequence.(iii) Effect exerted on DNA sequences. Cis- and trans-lncRNAs: the former regulate the expression of genes in close genomic proximity, while the latter regulate the expression of distant genes.(iv) Mechanism of function. According to the function that they exert, lncRNAs are categorized into the following groups: (a) Transcriptional regulation. This group involves lncRNAs that are implicated in chromatin remodeling and transcriptional interference, including enhancer lncRNAs (eRNAs) that are transcribed from enhancers; (b) Post-transcriptional regulation. This group comprises lncRNAs that participate in splicing regulation and post-transcriptional regulation (mRNA stability); (c) Translational control, by facilitating or repressing this process; (d) Other functions, such as protein localization, telomere replication and RNA interference.(v) Targeting mechanism. This is a heterogeneous group of lncRNAs due to the multiple mechanisms of action and interactionsubstrates that they present or recognize, respectively. There are several subdivisions in this category: (a) signal dependency as lncRNAs show cell type-specific expression and response to diverse stimuli; (b) decoy role, since certain lncRNAs can bind and titrate away an RNA or protein target, without any further function(s); (c) guiding ability as they bind proteins and then direct the localization of the ribonucleoprotein complex to specific targets; (d) scaffolding capacity, since such lncRNAs serve as central platforms for the concurrent binding of multiple proteins to form ribonucleoprotein complexes. LncRNAs can also be grouped together based on the nature of the interactions that they establish with their targets: RNA–RNA pairing, RNA–DNA hybrids, RNA structure-mediated interactions and protein linkers.Due to the many functions that they perform and the variety of biological processes in which they are implicated, a classification of lncRNAs has been proposed according to the following criteria ,17:(i) GDNA serves as a repository for genetic information; therefore, all living cells rely on a complex protein network, termed DDRR, to preserve its integrity ,2. GivenDSBs) in the DNA helix [Two broad categories of genetic aberrations occur and accumulate in the genome, namely chemically induced changes at the nucleotide level and single- or double-strand breaks , unrepaired mutations leading to stalled replication forks and ionizing radiation [DSBs can arise from a variety of endogenous or exogenous sources, such as oncogene-induced replication stress, reactive species  lncRNA is a partial antisense to the TP53 gene, which has the ability to recruit and stabilize RNF8 at DSBs . AdditioThe p53-upregulated “Metastasis-associated Lung Adenocarcinoma Transcript 1” (MALAT1) lncRNA ,37 is anFinally, “Telomeric Repeat-containing RNA” (TERRA) is a lncRNA with a dual role. On one hand, it interacts with LSD1 to regulate meiotic recombination 11 (MRE11) activity at uncapped telomeres . On the INK4A locus) [ATM activation leads to the induction of the ANRIL lncRNA (antisense noncoding RNA from the A locus) . The preA locus) .“LncRNA-Jade family plant homeodomain (PHD) finger” (lncRNA-JADE) is another ATM-induced target in response to DNA damage. Specifically, lncRNA-JADE upregulates JADE1, a key factor of the HBO1 histone acetylation complex, thus triggering histone H4 acetylation during DDRR . It has The lncRNA “Non-coding RNA Activated by DNA damage” (NORAD) was found to interact with RNA Binding Motif Protein X-Linked (RBMX) and control its ability to assemble a ribonucleoprotein complex that includes critical genome instability suppressors such as topoisomerase I (TOP1), ALYREF and the PRPF19–CDC5L complex . SilenciMultiple lncRNAs have an established role as effectors in the DDRR signaling network. Of particular interest are those targeted by p53. PINT is a lincRNA that acts as a transcriptional target of p53 and a positive regulator of cell proliferation and survival in mouse cells, while it has been shown to function as a negative regulator in human cells ,49. In bMALAT1 is also upregulated by p53 in response to DNA damage, similarly to TUG1. Apart from its role in damage sensing, numerous other proteins that are involved in transcription, RNA processing, translation, protein degradation and metabolism have been found among its various partners . MoreoveAnother DNA damage-induced lncRNA named “P21 Associated NcRNA DNA damage Activated” (PANDA) is also expressed in a p53-dependent manner and represents an antisense-transcribed lncRNA of approximately 5 Kb that is located upstream of CDKN1A . LincRNAThe WRAP53 locus encodes for three isoforms , of which WRAP53α has been demonstrated to interact with p53 mRNA, affecting, in turn, p53 protein levels . AnotherExpression of the “Transcribed in the Opposite Direction of RAD51” (TODRA) lncRNA is controlled by the same regulatory region that induces RAD51, but is transcribed reciprocally to RAD51, in an E2F1-dependent manner . TODRA cAnother lncRNA that stabilizes RAD51 is the “long noncoding RNA Radiation Induced” (lnc-RI) . Lnc-RI “BRCA1-associated RING Domain protein 1 9′L” (BARD1 9′L) is transcribed from an alternative intronic promoter of the BARD1 gene and upregulates the expression of an oncogenic BARD1 isoform. Normally, BARD1 stabilizes the BRCA1 protein, thus facilitating HR repair. By promoting the expression of an oncogenic isoform, BARD1 9′L interferes with this process in certain malignancies and is accompanied by reduced RAD51 foci generation and decreased nuclear accumulation of the BRCA1 protein ,62.Two estrogen-regulated long noncoding RNAs, “CCND1-UPstream Intergenic DNA repair 1 and 2” (CUPID1 and CUPID2), are expressed from a bidirectional promoter from an intergenic region at 11q13 . CUPID1 “DLX6 Antisense RNA 1” (DLX6-AS1 or Evf 2) is a lncRNA that interacts with Brahma-related gene-1 (BRG1) and DLX1. It has an inhibitory role on RNA-dependent chromatin remodeling . NotablyThe recently discovered AERRIE lncRNA was shown to be expressed preferentially in endothelial cells and facilitate DDRR . SpecifiCCND1 are expressed in response to ionizing radiation from the Cyclin D1 (CCND1) promoter locus [CCND1 subsequently interact with the RNA-binding protein “Translocated in Liposarcoma” (TLS) to suppress CCND1 transcription through inhibiting the histone acetyltransferase complex CBP/p300, resulting in cell proliferation cessation [A group of lncRNAs termed lncRNAer locus ,35. lncRessation ,35.KIP1 has been shown to be under epigenetic regulation in mammals through an imprinted control region (ICR) [KIP1 is expressed from the maternal allele, as the paternal one is suppressed by a lncRNA termed “Long QT Intronic Transcript 1” (LIT1) [KIP1, in the same chromosomal arm [KIP1 expression levels, suggesting that LIT1 lncRNA could block p57KIP1-dependent cell-cycle checkpoint [Expression of the CDKI p57on (ICR) . Specifi” (LIT1) . LIT1 lnomal arm . We haveeckpoint .The initial observations that a large proportion of the eukaryotic genome is transcribed but, eventually, only a small fraction of the generated RNAs are translated into protein “players” seemed intriguing at the time . Why wouAt first, ncRNA was considered to be “dark matter”, but evidence progressively came to light unveiling an increasing range of cellular functions that rely on the presence of ncRNAs for normal execution ,5,9. NowOf particular importance are those lncRNAs involved in the DDRR network due to its vital role in preserving the integrity of the eukaryotic genome. Taking into account the rapidly increasing numbers of discovered and/or functionally characterized lncRNAs against the protein components of the DDRR network, it is tempting to speculate that lncRNAs engaged in the DDRR may, in fact, outnumber the protein factors participating in it. In any case, the intriguing question of why RNA transcripts are deployed in this critical network still remains unanswered. To address the role of lncRNAs in cellular physiology, several cell systems have been implemented, such as stem cells or induced pluripotent stem cells (iPSC), uncovering their dynamic role in cell fate determination . To thisA plausible hypothesis regarding lncRNA evolution could envisage that, since RNA once served as an ancestral life-coding molecule , ncRNAs DDRR impacts multiple cellular functions; however, addressing how such complex programs are orchestrated still remains a challenge ,76. The Within this context, the advent of high-throughput technologies has already generated large numbers of lncRNA entries in repository databases, many of which are still awaiting functional characterization. Nevertheless, the already characterized lncRNAs have signified their vital contribution to cellular physiology and the impact that their deregulation has on disease onset ,73. As s


SARS-CoV-2 has an exonuclease-based proofreader, which removes nucleotide inhibitors such as Remdesivir that are incorporated into the viral RNA during replication, reducing the efficacy of these drugs for treating COVID-19. Combinations of inhibitors of both the viral RNA-dependent RNA polymerase and the exonuclease could overcome this deficiency. Here we report the identification of hepatitis C virus NS5A inhibitors Pibrentasvir and Ombitasvir as SARS-CoV-2 exonuclease inhibitors. In the presence of Pibrentasvir, RNAs terminated with the active forms of the prodrugs Sofosbuvir, Remdesivir, Favipiravir, Molnupiravir and AT-527 were largely protected from excision by the exonuclease, while in the absence of Pibrentasvir, there was rapid excision. Due to its unique structure, Tenofovir-terminated RNA was highly resistant to exonuclease excision even in the absence of Pibrentasvir. Viral cell culture studies also demonstrate significant synergy using this combination strategy. This study supports the use of combination drugs that inhibit both the SARS-CoV-2 polymerase and exonuclease for effective COVID-19 treatment. In this paper, the hepatitis C virus inhibitors Pibrentasvir and Ombitasvir are found to inhibit the SARS-CoV-2 exonuclease and are shown to have therapeutic potential when combined with SARS-CoV-2 polymerase inhibitors in viral cell cultures. Thus it requires an RNA-dependent RNA polymerase (RdRp) to replicate and transcribe its genome. Because of its large genome (~30 kb) and error-prone RdRp, SARS-CoV-2 also possesses a 3′–5′ exonuclease for proofreading to maintain the integrity of the genome3. The replication complex of coronaviruses consists of several viral proteins, including the RdRp itself  and its two accessory proteins (nsp7 and nsp8)6, and the exonuclease (nsp14) with its accessory protein (nsp10)7.Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which is responsible for COVID-19, is a positive-sense single-stranded RNA virus11. Of particular interest are those that are able to be incorporated into the replicated RNA by the viral RdRp where they halt or slow further replication. These include, among several others, nucleotide analogues such as Sofosbuvir , a nucleotide analogue, is the only small-molecule antiviral drug approved by the Food and Drug Administration (FDA) for COVID-19 treatment, although it was shown to have limited benefit in the World Health Organization’s Solidarity Trial. Lack of oral bioavailability and short half-life reduces RDV’s clinical effectiveness. With the appearance of new SARS-CoV-2 variants that pose a threat to public health and may escape the immune response to current vaccines, it is more critical than ever to develop antivirals that can treat COVID-19 effectively. We have investigated a library of nucleotide analogues targeting the SARS-CoV-2 RdRp, several of which have been FDA approved for other viral infections including HIV/AIDS and hepatitis B and Cvir Fig. , an immevir Fig. , a delaytor Fig. 10,11. Revir Fig. , Molnupivir Fig. , f, are 10 An in silico study has proposed combining RdRp and exonuclease inhibitors as a strategy to combat COVID-1917; however, no successful demonstration of such a combined drug strategy has been reported. Hepatitis C virus (HCV) and SARS-CoV-2 are both positive-sense single-stranded RNA viruses. Viruses with small genomes that encode relatively few proteins, such as HCV, must possess multifunctional proteins18. HCV NS5A is a good example because it binds lipids, Zn++ and RNA, as well as participates in phosphorylation and cell signaling events18. Zn++ bound to the NS5A protein coordinates the binding of RNA. The SARS-CoV-2 genome is threefold larger than that of HCV. Although there is no counterpart of NS5A in SARS-CoV-2, the analogous NS5A functions are likely distributed among proteins nsp1 to nsp16 of the coronavirus19. We have previously shown that the NS5A inhibitors Daclatasvir and Velpatasvir inhibit both the SARS-CoV-2 polymerase and exonuclease activities21. The combination of Velpatasvir with Remdesivir was shown to have a synergistic effect relative to Remdesivir alone in inhibiting SARS-CoV-2 replication22. Here, we further examined whether the NS5A inhibitors Pibrentasvir, Ombitasvir, and Daclatasvir  or induce errors in the replicated RNA .Using the molecular assays that we have developed previouslyPibrentasvir and Ombitasvir inhibit the SARS-CoV-2 exonuclease in a concentration-dependent manner, as shown in Fig. 16. The Sofosbuvir (S)-terminated RNA and Remdesivir (R)-terminated RNA  is a G analogue25. As expected, Favipiravir-RTP was incorporated into RNA opposite C or U in the template strand  and nsp7 and nsp8 proteins (RdRp cofactors)15. However, if the exonuclease is able to remove the incorporated nucleotides including Remdesivir from this delayed termination RNA product, then the inhibitory activity of Remdesivir for SARS-CoV-2 will be reduced. Therefore, we investigated whether Pibrentasvir is efficient in preventing the excision of Remdesivir from delayed Remdesivir terminated RNA through inhibiting SARS-CoV-2 exonuclease activity. In Fig. The nucleotide inhibitor Remdesivir exhibits delayed termination during the polymerase reaction, which has been suggested as one of the possible mechanisms for inhibiting replication of SARS-CoV-211. As an acyclic nucleotide, Tfv-DP lacks a normal sugar ring configuration, and thus we reasoned that it is less likely to be recognized by SARS-CoV-2 exonuclease. We, therefore, investigated whether Tfv can be removed by the exonuclease from the extended RNA. As shown in Fig. We previously showed that Tenofovir diphosphate (Tfv-DP) can be incorporated into RNA as an obligate terminator by the SARS-CoV-2 RdRp complex27, were infected at a multiplicity of infection (MOI) of 0.1 and treated with the drugs under investigation alone or in combination. After 2–3 days, culture supernatant was harvested and titered in Vero E6 cells to quantify the infectious viruses as plaque-forming units (PFU). The NS5A inhibitors Pibrentasvir, Ombitasvir, and Daclatasvir inhibited SARS-CoV-2 replication in a dose-dependent manner  compared to other repurposed RdRp inhibitors, such as Sofosbuvir (6.2 μM), Tenofovir (4.3 μM) and Favipiravir (7.8 μM) , Ombitasvir (0.1 μM) and Daclatasvir (0.5 μM) and the results are listed in Table 50, EC90, and EC99 values for the RdRp inhibitors in the presence and absence of the exonuclease inhibitors. Figure vs percent viral inhibition, for easier visualization of the inhibitor effects. Pibrentasvir improved RDV’s efficacy at the 90% and 99% levels six- and threefold, respectively  in the presence of Pibrentasvir , RDV (a delayed chain terminator), and Favipiravir (an error-prone nucleobase) were tested at various concentrations along with the ECtasvir 0. μM, Ombitasvir 0. μM, Ombitasvir 0. μM, Ombitasvir 0. μM, Ombitasvir 0. μM, Ombitasvir 0. μM, Ombi9% Table . Daclatatasvir 0. μM, Ombitasvir 0. μM, Ombi29, which represents synergistic, additive, and antagonistic effects as scores above 10, between 10 and −10, and below −10, respectively. The results show that Ombitasvir displayed synergistic effects with RDV . The Mg++ ion coordinates with amino acid residues Asp-90, Glu-92, Glu-191, and Asp-273 and the 3′ terminus of the RNA. Because the NS5A inhibitors interfere with this coordination, they are likely to prevent nucleotide excision from the RNA 35. The nucleotide inhibitors Remdesivir and Sofosbuvir are incorporated into the replicating RNA to inhibit further polymerase reaction, but they are rapidly removed by the exonuclease in the absence of exonuclease inhibitors such as Pibrentasvir and Ombitasvir. However, in the presence of these inhibitors, any nucleotide inhibitors incorporated into the RNA will not be rapidly excised by the exonuclease and their inhibitory effect on overall viral RNA replication will be substantially enhanced. Recently, the coronavirus exonuclease has also been shown to promote viral recombination which can increase the emergence of novel strains36, providing additional incentive for targeting this enzyme with antivirals.NS5A inhibitors enhance the antiviral activity of RDV, Tenofovir, Sofosbuvir, and Favipiravir, likely due to their inhibition of exonuclease activity. The combinations of drugs described here, especially the orally available antivirals, have the potential to progress to COVID-19 clinical trials. We show that by combining Pibrentasvir or Ombitasvir with Remdesivir, Sofosbuvir, Tenofovir, or Favipiravir, higher inhibitory activity for SARS-CoV-2 was achievable at lower doses, bringing the nucleotides’ pharmacological parameters more in line with their pharmacokinetic exposures , by a cellular enzyme, hypoxanthine guanine phosphoribosyltransferase, and cellular kinases  with the RNA substrate and Favipiravir-RTP was determined, providing insight into the mode of action of this drug45. Here, we demonstrated that the excision of Favipiravir nucleotide from the 3′ end of RNA by the SARS-CoV-2 exonuclease complex is greatly reduced in the presence of Pibrentasvir  by cellular enzymes, is currently in a COVID-19 clinical trial46. Here, we showed that the excision of NHC from the 3′ end of RNA by the SARS-CoV-2 exonuclease complex is substantially reduced in the presence of Pibrentasvir 25. The active triphosphate form of AT-527 is structurally similar to that of Sofosbuvir with the only difference being that it has a guanine base in place of uracil. AT-527 inhibits SARS-CoV-2 by targeting both the RdRp activity and the Nidovirus RdRp-associated nucleotidyltransferase (NiRAN) activity of nsp12, which are essential for viral RNA replication and transcription47. AT-527 is currently in COVID-19 clinical trials. Here, we demonstrated that the excision of AT-527 from the 3′ end of RNA by the SARS-CoV-2 exonuclease is largely abrogated by the exonuclease inhibitor Pibrentasvir  and NHC triphosphate were purchased from MedChemExpress , Sofosbuvir triphosphate (SOF-TP) was purchased from Sierra Bioresearch , Tenofovir diphosphate (Tfv-DP) was purchased from Alfa Chemistry , and Favipiravir triphosphate and AT-9010 were purchased from NuBlocks LLC . Pibrentasvir was purchased from Cayman Chemical Company  and Ombitasvir, Daclatasvir, Ritonavir and Lopinavir were purchased from Selleckchem . UTP, GTP and ATP were purchased from Fisher Scientific. The RNA oligonucleotides (template-loop-primers) were purchased from Dharmacon .The RdRp of SARS-CoV-2, referred to as nsp12, and its two protein cofactors, nsp7 and nsp8, shown to be required for the processive polymerase activity of nsp12, were cloned and purified as described48. The portion of the RNA encoding nsp10 and nsp14 was reverse transcribed into cDNA using gene-specific primers (see below) and SuperScript III Reverse Transcriptase (Thermo Fisher). The nsp10 and nsp14 coding sequences were PCR amplified using forward and reverse gene-specific primers flanked by BamHI and XhoI recombination sites, respectively. PCR products were digested with BamHI and XhoI and subsequently ligated into BamHI and XhoI-digested pRSFDuet-His6-sumo vector and sequence verified. pRSFDuet-His6-sumo is a modified pRSFDuet-1 vector (Novagen) bearing an N-terminal His6-SUMO-tag cleavable by the ubiquitin-like protease (ULP1).The 3′-exonuclease, referred to as nsp14, and its protein cofactor, nsp10, were cloned and expressed based on the SARS-CoV-2 genome sequence. The pRSFDuet-1 plasmids (Novagen) encoding SARS-CoV-2 nsp14 or nsp10 engineered with an N-terminal His-SUMO tag were prepared as follows: SARS-CoV-2 RNA isolated from the supernatant of SARS-CoV-2-infected Vero E6 cells was provided by Benjamin R. tenOeverList of PCR primers used:NSP14_pRSF_BamHI_fwCGCGGATCCGCTGAAAATGTAACAGGACTCTTTAAANSP14_pRSF_XhoI_revCCCGCTCGAGCGGTCACTGAAGTCTTGTAAAAGTGTTCCAGAGGRT_primer_NSP14TTCTTGGCTATGTCAGTCATAGAACAAACNSP10_pRSF_BamHI_fwCGCGGATCCGCTGGTAATGCAACAGAAGTGCCTGCCNSP10_pRSF_XhoI_revCCCGCTCGAGCGGTCACTGAAGCATGGGTTCGCGGAGTTGATCRT_primer_NSP10GATGTTGATATGACATGGTCGTAACAGCEscherichia coli BL21-CodonPlus(DE3)-RIL (Stratagene). The bacteria were grown in Luria–Bertani medium supplemented with 50 mg/mL kanamycin at 37 °C to an OD600 of 0.6, and induced with 0.4 mM isopropyl β-d-1-thiogalactopyranoside and 50 μM ZnCl2 overnight at 18 °C. Cells were collected via centrifugation at 5000×g and equal volumes of the nsp14 and nsp10 bacterial cells were then mixed for nsp14-nsp10 protein complex purification. The cells were lysed via sonication in Lysis Buffer . After centrifugation at 40,000×g, the supernatant was loaded onto 5 mL Nickel Sepharose 6 fast flow resins  in a gravity flow column. The target protein was eluted using Lysis Buffer supplemented with 500 mM imidazole. The eluted protein was incubated with ULP1 (lab stock) during dialysis at 4 °C overnight against a buffer containing 20 mM Tris-HCl, pH 7.5, 20 mM imidazole, 150 mM NaCl, 100 µM ZnCl2, and 5 mM β-mercaptoethanol. Then the sample was loaded onto the HisTrap FF column  to remove the His-SUMO tag, and the flow-through was collected. The target proteins were further purified through a Superdex200 10/300 gel filtration column  in a buffer containing 20 mM HEPES, pH 7.4, 150 mM NaCl, 1 mM MgCl2, and 1 mM dithiothreitol. The fractions corresponding to the nsp14/nsp10 complex were detected by SDS-PAGE and collected. The protein sample was flash-frozen in liquid nitrogen and stored at −80 °C.The nsp14 and nsp10 proteins were expressed in 20 in 1× reaction buffer was added to the annealed RNA template-loop-primer solution and incubated for an additional 10 min at room temperature. Finally, 5 µL of a solution containing 0.2 mM RDV-TP or 2 mM SOF-TP in 1× reaction buffer was added, and incubation was carried out for 2 h at 30 °C. The final concentrations of reagents in the 20 µL extension reactions were 2 µM nsp12/nsp7/nsp8, 5 µM RNA template-loop-primer, 50 µM RDV-TP, and 500 µM SOF-TP. The 1× reaction buffer contains the following reagents: 10 mM Tris-HCl pH 8, 10 mM KCl, 2 mM MgCl2 and 1 mM β-mercaptoethanol. Desalting of the reaction mixture was performed with an Oligo Clean & Concentrator kit (Zymo Research) resulting in ~10 µL purified aqueous RNA solutions. 1 µL of each solution was subjected to MALDI-TOF MS (Bruker ultrafleXtreme) analysis. The remaining ~9 µL extended template-loop-primer solutions were used to test exonuclease activity.In total, 10 µL of 10 µM RNA template-loop-primers  in 1 × RdRp reaction buffer was annealed by heating to 75 °C for 3 min and cooling to room temperature. In all, 5 µL of 8 µM RdRp complex (nsp12/nsp7/nsp8)2 and 0.2 unit Therminator II in 1× ThermoPol buffer was incubated in a thermal cycler using the following protocol . Desalting of the reaction mixture was performed with an Oligo Clean & Concentrator kit (Zymo Research) resulting in ~10 µL purified aqueous RNA solutions. 1 µL of each solution was subjected to MALDI-TOF MS (Bruker ultrafleXtreme) analysis. The remaining ~9 µL extended template-loop-primer solutions were used to test exonuclease activity.The RNA template-loop primer (5′-UUUUCUCCGCGUAGUUUUCUACGCG-3′) was annealed at 75 °C for 3 min and cooled to room temperature for 25 min in 1× ThermoPol buffer before adding the other ingredients. The final 20 µL mixture containing 5 µM of the RNA template-loop primer, 250 µM, 500 µM or 1 mM AT-9010, 2 mM MnCl20 in 1× reaction buffer was added to the annealed RNA template-loop-primer solution and incubated for an additional 10 min at room temperature. Finally, 5 µL of a solution containing ~2 mM Fav-RTP or 0.4 mM NHC in 1× reaction buffer was added and incubation was carried out for 3 h at 30 °C. The final concentrations of reagents in the 20 µL extension reactions were 3 µM nsp12/nsp7/nsp8, 5 µM RNA template-loop-primer and ~500 µM Fav-RTP or 100 µM NHC-TP. The 1× reaction buffer contains the following reagents: 10 mM Tris-HCl pH 8, 10 mM KCl, 2 mM MgCl2, and 1 mM β-mercaptoethanol. Desalting of the reaction mixture was performed with an Oligo Clean & Concentrator kit (Zymo Research) resulting in ~10 µL purified aqueous RNA solutions. In all, 1 µL of each solution was subjected to MALDI-TOF MS (Bruker ultrafleXtreme) analysis. The remaining ~9 µL extended template-loop-primer solutions were used to test exonuclease activity.In all, 10 µL of 10 µM RNA template-loop-primers  in 1 × RdRp reaction buffer was annealed by heating to 75 °C for 3 min and cooling to room temperature. 5 µL of 12 µM RdRp complex (nsp12/nsp7/nsp8)2, 10 mM DTT, 50 mM KCl) was incubated at 45 °C for 3 h. Desalting of the reaction mixture was performed with an Oligo Clean & Concentrator kit (Zymo Research) resulting in ~10 µL purified aqueous RNA solutions. In total, 1 µL of each solution was subjected to MALDI-TOF MS (Bruker ultrafleXtreme) analysis. The remaining ~9 µL extended template-loop-primer solutions were used to test exonuclease activity.The RNA template-loop primer (5′-UUUUCAUCGCGUAGUUUUCUACGCG-3′) was annealed at 75 °C for 3 min and cooled to room temperature for 25 min before adding the remaining ingredients. The final 20 µL mixture containing 5 µM of the RNA template-loop primer, 500 µM Tenofovir-DP and 200 units Superscript IV Reverse Transcriptase in 1× reaction buffer (50 mM Tris-HCl (pH 8.3), 4 mM MgCl20 in 1 × reaction buffer was added to the annealed RNA template-loop-primer solution and incubated for an additional 10 min at room temperature. In all, 5 µL of a solution containing 0.2 mM RDV-TP and 0.2 mM UTP in 1× reaction buffer was added and incubation was carried out for 1 h at 30 °C. The final concentrations of reagents in the 20 µL extension reactions were 2 µM nsp12/nsp7/nsp8, 5 µM RNA template-loop-primer, 50 µM RDV-TP, and 50 µM UTP. The composition of 1× reaction buffer was as described above. Desalting of the reaction mixture was performed with an Oligo Clean & Concentrator kit (Zymo Research) resulting in ~10 µL purified aqueous RNA solutions. In all, 1 µL of each solution was subjected to MALDI-TOF MS (Bruker ultrafleXtreme) analysis. The remaining ~9 µL extended template-loop-primer solutions were used for the second extension reaction.In all, 10 µL of 10 µM RNA template-loop-primers (5′-UUUUUACAUCGCGUAGUUUUCUACGCG-3′ for RDV-TP + UTP extension) in 1 × RdRp reaction buffer was first annealed by heating to 75 °C for 3 min and cooling to room temperature. In total, 5 µL of 8 µM RdRp complex (nsp12/nsp7/nsp8)20 in 1× reaction buffer was added to the annealed RNA template-loop-primer solution and incubated for an additional 10 min at room temperature. In all, 5 µL of a solution containing 80 µM GTP and 80 µM UTP in 1× reaction buffer was added and incubation was carried out for 1 h at 30 °C. The final concentrations of reagents in the 20 µL extension reactions were 2 µM nsp12/nsp7/nsp8, ~4 µM RNA template-loop-primer, 20 µM GTP, and 20 µM UTP. The composition of 1 × reaction buffer was as described above. Desalting of the reaction mixture was performed with an Oligo Clean & Concentrator kit (Zymo Research) resulting in ~10 µL purified aqueous RNA solutions. 1 µL of each solution was subjected to MALDI-TOF MS (Bruker ultrafleXtreme) analysis. The remaining ~9 µL extended template-loop-primer solutions were used to test exonuclease activity.Then, 10 µL of ~16 µM Remdesivir (R) and uridine extended RNA template-loop-primers (5′-UUUUUACAUCGCGUAGUUUUCUACGCGRU-3′ for second extension) in 1× RdRp reaction buffer was annealed by heating to 75 °C for 3 min and cooling to room temperature. 5 µL of 8 µM RdRp complex (nsp12/nsp7/nsp8)4-hydroxycytidine (Nhc), AT-9010 and Tenofovir (Tfv) terminated RNAs, as well as the RDV delayed terminated RNA products from above, were annealed by heating to 75 °C for 3 min and cooling to room temperature in 1× exonuclease reaction buffer. To a 14 µL solution of 71.4 nM exonuclease complex (nsp14/nsp10) in 1× exonuclease reaction buffer, 1 µL of DMSO with or without various concentrations of Pibrentasvir was added and incubated for 15 min at room temperature. Then, 5 µL of the annealed RNA (2 µM) in 1× exonuclease reaction buffer was added to the exonuclease/Pibrentasvir mixture and incubated at 37 °C for 15 min. The final concentrations of reagents in the 20 µL reactions were 50 nM nsp14/nsp10, 500 nM RNA, 0-20 µM Pibrentasvir and 5% DMSO. The 1× exonuclease reaction buffer contains the following reagents: 40 mM Tris-HCl pH 8, 1.5 mM MgCl2, and 5 mM DTT. After incubation for 15 min, each reaction was quenched by adding 2.2 µL of an aqueous solution of EDTA (100 mM). Following desalting using an Oligo Clean & Concentrator (Zymo Research), the samples were subjected to MALDI-TOF MS (Bruker ultrafleXtreme) analysis. SARS-CoV-2 exonuclease reactions in the presence of Ombitasvir, Daclatasvir, Ritonavir, and Lopinavir were conducted analogously to the experiments with Pibrentasvir.The U-terminated RNA, A-terminated RNA, G-terminated RNA, the Remdesivir (RDV), Sofosbuvir (SOF), Favipiravir (Fav), NThe A-terminated RNA (2 µM) and Tenofovir extended RNA (2 µM)  and minimized in terms of energy by Density Functional Theory (DFT), with the Becke-3-Lee Yang Parr (B3LYP) method and the standard 6-31 G* basis set, available in Spartan’18 software  (https://www.wavefun.com/). Since the 3D structure for the SARS-CoV-2 nsp14 exonuclease was not available, a structural model was built via the online tool Swiss Model software  (https://swissmodel.expasy.org/) using the crystallographic structure of SARS-CoV nsp14 as a template (Protein Data Bank (PDB) code: 5C8T)49 and the following SARS-CoV-2 nsp14 amino acid sequence:The chemical structures for Pibrentasvir, Ombitasvir, Elbasvir, Daclatasvir, Ledipasvir, and Velpatasvir  and Global Model Quality Estimation (GMQE) values.AENVTGLFKDCSKVITGLHPTQAPTHLSVDTKFKTEGLCVDIPGIPKDMTYRRLISMMGFKMNYQVNGYPNMFITREEAIRHVRAWIGFDVEGCHATREAVGTNLPLQLGFSTGVNLVAVPTGYVDTPNNTDFSRVSAKPPPGDQFKHLIPLMYKGLPWNVVRIKIVQMLSDTLKNLSDRVVFVLWAHGFELTSMKYFVKIGPERTCCLCDRRATCFSTASDTYACWHHSIGFDYVYNPFMIDVQQWGFTGNLQSNHDLYCQVHGNAHVASCDAIMTRCLAVHECFVKRVDWTIEYPIIGDELKINAACRKVQHMVVKAALLADKFPVLHDIGNPKAIKCVPQADVEWKFYDAQPCSDKAYKIEELFYSYATHSDKFTDGVCLFWNCNVDRYPANSIVCRFDTRVLSNLNLPGCDGGSLYVNKHAFHTPAFDKSAFVNLKQLPFFYYSDSPCESHGKQVVSDIDYVPLKSATCITRCNLGGAVCRHHANEYRLYLDAYNMMISAGFSLWVYKQFDTYNLWNTFTRLQ(NCBI RefSeq YP_009725309.1)https://www.ccdc.cam.ac.uk/solutions/csd-discovery/components/gold/). Hydrogen atoms were added to the protein structure according to the data inferred by software on the ionization and tautomeric states. Since there are no crystallographic structures for drugs associated with SARS-CoV-2 nsp14 exonuclease, the standard function ChemPLP was used for the molecular docking calculations, selecting an 8 and 10 Å radius spherical cavity around the active binding site. The figures for the docking poses of the largest docking score value were generated with PyMOL Delano Scientific LLC software  (https://www.pymol.org/2/).The molecular docking calculations were performed using GOLD 2020.2 software  . All procedures related to virus culture were handled in a Biosafety Level 3 (BSL3) multiuser facility according to WHO guidelines. Virus titers were determined as PFU/mL. Virus stocks were kept in −80 °C freezers.African green monkey kidney cells  and the human lung epithelial cell line  were cultured in high glucose DMEM and low glucose DMEM medium, both complemented with 10% FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin at 37 °C in a humidified atmosphere with 5% CO4 cells in 96-well plates were treated for 3 days with various concentrations (semi-log dilutions from 1000 to 10 µM) of the antiviral drugs. Then, 5 mg/ml 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) in DMEM was added to the cells in the presence of 0.01% N-methyl dibenzopyrazine methyl sulfate (PMS). After incubating for 4 h at 37 °C, the cells were measured in a spectrophotometer at 492 nm and 620 nm. The 50% cytotoxic concentration (CC50) was calculated by a nonlinear regression analysis of the dose-response curves.Monolayers of 1.5 × 105 cells/well) in 48-well plates were infected at a MOI of 0.1 for 1 h at 37 °C. The cells were washed, and various concentrations of compounds were added to DMEM with 10% FBS. Forty-eight to seventy-two hours supernatants were collected and the harvested virus was quantified as PFU/mL by titering in Vero E6 cells. A variable slope nonlinear regression analysis of the dose-response curves was performed to calculate the concentration at which each drug inhibited the virus production by 50% (EC50), 90% (EC90), and 99% (EC99). Analysis of drug combinations were performed using SynergyFinder 2.0 software29 with the HSA method, in which the percentage of inhibition was plotted for curves of drugs alone and in combination.Calu-3 cells (5 × 104 cells/well) in 96-well plates were infected with serial dilutions of supernatants containing SARS-CoV-2 for 1 h at 37 °C. Fresh semi-solid medium containing 2.4% CMC was added and the culture was maintained for 72 h at 37 °C. Cells were fixed with 10% formalin for 2 h at room temperature and then stained with crystal violet (0.4%). Plaque numbers were scored in at least three replicates per dilution by independent readers, who were blinded with respect to the source of the supernatant. The virus titers were determined as PFU/mL.Monolayers of Vero E6 cells (2 × 10R2 values ≥0.9. Statistical significance differences, with P values <0.05, were assessed using Student’s t tests to compare the pharmacological parameter values in the presence of RNA polymerase inhibitors alone and combined with HCV NS5A inhibitors.The assays were performed blinded by one professional, codified, and then read by another professional. All experiments were carried out at least three independent times, including a minimum of two technical replicates in each assay. The dose-response curves used to calculate pharmacological parameters were generated by variable slope plot from Prism GraphPad software 8.0. The equations to fit the best curve were generated based on Further information on research design is available in the Supplementary InformationDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2Reporting Summary


According to the central dogma of molecular biology, genetic information is passed from DNA to RNA to protein. The transmission of information from DNA to RNA is called transcription and is carried out by the RNA polymerases (RNA pols). The RNA pols from bacteria to eukaryotes are multimeric enzymes which show a high degree of conservation in terms of their structure and functionality  Figure . NotablyLei and Burton compares the three-dimensional structure of archaeal, bacterial and eukaryotic RNA pols. Their analyses revealed that the enzyme, in all three domains of life, are of two-double-ψ-β-barrel (2-DPBB) type. In addition to 2-DPBB, the catalytic core of multisubunit RNA pols is comprised of a conserved bridge helix and trigger loop. Lei and Burton propose that the 2-DPBB family of multisubunit RNA pols might have evolved prior to the last universal common cellular ancestor during evolution.Despite variation in structure and subunit composition, RNA pols from different organisms harbors conserved features . The artNottebaum and Weinzierl describe that several of the key motifs in the active center of Nanoarchaeum equitans RNA pol contain unusual and radical substitutions expected to be harmful to the catalytic activity. However, the authors reconstituted a RNA pol complex in vitro with transcription activity, concluding that sequence changes do not adversely affect catalytic activity, even if they are unusual and localized in key motifs. Moreover, they identified a stringent atypical requirement for fluoride ions for maximal RNA pol activity, proposing a model where more “conventional” archea will not use it.Nanoarchaea is a highly diverged archaeal phylum with many unusual biological features. Barba-Aliaga et al. focuses on nuclear RNA pols in eukaryotes and summarizes their evolutionary origin and functional reasons that could have led to their multiplicity. Furthermore, authors discuss the regulation and the homeostasis of the different RNA products that they synthesise. The authors present several studies to show how the coordination between RNA pols activities is necessary for cellular processes, such as the influence of RNA pols on the translation machinery synthesis (ribosomes and tRNAs) or how eukaryotic RNA pols transcription regulation occurs with respect to the changes in cellular volume.The work by Fernández-Parras et al., investigates the transcriptional regulation of the RNA pols common subunits genes in olive tree cultivar  and shows that they are spatio-temporally regulated, as well as regulated by biotic and abiotic stresses. This work opens questions about the existence of multiple RNA pols variants in polyploid organisms.Plants are the only known eukaryotic organisms containing two addition RNA pols (IV and V), in addition to RNA pol I-III, which evolved from RNA pol II . The worGarrido-Godino et al., focuses on the knowledge of biogenesis of RNA pols in yeast. The authors review the mechanisms and proteins (assembly and transport factors) involved in these processes and make the comparison with human factors described previously. In addition, the manuscript by Turowski and Boguta summarizes the current knowledge on the biogenesis of the RNA pol I and III and focuses on the model of their co-translational assembly, based on recent publications, showing the importance of Rpb10, Rpc19 and Rpc40, and of the Rbs1 protein in the assembly of RNA pol I and III complexes.A model for the biogenesis of eukaryotic RNA pol II has been proposed based on the bacterial RNA pol formation, a sequential process involving participation of several subassembly complexes that leads to the complete enzyme assembly in the cytoplasm before its nuclear import . HoweverLata et al. reviews the current knowledge regarding the functional impact of specific mutations, possible Pol III-related disease-causing mechanisms, and animal models that may help to better understand the links between Pol III mutations and disease. Similarly, a large number of genetic diseases associated with RNA pol I mutations exists. They are collectively called ribosomopathies. The understanding of the precise mechanistic of Pol I transcription opens broad perspectives in health-related research areas.Notably, mutations of RNA pols lead to diseases and disorders. Some of these are suggested to be associated with RNA pol III assembly. Although the causal relationship between RNA pols mutations and disease development is widely accepted, the associated molecular mechanisms are poorly understood. The work by Azouzi et al. nicely review recent advances in the field of RNA pol I transcription elongation, revealed using nucleotide resolution techniques. These advances showed the connection between the production of rRNA and nascent rRNA folding. Indeed, rRNA folding during transcription seems to be an anti-pausing mechanism favoring transcription elongation because rRNA secondary structures prevent backtracking. Furthermore, they also discuss mechanisms involved in RNA pol I termination. Based on recent discoveries by in vivo.Delgado-Román and Muñóz-Centeno proposed that RNA pols I and III activities should be connected to regulate cell cycle progression. How cell cycle regulation is affected by the balance between the three RNA pols products and RNA pols assembly is discussed. The authors focus on ribogenesis, a process that requires the activity of all three RNA pols (There is an enormous interest in deciphering how RNA pols integrate the information that cells receive and how RNA pols are coordinated and communicated to regulate gene expression in response to physiological and pathological conditions. In this regard, RNA pols , and disRNA pols .González-Jiménez et al. propose that phosphorylation may have a role in the coordination of the three transcription machineries. Various studies have reported that several subunits of RNA pol I, II and III are susceptible to phosphorylation (for instance, Dwyer et al. proposes a novel mechanism of splicing-mediated regulation of transcription by RNA pol II through modulating the gene architecture. The transition in topology of a gene from linear to a loop during cotranscriptional splicing and the mechanism of enhancement of transcription by the looped structure is being discussed.Since their discovery, the biological significance of introns in the eukaryotic genome has remained an enigma. A number of studies in a diversity of eukaryotes have revealed that the process of splicing, which removes an intron from a primary transcript, is often a positive regulator of transcription . The artO’ Brien and Ansari focuses on a rather unexpected role of TFIIB during viral pathogenesis. The article describes in detail the targeting of TFIIB by viral transcriptional regulators during pathogenesis. Likely reasons for preferred targeting of TFIIB over other general transcription factors by viruses are discussed. This makes TFIIB a potential target of antiviral therapies.Transcription by RNA pol II in eukaryotes is facilitated by a number of transcription factors. TFIIB is one such essential general transcription factors . The artLinzer et al. discuss multiple aspects of TFII-I participation in the transcription cycle. In addition to affecting initiation from a subset of promoters, TFII-I is involved in transcription elongation by regulating pausing of RNA pol. The involvement of this factor in cancer, neurological and immunological disorders in humans, development in mice, and induction of pluripotency is discussed.TFII-I is another transcription factor of RNA pol II . It was mfd (Lindsey-Boltz and Sancar discusses the recent advances in three-dimensional structure and single molecule studies pertaining to mfd. These studies have revealed that Mfd binds stalled RNA pols even in the absence of UV damage and helps the pol operate in hard-to-transcribe regions. The possibility of mfd-RNA polymerase interaction contributing to both, promotion and prevention of mutagenesis in a context-dependent manner, is discussed.It was known since a long time that the transcriptionally active UV-damaged regions of genome are repaired more efficiently than the non-transcribed regions. The factor responsible for the transcription-coupled repair of damaged DNA in prokaryotes is mfd . The artThe articles in this special issue cover some, but not all, lesser-known aspects of RNA pols and highlight that many mechanistic, structural and/or evolutionary aspects of RNA pols, among others, remain unexplored or are still not well investigated. Futures investigations on RNA pols will greatly help to understand gene expression regulation, where transcription is the bottle neck.