Epitranscriptomic abnormalities, which are highly prevalent in primary central nervous system malignancies, have been identified as crucial contributors to the development and progression of gliomas. RNA epitranscriptomic modifications, particularly the reversible modification methylation, have been observed throughout the RNA cycle. Epitranscriptomic modifications, which regulate RNA transcription and translation, have profound biological implications. These modifications are associated with the development of several cancer types. Notably, three main protein types—writers, erasers, and readers, in conjunction with other related proteins, mediate these epitranscriptomic changes. This review primarily focuses on the role of recently identified RNA methylation modifications in gliomas, such as N6-methyladenosine (m6A), 5-methylcytosine (m5C), N7-methylguanosine (m7G), and N1-methyladenosine (m1A). We delved into their corresponding writers, erasers, readers, and related binding proteins to propose new approaches and prognostic indicators for patients with glioma. Glioma is a widely prevalent primary malignant tumor of the central nervous system, comprising up to 77% of all primary brain malignancies , 2, and Recent studies demonstrated the crucial role of epitranscriptomic modifications in regulating tumor occurrence and development . This reEpigenetics is a complex process involving various modifications such as histone modification, chromatin remodeling, nucleosome localization, DNA methylation, and RNA modification. In particular, RNA modifications encompass over 170 covalent modifications, mostly methylation modifications, including m6A, m5C, and m7G methylation. Thus, a comprehensive understanding of various epitranscriptomic modifications and their roles in glioma pathogenesis is crucial for developing novel and effective diagnostic and therapeutic strategies for different diseases . MethylaIn this review, we present an overview of recent research progress on several common RNA methylation modifications and their corresponding regulatory enzymes in gliomas. Hence, this review aims to discuss the potential research directions for the study of RNA modification and its potential role in the origin, diagnosis, and treatment of glioma to improve patient outcomes and develop more effective therapies.The flowchart of this review Fig. . A comprIn the early 1970s, Desrosiers et al.  identifiRNA m6A modifications involve three types of proteins: methyltransferases (writers), demethylases (erasers), and m6A binding proteins (readers) . The sixMETTL3, the first identified m6A methyltransferase, contains a gene located on 14q11.2, comprising 10 exons. It modulates the expression of oncogenes and tumor suppressor genes at the post-transcriptional level, including mRNA stability and translation. Visvanathan et al.  reportedHowever, some studies have shown that METTL3 and a highly m6A-modified state may suppress tumor development. Inhibiting m6A enrichment via METTL3 knockdown resulted in enhanced growth, self-renewal, and tumor progression of glioma stem-like cells (GSCs), as reported by Cui et al . Ji et aMETTL14, located on chromosome 4q26 and comprising 12 exons, shares 43% sequence similarity with METTL3. METTL14 knockdown promotes a malignant phenotype by upregulating the expression of oncogenes  and downregulating the expression of tumor suppressor genes . These findings highlight the critical role of METTL14 in glioma progression and emphasize its potential as a therapeutic target . Miao etWTAP, also known as Mum2, interacts with the Wilms’ tumor 1 gene and plays a coordinating role in RNA methylation. Although WTAP lacks catalytic functions, it binds to RNA and recruits METTL3 and METTL14 for methylation , 32. EarRNA-binding motif protein 15/15B (RBM15/RBM15B), also referred to as OTT or SPEN, is adjacent to the methylated RRACH motif in the mRNA transcriptome and can bind to the METTL3-WTAP complex. It is recruited to a specific site for methylation, so it is also considered a methyltransferase . Chai etKIAA1429 is a pivotal m6A methyltransferase-related protein and is the most abundant component of the methyltransferase complex (MTC). As a scaffold for coordinating core components on RNA substrates, KIAA1429 mediates m6A methylation on specific sequences near the 3′-UTR and termination codon. The knockdown of KIAA1429 in A549 cells resulted in a significant four-fold reduction in the median peak value of m6A compared to the knockdown of METTL3 or METTL14 alone, indicating its essential role in mammalian methylation , 37. IntZC3H13, also termed KIAA0853 or Xio, was recently identified as a methylation regulator. It regulates m6A methylation by forming a ZC3H13-WTAP-virilizer-hakai complex in the nucleus . Chow etTwo enzymes, FTO and ALKBH5, have been identified to participate in the m6A demethylation process , 40. BotFTO, the earliest reported m6A demethylase, provided the first evidence of the reversibility and dynamics of m6A modification . FTO is In contrast, various pieces of evidence challenge the notion of cancer-promoting effects of FTO in gliomas. Tao et al.  reportedALKBH5 is a non-heme iron (II)/ketoglutarate-dependent dioxygenase with iron-dependent active expression and is localized in the nucleolar patches and subcellular organelles within the nucleus. ALKBH5 may be involved in the alternative splicing of RNA precursors, and its knockdown can promote mRNA nucleation , 52. DonThe m6A reader proteins can be broadly classified into three primary groups, with the first being the YTH protein family, which contains an evolutionarily conserved YTH (YT521-B homolog) structural domain. Insights from an RNA pull-down experiment showed that proteins containing the YTH domain are universal m6A binding agents . YTHDF1 Another group of m6A reader proteins comprises insulin-like growth factor mRNA-binding proteins (IGF2BP 1–3). IGF2BP improves the stability and storage of target mRNA in an m6A-dependent manner . The thiAmong the proteins with YTH domains, YTHDF1, YTHDF2, and YTHDF3 play pivotal roles in gliomas. YTHDF1 and YTHDF3 synergistically enhance the translation efficiency of their target RNA , while YYTHDF2 is the first m6A recognition protein to be discovered and has since been studied extensively. It recruits the splicing factor of the precursor mRNA and regulates mRNA splicing and decay. Fang et al.  reportedYTHDC1 and YTHDC2 are members of the YTH domain-containing nuclear proteins that regulate RNA splicing and export processes modulated by m6A and are predominantly located in the nucleus. YTHDC1 primarily regulates mRNA splicing and YTHDC2 promotes mRNA degradation. Previous studies have highlighted the pivotal role of YTHDC1 in m6A-mediated alternative splicing . A lucifYTHDC1 reduces the expression of VPS25 and inhibits glioma proliferation through the JAK-STAT signaling pathway . Li et aYTHDC2 is a binding protein of the YTH protein family and the only member with ATP-dependent RNA helicase activity. YTHDC2 plays a role in the different methylation levels observed in uterine corpus endometrioid carcinoma, adrenocortical carcinoma (ACC), and endocervical adenocarcinoma (CESC), leading to different prognoses and levels of immune cell infiltration . FurtherIGF2BPs are mRNA-binding proteins containing a KH domain that can maintain the stability of their target mRNAs and prevent their degradation by binding to the m6A methylation site and acting as a recognition protein , 75. IGFThe heterogeneous nuclear ribonucleoprotein (HNRNP) family, also known as the m6A methylated binding proteins, includes HNRNPA2B1 (heterogeneous nuclear ribonucleoprotein A2B1), HNRNPC (heterogeneous nuclear ribonucleoprotein C), and HNRNPG (heterogeneous nuclear ribonucleoprotein G), which play important regulatory roles in RNA processing, maturation, and gene expression. hnRNPs have RNA-binding domains that contain RNA recognition motifs, K-homology domains, and arginine/glycine-rich boxes . As a nuEukaryotic initiation factor 3 (eIF3) can also function as an m6A recognition protein by binding to bases that undergo m6A modification in the 5′-UTR of RNA. This action facilitates mRNA translation and recruits the 43S complex for protein translation in a cap-independent manner . eIF3b km5C RNA methylation refers to the covalent modification of the fifth carbon atom of the cytosine ribose nucleotide in RNA molecules. Initially identified in rRNA in the 1970s, this modification was subsequently observed in tRNAs, mRNAs, and lncRNAs –98. m5C Sadenosylmethionine (SAM) serves as a methyl donor, enabling the m5C methyltransferase to transfer a methyl group to cytosine, yielding 5-methylcytosine . CurrentThe human NSUN family consists of seven members: NSUN1–NSUN7, all of which contain the Rossman folding catalytic domain and one SAM-binding site. These proteins catalyze methyl group transfer using a similar mechanism, which involves the formation of a covalent intermediate via covalent binding between the cysteine of the methyltransferase and the cytosine of the RNA. This is followed by the nucleophilic addition of the electron-rich cytosine ring to the methyl group on the SAM to complete methylation. Predominantly found in the nucleus . NSUN1 hThere are a few reports on the role of the NSUN family in gliomas. Many studies  have claDNMT2 is a tRNA and miRNA methyltransferase mainly located in the nucleus. Unlike the NSUN family of proteins that utilize two catalytic cysteines at the active site, DNMT2 employs a single cysteine at its active site, similar to other DNA methyltransferases , 114. DNTET1, TET2, and TET3 belong to the ten-eleven translocation (TET) family, representing dioxygenase dependent on Fe (II) and α-ketoglutaric acid (α-KG). Although TET3 is found in both the nucleus and cytoplasm, TET1 and TET2 are primarily located in the nucleus. The TET family was first identified as a family of DNA dioxygenases. Fu et al.  found thDiminished DNA repair resulting from TET1 deficiency can lead to genomic instability, which may account for the low survival rates of patients with glioma , 120. InThe biological function of RNA modification is primarily related to protein binding. The m5C methylation-binding proteins include YBX1 (Y-box binding protein 1) and Aly/REF export factor (ALYREF).YBX1 is an m5C-binding protein that regulates mRNA stability in the cytoplasm and is involved in the proliferation, differentiation, and malignant transformation of tumor cells . YBX1 exALYREF is a key component of the TREX mRNA transporter complex. ALYREF acts as an m5C methylation recognition protein and can specifically bind to m5C-modified mRNA in the nucleus to form mRNP complexes that promote nuclear mRNA export , 127. Rem1A refers to the methylation of the N1 site of adenosine bases in RNA molecules. Abundant m1A modifications have been observed in tRNA and rRNA; however, m1A modification in mRNA is lower, approximately one-sixth of that of m6A . m1A58 mCurrently, METTL1 is the most well-recognized m7G regulator, which binds with its cofactor, WD repeat domain 4 (WDR4), to catalyze m7G modifications in tRNA, miRNA, and mRNA . m7G hasRNA guanine-7 methyltransferase (RNMT) and its cofactor RNMT-activated small protein (RAM) participate in the m7G modification at the 5′ end of mRNA . B7-H6 pIn addition to protein-coding RNAs, noncoding RNAs are also modified in gliomas. Modifications of noncoding RNAs affect the binding of RNA-binding proteins to noncoding RNAs. For miRNAs, HNRNPC directly recognizes and binds to the pri-miR-21 site with m6A modification, subsequently enhancing the expression of miR-21. Therefore, miR-21 promotes cell migration and invasion in glioblastomas . has-mirCanonical non-reader RBPs associated with RNA methylation also interact with modification-containing transcripts without directly recognizing m6A bases. RBPs target adenosine- and uridine-rich elements (AREs) present in the 3′-UTR of RNA. Approximately 8% of human transcription products contain AREs  and the HuR is a member of the ELAV family of RBPs, known to selectively recognize and bind AREs. It regulates miRNA expression through the AUUUA motif in pri-miRNA. Characterized by its three RNA recognition motif domains, HuR is predominantly located in the 3ʹ-UTR of mRNA near the miRNA binding sites . It was Tristetraprolin (TTP), also referred to as ZFP36, is a well-known RBP that regulates mRNA degradation by recognizing AREs in the 3′- UTR of mRNA . This prMalignant progression and high recurrence rates make gliomas the most lethal primary brain tumors; therefore, understanding the molecular mechanisms of glioma development is imperative to address these challenges. In recent years, RNA modification has emerged as a key area of research owing to its extensive influence on RNA metabolism and function, thus presenting as a promising target for developing novel therapeutic approaches for glioma treatment. Aberrations in epitranscriptomic modification are considered to be one of the key factors driving tumor progression. Among them, methylation modification has the characteristics of being dynamic and reversible, playing an important role in cancer. In this review, we discussed the role of RNA methylation modifications in gliomas and provided evidence from current studies. We suggest that RNA modification writers, erasers, and readers can serve as potential biomarkers for glioma origin, diagnosis, and prognosis, as well as potential drug targets for therapy. Epigenetic regulators such as HDACs, DNMTs, and EZH2 have been confirmed to have significant potential as cancer treatment targets. However, targeting epitranscriptomic factors alone does not always achieve ideal treatment efficacy; it often needs to be combined with other antitumor therapies to achieve optimal results , 154.A multifaceted approach is essential to transition basic research findings into clinically relevant interventions, such as: diagnostic and prognostic biomarkers, targeted therapies, personalized medicine, monitoring treatment response and so on, which are shown in Fig. Furthermore, METTL3, FTO, and YTHDC1 were higher in IDH-mutant LGG and GBM than in wild-type gliomas; however, this did not mean that the RNA was more or less methylated (more than m6A/m5C) in IDHm gliomas . MoreoveIn summary, this review highlights the potential value of RNA modifications in the diagnosis and treatment of gliomas. Delving deeper into the distinct mechanisms of RNA modification on gliomas is an auspicious future research direction that will refine our understanding of the role of RNA modification in glioma, which may provide more possibilities for the early diagnosis and effective treatment of this life-threatening disease.


The COVID-19 pandemic led to major service disruptions in the healthcare sector, especially regarding sexual and reproductive health services. However, the impact of the pandemic on Canadian adolescents is relatively unknown. This study aimed to investigate the impacts of the COVID-19 pandemic and associated public health measures on the sexual and reproductive health (SRH) of adolescents in Alberta, Canada.A qualitative study using an interpretive description (ID) approach and community-based participatory research principles was conducted to capture the subjective experience and perceptions of adolescents and service providers. With the collaboration of the Adolescent Advisory Group and community partners, 18 adolescents and 15 service providers were recruited for the study through purposive sampling. Findings from the qualitative interviews were analyzed using thematic analysis.Three major themes emerged from the analysis: (1) COVID-19 SRH experience, (2) barriers to SRH, and (3) adolescent SRH strategies. Our findings highlight numerous barriers and challenges that prevented adolescents from accessing SRH education, products, and services.The COVID-19 pandemic had a profound impact on the SRH and the well-being of adolescents. Our study reflects the need for diverse SRH strategies to maintain continued access to SRH resources during disruptive events, such as the pandemic.The online version contains supplementary material available at 10.1186/s12978-023-01712-x. Access to sexual and reproductive health (SRH) services is a basic human right. All individuals require access to appropriate SRH services to maintain their optimal sexual and reproductive health. Adolescents require special guidance, support, and youth-friendly services in matters of SRH as they enter puberty and explore their sexual identity. However, during the COVID-19 pandemic, many health and SRH services were suspended. Access to SRH products and services became difficult due to public health restrictions, which has possible negative consequences for adolescents’ SRH. The experiences of adolescents during the COVID-19 pandemic regarding their SRH are not reported in Alberta, Canada. Therefore, we explored the impacts of these public health restrictions on adolescents’ SRH. We performed qualitative interviews with adolescents and SRH service providers to know their perspectives on how the pandemic influenced the SRH of adolescents. This paper provides insights into the barriers faced by adolescents while accessing SRH services during the pandemic, as well as their perceptions of digital strategies, such as mobile applications, and other recommendations for supporting SRH education and services. Based on the study findings, an adolescent-friendly mobile application will be developed to provide a virtual platform connecting adolescents to SRH educational resources, services, and support.The online version contains supplementary material available at 10.1186/s12978-023-01712-x. Globally, the COVID-19 pandemic led to drastic changes in the access and delivery of healthcare services, influencing the physical, psychological, social, and emotional well-being of individuals, and profoundly impacted adolescents . The WorA Canadian Community Health Survey (2015/2016) reported that more than half (54.1%) of 15–24-year-olds in Canada were sexually active and at risk of unintended pregnancy and/or STIs . AdolescAdolescents often experience a range of intersecting barriers making it difficult to access appropriate SRH services  , 12. TheWhat are the perspectives of adolescents and service providers on how the pandemic has influenced the SRH of adolescents in Alberta?How did pandemic-related public health measures shape access to SRH services by adolescents?What types of services, programs, or supports for SRH did adolescents use during the COVID-19 pandemic?The impacts of the pandemic are still unfolding and there are potential long-term consequences that will shape adolescents’ SRH. However, the impacts of these measures on the SRH of adolescents in Alberta, Canada is relatively unknown. Understanding the effects of these measures is a crucial step towards informing effective services, supports, and strategies to improve adolescents’ access to SRH services. Therefore, the overall purpose of our study was to investigate the impact of the COVID-19 pandemic and associated public health measures on the SRH of adolescents in Alberta. Specifically, the study addressed three research questions:We used Levesque’s conceptual framework of access to health care  to underWe conducted this study in two phases over 2 years (2021–2023). The first phase of the study consisted of qualitative interviews with adolescents and care providers, whereas the second phase consisted of co-designing an adolescent specific SRH website based on the knowledge generated from the interviews. In this manuscript, we are presenting the findings from the qualitative interviews.To enhance the integration of research findings into practice, programming, and policy and to promote the co-creation of knowledge within the study, we used an interpretive description (ID) approach combined with integrated knowledge translation (IKT) . ID faciWe used a multi-faced community-based strategy to recruit adolescents, which involved social-media marketing campaigns, university-wide weekly email campaigns, and promotion of the study in the university campus and clinics. In collaboration with our community partners who provide care to vulnerable youth, we recruited participants from various backgrounds, socioeconomic standing, and gender identities to ensure adequate representation. We also recruited SRH service providers through Adolescent Medicine Clinic and Birth Control Centre in Edmonton.Participants were included in the study based on the following inclusion criteria: (1) Adolescents aged 15–19 years old who were seeking or had accessed SRH services in the past 5 years or healthcare professionals who provided SRH care to youth, (2) living in Alberta, and (3) fluent in English. We included older adolescents (15–19 years) in this study as their SRH needs are different from younger adolescents (10–14 years).We used purposive sampling to deliberately choose participants based on the inclusion criteria, who would provide relevant and rich descriptions of their SRH experiences. Informed consent was obtained from all participants and additional parental consent was obtained if it was determined that adolescents lacked decision-making capacity. A total of 18 adolescents and 15 healthcare providers were recruited for the study. This sample size was determined a priori and deemed adequate to gain a deeper understanding of participant viewpoints while acknowledging that there may be differences in perceptions and outliers . Due to Data collection was conducted from 2021 to 2022. Ethics approval was obtained from the University Research Ethics Board. Data were collected through a semi-structured interview guide  Methodological coherence; ensured congruence between the research questions and components of ID, (2) appropriate and diverse sampling to ensure efficient and effective saturation of categories with optimal quality, (3) collected and analyzed data concurrently, (4) developed a coding system that was discussed and verified with research team members including AAG, (5) recorded field notes and evaluated the quality of the interviews consistently by analyzing the participant’s responses to the questions and modifying the questions accordingly.See Tables Three broad themes emerged from the adolescents’ and service providers' qualitative interviews: (1) COVID-19 SRH experience, (2) barriers to SRH, and (3) adolescent SRH service access strategies. These themes were then further divided into sub-themes, which are presented below.The COVID-19 pandemic influenced many institutions including adolescents' access to sexual and reproductive health. We found that adolescents had both positive and negative experiences during the pandemic.…It was definitely a lot harder to find that kind of access as well because of I assume, less staff working and more pandemic measures, so, while I assume it should have taken about a week it took a month to get the service that we did. (Adolescent Participant-1)Many adolescents reported both positive and negative changes in their SRH services before and during the pandemic. These changes were associated with the challenges of accessing walk-in clinics and longer times to seek SRH services:Service providers commented on the de-prioritization of SRH services during the pandemic: "I think this kind of got pushed aside – like sexual health like wasn’t people’s priority, of course, with COVID going on." .Definitely not having the walk-in impacted youth, not providing the open access, you know, all the screening tools, the mask. It just became a whole ordeal just to even come in and pick up their birth control or, you know, get emergency contraceptives, or get tested. It was, you know, a whole lot, where it was just before, it was easy. They’d just come, they could go. So definitely, our access, and the walk-in, not having that available for them was probably that. Another service provider also mentioned:I needed to talk to my doctor about something and I wanted her to like look at something or like have a visual she only does phone appointments now most of the time, and then, if she didn't do for an appointment it's like she's only in office, I think, like two days a week, and that's changed definitely because she used to be an office I think. (AP-9)As consultation moved from in-person to online, some adolescents expressed their dissatisfaction with online services, especially with matters regarding counselling and family planning. They reported feeling uncomfortable with a healthcare provider in a virtual setting:Alternatively, many adolescents indicated online services to be a positive outcome of the pandemic: “Discussing with my doctor over the phone was very efficient and faster than it would have been in person” (AP-14).Some participants stated that there was no impact of the pandemic on their SRH: “Personally, no. I don’t think it’s done anything like about my reproductive health, really. Like before and after, or even during, it was nothing’s really changed.” (AP-11).Several adolescents reported the easy accessibility of using alternate (online) services for their SRH needs and highlighted the use of newer resources like mobile applications to support their SRH. Although some adolescents appreciated the online services, many struggled with lengthy waiting times and securing SRH products (contraceptives). They recalled the ease of obtaining free contraceptives and medical advice from clinics on a walk-in basis before the pandemic: “It’s also a lot harder to find free like birth control, like condoms or whatever versus before I just would pick them up in my local clinic.” (AP-4).There's definitely a decrease in in-person services and I also think that there's much more of a wait time when it comes to picking up stuff like birth control or asking basic questions that I could easily have asked my physician before the pandemic. (AP-10)Another participant also expressed:Service providers talked about the challenges that were introduced in the system during the pandemic: "During the worst of the pandemic, there was only one nurse. It was by appointment only, which kids don’t do great with appointments. And it was symptomatic testing only." (Intake Coordinator).So, staff shortages were – were typically because of redeployment to COVID-19 efforts, and so when your staff is doing something else, then you have limited access in terms of all of it Additionally, workforce shortages because of redeployment to COVID-19 response further compromised SRH operations:We explored the barriers that adolescents encountered during the pandemic as many challenges were brought on by the closure of schools, limited hours of stores, and the absence of support systems.Many adolescents indicated that the majority of their SRH education was obtained through the school. Formal classes on sexual education (sex-ed), as well as other courses, helped them to gain an understanding of their bodies and SRH, which got interrupted. As participant 14 mentioned: “I mean most of my information has come from school resources such as reproductive health classes and biology classes, so that is my main means for understanding reproductive and sexual [health].”During the pandemic, you know, due to the closure of schools, it affected the information I used to get, and you know, libraries. Like in most cases, I used to go to the library to, you know, have access to library materials to read more about sexual education and reproduction. (AP-12)Another participant expressed:So, before the pandemic, our clinic did a lot of outreach, so we’d go to like – it’s at the high school for kids that struggle in regular high school. We would go there. We would go to other little places around the community, and then with COVID, that obviously stopped. A service provider commented on how the pandemic led to the suspension of SRH outreach education initiatives:It definitely like affected it, because I missed out on my grade ten and eleven – well, sorry, eleven and half of twelve school year, so it was like – like that’s like prime age for kids to start like exploring, and like I didn’t get that sex ed during those years, so like it was definitely more of a figure it out yourself kind of thing that happened, to like the age group that I belonged in, because we didn’t get taught it, because it wasn’t a priority, so they didn’t teach it over Zoom. (AP-16)During the later stages of the pandemic when educational institutions switched to online platforms, SRH education was not given a higher priority:Participants reported that reduced hours of stores during the pandemic decreased access to SRH products, like contraceptives and menstrual hygiene products: “I guess during the pandemic it definitely got a little bit harder, because of like the mandates and stuff like that, some stores were closed (AP-11).The ability to go to the store became a little bit harder because of limited capacities and things a lot of stores have only 25% capacity or 30% capacity, so it was hard just having to like wait in lines and things but that's just like a general inconvenience. (AP-17)Another participant expressed:Another participant reported difficulties securing contraceptives: “Like if I was getting contraceptives, it was definitely harder to get during the pandemic because stores were closed down.” (AP-11).Reduced supply of SRH products during the pandemic, such as contraceptives was also reported: “Lower store capacities or stock of items, especially when like toilet paper and, like other hygiene products things became a little bit harder.” (AP-17).Like it's a lot harder to find a group like I said and to find people that you can rely on to support you as well, I felt like it was like I was discouraged during the beginning of the pandemic and during peak or like when there was more cases to go and get tested. (AP-8)Many adolescents expressed the loss of social support and connectedness during the pandemic. Social distancing mandates made it difficult to meet peers and share common concerns:Another participant talked about losing other forms of support, such as counselling: “Yeah, kind of both, because the only counselling I would have had access to would have been school, and when schools went online, there was really no way to do that.” (AP-16).So I found that has really impacted me and my sexual and reproductive health, during the pandemic I can't really talk about that, before because I don't think I would have gone through the journey that I've gone through with my sexuality if it wasn't for this pandemic but I do know that resources have been a lot harder for LGBTQ+ individuals during this time. (AP-4)One participant expressed the difficult journey of exploring their sexual orientation during the pandemic without any support:We inquired about adolescents’ perceptions of the development of a mobile application to access SRH resources and what strategies proved to be useful to them during the pandemic. Participants recommended strategies that they believed would be more beneficial for the adolescents.Yeah, I definitely would recommend that (mobile applications) I think would be really useful, especially because I'm on websites sometimes the university website sometimes just in general websites on the Internet, it can be hard to navigate them like they aren't made in a way that it's easy to know where things are located. (AP-1)The majority of the participants in this study supported the idea of developing a mobile application for SRH resources and indicated that it would increase access to SRH resources. One participant stated their preference for mobile applications over other digital platforms:Like there’s actually a lot of good technology out there that I knew about and could use. Most of the time it was tech problems on the school side. Like there was lots of problems getting – if there was two classes, getting both teachers to log in, because the person who made the Google lead had to let them in. I couldn’t let them in. So, like there was like those little issues. Service providers shared their experiences of using digital strategies for SRH education:Oh yes, and mobile apps I tend to be a bit more wary because there is a lot of misinformation and bad apps, I guess, but the online websites if it looks credible than I do, listen to what they say and they have helped me get more information on productive health and, what is needed to ensure that I'm being healthy and safe. (AP-14)On one hand, participants expressed that mobile applications would provide adolescents with the privacy to seek SRH help confidentially: “That's really cool and it could be super useful, especially for like people who are in families that don't feel comfortable talking about those things are no” (AP-13). On the other hand, some had concerns regarding the safety features of such applications and the possibility of receiving unreliable information:Maybe simplifying the online website would be helpful, so making the interface easier to navigate and having more information summarized - because sometimes some aspects aren't very clearly explained so just summarizing where things and having an easier interface would be very helpful. (AP-14)We explored various strategies and recommendations from participants that would help in developing targeted interventions to support adolescents’ SRH. Participants talked about various features to facilitate the delivery of SRH resources and provide a holistic platform for adolescents to receive support. One participant indicated the importance of a simple interface design:Just a mobile app that makes information easy to consume because some people are like shy to look things up and just some maybe anonymous forums like so we can have this community of adolescents, to discuss freely about their problems rather than being embarrassed by having their public identity revealed so just some forums and some information and maybe some clinicians, if possible. (AP-7)Another participant recommended anonymous forums where adolescents with similar interests can freely discuss SRH with care providers:I did a lot of texting with our youth patients during the pandemic. And I found that that was a really helpful way to sort of deliver a lot of information at once delivered in a way that's like immediate and accessible to the youth. Along with videos, Zoom, like virtual sessions were also effective, because again, I can reach more rural. And we made a frequently asked questions PDF because usually we get really similar questions... And so that was really helpful because they can just read that and get their questions answered. (Partner Notification Nurse)Service providers highlighted how they supported adolescents’ SRH by implementing various strategies:I think that an app or like a website with like STI testing centers or different clinics and areas that you can go and get you know condoms birth control, whatever we can get all of this help just one app or one website that has all of that information there with very minimal searching. I think that would be something that would really benefit society because it's very hard to find information. (AP-4)Many participants also expressed seeing various features to help them get quick access to resources and nearby SRH services:I think like it’s the younger generations that are really doing well with like the virtual appointments. I think if they could find a way to make access to that more easier, I think kids would be more comfortable getting tested, instead of having to – you know, it’s intimidating for a 15 or 16-year-old kid to walk into a health building all by themselves, try to register themselves, sign in, go to the right desk, answer all these medical questions. So, it’d be nice if there was a more comfortable environment for them, perhaps. One service provider indicated how virtual strategies can target adolescents effectively and reduce barriers to their SRH and well-being:This study evaluated the impact of the COVID-19 pandemic on the sexual and reproductive health of adolescents and explored the factors that supported and promoted their SRH. We found that the COVID-19 pandemic had significant impacts on various aspects of the SRH of adolescents, including but not limited to access to SRH education, services, and products.The pandemic disrupted major sectors, including preventive healthcare, leading to the loss of basic SRH rights . DivertiAdolescents in our study reported the challenges of securing SRH products timely , which was brought on by the closure of schools, stores, and in-person clinics. The unavailability of contraceptives   and in-pAdolescents faced a major setback when physical distancing requirements prevented them from meeting their social circles and receiving support from their peers, counsellors or service providers. Loss of support systems and educational and extracurricular activities increased anxiety among adolescents, leading to increased risky behaviours, including unprotected sexual activity . A few aThe shift from in-person to online SRH services  was well appreciated by many adolescents in this study, as it not only prevented longer waiting times in high-risk COVID-19 environments but also made services accessible at the fingertips. Service providers also reported that online services improved access and gave them an opportunity to connect with adolescents. Regardless of the ease of access, some participants expressed their discomfort and dissatisfaction with meeting healthcare providers in a virtual setting. Although telemedicine created ease for some SRH issues, many other reproductive health issues necessitated physical examinations  or laboratory investigations  . Our finThe pandemic caused a massive shift from in-person SRH care to self-management through telemedicine and digital resources . AdolescThis study is the first to report the experiences of adolescents regarding their SRH during the COVID-19 pandemic in Alberta, Canada. We collected and analyzed data from both adolescents and service providers, which yielded a comprehensive understanding of the challenges faced by adolescents. Also, we recruited participants from various backgrounds to ensure a better representation of the sample.Due to the sample size and geographical limitations, the results may not be generalizable to the entire population. Data were collected only from Alberta, and the results could vary in other provinces and territories. We did inquire about adolescents’ current level of education but did not inquire about their school system  as this could highlight the similarities or differences in the provision of SRH education across education systems. We did not collect data on participants’ level of sexual activity or contraceptive methods; this can be beneficial in future to determine the impact of public health restrictions on sexual activity and/or unintended pregnancies. While our study focused on the SRH experiences of adolescents aged 15–19 years, we acknowledge the limitation that some participants might have referenced SRH services accessed as early as age 10, and we did not consistently specify the exact age or nature of the service at the time of their experiences. Studies in the future can address the nature of SRH services accessed in different age groups. Also, we did not analyze the impact of the pandemic in relation to the socio-demographic characteristics of the participants, which could provide multi-dimensional perspectives on SRH experiences. Future research can explore using the principles of intersectionality and health equity in determining the SRH experiences of adolescents during disruptive events.In conclusion, the COVID-19 pandemic had a profound impact on adolescents' access to SRH education and services. School closures, limited store hours, and a lack of social support contributed to an overall decline in SRH well-being. Adolescents faced numerous barriers in obtaining essential SRH products and services and experienced a knowledge gap because of the pandemic. Service providers also experienced barriers in reaching adolescents for SRH education and services during the pandemic. Most participants supported the development of mobile applications as they can increase access to SRH resources. By incorporating these insights, targeted interventions can be developed to enhance SRH well-being and empower adolescents to navigate their SRH confidently during public health crises. Future research and policy efforts should focus on developing strategies that accommodate both in-person and remote services, ensuring that adolescents can access SRH services according to their needs and preferences, and fostering social support networks for adolescents in times of crisis.Additional file 1. Interview guide for adolescents.Additional file 2. Interview guide for service providers.


Cauliflower mosaic virus replication complexes are condensates that differ from those of most viruses, as they are nonmembranous assemblies that consist of RNA and protein, mainly the viral protein P6. Although these viral factories (VFs) were described half a century ago, with many observations that followed since, functional details of the condensation process and the properties and relevance of VFs have remained enigmatic. Here, we studied these issues in Arabidopsis thaliana and Nicotiana benthamiana. We observed a large dynamic mobility range of host proteins within VFs, while the viral matrix protein P6 is immobile, as it represents the central node of these condensates. We identified the stress granule (SG) nucleating factors G3BP7 and UBP1 family members as components of VFs. Similarly, as SG components localize to VFs during infection, ectopic P6 localizes to SGs and reduces their assembly after stress. Intriguingly, it appears that soluble rather than condensed P6 suppresses SG formation and mediates other essential P6 functions, suggesting that the increased condensation over the infection time-course may accompany a progressive shift in selected P6 functions. Together, this study highlights VFs as dynamic condensates and P6 as a complex modulator of SG responses.Biomolecular condensation is a multipurpose cellular process that viruses use ubiquitously during their multiplication.  Background: Regulating RNA abundance and its availability for translation is one of the main struggles between virus and host during plant virus infections. The pararetrovirus Cauliflower mosaic virus (CaMV) shields its nucleic acids, proteins, and particles from its host antiviral pathways by establishing large, amorphous condensates, termed viral factories. These condensates consist mainly of 1 multifunctional viral protein, the P6 protein. We have previously found that host RNA decapping proteins, canonically associated with phase-separated RNA granules, localize within these viral factories, and aid in the translation of viral RNA. The interplay between virus replication and RNA granule biology is conserved among eukaryotes and likely represents an ancient mechanism.Question: Our first aim was to further characterize the viral factory of CaMV and observe the behavior of host proteins within these condensates. Our second aim was to dissect the interaction of the viral P6 protein with RNA granules and RNA granule proteins, as well as its effect on translation in the context of condensation.Findings: Unlike the rigid P6 matrix, RNA granule proteins can rapidly shuffle between viral factories and the surrounding cytoplasm. Several RNA granule proteins can bind viral RNA; however, their binding capacities seem to be specific for certain viral RNA species. Stress granule proteins preferentially bind to the noncoding highly abundant 8s RNA, which may dampen stress granule responses in the plant. The P6 protein, in contrast, strongly localizes to stress granules and hinders their establishment due to its remarkable ability to enhance global translation levels. Importantly, the efficiency with which P6 exerts its functions is in part coupled to its condensation level, which likely represents a self-attenuation mechanism of CaMV during prolonged infections.Next steps: We have merely scratched the surface of the viral factory-localized host proteome and RNAome. Are there common features of the plant proteins and RNAs that accumulate within the viral factories? Elucidating which host factors are co-opted by the virus will further our understanding of virus disease in plants.The post-transcriptional fate of mRNA is controlled by an extensive network that mediates RNA processing, translation, and ultimately degradation . An intrThe most extensively studied cytoplasmic RNA granules in plants and other organisms are SGs and PBs, which are both implicated in the transient storage of nontranslating RNA . An antiArabidopsis thaliana), is the closest homolog of the mammalian SG nucleation factor TIA-1 (T-cell intracellular antigen 1)  , and wastigen 1) . A secontigen 1) , anothertigen 1) . AssuminCauliflower mosaic virus (CaMV) is a double-stranded DNA pararetrovirus that replicates in large amorphous cytoplasmic inclusion bodies referred to as viral factories (VFs). These VFs have been described as nonmembranous electron-dense protein/RNA-rich structures like RNA granules. Even though VFs are considered to be functionally associated with viral genome replication, translation and particle assembly , LSM1a (like SM-protein and VCS (varicose) localize to VFs and support viral translation . This raVFs are essential structures in CaMV infection and are largely built by P6, but the aggregation dynamics and mobility of P6 within VFs are unclear. We monitored VF size and changes in shape along the course of infection using the transgenic Arabidopsis marker lines 35S:P6-GFP and 35S:P6-mRFP. In the absence of infection, P6-GFP formed numerous foci spanning a broad size range similar to that observed previously , while PWhile having a markedly different degree of condensation under uninfected conditions, P6-GFP and P6-mRFP behaved similarly, as they abundantly accumulated in VFs during infection. To estimate the mobility of P6 in these structures, we performed fluorescence recovery after photobleaching (FRAP) analysis of P6-GFP- and P6-mRFP-tagged VFs following CaMV infection as well as P6-GFP condensates in the absence of infection . P6 was We recently showed that PB components localize to VFs during CaMV infection . Here, wWe also analyzed the previously described Arabidopsis eIF4A-GFP line, which is reported to assemble SGs in response to heat stress . We confTo evaluate this issue, we chose arsenite treatment as another commonly used stressor leading to stalled translation and SG assembly . We deteIt is interesting that CaMV induces the formation of SG-like foci in addition to VFs, despite the enhanced translation levels in infected tissues  Hoffman, which iA hallmark characteristic of canonical PBs and SGs is their sensitivity to cycloheximide CHX), a drug that inhibits translational elongation and locks ribosomes on RNA; this inhibits ribosome runoff, thereby reducing the availability of mRNA for granulation. Accordingly, we observed both the disassembly of DCP5-GFP-positive PBs and the prevention of arsenite-induced GFP-RBP47b SG formation in the presence of CHX  to C. InHX, a druThe insensitivity of SG proteins to CHX regarding their association with VFs suggests they might play a role other than the translational repression of transcripts. Furthermore, the unconventional overlap of PB and SG components points to an aberrant RNA granule character that is usually associated with the relative immobility of RNA granule proteins compared to SGs and PBs in liquid phase . HoweverTo further explore the analogy between VFs and canonical SGs, we compared their component dynamics during heat and arsenite stress. Upon heat treatment, G3BP7 still recovered quickly in both VFs and SGs, although a fraction of the protein became immobile . In contConsidering that eIF4A foci assemble only upon heat but not arsenite stress, in contrast to G3BP7 and RBP47b, and that SGs induced by heat and arsenite can differ in many aspects , we also35S8S leader RNA but not the protein-coding full viral 35S RNA or control rRNA  35S RNA.A primary function of SG and PB components is RNA regulation through RNA binding, prompting us to address if any of these components could be detected in association with viral RNA during infection. First, we infected GFP-RBP47b and free GFP control Arabidopsis plants and harvested symptomatic tissue at 21 dpi for GFP-based coprecipitation. We detected copurification of the viral protein P6 but not P4 using immunoblot analysis , and introl rRNA . To redut of 35S . However control . Next, w with 8S  to F. Im with 8S . Moreove with 8S  suggesteUBP1b and UBP1c suggesting some nonredundant functions (rbp47a ubp1b ubp1c) to address their importance for CaMV accumulation. However, these mutations had no evident effect, as even the slight reduction in CaMV accumulation initially observed in ubp1b and ubp1c was absent in the combinatorial triple mutant  lines of 5 SG marker proteins for CaMV infectivity. CaMV infection success was high in Col-0, G3BP7, and eIF4A but dropped substantially in RBP47b, UBP1b, and RBP45c . InteresAs observed in 35S RNA translation   and fuse protein .N. benthamiana  at 22 °C day temperature (20 °C night temperature) and 65% relative humidity for crossing, propagation, and transient expression assays. For infection experiments, plants were grown under short-day conditions  at 22 °C day temperature (19 °C night temperature) and 65% relative humidity. Light spectra in both conditions ranged from 400 to 720 nm. All T-DNA and marker lines used in this study were in the Arabidopsis accession Columbia (Col-0) background, which was used as a control for all experiments  and the constructs analyzed after 72 h.Clones containing l-0 cDNA  and insel-0 cDNA  and usedl-0 cDNA  and pUBNl-0 cDNA . Transgel-0 cDNA ; all linD32:FLUC  to creat18S ribosomal DNA. RT-qPCR analysis was performed with Maxima SYBR Green/Fluorescein RT-qPCR Master Mix (Thermo Fisher Scientific) using the CFX Connect Real-Time PCR detection system (Bio-Rad) with gene-specific primers  and Cabb-BJI (BJI) were vacuum-infiltrated with 1 mM sodium arsenite and incubated at room temperature (RT) for 2 h, heat shocked for 30 min at 38 °C, or left untreated as a control, followed by harvesting in liquid N and storage at −80 °C until processing. Polysome extraction and profile analysis were performed exactly as described in 2, 1% TX-100, 40 U Ribolock mL−2, and protease inhibitor cocktail, Roche) followed by clearing at 17,000 × g for 10 min at +4 °C. For FA cross-linking, the tissue was vacuum infiltrated with 1% FA in PBS, cross-linked for 10 min, quenched with 0.125 M glycine PBS, and processed as described above. The lysates were rotated with 25 µL anti-GFP magnetic agarose beads (Chromotech) for 60 min +4 °C, washed 5 times with 1 mL buffer, and eluted with Laemmli sample buffer for immunoblot analysis and TRIzol for RNA isolation. For FA cross-linked RNA, input samples and beads were incubated with protease K for 30 min at +50 °C before isolating RNA using TRIzol. Isolated RNA was treated with DNase, purified, and used for cDNA synthesis followed by RT-qPCR. For in vitro pull-down assays, we generated an RBP47b deletion mutant lacking 100 amino acids from the N-terminus that constitutes the prion domain  and incubated with total RNA extracted from CaMV infected plants diluted in IP buffer rotating for 30 min largely as in Pull-down of GFP-fusion proteins from plants was carried out by grinding 1 g of leaf tissue in 2 mL of buffer  according to the manufacturer's instructions.The FLUC-linker-RLUC and FLUC-3Xstop-RLUC were coexpressed with the indicated P6s and controls in To detect proteins, immunoblot analysis was performed essentially as described , with ang for 10 min +4 °C to obtain a supernatant with the soluble P6 and a pellet containing the condensate P6. The pellet was reconstituted in the same buffer using a volume corresponding to the original input. After adding Laemmli sample buffer, all samples were used in immunoblot analysis.For fractionation of P6 between the soluble and condensate fragments, we homogenized leaf tissue in buffer  to obtain the total sample, followed by centrifugation at 17,000 × Micrographs from leaf abaxial epidermal cells were taken under a Zeiss LSM 800 microscope. GFP and RFP signals were detected at 488 nm/490 nm to 552 nm and 561 nm/569 nm to 652 nm, respectively. Covisualization was achieved through sequential scanning mode. In FRAP analysis, imaging sensitivity was set below saturated fluorescence levels, a 3-s prebleach imaging was followed by bleaching GFP at 488 nm and RFP at 561 nm and, fluorescence recovery was recorded over a 60 s time-course by image acquisition every 1 s. Images were processed with ZEN black software (Zeiss) and ImageJ version 1.53 s. For quantification, Z-stacks were Brightness increased, and a “Gaussian Blur” filter (sigma = 1) was applied. A mask was generated through thresholding, and foci were analyzed using the “Analyze Particles” tool with the settings . Colocalization analysis for RBP47b with P6 was performed using the Plugin JACoP . The rest-test with R v4.0.2. One-way ANOVA followed by a post hoc Tukey HSD test (α = 0.05) was performed with R v4.0.2 and the R-package “agricolae” . Test statistics are shown in Boxplots/violin plots were constructed with R v4.0.2. The box represents the interquartile range (IQR), the solid lines represent the median. Whiskers extend to a maximum of 1.5× IQR beyond the box. Data were tested for normality using the Shapiro–Wilk test. Statistical comparisons of 2 groups were performed by Welch Two Sample Sequence data from this article can be found in the EMBL/GenBank data libraries under the following accession numbers: LSM1a (AT1G19120), DCP5 (AT1G26110), RBP45a (AT5G54900), RBP45b (AT1G11650), RBP45c (AT4G27000), RBP47a (AT1G49600), RBP47b (AT3G19130), RBP47c (AT1G47490), UBP1a (AT1G54080), UBP1b (AT1G17370), UBP1c (AT3G14100), G3BP7 (AT5G48650), eIF4A (AT1G54270), and eIF3g (AT3G11400).koad101_Supplementary_DataClick here for additional data file.


Ovarian follicles undergo a series of dynamic changes following the ovulatory surge of luteinizing hormone including cumulus expansion, oocyte maturation, ovulation, and luteinization. Post-transcriptional gene regulatory events are critical for mediating LH follicular responses, and among all RNA isoforms, circular RNA (circRNA) is one of the most abundant forms present in cells, yet they remain the least studied. Functionally, circRNA can act as miRNA sponges, protein sponges/decoys, and regulators of transcription and translation. In the context of ovarian follicular development, the identity and roles of circRNA are relatively unknown. In the present study, high throughput RNA sequencing of granulosa cells immediately prior to and 4-h after the LH/hCG surge identified 42,381 circRNA originating from 7712 genes. A total of 54 circRNA were identified as differentially expressed between 0-h and 4-h time points , among them 42 circRNA were upregulated and 12 circRNA were downregulated. All differentially expressed circRNA between the 0-h and 4-h groups were subjected to circinteractome analysis and identified networks of circRNA-protein and circRNA-miRNA were further subjected to “micro-RNA target filter analysis” in Ingenuity Pathway Analyses, which resulted in the identification of miRNA targeted mRNAs. A comparison of these circRNA target mRNAs with LH-induced mRNAs identified Runx2, Egfr, Areg, Sult1el, Cyp19a1, Cyp11a1, and Hsd17b1 as targets of circKif2, circVcan, circMast4, and circMIIt10. These newly identified LH/hCG-induced circRNA, their target miRNA and protein networks provide new insights into the complex interactions associated with periovulatory follicular development. Ovarian follicles undergo dramatic developmental and metabolic changes during the periovulatory period. During this time, the ovulatory surge of luteinizing hormone (LH) triggers a number of molecular and cellular events to initiate cumulus expansion, oocyte maturation, ovulation, and luteinization of the follicular wall . LH-mediCircular RNA are covalently closed RNAs and are generated by lariat-driven circularization, cis-acting elements such as Alu/Sine/Line elements, trans-acting splicing factors, and RNA binding proteins (RBP) that direct back splicing . Recent Interestingly, circRNA have been widely implicated in cell proliferation, epithelial-mesenchymal transitions, pluripotency, and early lineage differentiation . MoreoveMural granulosa cells contained 42,381 different circRNA originating from 7712 genes based on the presence of a back spliced isoform . Based oHeatmaps of the abundant (1658) circRNA and their linear isoforms were consistent across the triplicate samples at the 0-h and 4-h time points .Of the abundant (1658) circRNA evaluated at 0-h post-hCG, 791 circRNA were found at higher or in equal counts than their linear RNA isoform (fold change ≥ 1) and 867 circRNA were found to have lower counts than the linear RNA isoforms . Of the Lhcgr, Tgfbr, Map2k1, Bmpr1b, and Fgfr2 were well-established ovarian genes with a prominent role in ovarian follicular development and ovulation  were subjected to IPA analysis (using native gene names). IPA analysis at the 0 h time point identified different pathways, such as NGF, FGF, TGF-β, ERK/MAPK, JAK/STAT, PTEN, and 12 other signaling pathways A. IPA anvulation C. As cirHeatmaps of the differentially expressed circRNA were consistent across the triplicate samples between the 0-h and 4-h time points . Fifty-fhttps://circinteractome.nia.nih.gov/). Using the matching human circRNA sequence to our identified mouse circRNA sequences resulted in a network of mouse circRNA/corresponding human circRNA/miRNA and a protein network  genes, and when further curated based on at least 100 counts per million, this number of circRNA still remained at a robust level. When mural granulosa cell circRNA were compared to their corresponding linear mRNA form, over 50% were found to exhibit levels exceeding that of the linear form. One might interpret this as evidence that circRNA are critical players in post-transcriptional gene regulation. However, it is also important to recognize that circularized RNA molecules are more resistant to exonucleases, and thus their presence alone could simply be a result of stochastic RNA synthesis and degradation pathways. In addition, we determined that the LH surge elicited a rapid change (within 4-h) in the abundance of a small number of RNA transcripts, most of which increased. Subsequent bioinformatic analyses of these selected circRNA indicated that they had the ability to bind to a small number of enriched proteins and miRNA partners related to processes well known to be critical in the growth/development and maturation of the ovarian follicle ,38,39,40circRNA molecules have many modalities through which they might modify cellular function ,7,8,9,10Fmr1 (gene coding FMNRP) lead to sub-fertility in female mice [C. elegans leads to a failure to ovulate and infertility [The functional relevance of all LH-induced circRNA was determined through circinteractorme studies. This resulted in a list of RBPs and interacting miRNAs. The RBPs identified have known roles in ovarian physiology; for instance, AGO2 is essential for early embryonic development; i.e., the destruction of oocyte transcripts and the activation of zygotic transcripts . IGFBP1 ale mice . FMNRP bale mice . DGCR8 iertility . An overertility . IGF2BP1ertility . IGF2BP2ertility . In totaThe functional relevance of all miRNA interacting with LH-induced circRNA was determined by microRNA filter analysis. A network of miRNA and their target mRNA was identified. This list was compared with LH-induced genes identified from our studies and previous reports on granulosa cell differentiation, cumulus expansion, and ovulation ,23,24,25In conclusion, this work identified a small number of circRNA transcripts that were altered by the LH surge; with the linear forms of some of these genes having clear and established roles in ovarian function. Whether the biogenesis of the circRNA observed here is important for the subsequent actions by the circRNA, through a sponging mechanism or other mechanisms, or whether the biogenesis of the circRNA was merely to reduce expression of linear isoform, remains to be evaluated. The circRNA identified in this experiment provide a foundation for these future functional studies, which will ultimately address the importance of this unique form of RNA in the ovulatory process.All procedures involving animals were reviewed and approved by the Institutional Animal Care and Use Committee at the University of Kansas Medical Center and were performed in accordance with the Guiding Principles for the Care and Use of Laboratory Animals. Experiments were performed on wild-type female mice. Mice were maintained in a humidity and temperature-controlled environment with a 14-h light, 10-h dark cycle (7 am to 9 pm) with ad libitum access to food and water; 21-day-old mice were given an intraperitoneal (i.p.) injection of 5 IU of equine chorionic gonadotropin  46–48 h later. Mice were euthanized at 46-h after eCG alone (0-h), or following eCG + 4-h post-hCG administration. Ovaries and oviducts were collected by dissection distal to the uterotubal junction to ensure the complete removal of the oviduct without any disruption of the ovary.g; excess media was aspirated using a pipette and 500 µL of TRI Reagent  was applied to the cell pellet. Solubilized cell pellets were frozen at −80 °C overnight followed by the completion of the manufacturer’s RNA extraction protocol .Ovaries from three wild-type mice euthanized at 0-h and three wild-type mice euthanized at 4-h were used for the isolation of mural granulosa cells. Studies on total transcriptome changes (either upregulation or downregulation) at different time points  after hCG showed that approximately 78% of total transcriptomic changes of either upregulation or downregulation occur at 4-h . That ishttps://circexplorer2.readthedocs.io/en/latest/tutorial/pipeline (accessed on 3 October 2018)The extracted total RNA was subjected to RNA quality analysis using an Agilent 2100 Bioanalyzer  and samples with RIN values over 9 were selected for RNA sequencing analyses. RNA sequencing was performed on mGCs collected after only eCG injection (0-h) and 4-h post-hCG (n = 3 for each sample). Total stranded RNA sequencing was performed at a 100-cycle paired-end resolution. Samples were analyzed in biological triplicates. Sequencing generated between 58.3 and 78.7 million reads per sample. The read quality was assessed using FastQC software (v0.11.7)  and cleaThe resultant differentially expressed circular RNA native genes from these groups were subjected to Ingenuity Pathway Analyses to determine the functional pathways in which the native genes are involved. Differentially expressed circRNA compared with their linear mRNA form in the 0-h and 4-h groups  were subhttps://circinteractome.nia.nih.gov/ accessed on 25 May 2023) to predict the interacting proteins and miRNA. Circinteractome is a human database, so initially, we compared the circRNA sequence from our data with human circRNA via BLAST (https://blast.ncbi.nlm.nih.gov accessed on 26 May 2023). Based on the matching and the region of the matching sequence, the miRNA and proteins were identified. Out of 54 circRNA, we found 47 circRNA had matching human circRNA (All LH-induced circular RNA (circRNA 4-h vs. 0-h) were subjected to circinteractme analysis  for RNA sequencing. Regarding RNA sequencing data, edgeR software  was usedg method , establi


Respiratory infections are the major cause of death from infectious disease worldwide. Multiplexed diagnostic approaches are essential as many respiratory viruses have indistinguishable symptoms. We created self-assembled DNA nanobait that can simultaneously identify multiple short RNA targets. The nanobait approach relies on specific target selection via toehold-mediated strand displacement and rapid readout via nanopore sensing. Here we show that this platform can concurrently identify several common respiratory viruses, detecting a panel of short targets of viral nucleic acids from multiple viruses. Our nanobait can be easily reprogrammed to discriminate viral variants with single-nucleotide resolution, as we demonstrated for several key SARS-CoV-2 variants. Last, we show that the nanobait discriminates between samples extracted from oropharyngeal swabs from negative- and positive-SARS-CoV-2 patients without preamplification. Our system allows for the multiplexed identification of native RNA molecules, providing a new scalable approach for the diagnostics of multiple respiratory viruses in a single assay. Polymerase-chain-reaction-based methods used in clinic to identify respiratory viral infections cannot simultaneously detect multiple viruses or viral variants. Here nanopore nucleic acid sensing is coupled to programmable ribonuclease to simultaneously identify specific short RNA sequences of multiple viruses, without predetection steps such as sample purification or preamplification. Respiratory tract infections are the major cause of death from infectious diseases globally3. Many respiratory viruses induce comparable symptoms and cannot be clinically differentiated, making the identification of appropriate treatment challenging. It is estimated that 65% of infection-associated cases of pneumonia are potentially misdiagnosed, with 95% of these cases erroneously receiving antimicrobials4. The ongoing coronavirus disease 2019 (COVID-19) pandemic further highlights another unmet diagnostic need: the routine identification and screening of viral variants as they arise5.The diagnosis of infectious diseases plays a vital role in determining appropriate patient treatment6. Polymerase-chain-reaction-based diagnostic methods provide a sensitive approach for detecting viral nucleic acids in complex biological samples but suffer from limited multiplexing capabilities7. There is a need for robust diagnostic methods that can simultaneously detect multiple respiratory viruses and variants in a limited sample volume, which can be quickly reconfigured to detect additional variants as they arise. Newer nucleic acid detection methods, such as nanopore sensing, which can distinguish multiple nucleic acid species11 with a unique signature for each designed DNA nanostructure may be an alternative approach for multiplexed biosensing14. Various groups have shown that nanopore sensing after viral nucleic acid enrichment or amplification may be a suitable platform to challenge these diagnostics16.Currently, viral diagnostics rely on quantitative reverse transcription–polymerase chain reaction (qRT-PCR), followed by genome sequencing, to detect viral variants17. We employed programmable viral RNA cutting with ribonuclease (RNase) H to remove short RNA targets that uniquely identify the virus. The resultant RNA target is captured by the nanobait, which is immediately detected by nanopore sensing, without reverse transcription, preamplification or purification. By multiplexing several targets from the same virus in samples containing human RNA, we show that our method can increase specificity and throughput compared with existing platforms, and can pave the way for amplification-free RNA identification and diagnostics.Here, aiming to solve many of the limitations for diagnostic multiplexing, we developed an innovative method that employs a bespoke nanobait for the simultaneous identification of multiple respiratory viruses and variantsWe developed a workflow for the nanobait detection of target RNA, ranging from patient swabbing, nucleic acid extraction and programmable RNase H cutting of viral RNA Fig. . The RNA12 with a collection of short complementary oligonucleotides . The second current drop (ΔIlabel) indicated the presence of references R1 and R2 and labels a–e   , rhinovirus , influenza  and parainfluenza 1 (Supplementary Tables st) Fig. . We test22. The first site was WT SARS-CoV-2 isolated in Wuhan (B as per the PANGOLIN nomenclature)22. The alternative four targets were European strain B.1 and three variants of concern17, namely, B.1.1.7 (Alpha), B.1.351 (Beta) and B.1.617 (Delta), which were first detected in the United Kingdom, South Africa and India, respectively. As an example, we highlight the identification of the B.1.1.7 variant  or the respective spike is absent depending on which variant is present  virus and four variants . These nanobaits could identify multiple samples from pooled samples in complex backgrounds by nanopore sensing Fig. .Fig. 3NaWe pooled together five synthesized SARS-CoV-2 RNA targets to investigate the specificity and potential crosstalk between the nanobait and non-specific htRNA background. After the targets were added, all the sites were displaced and correctly identified using the nanopore measurements Fig. . A typicWe next aimed to optimize multiple parameters in designing an efficient target RNA identification system. One key parameter was the successful excision of the short RNA targets from viral RNA. We found that the location of the target RNA in the viral RNA secondary structure affected the concentration of free target RNA and consequently affected the displacement efficiency. A target in a highly complementary region would remain bound to the viral RNA after cutting and prevent detection. In contrast, the release of the target after RNase H excision increases when more unpaired bases were in the target region than within the secondary structure of the viral RNA. For future experiments, we can maximize the number of unpaired bases to increase the effective concentration of the target in solution and consequently aid detection.25). The three targets  had a decreasing percentage of unpaired bases . Subsequently, we designed oligos and employed RNase H cutting of all the three targets and quantified the displacement efficiency using nanobait with the three sites  were designed in the conserved regions of the genome that contained the highest percentage of unpaired bases  are shown in Fig. We additionally exploited a DNA flower as an alternative to monovalent streptavidin using patient samples processed with RNase H cutting, too. We observed comparable results with this DNA-based system  value of <20  can be measured by either a single nanopore running for a longer time or many nanopores in parallel. Here the detection time scales with 1/N, where N is the number of nanopores.Recent studies have developed a viral nucleic acid detection system using nanopores, which holds great promise for a rapid detection system<20 Fig. . Nanopor17. A nanobait is rapidly adaptable for new viral targets, as we demonstrated by discriminating emerging SARS-CoV-2 variants. Our study has the potential to enable amplification-free native RNA identification. The nanobait bypasses amplification sequence biases by detecting innate RNA diversity. Our results show that a nanobait can identify short and long RNAs and may find wider applications in the analysis of physiological and pathological conditions including cancer detection.Rapid programmability of diagnostic platforms is of paramount importance for detecting new viruses or their variants as they arise17. Finally, we successfully used the nanobait-based nanopore-sensing method in clinical samples and could accurately determine the presence or absence of SARS-CoV-2 in patient swabs. Nanobait circumvents the need for reverse transcription, amplification, or reaction purification, and therefore bypasses enzymatic biases and some additional steps. As the nanobait has proven to be specific and accurate for viral detection in patient samples, we think our platform can be employed for native RNA detection. The nanobait paves the way for a multiplexed amplification-free RNA detection method that is dependent only on the rapid single-molecule readout of the nanobait structure.In conclusion, we have demonstrated the simultaneous identification of nucleic acids from multiple viruses and SARS-CoV-2 RNA variants in a specific and rapid manner by combining DNA nanotechnology and nanopore sensing. We employed the easily programmable nanobait with strand displacement for discrimination between SARS-CoV-2 WT RNA from variant RNA, comprising three variants of epidemiological concern26. The samples were collected from patients with the COVID-19-like clinical picture and were tested with qRT-PCR after nucleic acid extraction. Briefly, after collection, swabs were placed into a labelled sample tube containing a lysis buffer ). The tube was gently agitated to ensure the even distribution of lysis buffer. The safety steps have been previously described and were performed in a certified CL2 laboratory26.Patient samples were collected by swabbing the back of the throat  of patients, as previously described26. The internal amplification control (MS2 (~6 × 104 PFU ml–1) per 10 ml of lysis buffer) was added in the top-up lysis buffer (25 µl per 10 ml of lysis buffer). The sample was eluted in 100 µl of nuclease-free water  and left to stand for 1 min before centrifugation for 1 min at 21,130×g  in a benchtop microfuge. The eluted samples were directly subjected to qRT-PCR. The remaining nucleic acid extracts were stored at −80 °C and further used for nanobait–nanopore sensing.The total nucleic acid was extracted using spin-column-based systems and as employed by standardized qRT-PCR testing26. Per reaction, the master mix contained 12.5 µl of 2× Luna Universal Probe One-Step reaction mix, 0.5 µl of 20 µM Wu forward primer (5′-ATGGGTTGGGATTATCCTAAATGTGA-3′), 0.5 µl of 20 µM Wu reverse primer (5′-GCAGTTGTGGCATCTCCTGATGAG-3′), 0.3 µl of 10 µM MGB Probe 3 fluorescein (5′-ATGCTTAGAATTATGGCCTCAC-3′), 0.5 µl of 10 µM of internal control forward primer for MS2 RNA, 0.5 µl of 10 µM internal control reverse primer for MS2 RNA, 0.3 µl of 10 µM internal probe (MS2 ROX), 1 µl of Luna WarmStart RT Enzyme Mix and 3.9 µl of nfH2O. Then, 20 µl of the master mix was aliquoted into each well of a 96-well plate and then combined with 5 µl of each extract. The MS2 internal extraction and amplification control that underwent the full extraction protocol was included as the negative extraction control in a minimum of two wells on the qRT-PCR plate. To determine potential contamination in the qRT-PCR process, 5 µl nfH2O was included as the qRT-PCR negative control. Then, 5 µl of spiked SARS-CoV-2 template plasmid was included in a single well as the qRT-PCR positive control. After adding 5 µl of each sample to its designated well, the plate was sealed with an optically clear plastic seal. The plate was centrifuged for 1 min at 2,000×g  at 4 °C and then inserted in the qRT-PCR machine  and the run was parametrized. Signals for fluorescein (FAM) and carboxyrhodamine (ROX) were acquired. ROX was used to detect the internal MS2 control and fluorescein was used to detect SARS-CoV-2 RNA. The assay was performed for 2 min at 25 °C, 15 min at 50 °C (for the reverse transcriptase), 2 min at 90 °C, before 45 cycles of 95 °C for 3 s followed by 60 °C for 30 s. The results were determined by the confirmation of correct positive controls (amplification of the plasmid), extraction and amplification controls of all the samples (ROX channel), no amplification in the negative controls and consistent mean values of controls. SARS-CoV-2 positivity was confirmed by amplification in the fluorescein channel with an appropriate sigmoidal curve with a CT value of ≤36. The CT values of MS2 and MGB probe 3 were maintained to track the quality and reproducibility of the assay44.SARS-CoV-2 detection was performed as previously described45. For the measurement with the absent target, the same protocol including guide oligos was used. The control measurements show no displacement, and hence, we can exclude any substantial cross-binding from guide oligos.For nanopore sensing, SARS-CoV-2 RNA controls, nucleic acid extracts (patient samples) or MS2 viral RNA were used further for detection with nanobait. First, we mixed guide oligos with the sample and heated it to 70 °C for 5 min. RNase H  was added, mixed and heated for 20 min at 37 °C to allow the enzyme to cut RNA in the DNA: RNA hybrid that effectively releases the target RNA. RNase H was thermally inactivated by incubation at 65 °C for 10 min. Guide oligos were validated to not form intramolecular structures, homo- or heterodimers using the NUPACK software21. For the DNA nanobait designs, the target sequences were selected to be in the conserved regions of a viral genome and had 40–60% GC content to form a stable 20 bp duplex. The toehold length was selected to be 6 nt long and have 40–60% GC content. We tested all the sequences for potential undesirable highly stable intramolecular interactions or homodimers using the NUPACK software (web application 2020)45. Then, we performed a cross-reactivity check between multiple sites employed in each experiment45.The length of target, toehold length and GC content were selected to ensure optimal hybridization2, pH 8.0) and heated to 90 °C for 5 min, then cooled down to 65 °C for 15 min, 45 °C for 15 min, 37 °C for 20 min, 25 °C for 20 min and finally to 4 °C for 20 min. Strand J4c was substituted by J4b to prepare 7WJb. For 7WJa, to avoid self-folding at site 43 on the nanobait, J1, J2, J3 J4a and C43 were mixed together before annealing.We designed a DNA flower as another label for SARS-CoV-2 RNA detection from the patient samples. Three DNA flowers specific for each SARS-CoV-2 target  were separately prepared. Taking 7WJc as an example, 4 μM DNA strand J1, J2, J3 and J4c (Supplementary Table 12 (some of which harboured reference structures and capture strands) and by adding partially complementary strands that were 3′-biotinylated for toehold-mediated strand displacement reaction. The linearized M13 DNA  was complemented by oligonucleotides, thereby creating a nicked double-stranded nanobait with two-terminal four deoxythymidine overhangs that prevent multimerization12. The mix contained 20 nM of linearized M13 DNA, 60 nM of oligonucleotides (three times excess to M13 DNA), 3′-biotinylated strands in the concentration of 180 nM, 10 mM MgCl2 and 1× TE . It was mixed by pipetting and spun down before heating to 70 °C for 30 s and cooled down over 45 min to ambient temperature. Excess oligonucleotides were removed using Amicon Ultra 0.5 ml centrifugal filters with 100 kDa cutoff with a washing buffer . If DNA flowers were employed as a label, the partially complementary strands that carry it were incubated in 10 mM MgCl2 for 2 h at ambient temperature, and subsequently, Amicon filtration was performed as described above. The asymmetry of the nanobait design allows for the unambiguous identification of the binding sites. The nanobait was stored until used for further experiments under 4–10 °C in 0.5 mM MgCl2, 10.0 mM Tris–HCl, pH 8.0. The nanobait design was checked by nanopore readout before each measurement.The DNA nanobait was assembled by mixing linearized single-stranded M13 DNA  with short complementary oligonucleotides2 and 100 mM NaCl. The mixture (5 μl) was incubated at room temperature (~10 min) until prepared for nanopore measurement. The difference in the target sequence composition and its physical characteristics might lead to variability in hybridization and hence the displacement efficiency of sensing sites21. We have used htRNA  as a background where indicated, to show that there are no non-specific signals induced by human native RNAs. For nanopore measurement, the sample was diluted to <0.5 nM nanobait  or 4.7 μl of RNase-H-cut patient sample was mixed with 0.3 μl of monovalent streptavidin (SAe1D3)18 (1 μM), 5 μl of LiCl (4.0 M) and 5.0 μl of LiCl (8.0 M). We have fabricated 14 ± 3 nm (mean ± standard deviation) nanopores12 using quartz glass capillaries with 0.5 mm outer diameter and 0.2 mm inner diameter (Sutter Instrument) by laser-assisted puller P-2000 (Sutter Instrument). The mix was pipetted in a nanopore polydimethylsiloxane chip, and all the measurements were performed at a constant voltage of 600 mV. Nanopore measurement details are shown in Supplementary Table The nanobait was mixed with a sample  in 10 mM MgClNanopore data analysis is explained in detail in Supplementary Section 27 was capable of the real-time analysis of nanopore data following the described procedure. Previously, we demonstrated that with around ten events, we reach 99% confidence in a positive detection of our designed DNA structures46.We verified that the convolutional neural network QuipuNet–1 in 1 mM MgCl2 and 10 μl was added to freshly cleaved mica, incubated for 1 min, rinsed with filtered Milli-Q water and then blow dried with nitrogen. Before scanning, the mica plate was affixed to the AFM sample stage using double-sided adhesive tape. Image visualization and analysis were performed using Gwyddion (version 2.60).AFM (Nanosurf Mobile S) imaging of nanobaits was performed in air in the non-contact mode. The nanobait structures were diluted to 1 ng μlt-test.For all the measurements, 99.9% confidence intervals for displacement efficiencies were calculated. Statistical significance between two sites without and with the target was tested using a two-sided Student’s Further information on research design is available in the Any methods, additional references, Nature Portfolio reporting summaries, source data, extended data, supplementary information, acknowledgements, peer review information; details of author contributions and competing interests; and statements of data and code availability are available at 10.1038/s41565-022-01287-x.Supplementary InformationSupplementary Sections 1–16, Figs. 1–19, Tables 1–30 and refs. 1–20.Reporting SummarySupplementary DataMethods compared with nanobait.


The Adenosine Deaminases Acting on RNA (ADAR) catalyze the posttranscriptional deamination of adenosine residues to inosine in double-stranded RNAs , preventing the overactivation of dsRNA sensor molecules and interferons. RNA editing is the cornerstone of innate immunity that distinguishes between self and non-self (virus), and it is essential for normal regulation of cellular homeostasis. Although much is already known about the role of ADAR proteins in RNA virus infection, the role of ADAR proteins in herpesvirus infection remains largely unexplored. In this review, we provide several lines of evidence from studies of different herpesviruses for another level of complexity in regulating the already intricate biphasic life cycle of herpesviruses. Viruses depend on the host cell for replication and constantly evolve in response to host defenses. Various host pattern recognition receptors , retinoic acid-inducible gene 1 (RIG-I)–like receptors, protein kinase R (PKR), interferon-gamma-inducible protein 16 (IFI16), etc.) recognize the so-called pathogen-associated molecular patterns (PAMPs), such as viral capsids, surface glycoproteins, and viral genomes and transcripts, and trigger signaling cascades that lead to the establishment of the replication nonpermissive state or initiate cell death . AlthMembers of the Adenosine Deaminase Acting on RNA (ADAR) protein family catalyze the conversion of adenosines to inosines (A-to-I editing), one of the most common forms of posttranscriptional RNA modification, and provide the molecular mark that distinguishes host RNA from foreign RNA . They suppress the hyperactivation of dsRNA sensors , OAS (2′-5′-oligoadenylate synthetase)/RNase L, and ZBP1 ) that could lead to autoimmunity . OnADAR proteins catalyze the C6 deamination of adenosine (A) to inosine (I) in double-stranded RNA (dsRNA), one of the most common posttranscriptional modifications of RNAs, known as A-to-I editing (reviewed in ). InosinADAR1 p150 has two Z-DNA binding domains, in contrast to p110 which has only one, and shows a broader range of targets compared to p110 . ADAR1 iIn humans, ADAR2 is highly expressed in the brain, arteries, lungs, and bladder and is responsible for site-specific editing (reviewed in ). The maThe role of ADAR proteins in viral infections has been studied for a great number of RNA and a few DNA viruses, and proviral and antiviral roles have been demonstrated. These studies are discussed in detail elsewhere , andHerpsesvirales (herpesviruses) are widespread in nature and have been discovered in a variety of animals. The order includes three distinct virus families: (a) the Orthoherpesviridae , (b) the Alloherpesviridae (the herpesviruses of fish and amphibians), and (c) the Malacoherpesviridae (the herpesviruses of mollusks). Herpesviruses are large dsDNA viruses that share several biological properties, including the establishment of latent infection as a mechanism for lifelong persistent infection and transmission [Members of the order smission ,35. The smission . Malacohsmission .The family Ortoherpesviridae encompasses viruses that infect mammals, birds and reptiles and includes nine clinically important human herpesviruses . Based on different biological properties the family is divided into three subfamilies: Alphaherpesvirinae, Betaherpesviriane, and Gammaherpesvirinae. Alphaherpesviruses have relatively broad host range, rapid productive phase, and establish latent infection in neurons, primarily in sensory ganglia. This subfamily contains viruses isolated from numerus different animals including important human pathogens herpes simplex virus 1  and HSV-2 (HHV-2) and varicella zoster virus . Betaherpesviruses have slow productive cycle and have very restricted host range. Usually, betaherpesviruses infect various cell types and tissues and establish latency in CD34+ hematopoietic progenitor cells and CD14+ monocytes or T lymphocytes. This subfamily contains numerous viruses including HCMV (HHV-5), human herpesvirus 6A and 6B (HHV-6A and HHV-6B), human herpesvirus 7 (HHV-7), murine cytomegalovirus (MCMV), rat cytomegalovirus (RCMV), etc., grouped in several genera. Gammaherpesviruses include human onocoviruses, Epstein–Barr virus , and KSHV (HHV-8). Latency is usually established in lymphoid tissues in either B or T lymphocytes. Interestingly, while alpha- and betaherpesviruses primarily enter productive phase in infected cells in culture, gammaherpesviruses predominate in latent phase .HSV-1 is an important human pathogen, a widely distributed and extensively studied virus known to cause cold sores. After productive infection in the mucosal epithelium in the oro-labial or genital area, HSV-1 reaches nearby neurons and migrates via axons to cell bodies in sensory ganglia, where it initiates latent infection. During latency, no viral progeny are produced, and only transcripts derived from the latency-associated transcript (LAT) locus are abundantly expressed . LATs arThe complexity of miRNA regulation of HSV-1 infection was recently further increased by the discovery of posttranscriptional editing of HSV-1 miRNAs in the latency phase. Zubković et al. found that miR-H2-3p, but not other miRNAs encoding HSV-1, is hyperedited in latently infected human and mouse ganglia, suggesting functional significance at this stage of infection ,54. miR-VZV is known to cause chickenpox (varicella) and shingles (zoster). VZV is a highly infectious virus transmitted by inhalation of aerosolized virions released from skin lesions. VZV initially infects mucosal cells in the upper respiratory tract, where it is recognized by dendritic cells and transported to lymph nodes, where infection spreads to T lymphocytes, which shed the virus into the skin. Latency is established in ganglia throughout the neuroaxis. The reactivated virus migrates anterograde to the dermatome where it can cause herpes zoster . Latent In contrast to HSV-1, the phenomenon of RNA editing of VZV transcripts was studied in its lytic phase. Using long-read nanopore sequencing technology of RNA extracted from lytic VZV infections, Prazsak et al. identified a number of novel VZV transcripts, including several transcripts derived from loci near the origin of replication (oriS), termed nroRNA (near-replication-origin) transcripts . One of GaHV-2 is an oncogenic virus that infects chickens causing major losses in poultry production. GaHV-2 induces aggressive T-cell lymphomas in only a few weeks leading to the death of the chicken. The genome of the virus is colinear with HSV-1, including internal repeats that contain a number of important genes , latency-associated transcript long non-coding RNA (LAT lncRNA), and three clusters of miRNAs ,59,60.Interestingly, Figueroa et al. recently identified another 5′-caped polyadenylated, alternatively spliced lncRNA, named ERL lncRNA . The traTaken together, current information on roles of ADAR in alphaherpesviruses is very limited. However, the above-mentioned studies clearly show that editing affects transcripts of these viruses, and functional studies are yet to be conducted.HCMV is a ubiquitously distributed virus that infects up to 60–100% of adults. HCMV is the leading cause of congenital disease and an important and life-threatening opportunistic pathogen in immunocompromised individuals, especially transplant recipients . HCMV esTo date, the role of ADAR in HCMV infection has been investigated in only one case, yet the study provided several important findings. Nachmani et al. found that ADAR1 p110, but not p150, is significantly upregulated in cells productively infected with HCMV in culture . InteresThis study did not address the possible editing of viral transcripts or viral miRNAs. However, in a study with a similar virus, murine CMV (MCMV), no evidence of edited MCMV miRNAs was found .EBV is a ubiquitous human virus that infects more than 90% of the adult population and causes no disease in the vast majority of healthy carriers . HoweverThe precursors of several of these miRNAs, including pri-miR-BHRF1-1, pri-miR-BART3, pri-miR-BART6, pri-miR-BART8, pri-miR-BART11, and pri-miR-BART16, have bene found posttranscriptionally edited at specific sites in cells latently infected with EBV ,72. The In addition to miRNA studies, massive RNA editing was observed in lncRNA transcripts derived from the EBV origin of replication, oriPtL and oriPtR, which have a role in promoting productive gene expression and DNA replication . These nKSHV is associated with at least three human malignancies: Kaposi’s sarcoma (KS), primary effusion lymphoma (PEL), and multicentric Castleman’s disease (MCD) . KSHV inKSHV—RNA Editing PhenomenaSequence heterogeneity and potential editing phenomena in herpesviruses were first noted for KSHV ,81,82, aKSHV—Editing-Independent Roles of ADARHerpesvirus infections, including KSHV, are known to trigger innate immune responses by activating host DNA and RNA sensors , leading to the induction of type I interferons and proinflammatory cytokines ,86. On tHerpesviruses of invertebrates are largely unknown, limiting our understanding of the diversity and evolution of nonmammalian, as well as mammalian, herpesviruses. Osterid herpesvirus-1 (OsHV-1) and Haliotid herpesvirus-1 (HaHV-1), which infect marine bivalves and abalones (sea snails), respectively, are the only two invertebrate herpesviruses isolated to date . HoweverNonetheless, using RNA analysis, Rosani et al. demonstrated the upregulation of ADAR1 homolog in the bivalve Crossostrea gigas and in the gastropod Haliotis diversicolor supertexta durign OsHV-1 or HaHV-1 infection, respectively, which correlated with the extensive hyperediting of viral and host RNAs ,90. The Over the years and through intensive studies, much has been learned about the biology and pathogenesis of herpesviruses, which has not only improved understanding but also enabled treatment of disease and improvement of health. The role of ADAR proteins in dsDNA viruses, including herpesviruses, is a rather neglected area of research. This is somewhat understandable considering that ADARs are dsRNA-binding proteins and that most studies have focused on RNA viruses. The goal of this literature review is to provide clues to another level of herpesvirus complexity that has not been adequately explored. We focus exclusively on the potential role of the ADAR proteins, although other RNA editing proteins, such as the activation-induced cytidine deaminase/apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like (AID/APOBEC) protein family also play important roles (reviewed in ).A-to-I editing (editome) in herpesvirus infections has been studied in detail in only a few cases. However, current evidence suggests that a substantial amount of editing of viral transcripts occurs during productive and latent infection in all viruses studied. While in RNA viruses editing could affect the genome, contribute to genome diversity, and impact fitness (hypermutations) and pathogenesis of the virus , sucIn herpesviruses, the functional consequences of editing are best seen in miRNAs. Editing of both KSHV and EBV miRNA precursor transcripts (pri-miRNAs) negatively affects Drosha processivity, limiting the amounts of these miRNAs and deregulating their direct targets ,72,84. MIn addition to transcripts encoding miRNAs, there are other transcripts with dsRNA structure that have been found edited in VZV, Ga- HV -2, KSHV, OsHV-1, and HaHV-1 infections, but clear evidence for a relevant biological role is much less obvious ,75,84,90Editing-(in)dependent functions of ADAR. The role of ADAR1, primarily ADAR p150, in suppressing antiviral signaling has been demonstrated for several RNA viruses  . HoweverSome open questions still need to be discussed. The studies described have provided us with only a small glimpse of the possible role of ADAR proteins and the complexity they bring to herpesvirus infection. There is still a large gap in basic knowledge about the editome in infected cells. For example, editing of many viruses, including HSV1 and HSV-2, is unknown, and the dynamics of editing during reactivation are unexplored for most viruses. Editing can affect the processing (biogenesis), stability, interactome, and function of RNAs, thereby uncovering editing will contribute to a better understanding of the basic biological properties of transcripts and their functions. The contribution of the two ADAR proteins, ADAR1 and ADAR2, to a variety of replication strategies, latency niches, and hosts remains to be better defined. In addition, how expression of the ADAR proteins is regulated during infection remains to be better understood .On the other hand, a body of evidence suggests that ADAR p150, the interferon-stimulated form of ADAR, is solely responsible for suppressing cytoplasmic sensors  and attenuating their antiviral response. However, which signaling pathways are involved ? what are the exact triggers of the signaling pathways? how these triggers link ADAR1 and dsRNA sensors? what is a role of various posttranscriptional and posttranslational modifications? and many other questions remain to be addressed.Finally, no herpesvirus gene product has yet been identified that mimics or directly affects the functions of ADAR, such as the VAI transcript of AdV , the NS1Overall, the biology of herpes viruses has been studied in great detail, but there is still much to learn, and the complexity of these viruses always offers new surprises.