Canada is one of the world’s most ethnically diverse countries, with over 7 million individuals out of a population of 38 million being born in a foreign country. Immigrant adolescents (aged 10 to 19 years) make up a substantial proportion of newcomers to Canada. Religious and cultural practices can influence adolescents’ sexual attitudes and behaviors, as well as the uptake of sexual and reproductive health (SRH) services among this population. Adolescence is a time to establish lifelong healthy behaviors. Research indicates an alarming gap in adolescents’ SRH knowledge, yet there is limited research on the SRH needs of immigrant adolescents in Canada.The purpose of this study is to actively engage with immigrant adolescents to develop, implement, and evaluate a mobile health  intervention . The interactive mobile app will aim to deliver accurate and evidence-based SRH information to adolescents.We will use community-based participatory action research to guide our study. This research project will be conducted in 4 stages based on user-centered co-design principles. In Stage 1 (Empathize), we will recruit and convene 3 adolescent advisory groups in Edmonton, Toronto, and Vancouver. Members will be engaged as coresearchers and receive training in qualitative and quantitative methodologies, sexual health, and the social determinants of health. In Stage 2 (Define and Ideate), we will explore SRH information and service needs through focus group discussions with immigrant adolescents. In Stage 3 (Prototype), we will collaborate with mobile developers to build and iteratively design the app with support from the adolescent advisory groups. Finally, in Stage 4 (Test), we will return to focus group settings to share the app prototype, gather feedback on usability, and refine and release the app.Recruitment and data collection will be completed by February 2023, and mobile app development will begin in March 2023. The mHealth app will be our core output and is expected to be released in the spring of 2024.Our study will advance the limited knowledge base on SRH and the information needs of immigrant adolescents in Canada as well as the science underpinning participatory action research methods with immigrant adolescents. This study will address gaps by exploring SRH priorities, health information needs, and innovative strategies to improve the SRH of immigrant adolescents. Engaging adolescents throughout the study will increase their involvement in SRH care decision-making, expand efficiencies in SRH care utilization, and ultimately improve adolescents' SRH outcomes. The app we develop will be transferable to all adolescent groups, is scalable in international contexts, and simultaneously leverages significant economies of scale.PRR1-10.2196/45389 Canada is one of the world’s most ethnically diverse countries, with over 7 million individuals born in a foreign country . GloballDespite the large number of immigrant adolescents in Canada, there is limited research on the SRH of this population. COVID-19 pandemic–driven changes in how services are provided, such as limiting in-person care and deferring new and walk-in patients, have created barriers for adolescents to access confidential SRH information and services. Individuals may want to avoid obtaining in-person care during this time to reduce the risk of viral transmission. Hence, it is imperative to promote and adopt innovative approaches such as digital health  to deliver culturally appropriate SRH and sexuality education to immigrant adolescents in Canada. Beyond pandemic concerns, there is evidence to suggest that mobile apps have the potential to promote adolescent health . This stThe transition from adolescence to adulthood is complex, as new responsibilities materialize and conﬁdence and independence emerge ,19. Yet,Globally, the SRH needs of adolescents have been largely unmet and pose a significant threat to the health and well-being of adolescents ,23-25. TMobile app platforms have the potential to advance SRH. Nearly 100% of youth in Canada aged 15 to 24 years use the internet daily or own a smartphone . The useIntersectionality places an explicit focus on differences among people and seeks to illuminate various interacting social factors that affect human lives, including social locations, health status, and quality of life . InterseA community-based participatory action research (CBPAR) framework will guide our study -61. CBPAFor a user-centered design, it is important that we engage the people for whom we are planning to design the product. Therefore, we will first develop and convene adolescent advisory groups (AAGs) in Edmonton, Toronto, and Vancouver. These cities have the highest percentages of immigrant populations from all geographic locations ,68. HaviTo explore the experiences, information needs, and challenges of immigrant adolescents related to SRH and how to address these challenges, we will conduct qualitative individual interviews or focus groups (FGs) following the CBPAR methodology ,60. SRH We will recruit immigrant adolescents aged 12 to 19 years who were born in a country other than Canada and who immigrated in the last 10 years or are part of a second-generation cohort. We will focus on this age group because we aim to understand the factors in early and late adolescence that predispose young people to subsequent sexual health risks. In addition, adolescents aged 12 to 19 years are generally mature enough to participate in individual interviews or FGs -72.We will use a multifaceted, community-based strategy to recruit immigrant adolescents from diverse ethnic groups. Public, community, and immigrant service agencies will support participant recruitment. In addition, social marketing campaigns  and snowball sampling will be used to recruit participants. Peer RAs and graduate RAs hired as part of this project will also assist with recruitment. We will also include non-English speaking participants. Our peer RAs are bilingual, and we will hire multilingual graduate RAs for this project who speak Canada’s most common languages other than English to provide safe interpretation services for participants who do not speak English fluently.We will conduct between 20 and 25 individual interviews or 3 to 4 FGs in each city. The participants for FGs will be divided into 2 groups (12 to 15 years and 16 to 19 years) to ensure that our data also capture the SRH needs of younger teens. The individual interviews or FGs will occur in person or over Zoom. The final sample size will be based on data saturation . IndividIn accordance with CBPAR, we will use the DEPICT  model for participatory analysis  74]. Da. Da74]. Informed by the Stage 2 findings and with integrated, continuous input from the AAGs, we will develop a prototype of a mobile app to house SRH content. The content of the app will stem from two primary sources: (1) evidence-based resources such as the Society of Obstetricians and Gynecologists of Canada, the Public Health Agency of Canada, and the World Health Organization; and (2) themes and experiences generated from individual interviews and FGs in Stage 2. The app will include age-specific information separately so that adolescents of different age groups can easily navigate the information related to them. In addition, information related to accessing SRH services will also be provided to connect adolescents to SRH services in their community.The AAGs and peer RAs will work with the app developer to iteratively revise and build the app to address questions of clarity, potential sources of bias or marginalizing design elements, ease of use, relevance, and other factors determined by the AAGs. The app will use an internal analytics system to run analysis on user demographics, feedback, and content views to gather information on how the app is being used. The app content will be approved by team members who have expertise in SRH to ensure evidence accuracy and appropriate evidence interpretation. We will initially develop the interventions in English only to consolidate the findings and best practice functionality. Once the app has demonstrated usability and effectiveness in English, we plan to apply for new funding to translate the app content into languages used by immigrant communities.At the end of the project, we will run several FGs to evaluate the usability, acceptability, and effectiveness of the app; seek further input for refining it; evaluate the effectiveness of the project; and seek direction for future research. For usability testing of the app, we will conduct (1) 6 FGs with 5 to 10 immigrant adolescent participants (n=30-60) in Edmonton, Toronto, and Vancouver (2 FGs in each city) and (2) 3 FGs with 5 to 8 service providers, policy makers, and members of the communities’ informal support channels (n=15-20).At the start of each FG, participants will be asked to download the app and use it on their own. Then, the researcher will begin the FG. Semistructured FGs will focus on questions about the app informed by a systematic search of over 180 usability evaluations : particiThis study will develop a holistic, prescriptive model that can be used to ensure the sustainability and scalability of mHealth apps in the public health care sector in Canada and other countries. Our long-term community partners in all 3 cities will support the sustainability of the app. These organizations will host the app, as they need an evidence-based tool to help immigrant adolescents increase their awareness and uptake of SRH services to improve SRH outcomes.This study received ethics approval from the University of Alberta Ethics Board (Pro0013664).Recruitment and data collection will be completed by February 2023, and we will begin co-designing the mobile app in March 2023. We expect to share a prototype of the app, gather feedback on its usability, and then refine and release the app in the spring of 2024. We will engage immigrant adolescents and service providers as partners in the development of the mobile app to ensure that immigrant adolescents are aware of this tool and will use the app, helping contribute to positive SRH outcomes. This study will also evaluate the usability of the mobile app. In the future, we will conduct pragmatic trials to evaluate the effectiveness of the app in improving the SRH outcomes of adolescents.This study aims to develop a mobile SRH app designed by and targeted to immigrant adolescents in Canada. Our exciting and novel adolescent-engaged methods and mobile app have the potential to rapidly address the unmet SRH needs of immigrant adolescents and to improve the SRH of immigrant adolescents across Canada. This app is important because it allows immigrant adolescents to independently access resources related to SRH and mitigates the impact of familial bias on teens’ ability to seek out such information. Further, young people are very comfortable utilizing mobile apps. This engaging modality will make it easier for immigrant adolescents to learn about safe SRH practices and may support them in having the knowledge and motivation to access services. The expected outputs of this study include:We will be able to identify the SRH needs of an underresearched population  in Canada.Voices of the full spectrum of gender diversity (including transgender and gender nonconforming adolescents), cultural knowledge, and agency will inform the mobile app.An innovative, adolescent-centered knowledge translation tool  will be developed to help improve the SRH of immigrant adolescents in Canada.The potential impact of this mobile app development includes increasing SRH knowledge and awareness, access to SRH services, and the use of contraception while decreasing unintended pregnancies and the need for abortion among adolescents. Our overall program of research is to build upon the active engagement of immigrant youth to develop, evaluate, and implement innovative and evidence-based digital knowledge translation tools and to build the resilience that immigrant youth need to successfully integrate into Canadian society. This research epitomizes creativity and innovation by merging the arts, sciences, and the engagement of youth in research.Targeting adolescents and using engaging modalities has a strong potential to effectively increase their involvement in SRH care decision-making, expand efficiencies in SRH care utilization, and ultimately improve adolescents’ SRH outcomes. We will disseminate the mobile app, along with other findings of our study, via community symposiums; public training sessions in Edmonton, Toronto, and Vancouver; and a website hosted by the University of Alberta Faculty of Nursing. Our findings will also be presented at academic conferences and published in open-access, peer-reviewed journals, advancing the body of knowledge on immigrant adolescents’ SRH needs.


Polycystic ovary syndrome (PCOS) is a complex, multifactor disorder in women of reproductive age worldwide. Although RNA editing may contribute to a variety of diseases, its role in PCOS remains unclear.A discovery RNA-Seq dataset was obtained from the NCBI Gene Expression Omnibus database of granulosa cells from women with PCOS and women without PCOS (controls). A validation RNA-Seq dataset downloaded from the European Nucleotide Archive Databank was used to validate differential editing. Transcriptome-wide investigation was conducted to analyze adenosine-to-inosine (A-to-I) RNA editing in PCOS and control samples.NUP43), Retinoblastoma Binding Protein 4 (RBBP4), and leckstrin homology-like domain family A member 1 (PHLDA) showing the most significant three 3′-untranslated region (3′UTR) editing. Furthermore, we identified 20 DRE sites that demonstrated a significant correlation between editing levels and gene expression levels. Notably, MIR193b-365a Host Gene (MIR193BHG) and Hook Microtubule Tethering Protein 3 (HOOK3) exhibited significant differential expression between PCOS and controls. Functional enrichment analysis showed that these 259 differentially edited genes were mainly related to apoptosis and necroptosis pathways. RNA binding protein (RBP) analysis revealed that RNA Binding Motif Protein 45 (RBM45) was predicted as the most frequent RBP binding with RNA editing sites. Additionally, we observed a correlation between editing levels of differential editing sites and the expression level of the RNA editing enzyme Adenosine Deaminase RNA Specific B1 (ADARB1). Moreover, the existence of 55 common differentially edited genes and nine differential editing sites were confirmed in the validation dataset.A total of 17,395 high-confidence A-to-I RNA editing sites were identified in 3,644 genes in all GC samples. As for differential RNA editing, there were 545 differential RNA editing (DRE) sites in 259 genes with Nucleoporin 43 (Our current study highlighted the potential role of RNA editing in the pathophysiology of PCOS as an epigenetic process. These findings could provide valuable insights into the development of more targeted and effective treatment options for PCOS. RNA editing is an epigenetic alteration of the RNA nucleotide sequence with nucleotide insertions, deletions, or substitutions . In mammPolycystic ovary syndrome (PCOS) is a complex multigenic disorder characterized by excessive androgen levels and ovarian dysfunction . It is tTo identify A-to-I RNA editing associated with PCOS, we performed a transcriptome-wide analysis of RNA-Seq data from ovarian GCs and validated the findings using a cross-cohort approach. Our findings revealed dramatic A-to-I RNA editing alterations in PCOS compared to controls and underlined their substantial role in the epigenetic regulation of PCOS.http://www.ncbi.nlm.nih.gov/geo) database was used for RNA editing event discovery and consisted of GCs from adult women, including five with PCOS and six controls without PCOS undergoing in vitro fertilization treatments (IVF) (https://www.ebi.ac.uk/), which contained GCs from four PCOS patients and four controls undergoing IVF or intracytoplasmic sperm injection  . In addiAfter the raw sequencing data were retrieved, FASTQC was first used to analyze the raw data for quality control. Adaptor and low-quality sequences were removed using FASTP Version 0.23.4 . RNA STAVariant calling was then conducted to identify candidates for RNA editing events. Single-nucleotide variation (SNV) was called from the BAM files by using VarScan (Version 2.4.3) as described in our previous study . The varTo quantify the gene expression levels, FeatureCounts Version 2.0.1 was used to calculate the pseudo-counts of gene expression from the BAM files, and EdgeR (Version 3.7) was then used to calculate the value of transcripts read per thousand bases per million mappings (TPM) for each gene , 24.To evaluate how RNA editing could contribute to the difference between PCOS and controls, the principal component analysis (PCA) of A-to-I RNA editing events was performed using the function prcomp of R (Version 3.6.3) and visualized using the ggplot2 (Version 2.2.1) package.p < 0.05 were considered significant and visualized using the online tools provided at the website (http://www.bioinformatics.com.cn/).To understand the possible functional relevance of RNA editing, gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways of edited genes were analyzed using Enrichr . Items whttp://rna.tbi.univie.ac.at/cgi-bin/RNAWebSuite/RNAfold.cgi) was used to predict secondary structures of single-stranded RNA sequences surrounding RNA editing sites (http://rbpmap.technion.ac.il) was used to predict the binding of RNA binding protein (RBP) to differential RNA editing sites  and p-value.To identify differential RNA editing, the intergroup RNA editing levels were compared using the generalized linear model (GLM), and the likelihood ratio test (LRT) was used. The Our analysis identified 17,395 high-confidence A-to-I RNA editing events in 3,644 genes in GCs , Retinoblastoma Binding Protein 4 (RBBP4), and leckstrin homology-like domain family A member 1 (PHLDA), including NUP43:chr6:149725375, RBBP4:chr1:32680141, and PHLDA1:chr12:76027474 (NR5A2), CUGBP Elav-Like Family Member 1 (CELF), and Filamin B, (FLNB), including NR5A2:chr1:200074554, CELF1:chr11:47481265, and FLNB:chr3:58166381 (TUBA1B), and MX Dynamin Like GTPase 1 (MX1), including TUBA1B:chr12:49128795 and MX1:chr21:41452734 . Among these sites, eight showed a positive correlation, while the remaining 12 exhibited a negative correlation. Noteworthy, the top three editing sites with positive correlations were found in Hook Microtubule Tethering Protein 3 (HOOK3), Rhomboid Domain Containing 2 (RHBDD2), and Transmembrane Protein 16 (TMEM165), including HOOK3:chr8:43021004 (r = 0.79), RHBDD2:chr7:75883398 (r = 0.8), and TMEM165:chr4:55412273 (r = 0.84) , and MIR193BHG, including, FLNB:chr3:58166381 (r = −0.89), FAM126B:chr2:200977846 (r = −0.84), and MIR193BHG:chr16:14330019 (r = −0.78) showed the top three significant negative correlations with gene expression  including the minimum free energy (MFE), the free energy of the thermodynamic ensemble (FE), the frequency of the MFE structure in the ensemble, and ensemble diversity is presented in Furthermore, we examined the expression levels of genes containing the identified editing sites and observed significantly lower expression levels of ADAR) and Adenosine Deaminase RNA Specific B1 (ADARB1). We observed a significant decrease in ADARB1 expression in PCOS compared to controls (ADARB1 expression and PCOS-associated RNA editing and identified six ADARB1-related sites in Scavenger Receptor Class B Member 2 (SCARB2), Charged Multivesicular Body Protein 3 (CHMP3), Ubiquitin Specific Peptidase 22 (USP22), FAM126B, Solute Carrier Family 47 Member 1 (SLC47A1), and Coiled-Coil Domain Containing 69 (CCDC69), including SCARB2:chr4:76159678, CHMP3:chr2:86504167, USP22:chr17:21000918, FAM126B:chr2:200978748, FAM126B:chr2:200978747, SLC47A1:chr17:19578434, and CCDC69:chr5:151181355, all with significantly downregulated RNA editing correlated to the decreased ADARB1 expression , Thioredoxin Domain Containing 15 (TXNDC15), RNA Polymerase I Subunit E (POLR1E), DEAD-Box Helicase 19A (DDX19A), and FKBP Prolyl Isomerase 11 (FKBP11) showed consistent changes between the two datasets, namely SCARB1:chr12:124783357, TXNDC15:chr5:134900418, POLR1E:chr9:37503395, DDX19A:chr16:70372975, AC013394.1:chr15:92889425, and FKBP11:chr12:48923107  from an independent study . By usinUnderstanding the molecular mechanism of PCOS could help develop new stratiges for diagnosis and treatment of PCOS , 30. SevTUBA1B:chr12:49128795 and MX1:chr21:41452734, while the role of the two in PCOS is unclear. However, TUBA1B, a critical gene in postmenopausal osteoporosis, has also been linked to BMI in children’s muscles and the overall survival of colorectal cancer patients  could promote GC apoptosis through the AKT-FOXO3 pathway . ApoptosADARB1 expression in PCOS correlated with seven downregulated DRE sites. In ovarian cancer, the downregulation of ADARB1 has been reported to have a potential role in the development of ovarian cancer (CCDC69 has been associated with immune infiltration and serves as a prognostic marker in breast and colon cancers (SLC47A1 are associated with type 2 diabetes (FAM126B, represented by FAM126B:chr2:200978748 and FAM126B:chr2:200978747, might potentially contribute to PCOS. Taken together, such findings indicated a substantial role of ADARB1 and RNA editing mediated by it in the pathophysiological process of PCOS.Our findings also reveal decreased n cancer . These e cancers , 60. Poldiabetes , 62. AddRBPs play a crucial role in regulating post-transcriptional processes, including RNA splicing, decay, and editing , 64. ImpIn conclusion, our study conducted a transcriptome-wide analysis of PCOS-associated A-to-I RNA editing sites and provided new insight into understanding the role of RNA editing in the pathogenesis of PCOS.The original contributions presented in the study are included in the article/FSK and ZJL contributed equally to this article. FSK contributed to drafting the manuscript. YPM, ZJL and JHW revised and improved the writing of the manuscript. YZ performed the data analysis. YHL, RFJ, BLZ, PWH and CYR contributed to the revision of the manuscript. JHC and YPM designed the study and revised the manuscript. All authors contributed to the article and approved the submitted version.


We develop candidate HIV-1 vaccines, of which two components, ChAdOx1.tHIVconsv1 (C1) and ChAdOx1.HIVconsv62 (C62), are delivered by the simian adenovirus-derived vaccine vector ChAdOx1. Aberrant adenovirus RNA splicing involving transgene(s) coding for the SARS-CoV-2 spike was suggested as an aetiology of rare adverse events temporarily associated with the initial deployment of adenovirus-vectored vaccines during the COVID-19 pandemic. Here, to eliminate this theoretically plausible splicing phenomenon from the list of possible pathomechanisms for our HIV-1 vaccine candidates, we directly sequenced mRNAs in C1- and C62-infected nonpermissive MRC-5 and A549 and permissive HEK293 human cell lines. Our two main observations in nonpermissive human cells, which are most similar to those which become infected after the intramuscular administration of vaccines into human volunteers, were that (i) the dominant adenovirus vector-derived mRNAs were the expected transcripts coding for the HIVconsvX immunogens and (ii) atypical splicing events within the synthetic open reading frame of the two transgenes are rare. We conclude that inadvertent RNA splicing is not a safety concern for the two tested candidate HIV-1 vaccines. The overall safety profile of licensed vaccines is typically thoroughly established, and their risk–benefit ratio is well in favor of the latter. As of January 2023, the administration of over 13.2 billion of vaccine doses against COVID-19 inevitably associated vaccines with undesirable rare adverse events (AEs) ,5. The bAdenoviruses are genetically stable viruses with a linear double-stranded DNA genome, which have been explored as vaccine vectors for several decades ,9. ChAdORNA splicing was first discovered during studies of human adenoviruses, which are known for their complex splicing patterns of transcribed virus-derived RNAs ,29,30,31We are developing a candidate vaccine with the aim to induce protective killer T cells against HIV-1 ,41. ThroThe ChAdOx1 vector is derived from simian adenovirus 23 (Y25) and has deletions of the E1 and E3 early regions. In addition, E4 region open reading frames (ORFs) 4, 6 and 6/7 were replaced by the corresponding regions of HAdV-5 to improve virus yield. The vaccine inserts were designed as follows. First, curated HIV-1 proteomes of full-size proteins within group M (major) of global isolates present in the Los Alamos National Laboratory HIV Sequence Database in September 2013 were aligned and computed into two mutually complementing amino acid sequences using the Mosaic Algorithm . Then, tThe rescue and preparation of the adenovirus stocks of vaccines ChAdOx1.tHIVconsv1 (C1) and ChAdOx1.HIVconsv62 (C62) were described before ,53. BrieThe adenovirus transcriptome was analyzed in human MRC-5 cells , which iTM reagent  at 1 mL per 107 cells, washed twice with 70% ice-cold ethanol and stored at –80 °C under 70% ethanol until use. On the day of sequencing, RNA was resuspended in sterile water, enriched for polyadenylated RNA and sequenced as described previously using the SQK-RNA002 kit and MINI06D R9 version of the flow cells  according to the vendors’ protocols [Total cellular RNA was extracted from harvested infected cells using the TRIzolrotocols .https://doi.org/10.5281/zenodo.36102480).Open-reading-frame-centric data analysis of the transcriptome was described before . BrieflyHuman A549 and MRC-5 cell lines are nonpermissive to the E1-deficient ChAdOx1 vector replication and were used to interrogate the vaccine-derived transcriptome. Cells were infected for 48 h and their total polyA RNA was enriched and sequenced using the nanopore technology. The overall sequence reads data are summarized in In the HEK293 cells supplying the HAdV-5 E1 function in trans to the ChAdOx1 E1-deficient vector, a full range of adenovirus transcripts were detected . Our ORFThere is a wide range of minor transcripts with unusual promoters, splicing pat-terns and/or polyadenylation sites derived from both C1 and C62 vaccines grown in HEK293 cells . These aIn the present work, we examined RNA transcriptomes of two candidate HIV-1 vaccines, ChAdOx1.tHIVconsv1 (C1) and ChAdOx1.HIVconsv62 (C62), using direct RNA sequencing to preempt any possible safety concerns about inadvertent splicing involving the engineered synthetic transgene ORFs, which could lead to the expression of unusual protein products and cause AEs ,38,59,60The presence of these very low-level transcripts should be seen in the context of the recent advances in RNA sequencing technologies. The short-read deep sequencing of fragmented RNA (RNAseq) has generated an accurate high-resolution image of the adenovirus transcriptome ,31; howeFurther reassurance of the safety of the C1 and C62 vaccines comes from the fact that the transcriptomes of these vaccines were very similar to ChAdOx1 nCOV-19 . During Direct sequencing of viral mRNA is above and beyond any previous or current regulatory licensing requirements for recombinant adenovirus-vectored vaccines. We believe that this approach to studying transcription from viral vaccines is a useful additional tool to increase our understanding of the transcriptomic repertoire of experimental engineered viral vectors and provide additional assurance regarding their safety profiles.The ChAdOx1-vectored C1 and C62 vaccine components are administered with a heterologous MVA HIVconsvX boost for the induction of protective T cells . This HI


Millions of adenosines are deaminated throughout the transcriptome by ADAR1 and/or ADAR2 at varying levels, raising the question of what are the determinants guiding substrate specificity and how these differ between the two enzymes. We monitor how secondary structure modulates ADAR2 vs ADAR1 substrate selectivity, on the basis of systematic probing of thousands of synthetic sequences transfected into cell lines expressing exclusively ADAR1 or ADAR2. Both enzymes induce symmetric, strand-specific editing, yet with distinct offsets with respect to structural disruptions: −26 nt for ADAR2 and −35 nt for ADAR1. We unravel the basis for these differences in offsets through mutants, domain-swaps, and ADAR homologs, and find it to be encoded by the differential RNA binding domain (RBD) architecture. Finally, we demonstrate that this offset-enhanced editing can allow an improved design of ADAR2-recruiting therapeutics, with proof-of-concept experiments demonstrating increased on-target and potentially decreased off-target editing. Human ADAR1 and ADAR2 edit millions of adenosines transcriptome-wide, altering RNA structure. Here the authors show that variations in RNA binding domains influence site-specific editing, enhancing ADAR2-targeted therapeutics. Inosine is perceived as guanosine by the internal cellular machinery, and hence editing can result in protein recoding7, alternative splicing10, and alterations in targeting and maturation of microRNA12. In parallel, editing can also alter the RNA secondary structure, and in doing so modulate the immunogenicity of self and viral RNAs within cells17. In accordance with the wide distribution of edited sites, abnormal dysregulation of A-to-I deamination has been associated with a broad spectrum of human diseases18, and targeting of ADAR enzymes is an emerging therapeutic strategy in cancer19.Millions of adenosines are deaminated into inosines transcriptome-wide20 in comparison to CRISPR-mediated DNA editing. Diverse approaches have been implemented in recent years to recruit ADAR enzymes towards specific substrates25. Although successful, these attempts often resulted only in partial efficiencies and in some cases also with considerable off-target effects26. Improving our understanding of the rules guiding inosine formation and of the factors determining enzyme specificity will pave the path toward the development of both more optimal editors and improved guides.Different adenosines throughout the transcriptome are edited at dramatically different efficiencies , begging the question of what governs enzymatic selectivity towards specific targets. Understanding the rules guiding these two enzymes to their diverse targets is of intense interest not only from biological and pathological perspectives, but also from therapeutic ones. In recent years, unraveling the rules dictating deamination via the ADAR enzymes has accrued substantial interest in the context of ongoing efforts to achieve targeted mRNA editing. Targeted editing is emerging as a therapeutic modality that may potentially offer a safer alternative to correct single-nucleotide mutations28, suggesting differences in the selectivity of these two enzymes. Second, RNA secondary structure plays a critical role. ADAR1 targets are nearly inevitably within long double-stranded RNAs29, and hence highly enriched in repetitive elements such as Alu and long interspersed elements32. ADAR2 targets tend to be in duplex regions interrupted by mismatches or loops36, and in-vitro work has shown that distal bulges can, at times, impact editing efficiency34. Yet, the structural rules governing editing—which would be of critical importance for predictive models—are not understood. In addition, for both ADAR1 and ADAR2, A-C mismatch harboring targets are particularly prone to undergo editing37. Finally, the sequence also plays a role in target selectivity. In-vitro editing assays with artificial RNA duplexes revealed that ADAR1 and ADAR2 prefer to edit adenosines depleted of G’s at the position preceding the target and show some bias for a G downstream of the target edited site39.Studies exploring the targeting efficiencies of ADAR1 and ADAR2 have revealed several general principles. First, the specificity of these two enzymes is only partially overlapping40, differ in their protein domain architecture. The catalytic domain, present on both ADARs, was shown to play a role in the definition of selectivity41. In addition to the catalytic domain, human ADAR1 contains either one or two Zα domains (dependent on the isoform) and three RNA binding domains (RBDs) whereas human ADAR2 contains two RBDs but no Zα domains. The RBDs participate in dsRNA substrate recognition and RNA binding42, and were suggested to partially mediate ADAR selectivity via both sequence-specific and non-specific mechanisms43. The Z domains, binding left-handed nucleic acids, have been implicated in allowing co-transcriptional binding of ADAR1 to nascent RNAs45 and more recently in preventing Z-RNA dependent activation of pathogenic interferon by Z-DNA binding protein 1 (ZBP1)48. Whether they have a role in defining substrate specificity is unclear.The factors underlying the differences in specificity between ADAR1 and ADAR2 are understood only to a limited extent. These two paralog proteins, which likely evolved via a gene duplication event roughly 700 million years ago49. Whether structural disruptions of ADAR2 targets lead to editing at a fixed offset, and what the mechanistic basis for this offset is, remains unknown.To systematically dissect how substrate selectivity by ADAR1 is governed by secondary structure, we previously screened ADAR1-mediated editing across thousands of sequence variants, which had been designed to systematically perturb the secondary structure along two highly double-stranded backbones. We discovered that the introduction of structural disruptions within an otherwise perfect double-stranded RNA structure gives rise to robust and predictable ADAR1-mediated editing at a fixed offset of 35 base pairs (bp) upstream from the disruptionHere, we systematically monitor how secondary structure modulates ADAR2 substrate selectivity, on the basis of systematic probing of thousands of synthetic sequences transfected into ADAR1-deleted cell lines exogenously expressing ADAR2. We find that similarly to ADAR1, structural disruptions give rise to symmetric, strand-specific induced editing at a fixed offset. However, in contrast to ADAR1 acting at a −35 bp offset, in the case of ADAR2, structural disruptions give rise to induced editing at an offset of −26 bp. We dissect the basis for the differences in offset between ADAR1 and ADAR2 via diverse mutants, domain-swaps and ADAR evolutionary homologs. We uncover that the difference in the offset is encoded by the differential RNA binding domain architecture of the two ADARs, yet that it is not determined by the number of RBDs. We demonstrate that this understanding of ADAR2 specificity can allow an improved design of ADAR2-recruiting therapeutics, yielding increased on-target editing, with some evidence also for reduced off-target editing. Our findings provide comprehensive insight into the features determining ADAR2 substrate selectivity and into the roles of the RNA binding domains of ADAR1 and ADAR2 in mediating differential targeting, and should facilitate the design of improved ADAR2-recruiting therapeutics.49. In brief, these sequence variants are based on two distinct backbones folding into a perfect hairpin structure: the endogenous mouse B2 element, serving as a more natural editing target, and a sequence complementary to the 3’ UTR of the fluorescent reporter mNeonGreen (mNG) transcript, serving as a completely synthetic target. In both cases, this hairpin consists of a 146-nt long stem and a 46-nt long loop , as a negative control. Subsequently, RNA was extracted, and the constant upper arm of each construct was reverse transcribed, PCR-amplified, and sequenced  . No editing was observed in ADAR1 KO cells transfected with the No-ADAR vector, corroborating that all deamination activity is triggered by the two exogenously overexpressed ADAR enzymes Fig. . In ADARts) Fig. . In addits) Fig. .28  arm, we next amplified and sequenced the ‘lower’ variable arm of each B2 construct from ADAR1-KO HEK293T cells overexpressing ADAR2  An ‘ADAR2-RBDs_ADAR1-deaminase’ variant, harboring the catalytic domain of ADAR1 fused to the two RBDs originating from ADAR2, and (2) An ‘ADAR1-RBDs_ADAR2-deaminase’ variant, harboring the catalytic domain of ADAR2 fused to the three RBDs originating from ADAR1. We next used the above-described human ADAR1-depleted system, into which we transfected B2 and mNG oligo libraries along with plasmids overexpressing these two ADAR variants. The two hybrids gave rise to deamination activity on both B2 and mNG positive control constructs Figure , B, albe54. Nonetheless, in both cases, the size of the offset remained fixed at roughly −26, similar to WT ADAR2 . On the basis of this collection, we next annotated each of the 40 nt flanking each editing site as being either closed (if it resided in a stem) or open (if it resided in a loop or in a bulge). Finally, we generated an editing metaplot, wherein we calculated the extent to which structuredness within a window of 80-nt centered around an editing site impacted editing levels. Remarkably, this analysis revealed two regions in which an open structure led to increased editing: A major window 35 nt downstream of the edited site and a more minor window 30 nt upstream of the edited site Figure . This an28. Among those sequences prediction of secondary structure could be established with high confidence for 7 sequences. An examination of ADAR2-mediated editing sites within these targets revealed that in 5 of the 7 most structures , the highly edited target harbored a mismatch 26 nt away  an arRNA as in (1) but harboring 3-bp mismatch 26 or 27 nt upstream from the target adenosine, and (3) an empty vector serving as a negative control  to elicit editing on five distinct targets harboring distinct consensus motifs located on four different endogenous transcripts: PPIB-ORF:UAG, GAPDH-UTR:UAG, SMAD4-UTR:CAG, PPIB-UTR:UAG and STAT1-ORF:UAU via recruitment of exogenously expressed ADAR2. For each of these targets, we designed three arRNA constructs: (1) a 151-nt long arRNA containing a C opposite to the target A located between two 75-nt stretches which are perfectly complementary to the endogenous transcript. Such constructs were used inrol Fig. . Consistrol Fig. . The relrol Fig.  and may In some clinical contexts, it could potentially be beneficial to induce editing only in cells expressing one of the two ADAR enzymes, for instance in order to achieve selectivity in brain tissues in which ADAR2 is expressed. Given the different offsets at which ADAR1 and ADAR2 induce editing, we sought to assess whether this could be leveraged to achieve such selective editing. Indeed, we found that an arRNA with a structural disruption at an offset of 35 nt selectively induced editing by ADAR1, and not by ADAR2, in comparison to a positive control lacking a structural disruption Fig. . ConversFinally, we sought to assess whether the introduction of structural disruptions at a 26 bp offset would not only increase on-target editing levels but also decrease off-target levels. To assess this, we amplicon-sequenced the GAPDH amplicon following targeted editing via either the ‘positive control’ or the ‘Mismatch 27’ arRNA. In this analysis, we only identified a single adenosine that was edited at levels exceeding 2% across either of these two samples. Remarkably, this position was edited at levels of 6.13% in the positive control sample, which decreased to 1.05% in the ‘Mismatch 27’ samples Fig. . This ofOverall, these findings lend support to the observations that structural disruptions lead to increased ADAR2-mediated editing at a fixed offset and provide a proof of principle that this rule can allow improved recruitment of ADAR2 towards target adenosines in therapeutic settings.The experimental design of the oligo-array libraries employed in this study had been primarily geared towards interrogating the impact of RNA secondary structure on editing. Nonetheless, the availability of measurements of editing levels across distinct sites and in varying sequence contexts allowed investigating the impact of sequence on editing, and the extent to which this varied across the eight ADAR variants interrogated here.56. The position immediately downstream displayed less of a bias, consistent with57.  tends to be substantially higher when the nucleotide opposite of the G at position −1 is a guanosine, and even more so an adenosine  will give rise to induced ADAR2-mediated editing at a fixed offset of 26 bp upstream of the disruption, contrasting with ADAR1 which induces editing at a 35 bp offset. Second, we uncover that these distinct offsets by the two ADARs are encoded via the distinct RBD domains of the two enzymes. Importantly, our analyses of GTEX-based data confirm the relevance of offset-mediated editing to endogenous sites within human tissues.Despite widespread interest in unraveling the determinants guiding the selectivity of ADAR1 and ADAR2, these have remained poorly understood and to a considerable extent unpredictable. It has been previously suggested that the basis for selectivity resides within mismatchesOur work uncovers interesting commonalities and differences between the two ADAR enzymes. Activity by both enzymes is induced at a fixed offset from structural disruptions. In both cases, there is substantial evidence for symmetricity, as is evident from comparing the upper and lower strand editing levels. Moreover, in both cases, the induction of editing is orientation-specific, with editing being induced on both strands upstream of the structural disruption. However, the size of the offset is different (−35 vs −26 nt). In addition, the magnitude of induction is also different, with more dramatic effects being typically observed for ADAR1 than for ADAR2. Finally, for ADAR1 in addition to the major peak at −35, we had also observed a more minor peak in editing activity 30 bp downstream of the edited site. We do not observe such a downstream peak for ADAR2. This may either reflect a difference in the mechanism driving induced editing, or the lower dynamic ranges which may limit us from clearly observing such a secondary, more minor peak for ADAR2.43, it is possible that the basis for the difference in selectivity between the two enzymes lies within such individual changes. Dissecting this systematically via genetic approaches is rendered challenging, given that mutations within RBDs oftentimes also abolish editing. Indeed, six additional RBD-disrupting ADAR mutants that we generated over the course of this study (data not shown) failed to show any substantial editing activity, consistent also with previous observations64. Our inability to obtain active mutants harboring only the catalytic domains and none of the dsRBDs (data not shown) also prevents us from formally ruling out the possibility that part of the offset might be directly mediated by the dsRBD itself. We anticipate that the structural dissection of these two enzymes bound to RNA targets will contribute towards answering this question.A major question left open by our study is the basis for the different offsets of ADAR1 and ADAR2. While based on the RBD swapping experiments it is clearly encoded by the RBD architecture, we rule out that this is a function of the number of RBDs, as offsets of 26 and 35 nt are achieved by variants and mutants with a distinct number of RBDs. Another possibility is that the difference in offset is not due to the difference in domains, but to the difference in the size of the linker between the RBD and the deaminase domain. However, we can largely rule out this possibility as well, because in our RBD swapping experiments between ADAR1 and ADAR2 we had maintained the original linkers, and the offset sizes segregated with the RBDs. Given that single amino acids in the RBD were shown to be important in RNA recognition and binding43. While our findings suggest that a 26 nt offset can also be maintained via ADAR mutants and variants harboring a single RBD, they do leave open the possibility that an offset of 26 nt could be the combined outcome of the RBDs of two ADAR enzymes acting as a dimer, given that both ADAR1 and ADAR2 act as homodimers68. Second, our studies resonate to some extent with findings that ADAR substrates are distributed periodically at ~50 bp intervals from each other69. Given that we find editing induced 26 nt upstream of structural disruptions on the top strand, but also at 26 nt upstream of the disruption on the lower strand  in a ‘GAG’ context, whereby it is known  that an upstream G plays an inhibitory role on editing. Finally, given that editing is a product of both sequence and structural preferences, it will be important to simultaneously perturb both, in order to understand to what extent these two dimensions act in an additive or synergistic manner. This dimension could not be profiled using our current design.Our study also suffers from several limitations. First, it was primarily geared to dissect nce Fig. , the seqnce Fig. , in contCollectively, our findings shed light on the mechanisms underlying the only partially overlapping target spectrum of ADAR1 and ADAR2, while advancing our technical toolkit to target these two enzymes towards clinically relevant targets.Full-length human ADAR2 (UniProt: P78563-2), ADAR2 RBD1 Deaminase, and ADAR2 RBD2 Deaminase coding sequences were amplified from the AAVS1-hADAR2, pYES-DEST52-hADAR2-dRBM1-Deaminase domain, and pYES-DEST52-hADAR2-dRBM2-Deaminase domain plasmids, respectively, using primers that included XbaI and EcoRI sites. Full-length human ADAR1 (UniProt: P55265-5) was amplified from the AAVS1-hADAR1 plasmid by primers containing XbaI and HindIII sites. All of those PCR products  and ADAR2-pcDNA3.1(-) plasmids were used as templates for PCR reactions (primers in Supplementary Data Octopus vulgaris (UniProt: A0A6P7SCW6_OCTVU), and Suricata suricatta (UniProt: A0A673T544_SURSU) were ordered from Twist Bioscience. Bacteria from glycerol stocks were inoculated and grown in ampicillin-supplemented LB liquid media, and plasmid DNA was extracted as previously mentioned.The pTwist CMV vectors containing the human-codon optimized sequences of ADAR from 70, which were obtained as gift from lab of Prof. Dan Stetson, were grown  in Gibco Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum, 1% Penicillin and Streptomycin, and 4ug/ml Puromycin. 5 × 105 cells were plated on a 6-well plate so that cells reached 70–90% confluency at the time of the second transfection. 24 and 48 hours after cell seeding, 1.6 μg of ADAR-expressing pcDNA3.1(-) plasmid and 4 μg B2 or mNG library DNA were transfected respectively according to Lipofectamine® 2000 DNA Transfection Reagent Protocol (Thermo Fisher Scientific). 24 hours later, cell harvesting was performed.ADAR1-knockout HEK293T cellsTotal RNA was extracted using Nucleozol (Macherey-Nagel), poly-A selected using oligo dT-beads (Dynabeads mRNA DIRECT Kit life tech), and DNase treated (Thermo Fisher Scientific). The upper constant arms or lower variable arms including 8 nucleotide barcodes of constructs were reverse transcribed, PCR amplified  supplemented with complete protease inhibitor cocktail (Roche). Lysates were incubated for 10 min on ice and subsequently centrifuged for 10 min at 13.000 g and 4 °C. 5x SDS-PAGE protein loading buffer was added to the supernatant. The samples were then incubated at 95 °C for 5 minutes. Tris-Glycine SDS-PAGE and wet immunoblotting were performed for subsequent analysis. Antibodies used were anti-mouse FLAG , anti-goat Actin HRP  and anti-mouse HRP .71 track of annotation downloaded from the UCSC genome browser72. We then filtered for pairs of Alu elements that were (1) located in UTRs, (2) oppositely oriented, (3) within 2000 nt from each other, and (4) lacked any other Alu elements in the window. The dsRNA structures of the Alu pairs were predicted using the FOLD73 program from the RNAStructure package73 with default parameters. The bpRNA tool was used to assign structural elements such as loop, bulge and stems to each nucleotide in the structure74. The editing level for each adenosine in those structures was calculated using pooled reads from the GTEX database75 . Only adenosines exhibiting editing levels above 3% were considered for further analysis. We then considered a window of 80 nt centered around each editing site, and annotated each position within this window—for each editing site—as being either closed (stem structure) or open (loop or bulge). For each position within this window, we then divided all editing sites into ones that harbor an open structure at that position, versus a closed one, and calculated the difference in mean editing levels. Statistical significance was determined using a t-test.To explore the effect of mismatches in specific locations within the dsRNA structure on ADAR1-mediated editing levels, we retrieved a list of Alu elements from the human genome using the Repeatmasker28. We were able to identify to map and retrieve editing levels on the basis of4 for 10 of the 17 targets. Genomic sequences of different lengths  from both sides of the editing sites were extracted, and the dsRNA structures were predicted using the FOLD program from the RNAStructure package73 with default parameters. Structures that included the highest number of additional editing sites previously described in these genomic locations4 with the lowest free energy were chosen for further analysis. As a minimal support for the relevance of the structure, we required that at least two editing sites be found across the predicted structure, retaining 8 of the 10 targets. In our analyses, we considered only the most highly edited site in each structure, and filtered out one case where this site was at the end of a stem/loop structure (and where the +26 position was a position predicted to base pair with a position upstream of the site). The bpRNA tool74 was used to assign structural elements such as loop, bulge and stems to each nucleotide in the structure.For ADAR2 analysis on the basis of the GTEX dataset, we began with 17 editing sites identified as ADAR2 targets in mice49.Gene fragments containing arRNAs and KpnI sites were ordered from Twist Bioscience. All sequences were KpnI-digested and cloned into the digested EPB104 backbone (Addgene plasmid # 68369) with transcription of arRNA driven by a U6 promoter. The list of TWIST gene fragments is described in Supplementary Data 5 cells were plated on a 6-well plate so that cells reached 70–90% confluency at the time of transfection. 24 hours after cell seeding, 1ug of ADAR1- or ADAR2-expressing pcDNA3.1(-) plasmid, 0.1 ug of pEGFP-N1 plasmid (for assessment of transfection efficiency), and 3ug of the corresponding arRNA-expressing plasmid were transfected according to Lipofectamine® 2000 DNA Transfection Reagent Protocol. 24 hours later, the medium was changed, and 12 hours later, cells were harvested.5 × 1076 and MultiEditR77.RNA isolation, DNase digestion, and reverse transcription were performed using NucleoZOL (Macherey-Nagel), Amplification Grade DNase I (Thermo Fisher Scientific), and MultiScribe Reverse Transcriptase cDNA synthesis kit (Thermo Fisher Scientific), respectively. The subsequent PCR with KAPA HiFi HotStart ReadyMix (Roche) was performed using transcript-specific primers , and DNase-treated (Thermo Fisher Scientific). The target UTR editing region was reverse transcribed, PCR amplified (primers in Supplementary Data Further information on research design is available in the Supplementary InformationPeer Review FileSupplementary Data 1Supplementary Data 2Description of Additional Supplementary FilesReporting Summary


Trichinella spiralis as a model to study miRNA secretion. T. spiralis muscle-stage larvae (MSL) secrete abundant miRNAs which are largely extravesicular. Here, we investigated how T. spiralis miRNAs might remain stable outside of cells. Using proteomics, we identified two RNA binding proteins secreted by T. spiralis larvae and characterised their RNA binding properties. One, a homologue of the known RNA binding protein KSRP, binds miRNA in a selective and sequence-specific fashion. Another protein, which is likely a novel RNA binding protein, binds to miRNA without exhibiting sequence specificity. Our results suggest a possible mechanism for miRNA secretion by T. spiralis and may have relevance for understanding the biology of extracellular miRNA more widely.Small non-coding RNAs such as microRNAs (miRNAs) are conserved across eukaryotes and play key roles in regulating gene expression. In many organisms, miRNAs are also secreted from cells, often encased within vesicles such as exosomes, and sometimes extravesicular. The mechanisms of miRNA secretion, how they are stabilised outside of cells and their functional importance are poorly understood. Recently, we characterised the parasitic nematode  Summary: The parasitic nematode Trichinella spiralis secretes microRNAs that are not contained within extracellular vesicles. Here we discover and characterize two proteins that may be involved in stabilising these microRNAs. Small (16-36-nt) non-coding RNAs (sRNAs) are key regulators of gene expression conserved across eukaryotes. Different pathways generate functionally distinct classes of sRNAs  but in gIntracellular functions and mechanisms of miRNAs have been extensively characterised across animal species . HoweverLack of understanding about the functions of extracellular miRNAs is accompanied by considerable uncertainty over the mechanism whereby intracellular miRNAs are targeted for secretion and stabilised when in the extracellular environment . The domHeligmosomoides polygyrus , the L1 stage. MSL remain in this state until the animal is predated on, whereby they are released in the digestive tract and develop into adults to complete the life cycle  were amongst those with high consistency between replicates  predicted to bind miRNAs   . The sizMethods) . The proin vitro. We found a significant depletion of C nucleotides in miRNAs enriched for TsKSRP binding but no enrichment for G content  TsPUF from secreted material and extracted bound RNAs, comparing to IP with naïve mouse serum as a control (P=3.4×10−7). The overall profile of non-miRNA reads was largely similar to those bound in vitro, with a slight enrichment of ribosomal RNAs  . Howeverlisation , and we In contrast, TsPUF contained the PAN domain, often found in extracellular proteins, and had a signal peptide, thus is likely to be secreted via the ER. Identification of this protein as an RNA binding protein was due to a small region that showed weak similarity to a PUF repeat; however, we showed that this is unlikely to reflect homology to the Pumilio family and may be either convergent evolution or a coincidence. Although a deletion encompassing the PUF repeat failed to bind RNA we cannot exclude that this interfered with the overall structure. Interestingly, it showed a very different profile of RNA binding to either TsKSRP or TsAGO, suggesting an entirely different mechanism of RNA binding and stabilisation. Structural characterisation of TsPUF and KSRP would be of great interest in resolving these different binding modes, particularly combined with biophysical measurements of RNA affinity and evaluation of the extent to which it can protect RNA from nuclease activity. Overall, TsPUF is a novel RNA binding protein but the exact mechanism responsible for RNA binding still awaits characterisation. We note that extracellular miRNAs are very common across organisms but there is so far limited evidence for canonical RNA binding proteins involved in their stabilisation ; indeed T. spiralis MSL. KSRP in humans is able to bind to a variety of RNA targets . All figures and analysis of the proteomic datasets were performed in RStudio. Protein abundance was defined as the mean normalised intensity value from mass spectrometry. To identify potential RNA-binding proteins in the MSL secretome, all secreted proteins were searched for RNA-binding domains. First, a literature search was performed to create a list of 59 canonical and non-canonical RNA-binding domains (Matrix assisted laser desorption ionisation time of flight (MALDI-TOF) mass spectrometry was performed on total worm extracts and secreted material from  domains   using Superscript IV reverse transcriptase (ThermoFisher Scientific) following the manufacturer's protocol. A Q5 high fidelity PCR reaction (New England Biolabs) was performed on the cDNA to amplify TsKSRP and TsAGO genes using primers encoding a c-myc tag at the 3′ end. The genes were ligated with a pET21a(+) vector, encoding a his-tag, via compatible restriction digestion sites. Plasmids were replicated and isolated from Escherichia coli DH5α for transformation into the expression host E. coli BL21. TsKSRP and TsAGO were batch produced via large scale culture of transformed BL21 and induction of protein expression using 1 mM IPTG at 18°C overnight. Cells were lysed with 2.5 mg/ml lysozyme via three freeze-thaw cycles, incubation on ice for 2 h and sonication. Ni-NTA affinity chromatography was performed to purify his-tagged TsAGO and TsKSRP from lysates, following the standard QIAexpressionist protocol.Total RNA extracted from E. coli DH5α for transformation into the expression host Pichia pastoris. TsPUF and TsPUF[-puf] were batch produced via large scale culture of transformed P. pastoris and induction of protein expression using methanol. Secreted recombinant proteins were collected from the supernatant of the yeast culture. Ni-NTA affinity chromatography was performed to purify his-tagged TsPUF and TsPUF[-puf] from the supernatant, following the standard QIAexpressionist protocol.A TsPUF gene, with sequences encoding a his- and c-myc tag at the N-terminus, was synthesised by GeneArt (ThermoFisher Scientific). The TsPUF mutant without the PUF domain (TsPUF[-puf]) was created by digesting the TsPUF gene with restriction enzymes that cut in positions either side of the sequence encoding the PUF domain and ligating it back together. Both TsPUF[-puf] and the wildtype TsPUF were ligated with a pPICZα vector via compatible restriction digestion sites. Plasmids were replicated and isolated from T. spiralis MSL for 30 min in PBS with 0.05% Tween 20 (PBST). The protein:RNA mixture was then incubated with anti- c-myc agarose beads from a Pierce c-Myc Tag IP/Co-IP kit (ThermoFisher Scientific) for 2.5 h. The protein:RNA:anti-c-myc mixture was washed through Pierce spin columns with PBST to remove anything unbound to the anti- c-myc agarose beads. TRIZOL was then used to elute the protein:RNA from the beads. Protein:RNA in TRIZOL was disrupted by repeated freeze-thawing in liquid nitrogen and vigorous vortexing. Chloroform was used to allow phase separation and subsequent precipitation of RNA in the aqueous phase with glycogen and isopropanol. The RNA pellet was washed with 75% EtOH and resuspended in ultrapure water. No-protein-control (NPC) reactions were performed in the exact same way, except with no recombinant protein incubated with the RNA.Recombinant protein (347 nM) was incubated with total RNA (156 nM) extracted from T. spiralis genome. Three sequences were selected in order to have a range of sequence lengths from 19-23 nucleotides long . Oligos were synthesised by Merck.RNA was used to prepare cDNA libraries with a Truseq sRNA library prep kit (Illumina), following the manufacturer's protocol. Single end sequencing of libraries was kindly performed by the MRC LMS Genomics Laboratory on a NextSeq2000 machine. Oligos were spiked in at the library preparation stage at a constant concentration (0.2% of the input RNA) to allow normalisation of reads from different libraries. To identify appropriate oligos, all human miRNA sequences were downloaded from miRbase  and thenT. spiralis miRNA, bowtie was used to find the position in the T. spiralis genome where the reads align. Bedtools  as an adjuvant. The mouse was then boosted with more protein/adjuvant mix 4 weeks later, and twice more 2 weeks apart before bleeding the mouse 1 week after the final boost. The supernatant was then collected from the blood and this was used as the TsPUF antiserum.A section of thigh muscle from an infected mouse was fixed in 10% neutral buffered formalin, embedded in paraffin and sectioned using standard techniques. The sections were deparaffinized and rehydrated via a series of washes with xylene and decreasing concentrations of ethanol. The sections were blocked with 1% bovine serum albumin (BSA) in PBS with 0.1% Tween 20 (PBST) and 10% naïve goat serum. A second block was performed (for mouse-on-mouse staining) using goat F(ab) anti-mouse IgG (Abcam) at 1:100 in PBST. Sections were then incubated with TsPUF antiserum (or naïve mouse serum) at 1:200 in PBST with 1% BSA. Goat anti-mouse alexa fluor 488 (Abcam) at 1:500 in PBST with 1% BSA was then used to stain the sections. The sections were then counterstained using mounting medium containing DAPI (Abcam). Immunofluorescence staining was visualised using a Leica SP8 - STELLARIS 5 Inverted Light Sheet Confocal Microscope. Localisation was determined by reference to the structure of infective larvae .T. spiralis MSL secreted material in PBS with 0.05% Tween 20 (PBST) for 1 h. The antiserum:secreted material mixture was then incubated with 50 ul Dynabeads Protein G magnetic beads (ThermoFisher Scientific) for 30 min. A magnetic plate was used for washing with PBST. TRIZOL was then used to elute the protein:RNA from the beads. RNA was then isolated following the same method used in the recombinant protein RNA immunoprecipitations.Immunoprecipitations were performed with 10 ul TsPUF antiserum (or 10 ul naïve mouse serum), incubated with 100 ug (protein concentration) 10.1242/biolopen.060096_sup1Supplementary informationClick here for additional data file.Trichinella spiralis muscle-stage larvae (MSL) and adult secreted material and total worm extracts.Table S1. Raw and processed data for the intensity level of all proteins identified by mass spectrometry in Click here for additional data file.Table S2. List of RNA-binding domains (RBDs) used in this study. List created by performing a literature search for canonical/non-canonical RBDs.Click here for additional data file.Trichinella spiralis muscle-stage larvae (MSL) than by the secreted material of adults. Abundance of all proteins is also shown .Table S3. Candidate RNA-binding domain-containing proteins which were more enriched in the secreted material of Click here for additional data file.Trichinella spiralis proteins.Table S4. Nematodes used in this study to analyse the conservation of Click here for additional data file.Trichinella spiralis miRNA read counts, normalised to total oligo spike counts, from small RNA sequencing of RNA immunoprecipitated using recombinant TsKSRP, TsAGO, TsPUF and TsPUF[-puf]. Data for no protein controls (NPC) are also shown. Two biological replicates performed.Table S5. Click here for additional data file.Trichinella spiralis miRNAs enriched by immunoprecipitation of recombinant TsPUF, relative to a no protein control (NPC). Only those miRNAs where log2(TsPUF/NPC) > 0 in two biological replicates were defined as enriched.Table S6. Homologues of Click here for additional data file.Trichinella spiralis miRNAs enriched by immunoprecipitation of recombinant TsKSRP, relative to a no protein control (NPC). Only those miRNAs where log2(TsKSRP/NPC) > 1 in two biological replicates were defined as enriched.Table S7. Homologues of Click here for additional data file.Trichinella spiralis miRNAs enriched by immunoprecipitation of recombinant TsAGO, relative to a no protein control (NPC). Only those miRNAs where log2(TsAGO/NPC) > 1 in two biological replicates were defined as enriched.Table S8. Homologues of Click here for additional data file.Trichinella spiralis miRNA reads, normalised to total oligo spike counts, from small RNA sequencing of RNA immunoprecipitated from T. spiralis secreted material in a native TsPUF pull-down. Data for a negative control (Neg) is also shown.Table S9. Click here for additional data file.Trichinella spiralis miRNAs enriched by immunoprecipitation of native TsPUF, relative to a negative control. Only those miRNAs where log2(TsPUF/Neg) > 0 were defined as enriched.Table S10. Homologues of Click here for additional data file.


The RNA oligomer contains a 2′–5′ phosphodiester bond, which typically forms during prebiotically plausible RNA synthesis. This non-canonical linkage helps prevent the formation of inactive complexes between self-complementary oligomers while decreasing the ligation efficiency. The system appears to possess an autocatalytic property consistent with exponential self-reproduction despite the limitation of forming a ternary complex of the template and two substrates, similar to the behavior of a much larger ligase ribozyme. Such a minimal, ribozyme-independent RNA self-reproduction may represent the first step in the emergence of an RNA-based genetic system from primordial components. Simultaneously, our examination of random RNA pools highlights the likelihood that complex species interactions were necessary to initiate RNA reproduction.The emergence of RNA self-reproduction from prebiotic components would have been crucial in developing a genetic system during the origins of life. However, all known self-reproducing RNA molecules are complex ribozymes, and how they could have arisen from abiotic materials remains unclear. Therefore, it has been proposed that the first self-reproducing RNA may have been short oligomers that assemble their components as templates. Here, we sought such minimal RNA self-reproduction in prebiotically accessible short random RNA pools that undergo spontaneous ligation and recombination. By examining enriched RNA families with common motifs, we identified a 20-nucleotide (nt) RNA variant that self-reproduces  via prebiotically plausible chemistry was identified by examining short random RNA pools. The oligomer exhibits a potential for exponential self-reproduction, similar to the behavior of much larger ribozymes.An RNA oligomer that self-reproduces  However, despite significant progress in improving non-enzymatic or ribozyme-catalyzed RNA polymerization,7–10 the self-replication of these systems remains challenging. Previous studies have therefore explored alternative, simpler mechanisms for RNA self-reproduction through the assembly of oligonucleotides.6,11,12 In view of recent clarification, we use the term “reproduction” to denote RNA copying in general by distinguishing canonical “replication” that follows template-directed polymerization chemistry.6,12The first genetic system before the emergence of life may have been based on RNA, because RNA can simultaneously carry genetic information and catalyze chemical reactions.13–16 For example, a ligase ribozyme derived from the R3C ligase ribozyme17 catalyzes the joining of two RNA substrates as a template to form a sequence identical to itself.13 This ribozyme is the simplest self-reproducing RNA known to date in terms of its length (61 nt) and the number of components (two fragments: 13 and 48 nt). However, the ribozyme is still relatively large and was rationally designed, and it remains unclear how such a ribozyme and its components could have been prevalent in prebiotically accessible RNA mixtures, which were likely dominated by shorter (up to ∼20 nt) and random oligonucleotides.18,19 The ligase ribozyme also requires 5′-triphosphate activation, which necessitates an additional set of complex reactions.20 Consequently, it has been proposed that the template-based self-reproduction of short RNA molecules independent of complex ribozymes may have emerged first in the RNA World.21,22RNA reproduction has been demonstrated for ligase and recombinase ribozymes.23–25 A recent study employed temperature cycling to overcome such template inhibition in the reproduction of chemically activated DNA.26 Despite these efforts with DNA, the self-reproduction of short RNA oligomers is currently missing. Moreover, temperature cycling would be incompatible with RNA because, unlike DNA, RNA easily degrades at high temperature, which is accelerated by divalent metal ions27,28 that commonly enhance ribozyme catalysis28,29 as well as template-directed RNA synthesis.30,31Self-reproduction of short nucleic acids has been studied mainly using DNA. Previous studies demonstrated the autocatalytic reproduction of chemically modified DNA oligonucleotides through template-directed ligation, although the reproduction was severely hindered by two tightly bound templates .30 which readily forms in prebiotically plausible environments,32,33 while recombination occurs directly—or in combination with spontaneous >p formation through hydrolysis of RNA, termed α/α′ mechanisms.31,34 Notable are recent studies that demonstrated that pools of short random RNA can undergo diverse intermolecular ligation and recombination, presumably in a templated manner.31,35 In these populations, RNA products that form efficiently or that self-amplify are expected to be enriched. Thus, a close examination of the enriched products may lead to the discovery of efficient RNA reproduction via template-directed ligation or recombination. The identification of such reproducing RNA would also provide insights into the likelihood of the emergence of self-reproduction out of random chemistry.Template-directed ligation of short RNA is achieved in the laboratory using terminal activation such as with 2′,3′-cyclic phosphate (>p),via template-directed ligation of two 10 nt substrates. The RNA contains a 2′–5′ phosphodiester bond, a linkage usually generated during non-enzymatic RNA synthesis.36–38 Partly due to the non-canonical linkage, the RNA circumvented its dimerization and displayed a potential for exponential reproduction in an isothermal environment, although the restricted formation of an active complex with the substrates limited the amplification. Its autocatalytic properties and structures are somewhat similar to those of the previously developed self-reproducing ligase ribozyme.13 These results demonstrate the first example of minimal RNA self-reproduction independent of a ribozyme and also help understand the dynamics of primordial, random RNA pools.In this study, we first examined spontaneous ligation and recombination reactions in pools of short random RNAs and found that they can be detected more quickly than previously demonstrated. We observed the enrichment of RNA families with common motifs in multiple RNA pools. Subsequent analyses of the most enriched products and their variants led us to find a short (20 nt) RNA oligomer that can self-reproduce 20), which was previously shown to undergo both ligation and recombination if pre-activated with >p.35 We prepared N20 and N20>p pools containing ∼3 × 1014 molecules to cover all possible ∼1012 sequences of 20 nt with redundancy (∼300 copies). Previous studies detected ligation and recombination in 16–20 nt random RNA pools (5–100 μM) only after incubation for months or longer times in ice.31,35 However, we found that, in the presence of high concentration (100 mM) of MgCl2, which promotes >p-mediated template-directed ligation and recombination,30,31 both N20 and N20>p pools (50 μM) generated detectable >20 nt products after just a 2 day incubation, as visualized by denaturing polyacrylamide gel electrophoresis (PAGE) (We investigated reactions in fully random 20 nt RNA (Ns (PAGE) . Note thca. 21–45 nt) in both N20 and N20>p pools from a denaturing polyacrylamide gel and subjected them to RT-PCR and high-throughput sequencing (HTS). The RT-PCR was performed using the SMARTer technology, via poly-A tailing and following template switching during reverse transcription. We detected PCR products for both N20 and N20>p pools only if they were pre-incubated for two days, confirming recombination and ligation during the incubation  with a sharp drop-off above 39 nt (20>p pool were predominantly 24–40 nt (98%) with a sharp peak at 40 nt (via ligation on >p of a hydrolyzed RNA.31 The nucleotide compositions in the <40 nt products of the random RNA pools displayed a slight enrichment in G at the both sides of a putative ligation junction between a cleaved RNA>p (<20 nt) and a 20-mer  and N20>p-f1 (43%). The enrichment of specific families was less clear when grouped by the 5′ end  can form through this mechanism by splitting f1-1 into the first 9 nt attached with >p  and the remaining 20 nt    so they 2% yield . It is iA (A11N). Incubation of A11N with B did generate a distinguishable product whose length is similar to—but slightly longer than—f1-1   for ligation with B. The fragment AA exhibited improved ligation but less efficiently so than AG, whereas AU and AC did not show enhanced ligation   signific (AG)  and the 3′ region  for 2 days in the absence or presence of 20 μM T or TG. Whereas T improved ligation between A and BS only slightly (∼1.4 fold)   instead of AG and TG  , demonstG Fig. S8. AlthougAG and BS could generate two possible phosphodiester bonds, either 3′–5′ or 2′–5′ linkages  incubation  and temperatures around 22 °C  or AG (FAM-AG) for 6 h (AG contained a monophosphate (-p) instead of >p at the 3′ end to preclude ligation to BS  forming a TG′·TG′ dimer  of AG was bound to BS, and thus most of the substrates were not freely available, which could explain the low percentage of the TG′·AG·BS complex formation and the limited self-reproduction of TG  with the self-reproduction equation by assuming p = 1, corresponding to exponential growth. This result indicates the potential of TG′ to undergo exponential self-reproduction. We estimated ka and kb as 0.0011 ± 0.000069 h−1 and kb = (0.0045 ± 0.00070) × 10−6 M h−1. The autocatalytic efficiency (ka/kb)42 of TG′ (2.4 × 105) is comparable to or lower than a much larger recombination or ligase ribozyme,13,14 while higher than DNA-based self-reproduction systems23–25 with the caveat that they have smaller reaction orders (p = ∼0.5).The high availability of G and BS . A commoion rate  and 5C. tes42 cf., as in aTG′ shares many similarities with the previously engineered 61 nt self-reproducing ligase ribozyme,13 although they catalyzed different ligation chemistries .45–47 Alternatively, as demonstrated for a self-reproducing ligase ribozyme,48 directed evolution with TG′ as the parent RNA may also identify highly efficient reproduction of oligonucleotides in a constant environment. It was shown that only a slight difference, including two critical mutations, was sufficient to convert the original ligase ribozyme13 , f1-1G was undetected even at a low frequency. On the other hand, biochemical analyses revealed the superiority of f1-1G to f1-1 for its formation through simple ligation of two substrate fragments (50,51 A previous study also reported the inefficient synthesis of some products isolated from random RNA pools.35 Altogether, our results highlight the difficulty of inferring dominant reactions in random RNA mixtures from the analyses of isolated sequences. Nevertheless, the information obtained from examining the random RNA products was valuable in the discovery of the minimal self-reproducing RNA, which exhibited its highest activity in the original environment where the random RNA pools were exposed (Fig. S12G, or TG′ in combination with random RNA mixtures would give more insights into the likelihood of the emergence of self-reproduction in a primordial RNA soup.Our results also give insights into the dynamics of short random RNA mixtures. From completely random pool of 20-mers, we identified a discrete class of related, enriched sequences of which f1-1 appeared to be a canonical representative. The fragment ragments . This di Fig. S12. Future The data supporting the findings of this study are available from the corresponding author upon reasonable request.R. M. and N. I. designed the project. R. M. performed experiments, analyzed data, and wrote the paper with comments from N. I.There are no conflicts to declare.SC-014-D3SC01940C-s001


We repeatedly sampled 54 colonies harboring Cladocopium C15 dinoflagellates, across three environmentally distinct reef zones  around the island of Moorea, French Polynesia over a 3-year period and spanning a reef-wide thermal stress event. By the end of the sampling period, 28% (5/18) of corals in the fringing reef experienced partial mortality versus 78% (14/18) of corals in the forereef. Over 90% (50/54) of colonies had detectable dinoRNAV infections. Reef zone influenced the composition and richness of viral mcp amino acid types (‘aminotypes’), with the fringing reef containing the highest aminotype richness. The reef-wide thermal stress event significantly increased aminotype dispersion, and this pattern was strongest in the colonies that experienced partial mortality. These findings demonstrate that dinoRNAV infections respond to environmental fluctuations experienced in situ on reefs. Further, viral productivity will likely increase as ocean temperatures continue to rise, potentially impacting the foundational symbiosis underpinning coral reef ecosystems.Viruses can affect coral health by infecting their symbiotic dinoflagellate partners (Symbiodiniaceae). Yet, viral dynamics in coral colonies exposed to environmental stress have not been studied at the reef scale, particularly within individual viral lineages. We sequenced the viral major capsid protein ( Viruses in natural environments can employ infection strategies along a continuum from lytic to persistent ; althougCoral reefs, climate-threatened generators of marine ecosystem services, are teeming with diverse microbial consortia, including viruses e.g., , 11). Vi. Vi11]).mcp, [The inability to track individual reef-associated viral lineages has constrained progress in characterizing viral prevalence and dynamics in coral holobionts. For coral-associated bacteria and Symbiodiniaceae , microbial dysbiosis has frequently been associated with increases or decreases in richness and increased dispersion –28 usingmcp, ) and tramcp diversity in the Symbiodiniaceae assemblages of 54 individual colonies of the thermally resistant coral holobiont, Porites lobata, from three reef zones  in Moorea, French Polynesia over a > 3-year period that spanned an extreme thermal stress event in March of 2019. Coral reef zones in Moorea are characterized by distinct environmental conditions [P. lobata typically associates with Cladocopium C15 Symbiodiniaceae [mcp amino acid diversity into the context of colony- and reef-scale parameters, we (1) quantify colony-level infection prevalence and persistence over time, (2) identify putative triggers of viral productivity and dysbiosis based on differences in abiotic factors , and (3) integrate colony health and viral infection data to generate hypotheses regarding the impacts of viral infection on coral holobionts.We tracked dinoRNAV nditions . For exanditions  and nutrnditions  than theiniaceae , 35, makiniaceae . By intePorites lobata were tagged on the north shore of Moorea, French Polynesia, spanning nine sites that encompassed three reef zones  of skeleton/tissue were sampled across the colony surface using bone cutters, placed in a sterile Whirl-Pak® (Nasco), and then preserved in DNA/RNA shield . Samples in DNA/RNA shield were kept on ice until returning to shore, at which point they were vortexed for 25 min at full speed with 5 ceramic beads and 1.35 g garnet matrix  and then frozen at −40 °C. An additional sample (~3 cm2 fragment) was collected for symbiont density measurements from a subset of these same colonies to compare densities over time and between reef zones. These samples were wrapped in aluminum foil and stored at −40 °C until further processing  gene was amplified from cDNA (generated from RNA), using a nested PCR protocol with primers from [DNA and RNA were extracted from the coral samples, which included mucus, tissue, and skeleton, using enzyme digestions and a ZymoBIOMICs DNA/RNA Kit . Dominaers from . Sequencers from  using thers from ). Brieflers from , 43. Parers from –46, ASVsers from  using Viers from ) and CD-ers from ).mcp analyses were conducted on samples that produced sequencing libraries containing at least 1,000 reads in R (version 4.1.0). Aminotype richness was calculated with phyloseq  using rarefied data. Linear models were used to assess the influence of reef type and time on aminotype richness using the equation richness~ reef type * time with stats . The influence of Symbiodiniaceae density on richness was also assessed, using the equation richness~ density. Normality of residuals was assessed visually with quantile-quantile plots and Shapiro-Wilk tests. Beta diversity analyses were conducted on rlog transformed count data [phyloseq; within-group distances (or dispersion) were assessed with permutation tests for homogeneity in multivariate dispersion (PERMDISP) using betadisper in vegan . Betweethods in ). PERMANthods in . Beta diPocillopora spp. harboring two Cladocopium symbionts in French Polynesia [ggtree [A phylogenetic tree was generated to depict relationships between abundant aminotypes (those with at least 10% relative abundance in at least one sample) and related sequences. Sequences included those associated with six coral species with diverse symbionts from the Great Barrier Reef , sequencolynesia , and sevolynesia , 52–54. olynesia  was usedolynesia  was usedolynesia . The tre [ggtree . BrancheR2 = 0.07, p < 0.001) and daily temperature ranges  differed significantly among reef zones, with the fringing reef having the highest temperatures (averaging 28.5 ± 1.1 °C and ranging from 21.5 °C to 31.3 °C), and the forereef having the lowest  and site . More forereef corals  experienced partial mortality than fringe reef corals . All focal coral colonies remained alive over the course of the >3-year sampling period, although 50% (27/54) of colonies exhibited partial mortality  of colonies and comprised less than 1% of the total Symbiodiniaceae community in each of these colonies  of aminotypes . Four colonies were excluded from sequencing and analyses due to poor RNA quality  of the samples, and high-quality sequence reads were obtained from 86%  is not known, and therefore these samples are referred to as ‘non-detection’ at the colony-level. It is possible that some Symbiodiniaceae cells across the landscape of a coral surface are infected, whereas others are not, potentially affecting detection. Other coral-Symbiodiniaceae holobionts do not always exhibit the high prevalence of dinoRNAV mcp detections as reported here. For example, under some conditions, dinoRNAV prevalence in Acropora hyacinthus-Symbiodiniaceae holobionts is more heterogeneous and episodic  with most being dominated by a single aminotype. Seventy-five percent of samples  and was highest in the fringing reef , and the forereef trended towards differing from the fringe and the back in composition as well  and back  reefs, but not in the forereef . Aminotype richness did not correlate with Symbiodiniaceae density . When samples from colonies with ambiguous health trajectories were excluded and timepoints were grouped according to their proximity to the reef bleaching event, dispersion was significantly higher during reef bleaching timepoints compared to ambient temperature timepoints , colony health trajectory significantly correlated with composition of dinoRNAV aminotypes . There was not an interaction between health trajectory and ambient versus bleaching timepoints .With all reef types considered together, aminotype dispersion varied across sampling timepoint Fig. . DispersPorites lobata colonies across multiple years and reef environments. Over 90% of the sampled colonies had detectable dinoRNAV infections, whose composition and diversity differed among reef zones, indicating that environmental conditions were deterministic in viral-host dynamics. DinoRNAV aminotype variability increased during the reef-wide thermal stress event, suggesting an increase in viral productivity, and this pattern was strongest in the colonies that exhibited partial mortality during the study. Such lineage-specific inferences advance coral reef virology by identifying host-virus interactions that may drive ecosystem impacts.Most shallow-water coral holobionts live in conditions that are close to the upper limit of their thermal tolerance , 63. As Porites lobata colonies are found in the fringe, back and forereef zones of Moorea and maintain a highly specific association with Cladocopium C15 Symbiodiniaceae in all zones  reported here . Symbiodiniaceae are vertically transmitted in Porites eggs, and this mode of transmission may contribute to high colony-level infection prevalence. The dinoRNAV detections in this study are most parsimoniously interpreted as representative of active infections, rather than endogenized viral elements (EVEs) in the Symbiodiniaceae genome. This is because (1) a genome-wide exploration of dinoRNAVs detected zero mcp EVEs in a Cladocopium C15 genome [mcp genes were successfully amplified from unfractionated coral cDNA but were not amplifiable from DNA purified from the same samples in parallel . The 778 detected aminotypes encompass at least three distinct groups of dinoRNAVs, based on phylogenetic and sequence similarity  93% 50/5 reportedP. lobata-Cladocopium C15 colonies. When timepoints were grouped according to their proximity to the reef bleaching event (timepoints of ambient temperature before and after 2019 versus the 2019 timepoints that fell during the bleaching event), dispersion of aminotypes was significantly higher during the reef bleaching timepoints. This pattern was primarily driven by colonies exhibiting partial mortality  suggest that temperature can increase dinoRNAV productivity with potential connections to host and colony health. Infection prevalence and fitness impacts at the cellular level in Symbiodiniaceae constitute an important next step to deciphering the functional roles of dinoRNAVs. Taken together, our findings provide the first characterization of coral-holobiont viruses at the reef scale and demonstrate that infection patterns of dinoRNAVs appear to be driven, at least in part, by environmental fluctuations that will intensify under global climate change.By quantifying dinoRNAV prevalence in individual colonies across years in a bleaching-resistant coral holobiont, this study shows that dinoRNAVs are a persistent component of a multi-partite symbiosis. Environmental conditions throughout the reef shaped the composition of viral ISMEJCOMMS-22-00210A-T-s01ISMEJCOMMS-22-00210A-T-s02ISMEJCOMMS-22-00210A-T-s03


PSME2, NAMPT, APOA1, APOA4, and Hsa-Let-7g) previously validated at 16–20 weeks for the prediction of sPTB ≤ 32 weeks might be useful in first-trimester nullipara. Sixty (60) nulliparous women (40 with sPTB ≤ 32 weeks) who were free of comorbidities were randomly selected from the King’s College Fetal Medicine Research Institute biobank. Total PCF RNA was extracted and the expression of panel RNAs was quantitated by qRT-PCR. The analysis employed, primarily, multiple regression with the main outcome being the prediction of subsequent sPTB ≤ 32 weeks. The test performance was judged by the area under the curve (AUC) using a single threshold cut point with observed detection rates (DRs) at three fixed false positive rates (FPR). The mean gestation was 12.9 ± 0.5 weeks (range 12.0–14.1 weeks). Two RNAs were differentially expressed in women destined for sPTB ≤ 32 weeks: APOA1 (p < 0.001) and PSME2 (p = 0.05). APOA1 testing at 11–14 weeks predicted sPTB ≤ 32 weeks with fair to good accuracy. The best predictive model generated an AUC of 0.79 (95% CI 0.66–0.91) with observed DRs of 41%, 61%, and 79% for FPRs of 10%, 20%, and 30%, including crown–rump length, maternal weight, race, tobacco use, and age.The first-trimester prediction of spontaneous preterm birth (sPTB) has been elusive, and current screening is heavily dependent on obstetric history. However, nullipara lack a relevant history and are at higher risk for spontaneous (s)PTB ≤ 32 weeks compared to multipara. No available objective first-trimester screening test has proven a fair predictor of sPTB ≤ 32 weeks. We questioned whether a panel of maternal plasma cell-free (PCF) RNAs ( Preterm birth (PTB) is the leading cause of perinatal mortality and neonatal morbidity worldwide. Over 15 million PTBs annually lead to more than a million childhood deaths and lifelong sequelae for many survivors ,3, theirCurrent screening for PTB is heavily dependent on obstetric history, but nullipara lack an obstetric history and are at higher risk for spontaneous (s)PTB ≤ 32 weeks compared to multipara . No avaiPSME2, NAMPT, APOA1, APOA4, and Hsa-Let-7g) measured at 16–20 weeks may be a good predictor of sPTB ≤ 32 weeks [Prior reports demonstrate a maternal blood test consisting of five plasma cell-free (PCF) RNAs  subject to the following conditions: nulliparous, healthy, and a pregnancy ending either at term or with sPTB ≤ 32 weeks. There were 20 controls and 40 cases. The racial distribution was the same for cases and controls: 50% self-described White and Black. Women who developed preeclampsia or had known comorbidities were excluded from this study. Gestational age was based on the first-trimester crown–rump length (CRL). RNA was extracted from 500 µL of EDTA plasma using a proprietary method . The mean total RNA extracted was 26.7 ± 9.5 µg/mL . RNA yield was assessed with a Nano spectrometer  and integrity confirmed by the Agilent Bio-analyzer . RNA expression was quantified by qRT-PCR, as described previously [mRNA RT: RNA samples were diluted with a master mix including dNTP mix, Omniscript Reverse Transcriptase, and Random Primer . mRNA was converted into cDNA at 37 °C for 60 min, per manufacturer instructions. miRNA RT: miRs were polyadenylated using the Invitrogen NCode miRNA First-Strand cDNA Synthesis Kit . The polyadenylated microRNA was reverse-transcribed to generate the first strand of cDNA per manufacturer’s instructions.PSME2, NAMPT, APOA1, APOA4, and Hsa-Let-7g) plus normalization and spike genes, so all three RNAs were run in the same well. One-µL RT samples were prepared for the preamplification mix reaction and underwent twelve cycles. Two µL of preamplification cDNA samples were diluted into 10µL PCR reaction mix, followed by RT PCR using SYBR Green Supermix . Threshold cycles  of qPCR reactions were extracted using QuantStudio™ Software V1.3 . Delta–delta CT method was used to calculate the expression of RNAs and then normalized.Preamplification and qPCR: Multiplex qPCR reactions used SYBR green and the ViiA 7 Real-Time PCR System . The primers were custom-designed and synthesized by Integrated DNA Technologies  . The proAll statistical analyses were performed by one author (HSC) using SAS 9.4 . Initial studies of laboratory tests are often modest in size, and metrics such as sensitivity, specificity, and negative/positive predictive values are highly influenced by disease prevalence. In contrast, the AUC is less affected by disease prevalence and provides an aggregate measure of performance across all classification thresholds and is one of the most useful parameters to evaluate a predictive model . An AUC Gaussian modeling was not possible with the sample size, so a variety of linear regressions were tested with and without log base 2 transformation of expression and conversion to multiples of the median (MoM) . The matp = 0.09). Three women smoked tobacco: 1 control and 2 cases. The mean (±SD (range)) GA at delivery was 39.99 ± 0.8 weeks (39.1–41.8 weeks) for controls and 27.9 ± 2.3 weeks (24.1–31.8 weeks) for cases.The mean MA (±1SD) was 32.1 ± 1.6 y and the mean GA was 12.9 ± 0.1 weeks (12.0–14.1 weeks). Two controls were sampled at 14.0 and 14.1 weeks; the remaining 18 controls and 40 cases were sampled ≤13.9 weeks. The mean MW was 72.9 ± 15.5 kg. There were no differences between the case and control groups in either the maternal characteristics or the gestation at sampling. There was a trend toward obesity in cases (p = 0.012).The five RNAs comprising the current panel were previously shown to have low expression in controls but overexpressed in cases ,10, and Two of the five panel RNAs, APOA1 and PSME2, were differentially expressed in women destined for sPTB ≤ 32 weeks . One subThe predictive accuracy of the significant models is listed in APOA1 = 0.73 (95% CI 0.60–0.87) provided a DR = 75%. This DR is similar to the RNA-only AUC = 0.76 (95% CI 0.65–0.87) achieved in a prospective cohort study at 16–20 weeks [APOA1 with CRL, MW, MA, race, and tobacco use achieved an AUC = 0.79 (95% CI 0.66–0.91) for sPTB ≤ 32 weeks with a DR = 79%. This is similar to the AUC = 0.83 (95% CI 0.74–0.92) achieved in the 16–20-week samples with a DR = 77% when race and a history of prior PTB were combined [No validated clinical screening test to date has provided even a ‘fair’ predictive accuracy  for sPTB ≤ 32 weeks in the first trimester of parous, much less nulliparous, women. The current study is the third validation study of this five RNA panel specifically selected based on their relationship to a unique set of myometrial RNAs overexpressed in women delivering <32 weeks . Like th20 weeks , suggestcombined . The facWhile medical history, CL, and fFN are used clinically to identify women at risk for sPTB, studies suggest the popularity of these tests is not based on their predictive accuracy, as all three have repeatedly been shown to be clinically poor tests ,5,6. RatOthers have sought biomarkers for sPTB in asymptomatic women ,18,19,20NAMPT, which in the second trimester is a predictor of preeclampsia and EOP [MMP8, SRPK1, S100A9, and S100A8. No validation study has been published to date. In the current first-trimester validation study and in our prior second-trimester cohort validation study [PSME2 was predictive of sPTB ≤ 32 weeks in both the first and second trimesters. Yet, in the first trimester, it did not add to the predictive accuracy of APOA1 and may be a sample size issue. Not being able to detect the expression of NAMPT cannot be explained by a laboratory failure, as APOA4 had the highest undetectable rates in the first trimester.There is reason to believe one or more RNAs comprising the current five RNA panel will be predictive of preeclampsia and/or EOP in the first trimester, just as occurred at 16–20 weeks . Del Vec and EOP . Their rThe clinical value of a screening test would be increased exponentially if there was an effective intervention available. Prophylactic progesterone is recommended in several scenarios. Additionally, while some professional organizations consider vaginal progesterone the standard of care for certain indications , there iAs encouraging as the association of differentially expressed PCF RNAs is with pregnancy pathology, there is a clear need for large, real-world prospective studies to confirm the reproducibility of RNA markers among laboratories, and their predictive accuracy when other pathologies are present. APOA1 at 16–20 weeks is a good predictor of EOP and not sPTB ≤ 32 weeks [APOA4 in immortalized human pregnant myometrial cells increases both intracellular calcium and cell contraction frequency [Interestingly, 32 weeks . These d32 weeks  may alte32 weeks . In silirequency . This suAPOA1 is predictive of sPTB ≤ 32 weeks at 12 weeks, but also of EOP at 16–20 weeks. Thus, the maternal disease phenotype may depend as much on the RNA transporter as it does on the contained RNA. In support of this hypothesis, Yoffe et al. [PCF RNAs rarely circulate naked; rather, they are associated with transporters such as extracellular vesicles, carrier proteins such as Argonaute 2 , or highe et al.  describee et al. , miR LetThe present validation study has several strengths. First, it is the third successful validation study of the five PCF RNA panel. Second, the samples were well documented and randomly chosen by an otherwise uninvolved party from a large biobank in a country previously not used for the testing of the panel. Third, the sample size, though limited, is similar to or larger than similar efforts from other laboratories. A fourth strength of the study is the use of a plasma RNA extraction method that increases total RNA yield from the nanograms extracted by existing commercial kits to microgram levels . No PCF The study also has limitations. First, subject selection occurred a decade ago, early in our experience with the plasma transcriptome. We intentionally excluded women who either developed preeclampsia or had other medical comorbidities, out of concern comorbid conditions in a limited sample size could have significant but nonidentifiable effects. As a result, we have no information about the applicability of the five PCF RNA panel in the first trimester to preeclampsia. Second, the case-control nature of the study engenders a high prevalence of cases and creates the potential for a biased sample selection. The insulated sample selection process from a very large biobank should have minimized sample selection bias. Further, the use of AUC rather than sensitivity, specificity, etc., helps offset the impact of high disease prevalence. A third potential weakness is a reliance on regression analysis, as overfitting could exaggerate the AUCs. However, the changing expression of the marker RNAs as gestation advances prevented us from testing first-trimester expression with AUC curves generated from our larger second-trimester samples. Lastly, there was a limited number of maternal characteristics available for inclusion in the regression models. However, this particular weakness would have acted to lower the composite AUC rather than falsely elevate it. The inclusion of additional characteristics such as socioeconomic status, prepregnancy diseases  and a family history of a prior preterm birth in the future may enhance accuracy ,37.Including the present study, there are now five validation studies ,10,18,20EU Patent No. 2646554 and US Patent No. 10,954,564.